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Asymptomatic carriers (ACs) of pathogenic biallelic mutations in causative genes for primary hemophagocytic lymphohistiocytosis (HLH) are at high risk of developing life-threatening HLH, which requires allogeneic hematopoietic stem cell transplantation (HSCT) to be cured. There are no guidelines on the management of these asymptomatic patients. We analyzed the outcomes of pairs of index cases (ICs) and subsequently diagnosed asymptomatic family members carrying the same genetic defect. We collected data from 22 HSCT centers worldwide. Sixty-four children were evaluable. ICs presented with HLH at a median age of 16 months. Seven of 32 ICs died during first-line therapy, and 2 are alive after chemotherapy only. In all, 23/32 underwent HSCT, and 16 of them are alive. At a median follow-up of 36 months from diagnosis, 18/32 ICs are alive. Median age of ACs at diagnosis was 5 months. Ten of 32 ACs activated HLH while being observed, and all underwent HSCT: 6/10 are alive and in complete remission (CR). 22/32 ACs remained asymptomatic, and 6/22 have received no treatment and are in CR at a median follow-up of 39 months. Sixteen of 22 underwent preemptive HSCT: 15/16 are alive and in CR. Eight-year probability of overall survival (pOS) in ACs who did not have activated HLH was significantly higher than that in ICs (95% vs 45%; P = .02), and pOS in ACs receiving HSCT before disease activation was significantly higher than in ACs receiving HSCT after HLH activation (93% vs 64%; P = .03). Preemptive HSCT in ACs proved to be safe and should be considered.

Chimeric antigen receptor (CAR) T-cell therapy is a new, effective treatment for patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukaemia (ALL). Tisagenlecleucel achieved a complete remission (CR) rate and minimal residual disease (MRD) negativity of 81% at 3 months in the pivotal study; overall survival (OS) was 76% at 12 months (Maude et al, 2018). Real world data confirmed similar outcomes, with 1-year OS of 77% and event free survival (EFS) of 52% (Pasquini et al, 2020). Relapse can occur in the form of CD19 negative or CD19 positive ALL. The latter is associated with lack of persistence of the CAR T product. B-cell aplasia (BCA) is an indirect measure of CAR T presence. Early ( In our centre, we administered maintenance therapy to a cohort of children with early loss of BCA. When compared to UK patients undergoing SCT for the same indication, we noted promising early outcomes. We report the findings here. We collected data on children with r/r ALL treated with tisagenlecleucel at Great Ormond Street Hospital (GOSH) from January 2018 to January 2021 who presented loss of BCA without evidence of disease (negative molecular or flow cytometry MRD) within 12 months from infusion. Loss of BCA was defined as peripheral B-cell count ≥0.10 x 10^9/L or bone marrow (BM) CD19+ events ≥0.1%. We compared outcomes of children who received maintenance as per UKALL 2011 protocol at GOSH to those who received SCT for the same indication from all UK paediatric centres. Fourteen patients from GOSH met the inclusion criteria. Four had loss of BCA after 6 months from CAR T infusion, none of them received additional therapy and they are all alive and in CR at a median of 535 days after CAR T infusion (Figure 1, A and B). Ten patients recovered B cells at Management of patients who experience early loss of BCA after CAR T is challenging and there are little data to support optimal treatment. In our experience, maintenance therapy compared favourably with SCT with similar rate of OS and EFS. Of note, 2/5 patients died of TRM in the SCT group highlighting the toxicity of this approach in such heavily pre-treated patients. On the other hand, maintenance is a well tolerated, low-cost treatment which can be easily delivered on an out-patient basis. Our preliminary data support investigation of this strategy in larger, prospectively-recruited cohorts of patients. Moreover, our preliminary data suggest that the time of loss of BCA is a crucial clinical parameter, as children who developed it within 2 months from infusion had the worst outcome, possibly reflecting prior therapy intensity and its impact on autologous T cells. Figure 1 Figure 1. Disclosures Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Ghorashian: UCLB: Patents & Royalties: CARPALL; Novartis: Honoraria.

Background 'Off-the-shelf' CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells. Investigational medicinal product (IMP) Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments. Trial sponsorship & approvals The study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised. Study aims and Objectives The study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information. Eligibility, infusion and outcomes The study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later. Conclusions Feasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. Disclosures Qasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.

Background: Pediatric patients younger than two years of age with acute myeloid leukemia (AML) commonly receive a chemotherapy-based myeloablative conditioning regimen before allogeneic hematopoietic stem cell transplantation (HSCT). The optimal choice of cytotoxic agents is still controversial. Methods: A retrospective EBMT-registry based study was conducted to investigate the impact of different chemotherapy-based conditionings on the outcomes in young children. Children younger than two years of age receiving a first HSCT of bone marrow (BM), peripheral blood stem cells (PBSC) or cord blood (CB) from matched siblings (MSD) or unrelated donors (UD) in first complete remission (CR1) between 2000 and 2019 were included. Busulfan/Cyclophosphamide (BuCy) and BuCy/Melphalan (BuCyMel) were the most frequent combinations on which this analysis focused. The primary endpoint was leukemia-free survival (LFS). Multivariate analysis adjusting for differences between the conditioning regimens and risk factors influencing outcome was performed using the Cox's proportional hazards regression model. Results: 289 patients (56% male) transplanted at a median age of 1.2 years (IQR 0.9-1.6) after BuCy (164, 57%) or BuCyMel (125, 43%) were included. 184 (64%) patients received BM, 71 (24%) CB and 34 (12%) PBSC from UD (201, 70%) and MSD (88, 30%). In-vivo T-cell-depletion (TCD) was performed in 160 (58%, missing data 14) of the HSCTs with anti-thymocyte-globulin (ATG, 153) or alemtuzumab (7). Ex-vivo TCD was performed in 13 (5%, missing data 3) of the HSCTs. Graft-versus-host-disease (GvHD)-prophylaxis was Cyclosporin-A-based in 90% of the HSCTs. Median follow-up (FU) was 4.9 years (95% CI 3.9-5.5). After a median FU of 4 years, 4-y-LFS after BuCyMel (74.3%, 95% CI 65.1-81.4) was significantly better compared to BuCy (59.7%, 95% CI 51.2-67.2), hazard ratio (HR) 0.56 (95% CI 0.35-0.90, P=0.02). Overall survival (4-y-OS) after BuCyMel (77.2%, 95% CI 68.1-84.0) was significantly better compared to BuCy (66.6%, 95% CI 58.0-73.8), HR=0.58 (95% CI 0.35-0.97, P=0.04). No significant differences were found in the probability of relapse (4-y-RI (whole cohort) 26.2% (95% CI 21.0-31.7), HR of BuCyMel 0.59 (95% CI 0.34-1.02), P=0.06), non-relapse mortality (4-y-NRM (whole cohort) 7.8% (95% CI 5.0-11.4), HR of BuCyMel 0.49 (95% CI 0.19-1.24), P=0.13) and incidence of acute grade II-IV GvHD at day 100 (day-100-aGvHD II-IV (whole cohort) 36.8% (95% CI 31.2-42.5), HR of BuCyMel 0.59 (95% CI 0.35-1.01), P=0.06). Incidence of chronic GvHD (4-y-cGvHD (whole cohort)) was 9.8% (95%-CI 6.3-14.2). The donor type had no significant influence on the outcome. Conclusion: Bu-based conditionings of HSCT for infants with AML at high risk of relapse offer a high probability of cure. Conditioning with three alkylators (BuCyMel) resulted in better LFS and OS compared with two alkylators (BuCy) without significantly increasing the risk of both NRM and aGvHD. Future trials will evaluate the impact of the more recently introduced alkylator Treosulfan within the conditioning of HSCT in pediatric AML. Disclosures Peters: Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Moraleda: Pfizer: Other: Educational Grants, Research Funding; Sanofi: Other: Educational Grants, Research Funding; MSD: Other: Educational Grants, Research Funding; ROCHE: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Takeda: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Sandoz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Educational Grants, Research Funding; NovoNordisk: Other: Educational Grants, Research Funding; Janssen: Other: Educational Grants, Research Funding; Celgene: Other: Educational Grants, Research Funding; Amgen: Other: Educational Grants, Research Funding. Biffi: BlueBirdBio: Consultancy, Other: Advisory Board. Corbacioglu: Gentium/Jazz Pharmaceuticals: Consultancy, Honoraria.

Introduction: Immunotherapies like Inotuzumab ozogamicin (InO), Blinatumomab and Chimeric antigen receptor T cell therapy (CAR-T) have revolutionized outcomes in patients with Relapse/Refractory Acute Lymphoblastic Leukaemia (R/R ALL) with event free survival (EFS) of 50% at 1 year (Maude et al. 2018). Optimal phasing of these agents has not been clearly defined and depends on antigen expression on blasts, CNS status, T cell number and prior disease response. InO, a CD22 targeting antibody-drug conjugate has proven efficacy in adults (Kantarjian et al., 2016) and children as a bridge to allogeneic stem cell transplantation (allo-SCT) with a favourable toxicity profile (Brivio et al. 2021). However, outcomes following the use of InO prior to CAR-T therapy are still not established. This study provides a retrospective analysis of children and young adults who received InO as part of pre-CAR-T management (before leucapheresis or as bridging therapy to CAR-T infusion). Methods: Patients aged 0-25years with R/R ALL were eligible for the study if they received InO pre-CAR-T therapy. Retrospective data collection was performed using a standardised form from CAR-T centres in the UK. Response to CAR-T therapy and/or relapse was evaluated at 1, 3, 6, 12 months or at last follow-up. Results were compared to a control cohort of R/R ALL patients treated with tisagenlecleucel but without preceding InO, over a contemporaneous period, at the largest paediatric CAR-T centre in the UK. Results: Fourteen patients from 5 paediatric and young adult centres in the UK received InO after screening for CAR-T therapy. InO was used pre-leucapheresis, as bridging and for both in 2, 11 and 1 respectively. Two were excluded from outcome analysis (1 adolescent with Trisomy 21 and transfusion induced hepatic siderosis died due to VOD while awaiting CAR-T production and 1 failed CAR-T manufacture despite achieving an adequate T cell harvest as per manufacturer guidance). Twelve (85%) patients were able to receive CAR-T infusion at a median of 1 month (range 0.7-5.6 months) from first InO dose. InO was well tolerated with 21% developing febrile neutropenia and grade 4 cytopenias. Use of InO pre-leucapheresis led to successful manufacture of CAR-T cells in 2/3 (66%). InO group was compared with 27 children who received CAR-T without preceding InO over the same time period. Table 1 summarises patient and CAR-T characteristics of the two groups which were comparable. Median follow-up of the InO and non-InO groups was 10 months (range 2.8- 30.1 months). In the InO group(n=12), 3 (25%) remained leukemia free at last follow up. Nine patients (75%) relapsed. All relapses occurred within 6 months from CAR-T infusion. Seven (58%) of those who relapsed died at a median of 7.8 months post CAR-T infusion and 2 (16.6%) were salvaged with further therapy. Subsequent therapy (alternate CAR-T and/or allo-SCT) was carried out in 4/12 (33%) patients in the InO group and in 5/27 (18.5%) in the non-InO group (p= 0.2). EFS was significantly higher in the non-InO group (53% vs 12%, p=0.0009), as was OS (86% vs 13%, p=0.004) (Figure 1). Conclusion: This study provides a direct comparison between two contemporaneously treated cohorts with R/R ALL receiving CAR-T therapy. InO prior to CAR T cell therapy was well tolerated despite the cohort being heavily pre-treated except for one patient who developed fatal VOD prior to CAR T cell infusion. InO was given for disease debulking, rather than to achieve MRD negativity and hence majority of the cohort (75%) received only 1 dose of InO in bridging rather than the usual schedule of three doses weekly per cycle. Outcomes for the non-InO group were comparable to those treated on the ELIANA study but the OS of the InO group was significantly lower (13% vs 86% p= 0.004). Potential reasons include an impact of InO on function of CAR-T cells in vivo or a deleterious effect of InO on the B cell compartment such that CAR-T cells prove less effective. Due to the retrospective nature of the study, it is possible there was an inherent bias to use InO in patients with more resistant disease, although the very comparable pre CAR-T bone marrow disease burden in each group suggests this was not the case. The small size of the cohorts may also have exaggerated differences in outcome. Ultimately, randomised controlled studies of InO pre-CAR-T are required before firm conclusions about its impact on outcomes post CAR-T therapy can be ascertained. Figure 1 Figure 1. Disclosures Sharplin: Kite Gilead: Honoraria; Novartis: Other: Travel Support. Nicholson: BMS/Celgene: Consultancy; Kite, a Gilead Company: Other: Conference fees, Speakers Bureau; Novartis: Consultancy, Other: Conference fees; Pfizer: Consultancy. Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Ghorashian: Novartis: Honoraria; UCLB: Patents & Royalties.

Reticular dysgenesis (RD) is a rare congenital disorder defined clinically by the combination of severe combined immunodeficiency (SCID), agranulocytosis, and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 were identified to cause the disorder. Hematopoietic stem cell transplantation (HSCT) is the only option to cure this otherwise fatal disease. Retrospective data on clinical presentation, genetics, and outcome of HSCT were collected from centers in Europe, Asia, and North America for a total of 32 patients born between 1982 and 2011. Age at presentation was

Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naive CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

Mutations in the tetratricopeptide repeat domain 7A (TTC7A)gene cause a severe form of very early-onset inflammatory bowel disease (VEOIBD). TTC7A has a crucial role in chaperoning the enzyme phosphatidylinositol-4-Kinase-3-alpha from the Trans-Golgi apparatus to the plasma membrane to facilitate phosphorylation of phosphatidylinositol (PI). The composition of the plasma membrane in particular levels of phosphorylated PI (PI-4P) are crucial for preserving epithelial cell polarity and survival . The clinical spectrum of the disease varies from multiple intestinal atresias (MIA) to severe autoimmune enterocolitis clinically evident by infantile-onset intestinal obstruction/failure, bleeding and diarrhoea. Furthermore, the disease can be associated with severe immunodeficiency or autoimmune phenomena owing to the central role of TTC7A in thymic architecture. Limited published data on TTC7A deficient patients suggests a median survival

In vivo T-cell depletion might contribute to the delayed immune reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late, and no antithymocyte globulin (ATG) on immune reconstitution and outcome. One hundred twenty seven children receiving UCBT in London or Utrecht were divided into 3 groups: early ATG (days -9 to -5; n = 33), late ATG (days -5 to 0; n = 48), and no ATG (n = 46). The no-ATG group received mycophenolate mofetile + cyclosporin A as graft-versus-host disease (GVHD) prophylaxis, while the ATG groups received cyclosporin A + prednisone. End points studied were survival, immune recovery, infections, and GVHD. The probability of survival was similar in all groups: no ATG, 71% ± 8%; early ATG, 68% ± 9%; and late ATG, 61% ± 7%. CD3(+), CD4(+), and CD4(+)-naive T-cell counts were significantly higher (P < .001) in the no-ATG group at 1, 2, 3, 6, and 12 months post-UCBT. In the no-ATG group, significantly fewer viral reactivations (P = .021) were noted. A higher probability of severe acute GVHD (aGVHD; 31%) was found in the no-ATG group compared with 18% (P = .018) for early-ATG and 5% (P < .001) for late-ATG groups. This was not associated with more chronic GVHD (cGVHD).

Introduction: The CARPALL study (NCT02443831) employed a novel CD19CAR (CAT-41BBz CAR) with a faster off rate than the Kymriah FMC63-41BBz CAR (CAT 3.1x10-3s-1, FMC 6.8 x 10-5s-1), with equivalent on-rate (CAT 2.2 x 105, FMC 2.1 x 105). We herein report updated outcomes and CAR T cell persistence with an additional 6 months follow up from a submitted manuscript (Ghorashian et al., Nat Med, submitted) Methods: Patients aged CAT-41BBz CAR T cells were generated by magnetic bead activation of leucapheresed PBMCs, lentiviral transduction, followed by bioreactor expansion and magnetic bead removal prior to cryopreservation. All patients received lymphodepletion (fludarabine + cyclophosphamide) followed by 1x106/kg CAR T cells. Presence of CAR T cells in the blood and bone marrow (BM) was assessed (flow cytometry and qPCR) monthly for 6 months, then 6 weekly to 1 year and then 3 monthly. BM MRD was assessed (IgH qPCR, flow cytometry) at the same time-points up to 2 years to establish durability of responses as a stand-alone therapy. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving molecular remission. Results: Of 17 patients recruited, 14 were treated due to manufacturing failure in 3 patients.The median age was 9 years (range 1-19 years). All patients had advanced ALL with a median of 4 prior therapy lines. 10 of 14 patients (71%) had relapsed post allogeneic SCT. Prior to lymphodepletion, 4 patients had >5% BM disease, 6 had disease between 5x10-2and 1x10-5, 4 were BM MRD negative having had recurrent isolated CNS disease. Median transduction efficiency was 31% (range 16.5 to 96.4%). 12/14 treated patients received the anticipated dose of 1x106CAR T cells/kg (2 received 0.9x106/kg). Considering all evaluable patients, (n=14 for CAR T cell persistence by qPCR, n=13 by flow) the geometric mean of Cmax was 128 912/µg DNA and of the area under the curve between D0 and D28 was 1,721,355 copies/ µg DNA (Table 1). At the point of maximal expansion, a median of 35% of circulating T cells were CAR+. Median half-life was 34 days (range 3-102). CAR T cells continued to be detectable by qPCR in 11 of 14 (79%) patients at last assessment and by flow cytometry up to 30 months post infusion in 8 of 13(61%). Median duration of CAR T persistence by flow was 261 days (range 7-917). 3 patients failed to have persistence of CAR T cells beyond 1 month. T cell mediated anti-CAR specific cytotoxic activity was detected in 2/2 evaluable patients. Updated persistence data will be presented at the meeting Cytokine release syndrome (CRS) occurred in 13 (93%, grade 1 n=9, grade 2 n=4). None developed ≥grade 3 CRS, had CRS-related ICU admission, or received Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γand IL-10. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously. One patient had grade 4 leucoencephalopathy presumed due to chemotherapy as well as grade 5 sepsis. Ten patients (71%) had grade 3-4 cytopenia persisting beyond day 28 or recurring afterthis. 12/14 (86%) patients achieved molecular complete or continuing complete remission at a median of 30 days post infusion (range 30-90 days, Table 2). At a median follow-up of 20.3 months, 4/14 (29%) evaluable patients remain MRD negative. 5 relapsed with CD19-disease, 1 with CD19+ disease. The median duration of EFS (based on death or morphological relapse) has not been reached, 12 month EFS = 52%, OS = 70% (Figures 1, 2 and Table 3). Conclusion: We noted excellent CAR T cell expansion and persistence in a ALL cohort treated with the fast off-rate CAT-41BBz CAR despite their lower BM disease at treatment compared to other studies. The kinetics documented for all evaluable patients showed a 5-fold greater CAR T cell expansion and 2-fold longer half-life than responders in published series utilising tisagenlecleucel in a similar ALL cohort (Mueller et al., Blood 2017). Patients had a favourable toxicity profile with no severe (grade 3-4) CRS and equivalent disease outcomes to the ELIANA study despite having similarly advanced disease (Maude et al., NEJM 2018292). These data suggest long lived CAR T cell persistence supports stand-alone therapy for ALL with durable responses. Disclosures Ghorashian: Celgene: Honoraria; novartis: Honoraria; UCLB: Patents & Royalties: UCLB. Kramer:UCLB: Patents & Royalties. Ciocarlie:Servier: Other: Financial Support. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Amrolia:UCLB: Patents & Royalties.

Introduction CAR T-cell therapies directed against CD19 or CD22 have shown remarkable activity in r/r B-ALL but relapse due to target antigen down-regulation/loss has been the major cause of treatment failure. To address this, we developed AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously. Preliminary results of this study showed an acceptable safety profile and encouraging efficacy in pediatric r/r B-ALL (all 6 patients treated in active doses ≥3 x 106 CAR T-cells/ Kg achieved complete remission (CR) with negative minimal residual disease (MRD) (Amrolia et al, Blood 2018 132:279). Here we present the updated results of CAR naïve patients treated at the active doses. Methods & Patients We constructed a bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. This second-generation CAR incorporated an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR. The cell product was manufactured on a semi-automated/closed process. Patients (aged 1‒24 years) with high risk relapsed (IBFM criteria) or refractory B-ALL, adequate performance score/organ function, an absolute lymphocyte count ≥0.5 x 109/L are eligible. Patients with CNS Grade 3 disease, active graft versus host disease are excluded. Patients receive lymphodepletion with 30 mg/m2/day fludarabine x 4 days and 500 mg/m2/day cyclophosphamide x 2 days prior to AUTO3 infusion. Three dose levels were explored (1 x 106, 3 x 106, and 5 x 106 cells/kg), CAR T cells are infused as a single (for Results As of the data cut-off date (June 17, 2019), 10 patients received AUTO3 at 3 x 106 cells/Kg (n= 5, of whom 1 received split dose) or 5 x 106 CAR T-cells/Kg (n= 5, all of them received single infusion). The median transduction efficiency was 15.5% (range 8.6‒39.3%). Median age was 8.5 years (range 5‒16 years) and 5 (50%) patients had prior haemopoietic stem cell transplant (HSCT). One patient (10%) had prior anti-CD19 CAR-T cells. The disease burden at Day ‒7 ranged from 0 to 38% (median 7.5%) blasts. Among the 10 treated patients, 2 have not completed the 30 days post-infusion DLT observation period as of the cut-off date. No deaths or DLTs were observed. MTD has not yet been reached. The most common grade (Gr) ≥3 adverse events were neutropenia (60%), anaemia (50%), pyrexia (40%), febrile neutropenia (40%) and thrombocytopenia (30%). Eight patients (80%) had Gr 1 cytokine release syndrome (CRS), one (10%) had Gr 2 CRS; no ≥Gr 3 CRS was observed. Only one patient was treated with tocilizumab and none required admission to ICU due to CRS. One patient (10%) experienced Gr 1 neurotoxicity; no ≥ Gr 2 neurotoxicity was reported. Among the 9 CAR naïve patients, 7 (4 in the 3 x 106 cells/Kg dose cohort, 3 in the 5 x 106 cells/Kg dose cohort) had a minimum of 8 weeks' follow up and were evaluable for efficacy analysis. All 7 patients achieved CR/CRi (100%) following AUTO3 infusion as well as molecular negative remission (100%). After a median follow-up of 8 months (range 2-12), emergence of MRD by PCR occurred in four patients, lack of persistence of circulating CAR T-cells was observed in 3 of the 4 patients. Three relapses were reported including one with CD19 negative/CD22 low expression at 1 year after treatment. One patient in ongoing molecular remission proceeded to HSCT. All the remaining 4 patients in ongoing CR/CRi maintain B-cell aplasia. The median CAR T-cell expansion (expressed as vector copy number per microgram of DNA) at peak was 102K (range 56-128). The median persistence of CAR-T cells in blood was 180 days (range 21-330). Updated data with longer follow up and additional patient data will be presented. Conclusion This interim data analysis demonstrates that AUTO3 at ≥3 x 106 cells dose achieved 100% molecular remission rate with a favourable safety profile, no ≥ Gr 3 CRS or ≥ Gr 2 neurotoxicity was reported. The most common cause of relapse was antigen positive relapse due to lack of CAR T cell persistence. Evaluation of patients with a modified manufacturing process is planned. Disclosures Amrolia: UCLB: Patents & Royalties. Clark:Autolus Ltd: Employment, Equity Ownership. Al-Hajj:Autolus Therapeutics: Employment, Equity Ownership. Cordoba:Autolus: Employment, Equity Ownership. Kotsopoulou:Autolus Therapeutics: Employment, Equity Ownership. Khokhar:Autolus Therapeutics: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Peddareddigari:Autolus Therapeutics: Employment, Equity Ownership.

Cidofovir is preemptively used for controlling adenoviremia and preventing disseminated viral disease in hematopoietic cell transplant (HCT) recipients but does not lead to resolution of viremia without T-cell immune-reconstitution. The lipid-conjugated prodrug of cidofovir, brincidofovir, has improved oral bioavailability and achieves higher intracellular concentrations of active drug. We present retrospective multicenter data comparing the kinetics of viremia and toxicities following preemptive treatment with and brincidofovir in children and adolescents diagnosed with HCT-related adenoviremia. Forty-one episodes (18 = brincidofovir; 23 = cidofovir) of antiviral therapy were observed in 27 patients. The 2 groups had comparable immune-reconstitution and viral burden. Major (≥2 log-reduction in 2 weeks; n = 13) and minor (≥1 to ≤2 log-reduction in 2 weeks; n = 2) virological responses were observed in 15 (83%) brincidofovir episodes compared to only 2 (9%) major virological responses with cidofovir (P < .0001). Brincidofovir mediated major responses in 9 of 11 cidofovir-unresponsive patients and resulted in complete responses (CR) despite significant lymphopenia (Brincidofovir vs cidofovir; CR = 13 (80%) vs 8 (35%); median lymphocyte count = 320/μl vs 910/μl; P < .05). One patient experienced abdominal cramps and diarrhea necessitating interruption of brincidofovir and none developed nephrotoxicity with brincidofovir. Thus, brincidofovir is well-tolerated and highly efficacious in controlling adenoviremia during the lymphopenic phase of HCT.

To determine whether in vivo T-cell depletion, which lowers GVHD, abrogates the antileukemic benefits of myeloablative total body irradiation–based conditioning and unrelated donor transplantation, in the present study, we analyzed 715 children with acute lymphoblastic leukemia. Patients were grouped for analysis according to whether conditioning included antithymocyte globulin (ATG; n = 191) or alemtuzumab (n = 132) and no in vivo T-cell depletion (n = 392). The median follow-up time was 3.5 years for the ATG group and 5 years for the alemtuzumab and T cell–replete groups. Using Cox regression analysis, we compared transplantation outcomes between groups. Compared with no T-cell depletion, grade 2-4 acute and chronic GVHD rates were significantly lower after in vivo T-cell depletion with ATG (relative risk [RR] = 0.66; P = .005 and RR = 0.55; P < .0001, respectively) or alemtuzumab (RR = 0.09; P < .003 and RR = 0.21; P < .0001, respectively). Despite lower GVHD rates after in vivo T-cell depletion, nonrelapse mortality, relapse, overall survival, and leukemia-free survival (LFS) did not differ significantly among the treatment groups. The 3-year probabilities of LFS after ATG-containing, alemtuzumab-containing, and T cell–replete transplantations were 43%, 49%, and 46%, respectively. These data suggest that in vivo T-cell depletion lowers GVHD without compromising LFS among children with acute lymphoblastic leukemia who are undergoing unrelated donor transplantation with myeloablative total body irradiation–based regimens.

In this retrospective collaborative study, we have analyzed long-term outcome and donor cell engraftment in 194 patients with Wiskott-Aldrich syndrome (WAS) who have been treated by hematopoietic cell transplantation (HCT) in the period 1980- 2009. Overall survival was 84.0% and was even higher (89.1% 5-year survival) for those who received HCT since the year 2000, reflecting recent improvement of outcome after transplantation from mismatched family donors and for patients who received HCT from an unrelated donor at older than 5 years. Patients who went to transplantation in better clinical conditions had a lower rate of post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment showed that stable full donor chimerism was attained by 72.3% of the patients who survived for at least 1 year after HCT. Mixed chimerism was associated with an increased risk of incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid donor cell chimerism < 50% was associated with persistent thrombocytopenia. These observations indicate continuous improvement of outcome after HCT for WAS and may have important implications for the development of novel protocols aiming to obtain full correction of the disease and reduce post-HCT complications.

Children with primary immunodeficiency diseases, particularly those less than 1 year of age, experience significant toxicity after hematopoietic stem cell transplantation, with busulfan- or melphalan-based conditioning. Treosulfan causes less veno-occlusive disease than busulfan and does not require pharmacokinetic monitoring. We report its use in 70 children. Children received 42 g/m2 or 36 g/m2 with cyclophosphamide 200 mg/kg (n = 30) or fludarabine 150 mg/m2 (n = 40), with alemtuzumab in most. Median age at transplantation was 8.5 months (range, 1.2-175 months); 46 (66%) patients were 12 months of age or younger. Donors were as follows: matched sibling donor, 8; matched family donor, 13; haploidentical, 4; and unrelated, 45. Median follow-up was 19 months (range, 1-47 months). Overall survival was 81%, equivalent in those age less or greater than 1 year. Skin toxicity was common. Veno-occlusive disease occurred twice with cyclophosphamide. Eighteen patients (26%) had graft-versus-host disease, and only 7 (10%) greater than grade 2. Two patients rejected; 24 of 42 more than 1 year after transplantation had 100% donor chimerism. The remainder had stable mixed chimerism. T-cell chimerism was significantly better with fludarabine. Long-term follow-up is required, but in combination with fludarabine, treosulfan is a good choice of conditioning for hematopoietic stem cell transplantation in primary immunodeficiency disease.

Severe combined immunodeficiency (SCID) carries a poor prognosis without definitive treatment by hematopoietic stem cell transplantation. The outcome for transplantation varies and is dependent on donor status and the condition of the child at the time of transplantation. Diagnosis at birth may allow for better protection of SCID babies from infection and improve transplantation outcome. In this comparative study conducted at the 2 designated SCID transplantation centers in the United Kingdom, we show that SCID babies diagnosed at birth because of a positive family history have a significantly improved outcome compared with the first presenting family member. The overall improved survival of more than 90% is related to a reduced rate of infection and significantly improved transplantation outcome irrespective of donor choice, conditioning regimen used, and underlying genetic diagnosis. Neonatal screening for SCID would significantly improve the outcome in this otherwise potentially devastating condition.

Introduction CAR T-cell therapies directed against CD19 or CD22 antigens have shown significant activity in pediatric patients with r/r B-ALL. Whilst complete response (CR) rates of 70‒90% have been observed, relapse due to target antigen downregulation or loss is the major cause of treatment failure. This Phase I/II study evaluates the safety and efficacy of AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously in order to reduce the likelihood of relapse due to antigen loss. Methods & Patients We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized and both CARs are in "2nd generation" format (incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR). The performance of the CD22 CAR was optimized by incorporating a novel pentameric spacer. The cell product was manufactured on a semi-automated and closed process using CliniMACS® Prodigy (Miltenyi Biotec). Patients (1‒24 years of age) with high risk relapse (IBFM criteria) or relapse post-allogeneic stem cell transplant (SCT), adequate performance score/organ function, and an absolute lymphocyte count ≥0.5 x 109/L are eligible. Patients with CNS 3 disease, active graft versus host disease, and clinically significant infection or serious toxicity from prior CAR T-cell are excluded. Patients receive lymphodepletion with 30 mg/m2/day fludarabine x 4 days and 500 mg/m2/day cyclophosphamide x 2 days prior to AUTO3 infusion. Three dose levels are being explored (1 x 106, 3 x 106, and 5 x 106 transduced CAR+ T cells/kg) and CAR T cells are infused as a single (for 25% blasts) dose based on leukemia burden. Bridging therapy is allowed during the manufacturing period. The primary endpoint of Phase I is the frequency of dose-limiting toxicities (DLTs) and key secondary endpoints include proportion of patients achieving a morphological/minimal residual disease (MRD) negative CR, disease-free survival, overall survival, as well as biomarker endpoints including AUTO3 levels and persistence in blood and bone marrow. Results As of the data cut-off date (July 16, 2018), 9 patients have been enrolled and 8 have received AUTO3. It was possible to generate a product in all patients and the median transduction efficiency was 16% (range 9‒34%). Median age was 7.5 years (range 4‒16 years) and 5 (63%) patients had prior SCT. One patient (13%) had prior anti-CD19 CAR-T cells and blinatumomab. The disease burden at Day ‒7 ranged from 0% to 90% leukemic blasts. Eight patients had a minimum of 4 weeks' follow up and were evaluable for safety and efficacy analysis. Three patients received ≤1 × 106 CAR T cells/kg as single dose, 1 patient received 2 × 106/kg as split dose, and 4 received 3 × 106 CAR cells/kg (3 single infusions, 1 split). No AUTO3 related deaths and no DLTs were observed. The most common grade (Gr) ≥3 adverse events were neutropenia (63%), febrile neutropenia (50%), pyrexia (25%), and anemia (25%). Five patients (63%) had Gr 1 cytokine release syndrome (CRS); no Gr 2 or higher CRS was seen. Five patients (63%) experienced neurotoxicity: 4 had Gr 1 and 1 patient (13%) had Gr 3 encephalopathy that was considered likely related to prior intrathecal methotrexate. No patients required ICU admission. Six of 8 patients achieved MRD negative CR, giving an objective response rate of 75% (95% CI 34.9‒96.8%) at 1 month. In patients treated at doses copies/µg DNA in blood at peak) compared to those receiving lower doses (median 10,243 copies/µg DNA). Conclusion This interim data analysis demonstrates for the first time the feasibility and safety of simultaneous targeting of CD19 and CD22 with AUTO3. Promising efficacy was demonstrated at a dose level of 3 × 106 CAR T cells/kg, as 4/4 patients achieved MRD complete remission with no antigen negative escape at this early stage. The study continues to enrol and updated follow up and additional patient data at higher dose levels, as well as cellular kinetics and additional biomarker analysis, will be presented. Disclosures Wynn: Orchard SAB: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Equity Ownership; Chimerix: Research Funding; Genzyme: Honoraria; Bluebird Bio: Consultancy; Orchard Therapeutics: Consultancy; Chimerix: Consultancy. Hough:University College London Hospital's NHS Foundation Trust: Employment. Vora:Amgen: Other: Advisory board; Medac: Other: Advisory board; Novartis: Other: Advisory board; Pfizer: Other: Advisory board; Jazz: Other: Advisory board. Veys:Servier: Research Funding; Pfizer: Honoraria; Novartis: Honoraria. Chiesa:Gilead: Consultancy; Bluebird Bio: Consultancy. Al-Hajj:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership. Cordoba:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership; Autolus Ltd: Patents & Royalties. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Kotsopoulou:Autolus Ltd: Equity Ownership; Autolus Ltd: Employment. Khokhar:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership. Pule:Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL. Peddareddigari:Autolus Therapeutics plc: Equity Ownership; Autolus Therapeutics plc: Patents & Royalties; Autolus Therapeutics plc: Employment.

Background UCART19 is an allogeneic, genetically modified CAR T-cell product (anti-CD19 scFv- 41BB-CD3ζ) manufactured from healthy donor T cells, in which TRAC and CD52 genes have been knocked out to allow its administration in non-HLA matched patients (pts). Aims Preliminary safety/anti leukemic and cellular kinetics data of UCART19 administered to pediatric and adult patients with R/R B-ALL are reported. Methods Data of patients included in the ongoing CALM study (adult) and PALL study (pediatric) have been pooled. CALM is a dose-escalation study (approximate dose level [DL] 1: 1x105 cells/kg,DL2: 1x106 cells/kg, DL3: 3x106 cells/kg) and PALL is testing a unique dose of 1.1 to 2.3x106 cells/kg. Eligible patients presented with a morphological disease (>5%) or MRD load ≥1x10-3. A lymphodepleting regimen (LD) combining cyclophosphamide (1500 mg/m² in CALM, 120 mg/kg in PALL) and fludarabine (90 mg/m2 in CALM, 150 mg/m2 in PALL) without (FC) or with alemtuzumab (FCA) (1 mg/kg) was administered one week before UCART19 infusion on Day 0 (D0). Cellular kinetics of UCART19 was assessed by flow cytometry (flow) in CALM and by vector copy number in PALL. Results As of 15 July 2018, 20 pts had received at least one UCART19 infusion, including 13 pts in CALM (6 at DL1; 6 at DL2 and 1 at DL3) and 7 pts in PALL. Seventeen pts had completed D28 evaluation (1 pt died at D15, 2 pts have not reached D28 yet). Prior to LD, blasts % in the bone marrow (median [range]) was 6% [0-68%] in PALL and 25% [0-96%] in CALM. Seventeen pts received LD with FCA and 3 pts received LD with FC. Safety was evaluable in 18/20 pts. Cytokine release syndrome (CRS) was reported in 17/18 pts and was mild and reversible in the majority of cases (2 G1, 12 G2, 2 G3, 1 G4). One pt died in context of CRS G4 and neutropenic sepsis. According to data available to date, the severity of CRS does not seem to follow the levels of serum cytokines (IL-6, IL-10 and IFNγ). Mild self-limited neurotoxicity events were reported in 6 pts (5 G1 and 1 G2). Two pts (1 infant and 1 adult) developed G1 acute skin GvHD (non-biopsy proven for 1 pt) that was reversible with steroids. Grade 1-4 viral infections were reported in 8/18 pts. The majority of events recovered, except in 2 pts who died after allo-SCT in context of prolonged cytopenia (defined as persistent G4 beyond D42 post UCART19 infusion): 1 child with BK virus infection and 1 adult with adenovirus infection. A further 4 pts developed prolonged cytopenia and all recovered after allo-SCT. Cellular kinetic data was available for 18/20 pts. UCART19 was detectable in blood from D7. Peak expansion was observed in 72% (13/18) pts between D10 and D17, mostly at D14, with a median persistence duration of 28 days. Expansion occurred at each dose tested; without consistent relationship between administered dose and magnitude of expansion. One patient treated at DL2 presented a high expansion and a long persistence (very low detection by flow at D120). UCART19 persistence could not be assessed beyond D42-D56 in 3 pts because the remaining CARs were ablated by the transplant conditioning regimen. No expansion was observed in 5/18 pts in whom early lymphocyte recovery was detected from D14. Of these 5 pts, 3 did not receive alemtuzumab. The role of clinical status, tumor burden and lymphodepleting regimen on UCART19 expansion is under investigation. Anti-leukemic activity was evaluable in 16/20 pts (1 pt died at D15, 1 pt was not assessed at D28, 2 pts have not yet reached D28). After UCART19 infusion, 88% of evaluable pts (14/16) achieved CR or CRi by D28 or D42 and 86% (12/14) of these pts were MRD negative (MRD- stands for < 1x10-4 copies) by flow or qPCR. Two out of 16 pts had no expansion and showed refractory disease. Among 12 pts achieving MRD-, 5 pts remain in molecular remission 4.5 to 16.4 months post UCART19. In total, 11 pts underwent allo-SCT (5 in PALL and 6 in CALM). Preliminary data suggests that in the majority of pts, anti-leukemic activity is linked with CAR expansion. Conclusion Pooled data of 20 pts show an acceptable and manageable safety profile of UCART19. Severe CRS was reported in 15%. Only 2 G1 cutaneous acute GvHD were observed and no severe neurotoxicity was reported. In this heavily pre-treated population, 88% pts of evaluable pts (14/16) achieved CR or CRi of which 86% (12/14) achieved MRD-. All pts who achieved MRD- had evidence of UCART19 expansion. Updated data, including data for the highest dose level in CALM study, will be presented (NCT 02746952, NCT02808442). Disclosures Benjamin: Servier: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Pfizer: Research Funding. Graham:Servier: Research Funding. Yallop:Pfizer: Consultancy; Servier: Other: Travel funding. Jozwik:Servier: Honoraria, Research Funding. Ciocarlie:Servier: Research Funding. Jain:Cellectis: Research Funding; Seattle Genetics: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; ADC Therapeutics: Research Funding; Astra Zeneca: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Research Funding; Servier: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Adaptive Biotechnologioes: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Pharmacyclics: Research Funding; Cellectis: Research Funding; Pfizer: Research Funding; Verastem: Research Funding; BMS: Research Funding; Servier: Research Funding; Infinity: Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maus:adaptimmune: Consultancy; novartis: Consultancy; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding; crispr therapeutics: Consultancy, Research Funding; kite therapeutics: Consultancy, Research Funding. Boissel:Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees. Larghero:Gilead: Consultancy. Baruchel:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Amgen: Consultancy; Servier: Consultancy; Celgene: Consultancy. Mohty:Takeda: Honoraria, Speakers Bureau; Molmed: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Janssen: Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Bloor:Janssen: Research Funding; AbbVie: Research Funding. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Konto:Pfizer: Equity Ownership. Veys:Pfizer: Honoraria; Servier: Research Funding; Novartis: Honoraria. Qasim:Bellicum: Research Funding; Autolus: Equity Ownership; Orchard: Equity Ownership; Servier: Research Funding.

PV and AL contributed equally Multiple studies from the EBMT registry and others have shown excellent survival rates after allogeneic haematopoietic stem cell transplantation (HSCT)for Wiskott-Aldrich syndrome (WAS) patients (Ozsahin et al, Blood 2008). The importance of myeloid engraftment for full disease correction has also been demonstrated (Moratto et al, Blood 2011). However, the vast majority of HSCTs in these studies were performed with (oral) busulfan/cyclophosphamide-based conditioning and in the early 2000 years or before. In 2005, the inborn errors working party (IEWP) of EBMT and ESID first recommended busulfan/fludarabine (BuFlu) or treosulfan/fludarabine (TreoFlu) based conditioning for primary immunodeficiencies such as WAS, with some centers deciding to add thiotepa (TT) to the conditioning. We performed a retrospective analysis via the EBMT and SCETIDE registries of WAS patients transplanted between 01/01/20006 and 12/31/2016 with these two regimens. The primary objective was to compare the overall (OS) and event-free survival (EFS) after HSCT with either BuFlu±TT or TreoFlu±TT conditioning. Secondary objectives included the influence of either conditioning regimen on acute and chronic GVHD, the degree of donor chimerism, incidence of secondary procedures after HSCT (2nd HSCT, stem cell boost, DLI, gene therapy or splenectomy) and rates of disease-specific complications after HSCT. At the time of this interim analysis, 174 patients were included, 92 (53%) with BuFlu±TT and 82 (47%) with TreoFlu±TT conditioning with a median age of 1.57 years (range 0.21-29.96) at HSCT and a median follow-up of 32.9 months (range 1.5-128.9). The donor was an HLA-matched sibling (MSD) in 30, a matched related donor (MRD) in 5, a matched unrelated donor (MUD, 9/10 or 10/10) in 105, a mismatched unrelated donor (MMUD, 5 years had a worse OS as compared to those 5 years or younger at HSCT (74.9% vs. 90.8%; log-rank test p=0.005). The type of donor had no influence on OS: 96.4% for MSD/MFD, 86.8% for MUD/MMUD and 87.7% for MMFD (log-rank test p=0.4). Whole blood chimerism was complete (>90% donor) in 60/75 evaluable patients (80%) at last follow-up or before secondary procedure (if a patient had one), 39/40 (98%) in the BuFlu±TT group and 21/35 (60%) in the TreoFlu±TT group. Twenty-six patients required a secondary procedure: stem cell boost in 4 patients, donor lymphocyte infusion in 9, 2nd HSCT in 15 and splenectomy in 1. Twenty-two of these 26 (84.6%) are alive and 14 of 16 with available chimerism data have a complete donor chimerism (>90%donor) at last follow-up. The 2-year cumulative incidence (CI) of secondary procedures was higher at 33.9% in the TreoFlu±TT versus 12.8% in the BuFlu±TT group (Gray's test p=0.017), and 2-year EFS (secondary procedure or death as event) was 61.4% in the TreoFlu±TT and 75.0% in the BuFlu±TT group (log-rank test p=0.2). Grade II-IV acute GVHD had the same incidence in both groups (100 day CI 24.4% vs. 26.3%; Gray's test p=0.849) and chronic GVHD of any grade was borderline more frequent in the TreoFlu±TT group (2 year CI 17.2% vs 6.7%; Gray's test p=0.054). The cumulative incidence of disease-specific complications occurring more than 6 months post HSCT (severe infections, bleeding, autoimmunity) was not different between the two groups (6.5% vs. 6.4%; Gray's test p=0.92). There was no malignancy reported after HSCT except for one EBV-post-transplant lymphoproliferative disorder (PTLD) 2.7 months after HSCT. In summary, HSCT with either BuFlu±TT or TreoFlu±TT conditioning reliably cures almost 90% of patients with WAS regardless of donor type. WAS-related complications are very rare events more than 6 months post HSCT. More patients required secondary procedures after treosulfan-based than busulfan-based conditioning. These data confirm the feasibility and efficacy of the regimens currently recommended by the IEWP. Disclosures Slatter: Medac: Other: Travel assistance. Chiesa:Gilead: Consultancy; Bluebird Bio: Consultancy. Kalwak:Sanofi: Other: travel grants; medac: Other: travel grants. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria. Sykora:Aventis-Behring: Research Funding; medac: Research Funding. Zecca:Chimerix: Honoraria. Veys:Pfizer: Honoraria; Servier: Research Funding; Novartis: Honoraria.

Introduction: Published studies of CD19 CAR T cells have shown unprecedented response rates in ALL but with a 23-27% incidence of severe Cytokine Release Syndrome (CRS) and 27-50% incidence of severe neurotoxicity which may limit broader application. We developed a novel second generation CD19CAR (CAT-41BBz CAR) with a lower affinity and faster off-rate but equivalent on-rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 10s vs 1260s) utilised in CTL019 currently under consideration by the FDA. Pre-clinical studies indicated T-cells transduced with CAT-41BBz mediate enhanced tumor clearance and show increased expansion in an NSG-NALM6 stress test model (Kramer et al., submitted). We here report interim results from a multi-centre, Phase I clinical study of autologous CAT-41BBz CAR T cells as therapy for high risk/relapsed paediatric ALL, CARPALL (NCT02443831) demonstrating efficacy with an excellent safety profile. Methods: Autologous T cells were activated with anti-CD3/CD28 beads, transduced with a SIN lentiviral vector encoding CAT-41BBz CAR and expanded for 4 days prior to magnetic bead removal and cryopreservation. Transduction efficiency was assessed using an anti-idiotype antibody. Serum levels of cytokines associated with CRS were measured using cytometric bead array. All patients received lymphodepletion with fludarabine 150 mg/m2 + cyclophosphamide 1.5g/m2 followed by a single infusion of CAR T cells at a dose of 1x106 CAR+ T cells. Patients were monitored for the presence of CAR T cells in the blood by flow cytometry and by qPCR for the 41BBz junctional region, as well as circulating B cell count monthly for 6 months and then 6 weekly to 1 year. Disease status was assessed in the bone marrow morphologically, by IgH qPCR, as well as by flow cytometric assessment of MRD at the same time-points to establish durability of responses as a stand-alone therapy. The primary end-points were incidence of grade 3-5 toxicity related to CAR T cells within 30 days and the proportion of patients achieving molecular remission. Results: We have enrolled 10 patients and treated 8 to date. Six of 8 had relapsed post myeloablative SCT. The median disease burden prior to lymphodepletion was 9% blasts (ranging from molecular CR to 74% blasts, Table 1). It was possible to generate a product meeting release criteria in all but 1 patient (90% feasibility). Median transduction efficiency was 18.1% (range 6.7 to 76.3%). All treated patients received the anticipated dose of 1x106 CAR T cells/kg. Cytokine release syndrome occurred in all patients (grade 1 n=4, grade 2 n=4), but to date none have developed ≥ grade 3 CRS, required ICU admission or therapy with Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γ and IL-10 and resolved spontaneously in all. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously, but no severe (≥grade 3) neurotoxicity was seen. Five patients had prolonged grade 4 neutropenia lasting > 30 days but this resolved in all by 2 months. Only 1 patient experienced significant infective complications in the context of pre-existing poor marrow reserve following allogeneic SCT. 6/7 (86%) evaluable patients achieved molecular remission at a median of 30 days post infusion (range 30-60 days, Table 1). One patient did not respond and died of CD19+ disease progression. At a median follow-up of 5.9 months (range 28-328 days), 4/7 evaluable patients remain in flow MRD negative remission of whom 3 show no evidence of molecular MRD at 1, 7.5 and 9 months. Two patients relapsed with CD19- disease at 3 and 4 months post infusion: 1 of these remains alive with disease at 11 months and the other died of disease progression. Reflecting our pre-clinical data with CAT-41BBz CAR, we have seen excellent CAR T cell expansion (median 65459 copies/µg DNA at 1 month, range 609 to 230112) and persistence at up to 11 months post-infusion (Figure 1). All 7 evaluable patients have ongoing CAR T cell persistence detectable by both flow and qPCR as well as ongoing B cell aplasia at last follow-up. Conclusions: These interim results with a novel low affinity CD19 CAR show similar remission rates to those reported by US studies in paediatric ALL with an improved safety profile. No severe (grade ≥3) CRS or neurotoxicity has occurred to date despite high tumour burden in 4 patients. Excellent CAR T cell expansion has been documented, as well as long duration of CAR T cell persistence and associated B cell aplasia. Disclosures Ghorashian: UCL: Patents & Royalties: UCL Business. Kramer: UCL: Patents & Royalties: UCL Business. Lucchini: Alexion: Membership on an entity's Board of Directors or advisory committees. Pule: Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL: Patents & Royalties: UCL Business.

Introduction: Most pediatric patients (pts) with ALL receive total body irradiation (TBI) for myeloablative conditioning of allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is unproven whether TBI can be replaced by chemotherapy (CHT). Methods: To compare the outcomes of TBI- versus (vs.) CHT-based conditioning, we performed a retrospective EBMT-registry based study. Children between 2 and 18 years of age (y.) after myeloablative conditioning for first allo-HSCT of bone marrow (BM) or peripheral blood SC (PBSC) from matched sibling (MSD) or unrelated donors (UD) in first (CR1) or second remission (CR2) between 2000 and 2012 were included. Propensity score weighting was used to consider the covariate "distribution of TBI recipients" for pts who did not receive TBI. Results: In total 3071 pts (CR1: 1504 (49%), CR2: 1567 (51%)) were included. CR1: 1045 pts (69%) received BM and 459 pts (31%) PBSC from MSD (760 (51%)) or UD (744 (49%)). CR2: 1067 pts (68%) received BM and 500 pts (32%) PBSC from MSD (675 (43%)) or UD (892 (57%)). Overall, conditioning was TBI- in 2647 (86%) and CHT-based in 424 pts (14%). Busulfan/Cyclophosphamide (Bu/Cy) and Bu/Cy/Etoposide (Bu/Cy/Eto) were the two most frequently applied CHT combinations in CR1 (68 (32%), 66 (31%)) and CR2 (68 (32%), 52 (25%)). The remaining conditionings included 5 different combinations of chemotherapeutics (other chemo). 1504 pts in CR1 were conditioned with TBI (1291), Bu/Cy/Eto (66) or other chemo (147) with a median follow-up of 4.4, 3.4 and 2.4 y. In weighted univariate analysis no significant differences were detected for LFS (5-y.-LFS, range: 62.4 to 67.5%) and relapse incidence (5-y.-RI, range: 24.0 to 29.0%). In pairwise testing, OS after Bu/Cy/Eto was significantly better compared with TBI (5-y.-OS, 78.7 vs. 66.8%, P=.006). Non-relapse mortality was significantly higher after other chemo (5-y.-NRM, 12.7%, P Other significant influencing factors on LFS were age (5-y.-LFS, 2-11 y. 65.8 vs. 12-18 y. 58.0%, P=.009), y. of HSCT (2008-2012 65.9 vs. 2000-2007 59.6%, P=.035) and donor type (MSD 64.9 vs. UD 59.5%, P=.007). RI was influenced by y. of HSCT (5-y.-RI, 2008-2012 21.7 vs. 2000-2007 26.8%. P=.026). OS was influenced by age (5-y.-OS, 2-11 y. 72.7 vs. 12-18 y. 61.7%, P In weighted multivariable Cox model clustered on EBMT centers, TBI-based conditioning in CR1 was associated with lower RI (HR 0.70, P=.047), lower OS (HR 1.54, P=.017) and higher NRM (HR 3.97, P 1567 pts in CR2 were conditioned with TBI (1356), Bu/Cy (68) or other chemo (143) with a median follow-up of 3.9, 3.2 and 3.1 y. In weighted univariate analysis highly significant differences of survival were detected. TBI-based conditioning resulted in highest 5-y.-LFS of 52.2% (Bu/Cy 41.0%, other chemo 18.4%, P Other significant influencing factors on LFS were age (5-y.-LFS, 2-11 y. 53.5 vs. 12-18 y. 43.0%, P In weighted multivariable Cox model clustered on EBMT centers, TBI-based conditioning in CR2 was associated with higher LFS (HR 0.48, P Conclusion: Conditioning by TBI demonstrated clear superiority in comparison to CHT for children with ALL undergoing HSCT in CR2. For pts in CR1, TBI- and CHT-based conditioning showed similar results. This retrospective data is currently re-evaluated in a prospective, randomized, international trial (ALL SCTped 2012 FORUM). Disclosures Büchner: Novartis Pharmaceuticals Corporation: Consultancy; Pfizer: Consultancy. Veys: Bellicum: Research Funding; Servier: Research Funding. Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

Hematopoietic stem cell transplantation (HSCT) is a highly successful treatment for severe congenital immunodeficiencies. However, some studies have suggested that children may experience cognitive difficulties after HSCT. This large-scale study assessed cognitive and behavioral function for the cohort of children treated by HSCT at one center between 1979 and 2003 to determine the frequency and severity of problems and to identify risk factors. A total of 105 patients were assessed on standardized measures of cognitive and emotional and behavioral function together with a control group of unaffected siblings. The average IQ for the cohort was 85 (95% confidence interval, 81-90), significantly lower than both the population average of 100 (P < .001) and unaffected siblings. Multivariate analysis indicated that the underlying genetic defect, diagnosis of adenosine deaminase-deficient severe combined immunodeficiency, and consanguinity were associated with worse outcome but that age at transplantation and chemotherapy conditioning were not. Children treated by HSCT for severe immunodeficiency have an increased risk of long-term cognitive difficulties and associated emotional and behavioral difficulties. The specific genetic diagnosis, consanguinity, and severe clinical course are associated with poor outcome. Long-term follow-up of these patients should include screening to identify and manage these problems more effectively.

Poor T lymphocyte reconstitution limits the use of haploidentical stem cell transplantation (SCT) because it results in a high mortality from viral infections. One approach to overcome this problem is to infuse donor T cells from which alloreactive lymphocytes have been selectively depleted, but the immunologic benefit of this approach is unknown. We have used an anti-CD25 immunotoxin to deplete alloreactive lymphocytes and have compared immune reconstitution after allodepleted donor T cells were infused at 2 dose levels into recipients of T-cell-depleted haploidentical SCT. Eight patients were treated at 10(4) cells/kg/dose, and 8 patients received 10(5) cells/kg/dose. Patients receiving 10(5) cells/kg/dose showed significantly improved T-cell recovery at 3, 4, and 5 months after SCT compared with those receiving 10(4) cells/kg/dose (P < .05). Accelerated T-cell recovery occurred as a result of expansion of the effector memory (CD45RA(-)CCR-7(-)) population (P < .05), suggesting that protective T-cell responses are likely to be long lived. T-cell-receptor signal joint excision circles (TRECs) were not detected in reconstituting T cells in dose-level 2 patients, indicating they are likely to be derived from the infused allodepleted cells. Spectratyping of the T cells at 4 months demonstrated a polyclonal Vbeta repertoire. Using tetramer and enzyme-linked immunospot (ELISPOT) assays, we have observed cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-specific responses in 4 of 6 evaluable patients at dose level 2 as early as 2 to 4 months after transplantation, whereas such responses were not observed until 6 to 12 months in dose-level 1 patients. The incidence of significant acute (2 of 16) and chronic graft-versus-host disease (GVHD; 2 of 15) was low. These data demonstrate that allodepleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT and may broaden the applicability of this approach.

The optimal approach to stem cell transplantation in children with immunodeficiency who lack a matched family donor is controversial. Unrelated donor stem cell transplantation gives equivalent outcome to mismatched family donor stem cell transplantation in severe combined immunodeficiency, whereas unrelated donors may be preferable in non–severe combined immunodeficiency children. However, unrelated donor stem cell transplantation with conventional conditioning regimens has been associated with significant treatment-related toxicity, particularly in non–severe combined immunodeficiency patients with preexisting organ dysfunction. We report the outcome of a series of 33 consecutive unrelated donor transplantations performed at our center in children with primary immunodeficiency using a reduced-intensity conditioning regimen between 1998 and 2001. We have compared these outcomes with a retrospective control cohort of 19 patients who underwent transplantation with myeloablative conditioning between 1994 and 1998. All children in both groups had primary engraftment. There was no statistical difference in the speed of immune reconstitution or incidence of graft-versus-host disease between the 2 groups. Overall survival was significantly better in the reduced-intensity conditioning group: 31 (94%) of 33 patients survived, compared with 10 (53%) of 19 in the myeloablative conditioning group (P = .014). We conclude that the reduced-intensity conditioning regimen results in improved survival and reduced transplantation-related mortality compared with myeloablative conditioning in high-risk patients undergoing unrelated donor transplantation.

To the editor: Marsh et al[1][1] summarize the international experience of stem cell transplantation for X-linked inhibitor of apoptosis protein (XIAP) deficiency. They demonstrate a poor outcome for patients with active hemophagocytic lymphohistiocytosis (HLH) entering transplant (no survivors)

CD40 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.

Non-malignant diseases (NMD) include a wide spectrum of either congenital or acquired disorders occurring in early and later childhood. The primary goal of conditioning in NMD is either the replacement of missing cells as in severe aplastic anaemia (SAA) or severe combined immunodeficiencies (SCID) or the exchange of a deficient blood cell compartment as in hemoglobinopathies, hemophagocytic syndromes or metabolic disorders. The right choice of a conditioning regimen before allogeneic hematopoietic stem cell transplantation (HSCT) is crucial for the outcome of patients with NMD, many of whom are often critically ill due to infections, organ dysfunctions, vascular complications or metabolic problems. As a consequence of a highly activated immune-system and no pre-treatment with chemotherapy, graft rejection is a known severe complication in these patients. To get up-to-date information on the most commonly used conditioning regimen for children with NMD, we performed an EBMT- registry-based analysis: 2187 patients from 39 countries with a congenital or acquired NMD below the age of 18 years who underwent first allogeneic HSCT between January 2010 and December 2014 registered in the EBMT database were included. 51% of children were transplanted before 4 years of age, 489 patients were less than 12 months old, and 61.5% were male. Majority of patients (32,2%) underwent HSCT for hemoglobinopathies (463 thalassemia major, 127 sickle cell disease), the second most common disease type was SCID with 17.4%, followed by other primary immunodeficiencies, chronic granulomatous disease and bone marrow failure syndromes (excluding Fanconi anaemia): 13.2%, 11% and 10.4%, respectively. The given conditioning regimen was under discretion of the treating physician. The most common conditioning regimen consisted of a combination of busulfan (bu)/fludarabine (flu) in 1063 patients, of treosulfan (treo)/flu in 422 patients; 473 patients received a combination of treo/flu/thiotepa (tt) and 229 patients received bu/flu/tt. The median total dose of the respective conditioning drugs was comparable (treo: 41 g/m2 or 42g/m2; bu: 160 mg/m2; tt: 201 and 205 mg/m2). In univariate analysis, overall survival (OS) was significantly different between the conditioning groups (p = 0.0032): 2y-OS 87.7% [84.3%-91.2%] for treo/flu/tt vs. 82.4% [78.5%-86.5%], 81.8% [77.8%-86.0%] for treo/flu, 80.8% [78.1%-83.5%] and 78.2% [75.2%-81.3%] for bu/flu and 81.3% [75.9%-87.0%] and 80.4% [74.9%-86.4%] for bu/flu/tt, respectively. Transplant-related mortality (TRM) was significantly different between the conditioning groups (p = 0.0015): day-100 TRM was lowest after treo/flu/tt: 4.8% [2.8%-6.7%] compared to 9.2% [6.3%-12.0%] after treo/flu, 7.9% [6.2%-9.5%] after bu/flu and 9.9% [5.9%-13.8%] after bu/flu/tt. TRM at 1 year post-transplant: 8.3% [5.5%-11.0%] for treo/flu/tt, 16.2% [12.2%-20.0%] after treo/flu, 16.9% [14.2%-19.4%] after bu/flu and 14.9% [9.7%-19.8%] after blu/flu/tt, respectively. In multivariate analysis, the HR was adjusted on age groups at HSCT, underlying disease, year of HSCT, stem cell source and donor types. 1-y OS was significantly better after treo/flu/tt (89.5 [86.5 - 92.6], CI 95%, p 0.0032) than with treo/flu, bu/flu or bu/flu, respectively. Day 100- and 1-year TRM was lower after treo/flu/tt compared to the other three conditioning combinations. A pairwise testing of the different treatment groups revealed no statistical significant difference in OS, TRM, disease-free survival (DFS) for bu/flu vs. treo/flu, bu/flu vs. bu/flu/tt and bu/flu/tt vs. treo/flu; a significant better OS, DFS and less TRM for treo/flu/tt vs. bu/flu, treo/flu/tt vs. treo/flu and a better DFS for treo/flu/tt vs. bu/flu/tt with a trend for better OS and less TRM for treo/flu/tt vs. bu/flu/tt. No statistically significant differences were found among the 4 conditioning regimens for acute or chronic GVHD. Conclusion: For children with NMD, a variety of conditioning regimen are in use to facilitate engraftment, prevent rejection, and enable sufficient cell function. Beside treo and bu, flu and tt are established drugs to eradicate lymphocytes. In this analysis, the combination of treo/flu/tt shows promising outcome results and should be further evaluated in diseases specific prospective trials. Disclosures Peters: Pfizer: Consultancy; Amgen: Consultancy; Jazz: Speakers Bureau; Novartis: Consultancy; Medac: Consultancy. Slatter:Medac: Other: Travel grants. Bader:Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Severe combined immunodeficiency (SCID) arises from different genetic defects associated with lymphocyte development and function and presents with severe infections. Allogeneic hematopoietic stem cell transplantation is an extremely effective way of restoring immunity in these individuals. Numerous multicenter studies have identified the factors determining successful outcome, and survival for SCID has shown great improvement. Advances in understanding the genetic basis of disease also mean that we increasingly tailor transplant protocols to the specific SCID form. Wherever possible, we attempt to transplant SCID patients without the use of cytoreductive conditioning, but it is clear that this is only successful for specific SCID forms and, although survival is good, in specific patients there are ongoing humoral defects. We aim to use matched related and unrelated donors (including cord blood) whenever possible and have limited the use of mismatched haploidentical donors. The development of autologous hematopoietic stem cell gene therapy provides another treatment of the X-linked and adenosine deaminase–deficient forms of SCID, and we discuss how we have integrated gene therapy into our treatment strategy. These developments together with the advent of universal newborn screening for SCID should allow for a highly favorable outcome for this otherwise lethal condition.

Hematopoietic Stem cell Transplantation (HSCT) is the definitive treatment for bone marrow failure in diskeratosis congenita (DC). Organ dysfunction (mainly lung and gastrointestinal tract) are often part of the disease and may be a limiting factor for or may affect the final outcome of HSCT. Scarce data or relatively small cohort studies are available in current literature on outcome of HSCT in this disease. We analyzed the outcome of 87 patients diagnosed with DC reported in the data base of the European Society for Blood an Bone Marrow Transplantation (EBMT) who underwent HSCT from 1979 to 2014. In particular we analyzed data on type of HSCT, characteristic of donors, source of cells, incidence of acute and chronic GvHD. Males were 76%. Median age at diagnosis of DC was 5.7 years (0-33 yrs), median age at HSCT was 11.2 years (range 0.56-41 yrs). Median time from diagnosis to HSCT was 19 months (0.62-304 mo). The cell source was bone marrow (BM) in 67%, peripheral blood (PB) in 22% and cord blood (CB) 11%. Twenty six percent of patients were engrafted from a matched related and 53% from a matched unrelated donor. Twenty one percent of subjects were engrafted from a mismatched (both related and unrelated) HSCT. Engraftment was documented in 95% of subjects: 4 % had primary graft failure and 10% lost the graft. Overall 35 patients (40%) died, and 52 were alive (60%) at last follow-up. Causes of death were: infections (43%), multi-organ failure (26%), rejection/oss of graft (14%), GvDH (8.5%) undefined (8.5%). In 32% of cases death was related to transplant. Lung injury was present in 17% of subjects who died. Acute GVDH (mainly grade 2-4) and chronic GvHD occurred in 39% and 30% of cases. Five year OS was 60 %. OS was not significantly different by calendar period (before and after year 2000: 50% vs 67% respectively, p=0.424), by age at transplant (below and above age of 12 years: 64% vs 55% respectively ; p= 0.564) and by source of cells (marrow 60%, cord blood 68%, and peripheral cells 46%; p= 0.687). Conversely, OS in HSCT from mismatched donor was inferior to that of transplants from matched donor (38% vs 65% respectively; p= 0.045). The use of radiotherapy or busulfan (any dose) in the conditioning vs other regimens did not impact on 5 y-OS ( 58% vs 62% respectively; p=0.898). HSCT from HLA matched donor can be considered a treatment option in DC. Transplant related mortality looks rater high. Multi organ failure and lung injury are amongst the main causes of death thus pointing to pre-transplant organ status as important determinant of HSCT outcome. Disclosures Dufour: Pfizer: Consultancy.

Background: The optimal approach for transplanting patients with primary immunodeficiency from a mismatched unrelated donor (MMUD) remains unclear. With reduced intensity conditioning, when bone marrow (BM) was used as the stem cell source in such patients we have previously observed a high rate of very low level donor chimerism (4 of 12 patients) requiring a second transplant procedure. The use of peripheral blood stem cells (PBSC) can overcome this but results in a high incidence of acute (≥ Gde II 10/21, Gde III-IV 5/21) and chronic (10/21, extensive 7/21) graft-versus-host disease (GVHD) (Rao et al in press). We hypothesized that the use of megadose CD34 selected stem cells from PB with addback of a T-cell dose akin to BM might facilitate engraftment through improved competition for the stem cell niche without severe GVHD. Methods: We prospectively analysed outcomes on 20 consecutive patients with primary immunodeficiency (SCID n=5, HLH n=3, Other PID n=12) transplanted from 8/10 (n=4) or 9/10 (n=16) HLA MMUD at our institution between 2011-2015. The mean age at transplant was 5.1 years. All patients received reduced toxicity conditioning regimens (12/20 Fludarabine/Melphalan/Alemtuzumab; 8/20 Fludarabine/Treosulphan/Alemtuzumab) with Cyclosporin (CsA) and mycophenolate mofetil (MMF) GvHD prophylaxis. CD34 selection of donor PBSC was performed using CliniMACs and the mean CD34+ dose was 20.1x106/kg. At day 0 either 108 CD3/kg (cohort 1, n=6) or 3x108 CD3/kg (cohort 2, n=14) T-cells from the CD34- fraction were infused with the graft. Mean follow up was 31.9 months. Results: In cohort 1, all patients engrafted and 2 developed high level donor mixed chimerism (both curative) in the myeloid and lymphoid lineages at last follow up. Two patients had Grade II acute GvHD and 1 had moderate chronic GvHD. In view of the slow immune reconstitution in this cohort, the T-cell addback dose was increased to 3x108/Kg in subsequent patients. In cohort 2, all patients achieved full donor haemopoiesis initially, 6/14 developed high levels of donor chimerism in both myeloid and lymphoid lineages later and 1/14 progressed with 10% donor T-cell engraftment but remains disease-free. The incidence of significant aGVHD was low (grade II n=4, no grade III or IV) and no patient developed cGvHD. Overall across both cohorts, 10 patients had viral reactivations and there were 5 deaths (3 viral complications, 1 pulmonary vasculopathy, 1 cGVHD lungs). The disease free survival at 2 years 7 months follow up was 70% which compares favorably with a previous cohort transplanted with unmanipulated BM as the stem cell source (Fig 1). Immune reconstitution was delayed (mean CD3 and CD19 counts at day 100 post-transplant was 245 x 109/L and 315 x 109/L) with similar kinetics in both cohorts (Fig 2), comparable to RIC transplant using unmanipulated BM. By 1 year post transplant 11/14 evaluable patients achieved normal CD3+T-cell numbers and 8/14 normal CD19+ B-cell counts. Conclusion: The use of megadose peripheral blood stem cells with T-cell addback in the mismatched unrelated donor transplant setting results in high rates of curative engraftment and a low incidence of acute and chronic GvHD following reduced toxicity conditioning. However, T-cell reconstitution remains delayed (presumably reflecting the effect of Alemtuzumab on the infused T-cells) with a high incidence of viral complications. Disclosures No relevant conflicts of interest to declare.

Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity. Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials. Disclosures Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

INTRODUCTION The prognosis for children with refractory acute myeloid leukemia (AML) treated with chemotherapy is dismal and data on the outcome after allogeneic haematopoietic stem cell transplant (SCT) are scanty with reported leukemia free survival (LFS) rates of 10-20%. Thus there is significant controversy about whether SCT is appropriate in such patients (pts). We performed a retrospective, national study to analyse outcomes and prognostic factors for children undergoing SCT for refractory AML in the UK. METHODS A retrospective analysis of all pts 5% blasts in the bone marrow (BM) or proven extramedullary disease (EM) was performed. Source data verification (SDV) was performed to ensure pts were indeed refractory. The primary end-point was 5 year LFS. Secondary end-points were Relapse Rate (RR), Treatment Related Mortality (TRM), Graft Versus Host Disease (GVHD) and Overall Survival (OS). The Kaplan Meyer method was used to estimate survival data and Fisher's exact and Mantel-Cox Log Rank tests were used to compare disease- transplant- and survival-related variables. RESULTS Following SDV, a total of 44 pts from 13 centres were included in the study. The median age at SCT was 11.5 yrs and the median number of prior lines of chemotherapy was 3. The median time from diagnosis to SCT was 197 days. 23 pts had primary refractory AML and 21 had relapsed refractory AML. 12 pts showed adverse risk cytogenetics, 26 standard risk and 6 favourable. EM disease was documented in 5 pts. 42 children had >5% myeloid blasts in the BM immediately prior to conditioning and refractory disease was confirmed by cytogenetics/molecular genetics in 23. 2 pts were in BM remission but had frank EM disease. 38 pts (86%) received myeloablative conditioning (14 TBI based) and 6 (14%) had reduced intensity conditioning (RIC). In vivo T cell depletion was used in 25 pts. 15 pts (35%) were transplanted from an HLA identical family donor, 15 from a matched unrelated donor and 14 (32%) a mismatched donor. BM was used as the stem cell source in 18 (41%), peripheral blood in 20 (46%) and cord blood in 6 cases (14%). Median follow up was 4 years 10 months. 5 pts never achieved engraftment and had disease progression. The remaining 39 pts engrafted at a median of 15 days post-SCT. 30 pts (68%) achieved a complete remission (CR) following SCT. TRM at 1 year was 18% (5 infections, 1 cardiac failure, 1 GVHD-related). Acute GVHD occurred in 23 pts and was severe (grade ≥III) in 8 (19%). The incidence of chronic GvHD was low (1 limited, 2 extensive). Relapse was the major cause of treatment failure and occurred in 17 pts (39%) at a median 2.3 months post SCT. At last follow-up, 18 pts remain alive and in continuous complete remission (CCR). In this cohort, the 5 year OS and LFS were both 43% (95%CI 31-61%) (Figure1). Outcomes in pts with primary refractory disease (9/23, 39% in CCR) and those with relapsed refractory AML (9/21, 43% in CCR) were equivalent. Outcome for pts with cytogenetic confirmation of refractory disease was not statistically different (7/23, 30% in CCR) from the overall group. Pts transplanted with ≤30% blasts in the BM had improved outcomes (5-year LFS 52% vs 27%, p= 0.05). Likewise, the development of aGVHD of any grade was associated with a significantly better LFS (5-year LFS 56% vs 30%, p= 0.05). Cytogenetics including monosomy 7 (n=7) and molecular risk classification did not translate into a significant prognostic factor for relapse. Since RIC was used in only 6 pts, the impact of the intensity of conditioning cannot be determined. CONCLUSIONS This is the largest series of outcomes for SCT for refractory paediatric AML reported to date. Our data indicate that for selected pts, particularly those with a lower disease burden, SCT offers a realistic chance of salvage in both primary refractory and relapsed refractory AML (5 year LFS 43%) with acceptable toxicity. The association of aGVHD with improved LFS suggest a possible role in engineering a graft-versus-leukemia effect in this patient group. Figure 1. Leukemia-free survival for pediatric patients with refractory acute myeloid leukemia undergoing allogeneic stem cell transplantation. Figure 1. Leukemia-free survival for pediatric patients with refractory acute myeloid leukemia undergoing allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Immunotherapy with allodepleted donor T cells (ADTs) improves immunity after T cell-depleted stem cell transplantation, but infection/relapse remain problematic. To refine this approach, we characterized the expression of surface markers/cytokines on proliferating alloreactive T cells (ATs). CD25 was expressed on 83% of carboxyfluorescein diacetate succinimidyl ester(dim) ATs, confirming this as an excellent target for allodepletion. Seventy percent of CD25(-) ATs expressed CD71 (transferrin receptor), identifying this as a novel marker to target ATs persisting after CD25 depletion. Comparison of residual alloreactivity after combined CD25/71 versus CD25 immunomagnetic depletion showed enhanced depletion of alloreactivity to host with CD25/71 depletion in both secondary (2 degrees) mixed lymphocyte reactions (P < .01) and interferon-gamma enzyme-linked immunospot assays (P < .05) with no effect on third-party responses. In pentamer/interferon-gamma enzyme-linked immunospot assays, antiviral responses to cytomegalovirus, Epstein-Barr virus, and adenovirus were preserved after CD25/71 allodepletion. CD25/71 ADTs can be redirected to recognize leukemic targets through lentiviral transfer of a chimeric anti-CD19 zeta T-cell receptor. Finally, we have established conditions for clinically applicable CD25/71 allodepletion under European Union Good Manufacturing Practice conditions, resulting in highly effective, reproducible, and selective depletion of ATs (median residual alloreactivity to host in 2 degrees mixed lymphocyte reaction of 0.39% vs third-party response of 62%, n = 5). This strategy enables further clinical studies of adoptive immunotherapy with larger doses of ADTs to enhance immune reconstitution after T cell-depleted stem cell transplantation. (Blood. 2010; 115: 396-407)

Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon-γ (IFN-γ) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN-γ in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN-γ response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.

Lymphoproliferative disease (LPD) is a recognized complication of primary immunodeficiency (PID) and immunodysregulatory syndromes. Historically, it has a very poor outcome. For patients surviving LPD, myeloablative hematopoietic stem cell transplantation (SCT) was the only cure for the underlying PID, with a high risk of developing posttransplantation complications, including recurrent lymphoproliferative disease. We describe 8 patients with a range of PID and immunodysregulatory syndromes complicated by LPD. After initial treatment of the LPD (including the use of anti-CD20 monoclonal antibody, rituximab, in 6 of the patients), all patients underwent reduced-intensity conditioning (RIC) SCT with prospective monitoring for Epstein-Barr virus (EBV) viremia. After transplantation, 3 patients received rituximab, and 3 patients received prophylactic EBV-specific cytotoxic T-lymphocytes. Only 1 patient developed recurrent LPD posttransplantation, which responded to rituximab. All patients who underwent transplantation survive free of LPD and are cured of their PID at a median follow-up of 4 years (range, 1-7 years). With careful monitoring and pre-emptive therapy, we advocate this RIC SCT approach to patients with PID who have pre-existing EBV-LPD.

We report serial transplantation procedures in 2 sets of brothers with X-linked primary immunodeficiency. The first boy in each family received a T-cell–depleted transplant from a mismatched donor. The recipients then acted as donors for T-replete transplantation of the “tolerized” graft into their HLA-identical brothers with the same disorder. Immune reconstitution was noted to occur at a significantly faster rate in the secondary recipients, and without the occurrence of graft-versus-host disease (GVHD), despite the presence of donor cells mismatched for 1 to 3 HLA antigens. This serial transplantation technique allows the primary recipient of HLA-mismatched donor cells to act as a functionally “HLA-matched” donor for subsequent affected siblings, and should be considered as a therapeutic option in families with congenital disorders.

The classical treatment algorythm of idiopathic severe aplastic anemia (SAA) forsees, if a sibling donor is not available in the family, a first line option represented by immunosuppressive therapy (IST) with ATG and Cyclosporine A (CsA). IST provides a very good survival rate but a fairly high rate of failures (no response, relapse, need for transplant). This is particularly important in childhood and adolescence where faulty hematopoiesis represents a relevant limitation of quality of life. The option of haematopoietic stem cell transplantation (HSCT) from matched unrelated donor (MUD) as front line treatment with no prior failed IST, has never been investigated so far. This study provides for the first time the outcome analysis of 29 consecutive SAA patients who received an upfront unrelated donor HSCT without prior IST, in 9 UK Centres and compares each of these patient with 3 matched controls from the data base of the SAAWP of the EBMT who, in a similar time-span, underwent Matched Sibling Donor (MSD) HSCT. Twenty four patients received HLA-A,-B,-C,-DQ-,-DRB1 matched MUD HSCTs whilst 5 received single antigen/allele Mismatched Unrelated Donor (MMUD) grafts. The conditioning regimen was FCC (Fludarabine, Cyclophosphamide and Alemtuzumab) with the addition of low dose (3cGy) TBI for the MMUD HSCTs. GVHD prophylaxis was with CsA +-Mycophenolate. The primary endpoints were overall survival (OS) and event free survival (EFS). Events were death, disease recurrence, second HSCT, clinical PNH and post-transplant malignancies. Complete remission (CR) was defined as Hb > 10 g/dl, neutrophil count > 1x 109/l and platelet count > 100x109/l. The 87 (3 for each MUD/MMUD HSCT) patients from the EBMT data base who underwent MSD HSCT as first line treatment were matched to the upfront MUD/MMUD cohort by age, gender, time from diagnosis to HSCT and source of stem cells. Their median follow-up was 1.6 years (range 0.01-9.39). In the upfront MUD/MMUD cohort there were 12 males (41%) and 17 females (59%). Median age at HSCT was 8.46 years (range 1.73-19.11), 72% was aged below and 28% above 12 years; median interval from diagnosis to HSCT was 0.39 years (range 0.19-1.35) with 72 % receiving bone marrow and 28% peripheral blood stem cells. The median follow-up was 1.7 years (range 0.19-8.49). The median time to neutrophil recovery (>0.5 x 109/l) was 18 days (range 9-29) and the median time to platelet recovery (>50 x 109/l) was 19 days (range 10-40). The 100 day cumulative incidence (CI) of grade II-IV acute GVHD was 10 ± 6% ; there was only one case of grade III-IV acute GVHD (frequency of 3.5%). The 1 year CI of chronic GVHD was 19 ± 8%. There were 5/29 cases (frequency of 17,2 %), 4 in the MUD and 1 the MMUD subset. Chronic GVHD was limited in all cases and restricted to skin. There were only 2 events in this cohort; one primary graft failure following a HLA-A MMUD HSCT who has now successfully received a second HSCT and one death due to idiopathic pneumonia syndrome. No post-treatment malignancies occurred at last follow-up. The other 27 patients are in CR at last follow-up. The median interval diagnosis-neutrophil recovery was similar in MUD/MMUD (0.39 years; range 0.19-1.35) vs MSD transplants (0,31 years; range 0,1- 0,45) (p=0.93). The 2 year OS was 96%±4% in the upfront MUD/MMUD cohort vs 91%±3% of the MSD cohort (p=0.30). The 2 year EFS was 92%±5% in the upfront cohort vs 87%±4% in MSD (p=0.37) (Fig 1). The 2 year CI of rejection was 4%±4% in the MUD/MMUD vs .1%±1% in the MSD group (p=0.48). This is the first controlled study indicating that in idiopathic SAA of childhood and adolecence upfront MUD/MMUD HSCT using FCC as conditioning regimen, has similar outcome to MSD HSCT. MUD HSCT may be considered a feasible and successful treatment option when children lack a MSD and a MUD is readily available. Figure 1 Figure 1. Comparison of EFS between upfront MUD/MMUD and front-line MSD HSCT. Events were: death, disease recurrence, second HSCT, clinical PNH and post-transplant- malignancies. Table 1 EFS N events 2-yrs Pr (%) (SE) p-value MUD/MMUD 29 2 92 (5) 0.37 MSD 87 11 87 (4) Disclosures Dufour: Pfizer: Consultancy.

Double UCBT (dUCBT) has been used in adults to reach an acceptable cell dose. For most children a single unit with a total nucleated cell (TNC) dose >3x107/Kg can be easily identified, but that is not always the case for heavier patients (pts). Use of dUCBT might decrease relapse and increase graft-versus-host-disease (GvHD). Data on dUCBT in children are scarce in the literature. A recent randomized study in children has described similar outcomes after double compared to single UCBT. Our study provides an overview of the use of dUCBT in the pediatric population reported to Eurocord. We retrospectively analyzed the outcomes of unrelated dUCBT in 177 children transplanted between 2002 and 2012 in 61 EBMT centres. Analysis was performed separately for pts with malignant (n=139) and non-malignant (NM, n=38) diseases. Among pts with malignancies, 76 had ALL, 40 AML, 6 MDS, 2 CML, 11 NHL, 3 Hodgkin Lymphoma and 1 Multiple Myeloma. Median age at dUCBT was 15 years (1.3-17.9) and median weight was 55 kg (13-97). Disease status at dUCBT was 1st complete remission (CR) (36%), ≥2nd CR (34%) or advanced (25%), and missing in 5% of the pts. In this group, 117pts received a myeloablative conditioning (MAC) and 22 a reduced intensity regimen (RIC). Cyclophosphamide+fludarabine+TBI was administered to 41% of the pts; 55% received ATG in the conditioning. Median number of collected TNC was 5.7x107/kg (3,6-12,8). Considering the unit with the higher number of HLA incompatibilities with the recipient, 56% had 2 mismatches. GvHD prophylaxis was cyclosporine-A (CSA) based in 93% of the pts (58% received CSA + mycofenolate mofetil). Median follow-up was 31 months. Cumulative incidence (CI) of neutrophil (PMN) and platelet (PLT) engraftment was 88% at 60 days and 64% at 180 days after dUCBT, and it was achieved with a median time of 24 and 45 days, respectively. Among the 122 pts with PMN engraftment, 85/94 with available data on chimerism were full donor and, of these, 20% had dual chimerism. CI of acute GvHD grade II-IV and grade III-IV at 100 days was 51% and 26%, respectively; it was significantly higher in pts who did not receive ATG (grade II-IV: 35% vs 67%, p=0.004; grade III-IV: 12% vs 37%, p=0.0075). Chronic GvHD was observed in 24/104 pts at risk (60% extensive; 2-year (yr) CI: 18%). The 2-yr CI of relapse was 31%. In univariate analysis, RIC, advanced stage at transplantation and a collected TNC dose lower than the median, were significantly associated with higher rates of relapse.The 2-yr CI of transplant related mortality (TRM) was 27%. Overall, 73 pts died: 35 of relapse, 15 of infections, 9 of GvHD and 14 of other causes. The 2-yr disease free survival (DFS) and overall survival (OS) were 42% and 45%, respectively. Among pts with NM disorders, 24 had bone marrow failure syndrome (BMFS) (10 Fanconi Anemia, 13 Acquired Aplastic Anemia and 1 other inherited BMFS), 2 hemoglobinopathies, 7 immune deficiencies and 5 metabolic disorders. Median age at dUCBT was 11 years (0.7-17.9) and the median weight was 40 kg (13-70). In this group, 27 pts received a RIC (40% TBI based), 10 a MAC (90% busulfan based), and 1 no conditioning regimen. ATG was administered to 82% of the pts and GvHD prophylaxis was CSA-based in 77%. The median number of collected TNC was 8.4x107/kg (1,2-11,2) and 60% of the grafts had ≥2 HLA mismatches with the recipient. Median follow-up was 39 months. Overall, 28 pts achieved PMN engraftment and 16 PLT engraftment, with a median time of 23 and 61 days, respectively. In univariate analysis, pts with BMFS compared to others had a significantly lower CI of PMN engraftment (58% vs 100%, p=0.002). Among the 10 pts who did not engraft, 3 had autologous reconstitution and 3 had a subsequent allogeneic HSCT. Forteen pts developed acute GvHD grade II-IV and 10/25 pts at risk had chronic GvHD (3 extensive). Overall 21 pts died (17 with BMFS): 9 of infections, 5 of GvHD and 7 of other causes. The 2-yr OS was 42% and it was significantly lower in pts with BMFS compared to those affected by other NM disorders (28% vs 70%, p=0.03). In pts with malignancies, despite a higher incidence of acute GvHD, DFS and OS seem to be comparable to those reported in the literature for single UCBT or HSCT from other alternative stem cell sources. In the NM disorders group, despite the high cell dose, dUCBT did not seem to improve results in pts with BMFS. This survey suggests that dUCBT is feasible in children and should be considered when a single unit with an adequate cell dose is not available. Disclosures No relevant conflicts of interest to declare.

Introduction In vivo T-cell-depletion generated by the use of Thymoglobulin (ATG) within the conditioning regimen might contribute to the delayed immune-reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late and no ATG on immune reconstitution and clinical outcome. Methods 127 Children receiving an unrelated UCBT in London or Utrecht between 2006 and 2011 were included and divided in 3 groups: late ATG (day -5 to 0, n=48), early ATG (day -9 to -5, n= 33) and no ATG(n=46). The no ATG group received MMF and CsA as GVHD prophylaxis, while the ATG groups both received CSA and prednison as prohylaxis, Endpoints studied were survival, early and late immune-recovery, infections and GvHD. Subset analysis CD3+, CD4+ and CD4+naïve, B and NK cell numbers were prospectively measured during post-transplant follow-up at 1, 2, 3, 6 and 12 months post-UCBT. Results The probability of survival was not significantly different between the groups: 71% +/- 8% (no ATG), 68% +/- 9% (early ATG) and 61% +/- 7% (late ATG). There were no significant differences in engraftment and primary or secondary graft failure. Immune reconstitution as defined by CD3+, CD4+ and CD4+ naïve T-cell counts were significantly higher (p Conclusion Cord blood transplantation without Thymoglobulin (ATG), in the context of MMF and CsA post-transplant prophylaxis, is associated with higher GVHD risk but with a surprisingly good immune reconstitution and with an engraftment and survival similar to the groups with ATG. The findings of improved immune-reconstitution, associated with lower viral reactivations, albeit at the cost of increased rates of acute GvHD (but not chronic GvHD), suggest that omitting ATG in cord blood transplantation may be important to prevent viral reactivations, especially in those at high risk for these. Disclosures: No relevant conflicts of interest to declare.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric malignancy. Hematopoietic stem cell transplantation (SCT) is the only curative approach. However, relapse after SCT remains the major cause of treatment failure. Unlike most other pediatric malignancies, JMML may be susceptible to a graft-versus-leukemia (GVL) effect, although, unlike chronic myeloid leukemia, reports of response to donor lymphocyte infusions (DLIs) remain scanty. This is the first report that describes the successful treatment of relapsed JMML with DLI in the absence of further chemotherapy and provides definite proof of a GVL effect in JMML.

Abstract 1958 Background: Hurler syndrome (HS), the most severe phenotype in the spectrum of Mucopolysaccharidosis type I, is caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase. HS is clinically characterized by a progressive and ultimately fatal multi-system deterioration with involvement of the central nervous system. At present, hematopoietic stem cell transplantation (HSCT) is the only treatment that prevents disease progression in the central nervous system and is therefore considered the treatment of choice in HS. Long-term follow-up of outcomes of HSCT for HS are sparse and risk factors for favorable long-term outcomes are still largely unknown. Therefore, an international multicenter study was initiated to describe the long-term outcomes of successfully transplanted HS patients. Methods: HS-patients transplanted between 1980 and 2007 within the leading transplantation centers in Europe and the United States were include in this study. Patient, donor, and transplantation-related variables which may influence long-term outcome were analyzed. Patients who were ‘alive and engrafted (donor-chimerism >10%)' with a follow up of at least three years after HSCT were included. The functional outcomes assessed for the various organ systems - orthopedic, cardiac, ophthalmologic, respiratory and audiologic - were analyzed using multivariate Cox proportional hazards and logistic regression models. Results: 197 Hurler patients were included from 8 different transplant centers. This is estimated to be about 70–80% of the successfully transplanted HS patients worldwide during that time period. These patients had a median age of 16 (2–80) months at HSCT with a median follow up of 88 (36–258) months after successful HSCT. Seventy-nine % of the patients received a graft from an unaffected (non-carrier) donor. Seventy-two % of the patients achieved full (>95%)-donor-chimerism and 28% mixed-chimerism. After HSCT, normal enzyme-levels (EL; according to the local reference range) were found in 75% of the patients while 25% had EL below lower limit of normal; either due to mixed-chimerism or carrier-donorship). Multivariate analyses ( table 1 ) showed having a “normal EL” after HSCT and younger (below the median age of 16mths) “age at transplantation” were associated with less serious orthopedic complications requiring surgical interventions; e.g. cord compression, genu-valgum surgery, carpal tunnel surgery. Genotype (double non-sense vs. any other genotype) was associated with a lower probability of requiring hip dysplasia surgery as well as with the occurrence of retinopathy. For other endpoints; e.g progression valve insufficiency, progression corneal clouding and development of retinopathy and the need for hearing aids having a normal EL as well as age at HSCT ( Conclusion: The long-term outcome of clinical manifestations in HS-patients after successful HSCT is promising although residual disease burden remains. Predictors, favorably influencing the long-term outcomes are suggested to be 1) enzyme level (normal vs. below LLN) after HSCT, 2) genotype and 3) age at HSCT. Achieving normal enzyme levels at an early age might significantly impact the prognosis of Hurler syndrome patients. Newborn screening (resulting in early HSCT), the use of non-carrier donors and achieving full-donor chimerism may be crucial in optimizing long-term outcomes. Table 1 . Multivariate analyses HR p-value Cord Compression Surgery normal enzymes (>LLN) 0,081 0,021 age at transplantation 0,075 0,025 Carpal Tunnel Surgery normal enzymes (>LLN) 0,021 0,011 age at transplantation 0,21 0,005 Hip Dysplasia Genotype (double nonsence vs. rest) 4,83 0,005 Genu Valgum Surgery normal enzymes (>LLN) 0,224 0,002 Mitral Insufficiency - Progression normal enzymes (>LLN) 0,32 0,044 age at transplantation 0,36 0,053 Corneal Clouding - Progression normal enzymes (>LLN) 0,21 0,015 age at transplantation 0,14 0,007 Retinopathy - Occurence Genotype (double nonsence vs. rest) 5,36 0,038 age at transplantation 0,05 0,008 Hearing Aids normal enzymes (>LLN) 0,29 0,011 Disclosures: No relevant conflicts of interest to declare.

Abstract 3292 Patients with refractory immune thrombocytopenia (ITP) have limited therapeutic options, with morbidity and mortality as often from infection as from bleeding. Thrombopoietin (TPO) mimetics allow an increase in the platelet count without causing immunosuppression. These agents may be especially useful in patients with secondary forms of ITP. We report three patients treated with TPO agonists for ITP post Allogeneic Stem Cell Transplants (SCT). Patient 1: MC, 37yrs, developed refractory ITP after a myeloablative allogeneic SCT for Accelerated Phase CML. He was unresponsive to standard treatments (figure 1a), requiring platelet transfusions 3x weekly for active bleeding. He started Eltrombopag 50mg/day in July 2009. He had a clinically significant increase in his platelet count (supported count increased to 40) so platelet transfusions were reduced to 2x weekly. Eltrombopag was increased to 75mg in November with a subsequent platelet increase to 90 and by December 2009 he was platelet independent, maintaining a platelet count 90–120 on Eltrombopag alone, 25mg/day, 5 days week. Patient 2: BT, 4yrs(12kg), underwent a myeloablative allogeneic PBSCT in September 2009 for JIA/HLH. She became thrombocytopenic in June 2010, presumed to be ITP. She had no response to prednisolone or rituximab and a only a minimal response to IVIG; requiring platelets 1x weekly. She started Eltrombopag 7mg OD in November 2010, escalated to 12mg by February 2011. This had a synergistic effect with IVIG and she became platelet independent after one month of Eltrombopag and IVIG, maintaining platelet counts 50–200 (figure1b). Patient 3: MG, 62yrs, underwent a reduced intensity allogeneic SCT in March 2010 for stage 3 Sezary syndrome. Post SCT he developed pancytopenia associated with EBV reactivation. He received daily GCSF, regular blood and weekly platelet transfusions. In July he developed GI bleeding from HSV colitis and his platelet transfusion requirements increased to alternate days. In October 2010 he was started on Romiplostim 250mcg, increased to 350mcg weekly, erythropoetin and GCSF. He had a clinically significant rise in his platelet counts and a reduction in his platelet requirements to 1x weekly within weeks of starting romiplostim. By December he was platelet independent, with counts persistently over 50, and romiplostim was stopped (figure1c). All three patients remain in remission at the time of submission of the abstract. Immune cytopenias are well-recognised post myeloablative allografts occurring in 5–37% of SCT recipients. The occurrence of ITP in SCT recipients has been reported, in which GVHD or the production of anti-platelet alloantibodies of recipient origin was considered to play major roles. Although small numbers, these case studies show efficacy of thrombopoietic agents (recently licensed for use in chronic ITP) in patients with thrombocytopenia post SCT refractory to standard therapies, with an early reduction and subsequent cessation in platelet transfusion requirements in all three patients. The lack of immune suppression with these agents may be particularly relevant in patients post SCT, in whom immune reconstitution is vital not only in preventing infections, but also in preventing rejection of the graft and in establishing a graft versus leukaemia effect. Given that TPO receptors are present on haematopoietic stem cells at all stages of development and that TPO provides a growth factor for stem cells, the use of these agents in patients treated for leukaemia requires caution. There was no evidence of progression of disease in these patients and TPO agents were only required for a period of time (patient 3 is off therapy and the others only receive minimal doses). Formal evaluation of these agents in allogeneic SCT is warranted. Disclosures: Cooper: GSK: Honoraria; Amgen: Honoraria.

Abstract 2337 Viral infections remain a significant problem following haematopoietic cell transplantation (HCT). Recovery of CD4+ T-cells only occurs with return of thymopoiesis 6–12 months after HCT. In an attempt to improve immune reconstitution and reduce viral infections following unrelated cord blood transplantation (CBT), serotherapy was omitted from conditioning. 26 children, median age 19 months (range 1–147) underwent unrelated CBT for immunodeficiency (13) or high risk/relapsed leukemia (13). Pre-transplant co-morbidities included lung aspergillosis (2), adenoviraemia (1), Paraflu3 respiratory tract infection (3). Conditioning was myeloablative in 25 patients. HLA matching was 6/6, 5/6 and 4/6 in 13, 14 and 3 cases respectively (4 double cords). GvHD prophylaxis was mostly cyclosporine/mycophenolate mofetil (24). The median number of nucleated, CD34+ and CD3+ cells infused was 8.12 × 107/kg (range, 4.7–17.72), 3.5 × 105/kg (range, 1.23–11.95) and 5.63 × 106/kg (range, 1.9–24.45). Median time to myeloid and platelet engraftment was 22 (range, 13–38) and 39 (range, 21–110) days. One patient died from conditioning related toxicity and 3 patients died after leukaemia relapse. 22 patients are alive and in remission with a median follow up of 12 months (range 3–66). 18 patients have full donor engraftment while 4 patients are mixed chimeras. Acute GvHD > grade I occurred in 16 patients (grade II n=11; grade III-IV n=5) but in general this responded to steroids and only 1 child developed cGvHD. There was no infection-related mortality, despite early adenoviraemia (4), CMV viraemia (4), HHV6 encephalitis (1) and RSV/Paraflu3 respiratory tract infections (3). The most striking finding was a rapid and sustained CD4+ T-cell expansion with conversion from naive to memory/effector phenotype. The median CD3+, CD4+ and CD8+ counts at 2 months post CBT was 0.85 ×109cells/L (range, 0.44–2.44), 0.55 ×109cells/L (range, 0.06–1.89), and 0.21 ×109cells/L (range, 0.01–1.21) respectively. As a comparison, the median CD4+ T-cell count at 2 months post a HCT from a matched sibling donor was 0.24 ×109cells/L in 23 children evaluated in our institution. In 3 patients who developed adenoviral/CMV reactivation virus-specific T cells were detected by ELISPOT/IFN-g assays as early as 45 days post transplant, and viral infections resolved rapidly. Unrelated CBT without serotherapy results in rapid “peripheral” expansion of naive/memory CD4+ T-cells, and restores cell-mediated immunity in children faster than any other HCT donor source. Disclosures: No relevant conflicts of interest to declare.

Abstract 3320 Poster Board III-208 Late mortality in children who have undergone hematopoietic stem cell transplantation (HCT) for severe combine immunodeficiency (SCID), non-SCID immune diseases and inborn errors of metabolism (IEM) has not been studied. The goals of the current analyses were to: 1) determine the probability of long-term survival after HCT in patients who survive the first 2 years after HCT; 2) identify risk factors for late deaths; and 3) determine excess mortality relative to rates in an age and sex-matched general population. Nine hundred and sixty patients with >95% donor chimerism or recovery of T-cell function who survived at least 2 years after their transplantation were included in the study. Two hundred and one patients had SCID, 407, non-SCID immune diseases and 352, IEM. Seventy percent of SCID transplant recipients received grafts from an HLA-matched sibling or mismatched relative. Fifty six percent of non-SCID and IEM transplant recipients received grafts from unrelated donors, 32% from a matched sibling and 12% from a mismatched relative. All transplantations occurred in 1980 – 2003; the median follow-up of surviving patients was 7 years. Median ages of long-term survivors were 7, 9 and 10 years for SCID, non-SCID immune diseases and IEM, respectively. Because of differences in biologic features and transplant strategies for SCID, non-SCID immune diseases and IEM, the disease groups were analyzed separately. The 7-year probabilities of overall survival were 93%, 96% and 90% for SCID, non-SCID immune diseases and IEM, respectively. No patient, disease or transplant characteristic was associated with late deaths in patients with SCID. For non-SCID immune diseases, late deaths were higher in recipients of T-cell depleted grafts (RR 4.63, p=0.003). For IEM, late deaths were higher after unrelated donor (RR 2.75, p=0.018) and mismatched related donor (RR 2.77, p=0.042) transplants compared to matched sibling donor transplant. There were 69 late deaths with 52 occurring 2 – 6 years after transplantation and 17 after 6 years. Causes of death in patients who died between 2 – 6 years included: chronic graft-versus-host disease [CGVHD] (n=12), infection including encephalitis (n=11), organ failure (n=12), post-transplant lymphoproliferative disease (n=4), primary disease (n=5), acute abdomen (n=1), status epilepticus (n=1), acute myeloid leukemia (n=1), accidental death (n=1) and not reported (n=3). Causes of death beyond 6 years included CGVHD (n=1), infection including encephalitis (n=3), organ failure (n=4), primary disease (n=2), acute abdomen (n=1), brain stem glioma (n=1) and not reported (n=5). The table below shows the estimated excess deaths per 1000 compared to an age- and sex-matched general population at 2 – 6 years and beyond 6 – 10 years after HCT. Though the risk of late deaths in this population is in excess of that for the general population for several years after transplantation, with extended follow up, the risk appears to decrease towards normal rates. Beyond 6 years after HCT, among patients transplanted for SCID and non-SCID immune diseases, the risk of mortality does not differ significantly from the general population whereas for patients with IEM, mortality rates continue to be higher than that in the general population. Screening programs aimed at identifying late complications together with planned intervention may improve long-term survival in these patients. Excess deaths per 1000 (95% confidence interval) SCID Non-SCID IEM 2 – 6 years after HSCT 54 (28, 79)* 41 (28, 53)* 95 (82, 109)* 6 – 10 years after HSCT 25 (0, 51) 16 (0, 39) 45 (27, 62)* * Significant differences in mortality risks compared to the general population Disclosures No relevant conflicts of interest to declare.

Abstract 664 Severe combined immune deficiency (SCID) diseases, as well as other severe T-cell deficiencies like Omenn syndrome, are considered pediatric emergencies. To date, hematopoietic stem cell transplantation (HSCT) is the only curative therapy for these children. Considering the urgency of these transplants, for patients lacking an identical sibling donor, the use of mismatched related donors (MMRD) and unrelated umbilical cord blood (UCB) are important alternatives. In a retrospective study, we compared the outcomes of 175 children with severe T-cell deficiencies (SCID=152, Omenn syndrome=23) receiving MMRD transplants and 74 (SCID=52, Omenn syndrome=15) receiving UCB transplants, reported to the SCETIDE and Eurocord databases, from 1995 to 2005. MMRD transplants were performed in 12 different centers and UCB transplants were distributed among 30 different centers. Only 5 centers performed the two techniques. The median age at transplant was 6.4 months for the UCB group and 6.5 months for the MMRD group (p=0,87). Median time elapsed between diagnosis and transplant was 47 days for MMRD (range 4-485) and 55 days (range 17-526) for UCB transplants (p=0.06). There were no statistically significant differences between the two groups regarding gender and incidence of failure to thrive, chronic diarrhea or respiratory impairment before transplant. Patients receiving UCB had more often B-negative phenotype (61% UCB vs. 41% MMRD; p=0.009), while patients receiving MMRD had a greater incidence of viral infections prior to transplant (59% MMRD vs. 36% UCB; p=0.004). The year of transplantation also differed between the two groups, as more patients receiving UCB were transplanted between 2000-2005 (65%) compared to MMRD (50%) (p=0.014). Regarding HLA (considering HLA-A,B-low resolution and DRB1-high resolution), in the UCB group, 67% of patients had 0 or 1 disparities while 33% had 2 or 3 disparities. In the MMRD group, 10% had 1 disparity and 90% had 2 or 3 disparities. Seven patients receiving UCB (16%) transplants and 30 patients receiving MMRD (17%) did not receive any preparative regimen prior to graft infusion. Conditioning regimen was non-myeloablative in 45% of MMRD transplants and 30% of UCB transplants. The remaining received myeloablative regimens. Graft-versus-host disease (GVHD) prophylaxis was cyclosporine A-based in the majority of the UCB transplants (n=71). T-cell depletion was used in all transplants with MMRD and 52 patients received immunosuppressants, mostly cyclosporine A. The median follow-up time was 84 months for patients given a MMRD and 59 months for UCB transplants recipients. Sixty-one patients receiving UCB transplants engrafted (82%), all with full donor chimerism in CD3+ cells. On the CD3- compartment 75% had full donor, 5% mixed and 20% had recipient chimerism. In MMRD group, 130 patients engrafted (74%), 98% with full donor chimerism in CD3+ cells. On CD3- cells, 33% had full donor chimerism, 48% were mixed and 19% recipient chimerism. Forty-six patients in the MMRD group and 7 patients in the UCB group needed to receive second transplants. The cumulative incidence of acute GVHD had no statistically significant difference between groups (33% UCB vs. 23% MMRD; p=0.13). However, patients receiving UCB transplants had more chronic GVHD (22% UCB vs. 11% MMRD; p=0.04). In a univariate analysis, 5-year overall survival was 62 ± 4% for MMRD and 58% ± 6% for UCB transplants (p=0.83). In a multivariate analysis adjusting for differences between the two groups, 5-year overall survival was not statistically different (p=0.54). Regarding T-cell reconstitution, we analyzed the total lymphocyte, CD3+ and CD4+ cell numbers and there were no statistically significant differences between the two groups comparing absolute numbers at 6, 12 and 24 months after transplantation. In conclusion, both unrelated cord blood and mismatched related donors are choices for transplants in severe T-cell deficiencies lacking an HLA-identical sibling donor. The choice of donor type will depend on centre's strategy regarding the possibility of efficiently deplete MMRD grafts of T-cells, or find a rapidly available UCB unit. Disclosures: No relevant conflicts of interest to declare.

Immunotherapy with allodepleted donor T-cells generated using a CD25 immunotoxin improves immune reconstitution after haplo-SCT, but leukaemic relapse remains a problem. To develop a rational approach to refining allodepletion, we characterised the phenotype of proliferating alloreactive T cells flow cytometrically. CFSE-labelled T cells were co-cultured with HLA-mismatched dendritic cells and serial FACS analyses performed to determine expression of CD25, CD69, CD71, HLA-DR, OX40, ICOS, CD95, CD45RA, CCR7, IFNγ, TNFα, and IL–2. By staining each sample with CD3, CFSE and CD25 we were able to determine the phenotype of proliferating CD25 negative alloreactive T cells. In 5 donors, after a 3 day culture, CD25 was expressed in a mean of 83% (range 67–89%) of the proliferating (CFSE-dim) alloreactive T cells, confirming CD25 as an excellent target for allodepletion. 70% (39–81%) of the proliferating alloreactive CD25 –ve population expressed CD71, and 62% (50–74%) expressed CD45RA, identifying these as markers to target alloreactive T-cells that would persist after CD25 based allodepletion. To optimize our CD25 based allodepletion, we compared allodepletion using a CD25 immunotoxin or immunomagnetic beads in 6 HLA mismatched donor-recipient pairs. There was no significant difference between residual alloreactivity to host in primary and secondary MLRs and IFNγ ELISPOT assays. We then compared CD25 vs CD25/71 immunomagnetic depletion in 8 HLA-mismatched donor-recipient pairs. Rested allodepleted cells were restimulated with host or 3rd party stimulators in 2° proliferation or IFNγ ELISPOT assays. The median residual reactivity to host in IFNγ ELISPOT assays was significantly lower after combined CD25/71 than CD25 allodepletion (14.1% vs 54.6%, p < 0.05). Likewise, in 2° MLRs, CD25/71 depletion resulted in significantly lower residual proliferative response to host than CD25 depletion (median 4.8% of the response of unmanipulated PBMC vs 9.9%, p < 0.01). Third party responses after CD25/71 allodepletion were equivalent to unmanipulated PBMCs from the same donors in both assays. CD25/71 allodepletion also gave lower residual responses to host in both assays than combined CD25/45RA allodepletion. In IFNγ ELISPOT assays, anti-viral responses to CMV and EBV were preserved after combined CD25/71 allodepletion (CMV: unmanipulated 151 spots vs.CD25/71 123 spots, EBV unmanipulated 100 spots vs. CD25/71 142 spots).Similarly, the frequency of CD8 +CMV and EBV–specific T-cells assessed by pentamer analysis was not significantly reduced compared to unmanipulated PBMCs. We conclude that CD25/71 allodepletion will selectively delete 94% of the proliferating alloreactive T cells and enhances the depletion of alloreactivity compared with CD25-based methods. This strategy may facilitate immunotherapy with larger doses of allodepleted donor T-cells after haplo-SCT, enhancing graft versus leukaemia and antiviral effects.

Childhood leukaemia which is refractory to chemotherapy or which relapses early following stem cell transplantation (SCT) has a very poor prognosis, with few patients becoming long-term survivors. Establishing limited graft-versus-host disease (GVHD) in an attempt to exert a graft-versus-leukaemia (GVL) response in these patients remains the only curative option. Second SCT procedures and/or donor lymphocyte infusions (DLI) have achieved some success but the pace of acute leukaemia is often too fast and the occurrence of GVHD/GVL too unpredictable to achieve this goal. We postulated that the addition of alpha interferon (alphaIFN) might induce/augment and control GVHD/GVL in this group of patients and so improve outcome. Thirteen patients underwent initial SCT, for cALL CR2 (n=1), Philadelphia positive ALL CR1 (5), MDS/AML (2), refractory AML (2), and CML (3), with unrelated (9) or sibling donors (4). Eight patients subsequently had a frank haematological relapse and 4 patients a molecular relapse of their leukaemia at a median of 10 months (2–18 months) from 1st SCT. Six patients underwent a 2nd SCT from the same donor, all with reduced intensity conditioned regimes; all received alphaIFN when GVHD did not occur after early withdrawal of immunosuppression. One (ALL CR2) received DLI 2 months after 2nd SCT for mixed chimerism. A further six patients received DLI plus alphaIFN, 3/6 together with low dose DLI (

Worldwide more than 400 patients with Hurler’s syndrome (HS), characterized by severe neurodegeneration, cardiac disease, skeletal abnormalities and death in early childhood, have undergone allogeneic-HSCT since 1980. Although, long term follow up of successfully transplanted children is very encouraging, the engraftment and survival results are very variable between the various studies, ranging from less than 25% up to more than 85%. We retrospectively analyzed the results of 146 patients transplanted in Europe from 1994–2004, to assess: 1) the effect of conditioning regimen and grafts (-manipulation) used on the “alive and engrafted” rate and 2) the transplantation-related morbidity/mortality. HSCT with a family donor was performed in 52 patients. An unrelated donor was used in 94 patients. The majority of patients received marrow (n=103). The rest received cordblood (n=23) or peripheral blood (n=20). Twenty-eight patients received a T-cell depleted (TCD) graft. Conditioning regimens used were grouped as follows: busulfan-cyclofosfamide 200mg/kg (n=68), busulfan- with high dose cyclofosfamide (either 240mg/kg or 260mg/kg; n=41), fludarabine-based myelo-ablative (n=19) and reduced intensity conditioning regimens (RIC: n=18). Fourteen patients received dose-adjusted busulfan. Engrafted was defined as a donor chimerism of more than 10%, and an alpha-L-iduronidase level of more than the lower limit of normal for the heterozygote individuals (>4.5 nmol/hr/mg). The “alive and engrafted” rate after first transplantation and overall “alive and engrafted” rate after one to three transplantations was 83/146 (57%) and 111/146 (76%), respectively. The median follow up was 39 mth (5–120mths). Multivariate analysis (confounders: age, sex, heterozygote donor, unrelated donor, stem cell source, HLA-disparity, conditioning regimen, TCD and busulfan targeting) on the primary endpoint “alive and engrafted” showed that RIC (RR 13,4: 2,6–67,1) and TCD (RR 5,7: 1,14–28,4) are individual risk factor for graft failure. Busulfan targeting suggests to be an individual protective factor (RR 0,27; 0,04–1,8); 12/14 (86%) were “alive and engrafted” after 1st HSCT. Thirthy three patients received a 2nd graft, of whom 26/33 (82%) are alive and engrafted: 16/21 using the same, 10/12 using a different donor, and 16/19 after myeloabaltive, 10/14 after RIC. Two of the 3rd HSCTs were successful. After 1st HSCT moderate to severe aGvHD (grade ≥2) occurred in 23/146 (16%) patients. Extensive cGvHD was seen in 2/114 (1.4%) patients, only. IPS/DAH was seen in 4/134 (2.3%) patients and VOD in 12/134 (9%) patients. Main cause of death (n=28) was infectious (n=15: mainly viral). Other causes of death: GvHD (n=3), Cardiac ECI (n=2), VOD (n=2), DAH (n=1), unknown (n=1), Hurler (n=4). The majority of the “alive and engrafted” patients have a donor chimerism of >95% (91/111; 82%), 11/111 (9,9%) between 75–95%, 6/111 (5,4%) between 50–75% and 3/111 (2.7%) between 10–50%. In summary, no stem cell source (BM, cordblood and PBSC) is superior and no conditioning regimen used is superior. RIC and TCD results in inferior engraftment rates. Second HSCTs are successful in more than 80%. Relatively low morbidity rates are seen. The engraftment of HSCT for HS can be optimized by avoiding T-cell depletion, RIC and probably by busulfan-targeting.

Worldwide more than 400 patients with Hurler’s syndrome (HS), characterized by severe neurodegeneration, cardiac disease, skeletal abnormalities and death in early childhood, have undergone allogeneic-HSCT since 1980. Although, long term follow up of successfully transplanted children is very encouraging, graft-failure, mortality and morbidity can limit its success. Although ERT (laronidase) is available for HS, a HSCT is still needed to abort severe neurodegeneration. We analyzed 21 patients who underwent a HSCT with ERT in Europe between 12/2003 to 4/2005, to assess: the effect of ERT on the “alive and engrafted” rate and the transplantation-related morbidity/mortality. Twenty-one patients received ERT (100 U/kg weekly) from diagnosis until approximately 7 weeks (range 0–20 wks) after successful HSCT. Clinical condition was, except for 1 patient, moderate/good before start of ERT. Thirty-one HSCTs were performed in these patients: 8 patients received a 2nd graft because of graft-failure of whom 2 received a 3th graft. For the first HSCT, 8 patients received full-graft marrow or PBSC, the others T-cell depleted (marrow/PBSC: n=3) or cordblood (n=10). Sixteen unrelated donors and 5 family donors (4sibs/1haplo) were used. Conditioning regimens used were conventional myeloablative (n=17) or treosulfan / fludarabine (n=4). Engrafted was defined as a donor chimerism of more than 10%, and an alpha-L-iduronidase level >LLN for heterozygote individuals (>4.5 nmol/hr/mg). Effect of ERT on the “alive and engrafted” rate was analysed in a multivariate analysis with a historic (transplanted 1994–2004) control group (n=148). Clinical condition before HSCT was good for all patients. The “alive and engrafted” rate after first HSCT and overall “alive and engrafted” rate after one to three HSCTs was 12/21 (57%) and 17/21 (81%), respectively. The median follow up was 8 mth (3–17mths). Two patients died: 1 after 2nd and 1 after 3th HSCT. HSCTs with full-graft were successful in 5/8, and for TCD 0/3 and cordblood 8/10. Five of 8 patients receiving a 2nd graft are alive and engrafted and 1/2 after 3rd graft. Multivariate analysis (confounders: age, sex, heterozygote donor, unrelated donor, stem cell source, HLA-disparity, conditioning regimen, T-cell depletion and busulfan-targeting) on the primary endpoint “alive and engrafted” showed that ERT doesn’t significantly influence the primary endpoint (RR 0,54: 0,15–2). After 1st HSCT moderate to severe aGvHD (grade ≥2) occurred in 1/21 (4,8%) patients. Extensive cGvHD wasn’t seen. IPS/DAH was seen in 1/21 (4,8%) and VOD in 2/21 (9,6%) patients. Cause of death was extensive cGvHD after 3rd and candida sepsis after 2d HSCT. The majority of the overall “alive and engrafted” patients are having a donor chimerism of >95% (15/17), 2 patients are mixed chimeric (75–95%). ERT-infusion-related toxicity was limited to mild reactions. In patients with HS, ERT with HSCT is well tolerated, with low morbidity rates, but had no effect (neither positive nor negative) on the engraftment in this group. Specifically for patients in a poor clinical condition preHSCT, ERT might be of advantage. A prospective study is needed to determine the longer term outcome of ERT on HSCT outcomes.