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The two first authors contributed equally. Introduction There is currently little data on the outcome of AML in adolescents and young adults (AyAs) after allogeneic HSCT. In this retrospective study from the SFGM-TC registry, we analyzed the outcome of AML patients classified in 3 groups according to the age at transplantation: children ( Patients and Method This study included 2240 patients from 43 centers among France, Belgium, Switzerland, Lebanon and Algeria, of whom 481 children (21.5%), 545 AyAs (24.3%) and 1214 adults (54.2%). All patients received a first allogeneic HSCT for AML between 2005 and 2015. Five-years OS, NRM, GRFS and EFS and cumulative incidence of relapse were compared between the three groups of patients. Results The median follow-up of the study was 7 years (range, 4-9.6). According to the 2016 WHO classification, children had the highest rate (31.8%) of adverse risk disease, followed by AyAs (25.2%) and adults (21.3%) (p=0.0001). Extramedullary disease was observed in 42.8%, 24% and 21.4 % of children, AyAs and adults (p=0.0001). Among patients in complete remission (CR), younger age was associated more frequently with advanced disease (29.9% ≥ CR2 for children, 23% for AyAs and 18.2% for adults) (p=0.0001). Donors were matched siblings in 36.4% of children, 36.7% of AyAs and 40.3% of adults, and matched unrelated in 29.1%, 33.4% and 35.7% respectively. Children had more mismatched unrelated donors (31.6%), compared to AyAs (25.3%), and adults (19.5%) (p=0.0001). Stem cell source was different between the 3 groups (p=0.0001): bone marrow (BM) was the main source for children (60.9 % of grafts) followed by cord blood (CB) (28.9%), whereas peripheral blood stem cells (PBSC) was the main source for adults (60 %), followed by BM (29.7%) and CB (10.2%). AyAs were transplanted with BM in 44.4% of cases, PBSC in 40.4% or CB in 15.2%. The intensity of the conditioning was myeloablative (MAC) in 93% of children, 79% of AYAs and 77.7% of adults, while a reduced-intensity regimen was used in 4% of children, 12.3% of AyAs and 14.1 % of adults (p=0.0001). The 5y-overall survival (OS) was higher in children (64%), compared to AyAs (54%) and adults (52%) (p=0.001) (figure). One-year cumulative incidence (CI) of relapse was comparable in the 3 groups: 27 % for children, 33% for AYAs and 28% for adults (p=0.19) (figure). The 5y-CI of EFS and the 5y-CI of NRM were better for children (56%) compared to AyAs (49%) and adults (47%) (p=0.017) respectively and 11%, 18% and 27% for NRM in each group respectively (p=0.0001). Regarding causes of deaths, GvHD related mortality was significantly higher in AyAs and adults with 3.1%, 5.1% and 7.7% in each group respectively. There was no difference in the CI of grade II-IV acute GvHD at day +100 between the 3 groups: 37.7%, 35.8% and 35% respectively (p=0.58) while the one-year CI of chronic GvHD was higher in adults (37%) and AyAs (31%) compared to children (17%) (p=0.0001) (Figure). AyAs had a significantly lower 5y-OS and 5y-EFS than children and AyAs had a greater risk of NRM than children in this study. Moreover the GRFS is significantly better in children with 5y-survival 26% for children, 18% for AyAs and 17% for adults (p=0.011) (figure). In univariate and multivariate analysis, independent factors associated with OS were age, TBI, cytogenetics and status of disease, HLA matching and donor age. Conclusion AyAs patients seem to have greater risk of NRM and cGvHD after HSCT than children. This study suggests that AyAs should be treated more like children and that donor age and HLA compatibility should be considered in the treatment strategy. Figure. Figure. Disclosures Chalandon: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel costs. Beguin:Kiadis Pharma: Consultancy. Peffault De Latour:Amgen Inc.: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Outcomes for adolescents and young adults (AYA) might be considerably different from children and older adults with acute leukemia (AL). Patients in this transitory period tend to be considered at higher risk for poorer outcomes. However, AYA is still underrepresented in most hematopoietic stem cell transplant (HSCT) studies. Recent evidence shows superior outcomes for AYA with acute lymphoblastic leukemia (ALL) using pediatric rather than adult first line chemotherapy regimens. The aim of this study is to analyze outcomes of umbilical cord blood transplant (UCBT) in AYA patients with ALL or acute myeloid leukemia (AML) after myeloablative conditioning regimen (MAC). Eligibility criteria included patients with AL aged 15-25 (median 19) years, transplanted between 2004 and 2016 in EBMT centers, with single or double UCBT as first allogenic transplantation after MAC regimen and reported to Eurocord. A total of 504 patients were included. Primary endpoint was overall survival (OS). Median follow-up for survivors was 38 months (1 month-11 years). Median time from diagnosis to UCBT was 11 months (1 month-17 years) (interquartile range 5-29 months). Diagnosis was ALL in 59% (n=297) and AML in 41% (n=207); 64% of patients were male. Disease status at UCBT were 42% in 1st complete remission (CR) (n=203), 2nd CR in 38% (n=183), and advanced disease in 20% (n=96). Fifty eight percent of patients (n=293) received single and 42% (n=211) double UCBT. Cyclophosphamide + TBI ± fludarabine (45%) was the most frequently used conditioning regimen. Anti-thymoglobulin (ATG) was used in 54% and GVHD prophylaxis was cyclosporine A + mycophenolate mofetil in 44% of patients. Median nucleated cell dose at collection was 3.63 (range 0.9-9.0) X107/Kg for single and 5.25 (range 2.3-10.6) X107/Kg for double UCBT. The 3-year OS was 45±2%; 3-year leukemia free survival (LFS) was 41±2% and refined GVHD free/relapse free survival (rGRFS) was 32±2%. OS was similar in ALL and AML (45±3% vs 45±4%, p-value=0.69). According to disease status at UCBT, 3-year OS was 54±4% for patients in CR1, 48±4% for CR2 and 20±4% for advanced disease (p-value Cumulative incidence function (CIF) for neutrophil engraftment at day-60 was 88% [95% confidential interval (CI) 85-90] in a median time of 24 days. CIF of acute GVHD grade II-IV at day-100 was 28% (95%CI 24-32) and out of the 233 patients with aGVHD, 73 (21%) had grade III-IV. The 1-year CIF of chronic GVHD was also 28% (95%CI 24-33) and out of the 124 patients with cGVHD, 52 (42%) had the extensive form. The 3-year CIF of transplant related mortality (TRM) was 31% (95%CI 24-33). The 3-year CIF of relapse was 28% (95%CI 24-32), and according to disease status at UCBT, relapse was 28% (95%CI 21-34) for patients in CR1, 24% (95%CI 18-31) for those in CR2 and 37% (95%CI 27-47) for advanced disease. Main causes of death were relapse (41%) and TRM (57%, mainly infections and GVHD). In multivariate analysis, disease remission at UCBT (HR 0.46, 95%CI 0.32-0.66, p-value This large series of AYA patients with AL provides detailed information on UCBT for this particular cohort with results comparable with other sources of HSCT. Due to the lack of information on pre-transplant chemotherapy (pediatric versus adult protocols), we were unable to evaluate its effect on outcomes. Nevertheless, this study contributes to a better understanding of HSCT for an age group that is not thoroughly described in most publications. Demonstrating the feasibility of UCBT in AYA patients is important to facilitate the decision of stem cell source selection when a more standard donor is not available. Disclosures Hough: University College London Hospital's NHS Foundation Trust: Employment. Angelucci:Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC; Celgene: Honoraria, Other: Chair DMC; Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Roche Italy: Other: Local (national) advisory board. Bader:Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Riemser: Research Funding; Cellgene: Consultancy; Novartis: Consultancy, Speakers Bureau.

The last three authors contributed equally to this work. Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) may improve long-term survival of patients with acute lymphoblastic leukemia (ALL) at high risk of relapse. However, ovarian failure is observed in 70 to 90% of patients who received myeloablative HSCT. Autotransplantation of cryopreserved ovarian cortex harvested before gonadotoxic treatments has been shown to re-establish the menstrual cycles and lead to the birth of healthy children (Donnez J. N Engl J Med, 2018). In patients treated for acute leukemia, concern has been expressed about the risk of leukemia recurrence after the ovarian autotransplantation due to the potential presence of residual leukemic cells in the ovarian tissue (Dolmans MM. Blood, 2010). To date, there were only few studies evaluating the presence of leukemic cells in cryopreserved ovarian tissue, and in most cases ovarian tissues were harvested at diagnosis of ALL or early after onset of chemotherapy. The present study prospectively investigated, in the context of fertility preservation, the presence of leukemic cells in cryopreserved ovarian samples harvested before allogeneic HSCT in patients in complete remission (CR) of ALL. Patients and Methods: From October 2015 to May 2018, all female patients with ALL who had a leukemia specific marker and underwent ovarian cryopreservation before allogeneic HSCT as part of preservation fertility program in 3 centers were included in the study. Consents were obtained from the guardians and/or age appropriate patients. The specific local ethical committees approved the study. Ovariectomy was performed after patients achieved CR, mostly in the weeks preceding HSCT. Ovarian cortex was separated into several fragments and cryopreserved as previously described (Poirot C. Human Reprod, 2002). When more than 14 cortical fragments were obtained, one was dedicated to molecular analysis, as well as the ovarian medulla. MRD quantification was performed by quantitative PCR of clonal rearrangements of immunoglobulin/T-cell receptor genes (Ig/TCR) or oncogenic fusion genes, according to EuroMRD and European Against Cancer (EAC) guidelines. MRD in ovarian samples were compared to MRD in bone marrow (BM) sample obtained at the same time point. Results: Fourteen patients were included in the study: 12 with B-cell precursor ALL, 1 with T-cell ALL and 1 with mixed phenotype acute leukemia. MRD marker was Ig/TCR rearrangement in 12 cases, M-BCR-ABL transcript in 1 case and genomic MLL-AF4 in 1 case. Median age at transplant was 18.2 years (range 1.1 - 35.2 years). Criteria for HSCT were poor early response to treatment (N=8), Philadelphia positive ALL (N=1), or previous relapse (N=5). With a median follow-up after HSCT of 8.1 months (range 0.8 - 29.8 months), all patients are disease-free. At the time of ovariectomy, 7/14 (50%) patients had undetectable MRD in ovarian samples, 6 had low positivity below 10-4 and one had positive MRD higher than 10-4. Unexpectedly of the 7 patients with positive ovarian MRD, 4 were undetectable in BM. One of the 7 patients with undetectable MRD in ovarian samples was positive in bone marrow. Concordant results were observed between the cortex and medulla samples in 10 of the 11 cases where both could be tested. Conclusions: To our knowledge, this series is the largest cohort evaluating MRD in ovarian samples from patients with ALL in complete remission after full chemotherapy regimen. We detected low levels of residual leukemic cells in ovarian tissues in half of the patients, some of them having no detectable MRD in BM at the time of ovariectomy, suggesting that leukemic cells could preferentially persist in ovary. Our results warrant further analyses of an extended cohort and longer post-transplant follow-up to better assess the potential presence of leukemic cells in ovarian samples and evaluate the impact of ovarian MRD status on post HSCT relapse. Still, autotransplantation of cryopreserved ovarian cortex could be discussed after allogeneic HSCT in patients with undetectable ovarian MRD. Table 1. MRD evaluation in bone marrow and ovarian samples. BM, bone marrow; BCP-ALL, B-cell precursor ALL; Ph-positive ALL, ALL with Philadelphia chromosome; NA: not available. Undetectable MRD means negative result obtained with a sensitivity of 10-4 or 10-5. BCR-ABL1 MRD results are expressed as the BCR-ABL1/ABL1 transcripts ratio. Disclosures No relevant conflicts of interest to declare.

Children with acute leukemia (AL) in need of an allograft and lacking an HLA-identical sibling and an HLA-matched unrelated donor, could benefit either from unrelated cord blood transplantation (UCBT) or T-cell depleted haploidentical (haplo) stem cell transplantation (SCT). Different techniques of T-cell depletion have been developed for improving immune reconstitution and for enhancing the graft-versus-leukemia (GvL) effect. Locatelli et al. (Blood 2017) reported remarkable results for children with AL given haplo-SCT after a novel method of graft manipulation based on selective, negative depletion of alfa-beta T and B cells. On behalf of Eurocord, CTIWP, and PDWP of EBMT, we compared outcomes of patients < 18 years with AL undergoing either single-UCBT (n=444) or haplo-SCT after alfa-beta T-cell and B-cell-depletion (n=99) from 2012 to 2016 using a myeloablative conditioning regimen (MAC). Median age at SCT was 6 (range 0.3-18) and 8 (range 0.6-18) years, and median follow-up was 36 and 14 months (p Conditioning regimen varied according to the type of transplant: for haplo-SCT recipients Fludarabine+TBI (28%) and Thiotepa+Busulfan+fludarabine (TBF) (24%) were the most frequent MAC. In UCBT, Busulfan+Cyclophosphamide (Cy), or Cy+Fludarabine+TBI or TBF were used in 19%, 15% and 13% of patients, respectively. Haplo-SCT recipients did not receive any additional pharmacological post-transplant graft-versus-host disease (GvHD) prophylaxis, while CyclosporineA (CSA)+ steroids and CSA+mycophenolate mofetil were used in 38% and 32% of UCBT recipients. The 60-day probability of neutrophil engraftment was 91% vs 93% (p According to donor type, no differences were found in overall survival (OS) (65% vs 68%, p=0.17) and leukemia-free survival (LFS) (61% vs 65%, p=0.24), while a lower GvHD-free, relapse-free survival (GRFS) was observed in UCBT compared to haplo-SCT (49% vs 60%, p In multivariate analysis, UCBT was associated with higher risk of NRM (HR 2.36, p Our results showed that RI, LFS, OS and cGVHD were comparable between UCBT and haplo-SCT recipients, while better NRM, aGVHD and GRFS were observed in patients transplanted from a family donor. These data confirm that alfa-beta T-cell and B-cell depleted haplo-SCT is a valid alternative for patients in need of an urgent allograft. Costs of manipulation in the haplo-SCT setting and costs of cord blood procurement should be further investigated. Technical expertise with this refined approach of graft manipulation and center experience are important to optimize patient's outcome. Disclosures Locatelli: Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Zecca:Chimerix: Honoraria. Bader:Medac: Patents & Royalties, Research Funding; Cellgene: Consultancy; Neovii: Research Funding; Riemser: Research Funding; Novartis: Consultancy, Speakers Bureau.

Introduction: Thrombopoietin receptor agonist eltrombopag (ELT) provides hematologic improvement in up to 50% of adults with acquired aplastic anemia (AAA) refractory to immunosuppressive therapy (IST). However, current data for ELT efficacy and tolerance in childhood AAA is very limited. Methods: We conducted a multicenter nationwide retrospective study on behalf of the French Reference Center for Aplastic Anemia. Patients under 18 treated with ELT after IST for AAA were included. Lineage responses were defined as follows: red cells transfusion independency or hemoglobin increase of 1.5 g/dL, platelets transfusion independency or a 20 G/L increase in platelets counts, doubling of the neutrophil count or an increase above 0.5 G / L. Results: We identified 12 children (2-17 years old, mean 9.5±5.9 years) treated for severe (7) /very severe (5) AAA with horse ATG and cyclosporine as first-line treatment between 2014 and 2018 in five different centers. Minor PNH clone was identified at diagnosis in two patients. All patients had a normal bone-marrow karyotype and FISH. None had a matched related donor. Median time between IST onset and eltrombopag initiation was 3.5 months [1-46]. All patients still met severe aplastic anemia criteria and needed RBC and platelet transfusions at ELT initiation. Mean hemoglobin, reticulocytes, platelets and neutrophils values were 7.3±0.8 g/dL, 24.5±24 G/L, 11±5.9 G/L and 0.8±0.7 G/L. Seven patients had received IST for more than 3 months and were therefore considered refractory to this treatment. ELT was indicated for an uncontrolled bacterial infection in 2 patients one and two months after IST initiation. One patient received an excessive dose of 12.5 mg/kg/day. Average ELT dosage for the other patients was 2±0.6 mg/kg/d. The median duration of treatment with ELT was 5.5 months [1-10]. After ELT initiation, nine patients (75%) reached hematological response for at least one cell line with no additional treatment. In these patients, hemoglobin increased on average by 3±1.3 g/dL, neutrophils by 2.5±3.5 G/L and platelets by 89.8±39.7 G/L. Seven patients (58%) achieved a trilineage response and were both RBC and platelets transfusions independent after 1 month (1), 3 months (3) and six months (7). Five patients (41.6%) reached a robust response (platelets > 50 G/L, hemoglobin > 10 g/dL, neutrophils > 1 G/L). All these trilineage responders were still on cyclosporine at the time of last assessment. ELT was withdrawn in 4 trilineage responders. Two of them still fulfilled robust response criteria 3 and 43 months after ELT withdrawal while the 2 others required occasional transfusions. The 3 others trilineage responders who were still receiving ELT remained transfusion independent 6 (2) and 11 (1) months after ELT initiation. Two patients (16.6%) had a unique lineage response: one was lost of view and one developed a myelodysplasia with RUNX1 mutation 3 months after ELT initiation. He is alive 8 months after HSCT. No clonal evolution was reported in the 5 other patients for whom cytogenetics follow-up was available. No response was achieved in 3 patients (25%), all of whom were treated for less than 3 months: ELT was withdrawn for deemed inefficiency in 2 patients (16.6%) after 1 and 2 months respectively and for toxicity (disseminated intravascular coagulation) in another patient after 2 months. These patients underwent HSCT. One patient died of graft failure 3 months after HSCT. Overall, eleven patients (91%) were alive 13.4±5.6 months after eltrombopag onset. Disseminated intravascular coagulation occurred in a 2 years patient who received a large ELT dosage (12.5 mg/Kg). No other grade III-IV toxicity was reported. Discussion/Conclusion: This is the first report of ELT for childhood severe AAA after IST. Eltrombopag was overall well tolerated. We observed a high rate of sustained trilineage response as reported in adult patients and only 1 case of clonal evolution which is a known complication in non-responsive AAA. Onset of the trilinear hematologic responses is progressive and achieved 3-6 months after ELT initiation. A longer follow-up of this cohort is mandatory to assess response durability and clonal evolution final risk. Nevertheless, we propose that ELT should be considered for severe AAA non-responsive to IST after 3 months of treatment especially if only an alternative donor is available. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute Graft versus Host Disease (aGvHD) occurs in 20-50% of pediatric patients who undergo allogeneic stem cell transplantation. Despite advances in HLA-typing methods and post-transplant immune suppression, it remains a significant cause of mortality and morbidity. Conditioning regimens include alkylating agents that exert their effects through their ability to directly or indirectly damage DNA. Unfortunately normal cells are also damaged due to their rapid proliferation cycles, increasing the probability of treatment-related toxicities. The most vulnerable targets among these damaged tissues are the skin, the intestinal epithelium, and the liver: In this study, our aim was to find biomarkers for aGvHD by focussing on how inter-individual differences in DNA repair mechanisms due to genetic variants in genes encoding DNA repair proteins might affect toxicity. Methods: The study included 115 children that had undergone allo-HSCT at four different centers. All patients received a Busulfan(Bu)/Cyclophosphamide(Cy) conditioning regimen. Pharmacokinetic-guided dose adjustment was performed for Bu to obtain a concentration at the steady state (Css) between 600 and 900 ng/ml. Patients received 16 doses of Bu followed by, i.v. Cy (200mg/kg total dose, 80% of patients) or CyVP16 (120mg/kg total dose). Cyclosporine was given as GvHD prophylaxis, and Methotrexate (MTX) or steroids were added to bone marrow and cord blood transplantation, respectively. ATG was given to 75% of patients. HLA matching was as follows: MRD = 37%; MUD = 21%; MMRD = 4%; MMUD = 38%. Acute GvHD was diagnosed according to the 1994 consensus conference up to day 180 post HSCT. Peripheral blood was collected and the DNA was extracted prior to transplant. Fifty-one single nucleotide polymorphisms (SNPs) within seventeen DNA repair genes were chosen for investigation. Cumulative incidence analysis of aGvHD 2-4 was performed using Kaplan-Meier analysis and log-rank test. Multivariate Cox regression was performed to estimate the impact of genotype on clinical outcome in the presence of other covariates. Results: The most significant finding came from one SNP (rs10764881, G>A) located in the promoter of the MGMT gene. Patients with rs10764881 GG genotypes had a lower risk for aGvHD 2-4 incidence (Figure 1). This variant was not associated with any other treatment related toxicities nor relapses. Multivariate analysis including MGMT rs10764881 with known aGvHD risk covariates did not influence the model. In addition, from the expression analysis that we performed on 24 lymphoblastoid cell lines rs10764881 GG carriers showed 1.5 fold higher expression compared to AA or AG carriers (Figure 2). We were able to confirm experimentally with luciferase reporter constructs the impact of this variant on promoter function. Plasmids, which included the promoter sequence with rs10764881 demonstrated higher luciferase protein levels, compared to plasmids with a promoter sequence excluding rs10764881 (p= 0.01), suggesting the presence of an enhancement element close to the variant region (Figure 3). Electrophoretic mobility shift assays confirmed the presence of a Glucocorticoid Receptor Element (GRE) near to this variant. To understand whether the enhancing effects are related to corticosteroids, keratinocytes transfected with the gene reporter plasmids were stimulated with dexamethasone and luciferase expression was examined as previously. Exposing the cells with dexamethasone significantly increased protein expression of luciferase in both plasmids compared to their non-treated plasmid construct. The highest response was seen from the plasmid containing variant rs10764881 G (Figure 3). However, clinically we did not find clear differences between steroid and non-steroids recipients against rs10764881 with regards to aGvHD vulnerability (Figure 4). Conclusions: We hypothesize that the reason why variant rs10764881 GG might have a protective effect against aGvHD is due to its higher expression levels, resulting in more efficient DNA repair, in turn diminishing the immune response, reducing inflammation and hence causing less aGvHD. Thus aGvHD rs10764881 GG could potentially be a biomarker for aGvHD protection. The effects of steroids on MGMT expression needs to be addressed in future studies. Figure 1 Figure 1. Disclosures Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding. Bittencourt: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel Grant; Amgen Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy.

Introduction Acute graft versus host disease (aGvHD) still remains one of the major causes of procedure-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Information on the outcome of paediatric patients experiencing this complication is limited. For this reason, we conducted a retrospective registry-based analysis on 2519 children who developed grade III-IV acute GVHD and were reported to the European Blood and Marrow Transplantation (EBMT) registry. Patients and methods Included in the study were children below age of 18 years who were transplanted between 2004 and 2014. Median year at transplantation was 2009. Of these children, 826 children had a non-malignant disorder, while 1689 were affected by malignancies. The donor was an HLA-identical sibling in 707 cases and an unrelated donor in 1081 cases. Umbilical cord blood (UCBT) was employed as stem cell source in 396 cases, while a relative other than a compatible sibling was utilized in 202 cases. Overall, 1281 patients were given bone marrow (BM), while 800 received peripheral blood stem cells (PBSC). Results Grade III acute GvHD occurred in 1607 patients (64% of the overall population), while grade IV acute GvHD was diagnosed in 908 (36%). Chronic GvHD occurred in 649 patients (26 % of the overall number of children who developed grade III-IV aGVHD). It was extensive in 333 (13%) and of limited severity in 269 (11%). At time of last follow-up, 1341 patients were alive (53%), while 1178 were dead (47 %). Fifty-seven patients were lost to the follow-up. Relapse of the original disorder occurred in 219 children (19.7% of patients experiencing grade III-IV aGvHD). Transplant-related causes were responsible for the death of 902 patients (76.5%), while 6 (0.5%) patients developed a secondary malignancy. The 3-year Kaplan-Meyer probability of overall survival (OS) was 46.7% (confidence interval 95, 44.1-49.5) and 50.9% (confidence interval 95, 47.1-54.9) in patients affected by malignant and non-malignant disorders, respectively. Patients who received as stem cell source BM had a better outcome in comparison to those who received PBSC (3-year OS, 53.2 vs 40.4%, P Conclusion These data indicate that the occurrence of grade III-IV aGVHD is associated with a dismal outcome also in paediatric patients. The main cause of fatality is represented by non-relapse mortality, while leukemia recurrence affected outcome of a lower number of children. Although the outcome of children experiencing grade III-IV aGvHD is improving over time, strategies aimed at preventing this immune-mediated complication and at optimizing its treatment are desirable. Disclosures Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

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.

CD4(+) T-cell functions that best correlate with CMV control were evaluated by studying the relationship between CMV infection and CMV-specific immune recovery as determined by proliferation assay and intracytoplasmic-IFNgamma assay. A total of 30 children (mean age: 8.30 years) who received an allogeneic hematopoietic SCT (HSCT) were included. In total, 13 recipients were seronegative before HSCT. None developed CMV infection or CMV-specific immunity. A total of 17 recipients were seropositive: (i) four patients spontaneously controlled CMV. The median of CMV-specific IFNgamma-secreting CD4 T cells was 9.13/microl at month 3 in these four patients and three of the four patients evidenced optimal proliferative responses since month 1; (ii) in 10 patients who received anti-CMV chemotherapy because of prolonged viremia, lower (P=0.016) IFNgamma responses (0.39/microl), together with delayed and/or depressed proliferative responses, were observed; (iii) finally, one patient with early CMV-associated disease had undetectable proliferative and IFNgamma responses until month 3. In conclusion, both intense IFNgamma responses and early proliferative responses seem to be associated with optimal CMV control.

Durant ces 30 dernieres annees, l’allogreffe de cellules souches hematopoietiques (CSH) est devenue une therapeutique courante de nombreuses hemopathies, avec un nombre de patients traites en augmentation constante. Les principales complications des allogreffes de CSH sont infectieuses en raison de la lourdeur des traitements immunosuppresseurs recus par les patients pendant des mois ou des annees et du fait de la perte des reponses immunes vis-a-vis des pathogenes apres la greffe. La reconstitution immune est un processus lent, dependant notamment du type de greffe et de la presence ou non d’une reaction du greffon contre l’hote (GvH) chronique. Des lors, la prevention des infections est un probleme majeur chez les patients receveurs d’une allogreffe de CSH, et la vaccination est l’une des strategies permettant de reduire le risque des infections potentiellement couvertes par les vaccins. Cet article resume les recommandations classiques de l’agenda vaccinal apres allogreffe et discute les benefices et les risques attendus de chaque type de vaccin, chez l’adulte et chez l’enfant, en se fondant sur les donnees actuelles de la litterature.

The properties of saliva led us to hypothesize that the salivary flow increase induced by gum chewing might protect the oral mucosa from lesions due to cancer chemotherapy. We conducted a multicenter randomized trial to evaluate the efficacy of chewing gum in preventing oral mucositis in 145 children receiving chemotherapy regimens expected to induce WHO grade 3-4 oral mucositis in at least 30% of patients. Patients were allocated at random to standard oral care with or without 5 gum pieces per day. No overall reduction in severe oral mucositis occurred in the gum arm (51%) compared with the standard arm (44%). VIDE, COPADM, and multidrug intensive chemotherapy caused severe oral mucositis in 75% of patients in both arms. In patients receiving less toxic regimens, a decrease in WHO grade 1-4 oral mucositis was noted in the gum arm compared with the standard arm (49% vs. 72%, P=0.03). In the multivariate analysis, the risk of oral mucositis was related only to the type of chemotherapy regimen, suggesting that further strategies for preventing oral mucositis could be mainly based on these criteria.

International audience; Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

International audience

International audience; Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P = 0·04) and duration of ciclosporin treatment (P < 0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90 d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

International audience; The main aim of the Leucémies de l'Enfant et l'Adolescent (LEA) project (Childhood and Adolescent Leukaemia) is to study the determinants (medical, socioeconomic, behavioural and environmental) of medium- and long-term outcomes of patients treated for childhood acute leukaemia (AL). The LEA study began in 2004 and is based on a French multicentric prospective cohort. Included are children treated for AL since January 1980 (incident and prevalent cases), surviving at month 24 for myeloblastic AL and lymphoblastic AL grafted in first complete remission or at month 48 for lymphoblastic AL not grafted in first complete remission. Information is collected during specific medical visits and notably includes the following data: socioeconomic data, AL history, physical late effects (such as fertility, cardiac function and metabolic syndrome) and quality of life. Data are collected every 2 years until the patient is 20 years old and has had a 10-year follow-up duration from diagnosis or last relapse. Thereafter, assessments are planned every 4 years. In active centres in 2013, eligible patients number more than 3000. The cohort has already included 2385 survivors, with rate of exhaustiveness of almost 80%. Data access can be requested from principal coordinators and must be approved by the steering committee.

To the editor: Fanconi anemia (FA) is a rare, phenotypically heterogeneous inherited disorder clinically characterized by congenital abnormalities, progressive bone marrow failure (BMF), and a predisposition to develop malignancies.[1][1] Hematopoietic stem cell transplantation (HSCT) is the only

The purpose of this study was to evaluate the impact of enteral nutrition on early outcome of patients after myeloablative allogeneic stem cell transplantation (allo-SCT). From January 2001 to January 2003, 22 patients agreed to receive enteral nutrition via a nasogastric feeding tube; the remaining 23 patients received parenteral nutrition (n=22) or standard oral feeding (n=1). Early complications and factors influencing 100-day overall survival (OS) were investigated. Patients who received enteral nutrition developed less often acute-grade III/IV graft-versus-host disease (18%) than those who did not (35%) (P=0.011). In addition, this group showed lower mortality from infection during the first 100 days after transplantation. In multivariate analyses, only the absence of enteral nutrition was found to adversely influence 100-day OS with a hazard ratio of 8.3. Enteral nutrition is a safe and effective method for feeding allo-SCT patients. A randomized trial is warranted to confirm its advantage on early patient outcome.

High-dose busulfan is an important component of myeloablative regimens. Variable drug exposure may occur following oral administration. Therefore, the use of intravenous busulfan has been advocated. Previous work has suggested a cumulative dosage of 16 mg/kg for haematopoietic transplantation in children less than 3 years of age, but only limited data are available in infants. Pharmacokinetics of intravenous busulfan administered at the suggested dosage were studied in 14 infants (median age 4.7 months). Busulfan plasma concentrations were measured by either GC-MS or HPLC-UV. In seven patients, the dose was decreased to target an area- under- the- curve of 600-1300 micromol min. The median total dose given was 13.8 mg/kg. All patients engrafted. Severe veno-occlusive disease occurred in one patient. Our study demonstrates that a cumulative dosage of 16 mg/kg is associated with higher exposure than expected in infants. We suggest an initial dose of 0.8 mg/kg followed by pharmacokinetically guided dose adjustment.

PURPOSE: To determine whether the use of maintenance therapy (MT) delivered after intensive induction and consolidation therapy confers any advantage in childhood acute myeloid leukemia (AML). PATIENTS AND METHODS: A total of 268 children with AML were registered in the Leucámie Aiquë Myéloïde Enfant (LAME) 89/91 protocol. This regimen included an intensive induction phase (mitoxantrone plus cytarabine) and, for patients without allograft, two consolidation courses, one containing timed-sequential high-dose cytarabine, asparaginase, and amsacrine. In the LAME 89 pilot study, patients were given an additional MT consisting of mercaptopurine and cytarabine for 18 months. In the LAME 91 trial, patients were randomized to receive or not receive MT. RESULTS: A total of 241 (90%) of 268 patients achieved a complete remission. The overall survival and event-free survival at 6 years were 60% ± 6% and 48% ± 6%, respectively. For the complete responders after consolidation therapy, the 5-year disease-free survival was not significantly different in MT-negative and in MT-positive randomized patients (respectively, 60% ± 19% v 50% ± 15%; P = .25), whereas the 5-year overall survival was significantly better in MT-negative randomized patients (81% ± 13% v 58% ± 15%; P = .04) due to a higher salvage rate after relapse. CONCLUSION: More than 50% of patients can be cured of AML in childhood. Either drug intensity or each of the induction and postremission phases may have contributed to the outstanding improvement in outcome. Low-dose MT is not recommended. Exposure to this low-dose MT may contribute to clinical drug resistance and treatment failure in patients who experience relapse.

International audience; We report herein the results we obtained and the limitations we experienced during the production and use of a bank of Epstein-Barr virus (EBV)-transformed human cytotoxic T lymphocytes (EBV-CTLs). To assess the feasibility and toxicity of this strategy, we selected and stored, in liquid nitrogen, 4 billion EBV-CTLs from each of the 13 selected donors. Subsequently, in a multicenter phase I/II study, 11 patients with EBV-associated lymphoma resistant to conventional treatments received 1-3 doses of 5 million EBV-CTLs/kg with 1-3 and 0-4 compatibilities for human leukocyte antigen (HLA)-I and HLA-II, respectively. Except for one event of fever after injection, no immediate or delayed toxicity, no graft versus host disease, and no graft rejection attributable to CTL infusion were observed. Three patients presented complete remission and 1 partial remission after treatment. Considering the clinical options currently available, and the constrains associated with CTL preparation and implementation, we conclude that CTL banks should consist of a reasonably small number of cell lines with documented specificities. This objective could be more easily achieved if the few homozygous donors for the most frequent HLA alleles of the targeted population could be made available for such a project.

The interplay between immune recovery, cytomegalovirus (CMV)-reactivation, CMV-driven immunity and graft-versus-leukaemia effect (GVL) was analysed in 108 children (median age: 8 years) who underwent haematopoietic-stem cell transplantation (HSCT) for acute leukaemia. Follow-up was 2 years unless death or relapse occurred. CMV-polymerase chain reaction (PCR) was programmed weekly until month +3 post-HSCT. Immunomonitoring consisted of sequential lymphocyte subset enumerations and analyses of T-cell proliferative and γ-interferon responses to CMV and to adenovirus. In the 108 recipients, the 2-year relapse rate (RR) was 25% (median time to onset 4·5 months; range: 24 d-17 months). CMV reactivation occurrence was 31% (median time to onset 26 d). Donor/recipient CMV serostatus did not influence RR. Among the 89 recipients disease-free after day +120, i) early CMV-reactivation before day +30 was more frequent (P = 0·01) in the relapse recipient group opposed to the non-relapse group. ii) CD8(+) /CD28(-) and CD4(+) CD45RA(-) T-cell expansions induced by CMV did not influence RR, iii) Recovery of anti-CMV and also anti-adenovirus immunity and of naive CD4(+) T-cells was faster in the non-relapse group (P = 0·008; 0·009 and 0·002 respectively). In contrast to adult acute myeloid leukaemia, CMV reactivation was associated with increased RR in this paediatric series. Accelerated overall immune recovery rather than CMV-driven immunity had a favourable impact on RR.

International audience; Minimal residual disease (MRD) is a major predictive factor of the cure rate of acute lymphoblastic leukaemia (ALL). Haematopoietic cell transplantation is a treatment option for patients at high risk of relapse. Between 2005 and 2008, we conducted a prospective study evaluating the feasibility and efficacy of the reduction of immunosuppressive medication shortly after a non-ex vivo T depleted myeloablative transplantation. Immunoglobulin (Ig)H/T-cell receptor MRD 30 d before transplant could be obtained in 122 of the 133 cases of high-risk paediatric ALL enrolled. There were no significant demographic differences except remission status (first or second complete remission) between the 95 children with MRD \textless10(-3) and the 27 with MRD ≥10(-3) . Multivariate analysis identified sex match and MRD as being significantly associated with 5-year survival. MRD ≥10(-3) compromised the 5-year cumulative incidence of relapse (43*6 vs. 16*7%). Complete remission status and stem cell source did not modify the relationship between MRD and prognosis. Thus, pre-transplant MRD is still a major predictor of outcome for ALL. The MRD-guided strategy resulted in survival for 72*3% of patients with MRD\textless10(-3) and 40*4% of those with MRD ≥10(-3).

Resume L’utilisation du methotrexate a hautes doses requiert une surveillance etroite de sa cinetique d’elimination. Celle-ci peut etre ralentie par differentes molecules, concourant a un tableau toxique parfois severe. Observations. – Nous rapportons le cas de deux adolescents ayant presente un retard d’elimination du methotrexate administre a hautes doses alors qu’ils recevaient de la ciprofloxacine. Le premier avait recu au prealable et sans probleme de multiples cures de methotrexate a hautes doses et le second a pu en recevoir sans probleme par la suite. Conclusion. – L’association methotrexate a hautes doses–ciprofloxacine est deconseillee.

PURPOSE: To investigate whether testicular disease in childhood B-cell lymphoma should continue to be considered a sanctuary site, as it is with other lymphoid malignancies such as acute lymphoblastic leukemia. PATIENTS AND METHODS: Seven hundred forty-two children with B-cell non-Hodgkin’s lymphoma were included in the LMB protocols of the French Society of Pediatric Oncology from February 1981 to May 1994. Thirty patients (5.3%) had testicular involvement at diagnosis. We describe the clinical presentation and outcome of these 30 patients, who were treated without local radiation therapy. RESULTS: Five patients underwent diagnostic orchidectomy. The median patient age was 8.5 years (range, 2 to 14 years), and their cancers were stage III (18 patients), stage IV (five patients), and B-cell acute lymphoblastic leukemia (seven patients). Five patients had central nervous system involvement. Twenty-eight patients (95%) achieved complete remission. Twenty-six patients are alive without progressive disease (median follow-up, 6.5 years). CONCLUSION: Testicular disease does not seem to confer a poor prognosis, and it is curable with intensive combination chemotherapy alone. Local treatment (surgery or radiation) is avoidable; therefore, gonadal function can be preserved.

Background: While therapeutic outcomes in patients (pts) with acquired severe aplastic anemia (SAA) has greatly improved in recent years, results remain poor for pts who are refractory to first-line immunosuppressive therapy (IST) and lack a matched unrelated donor. We conducted a prospective multi-center phase II uncontrolled trial to assess the efficacy and safety of cord blood transplantation (CBT) in refractory SAA pts (NCT 01343953). Patients and methods: Pts with SAA refractory to IST with anti-thymocyte globuline (ATG) and ciclosporin (CsA) were eligible in case of no existing matched unrelated donor but available one or two unrelated CB units containing alone or both together more than 4 x 107/Kg frozen nucleated cells/Kg (no more than 2 out of 6 HLA mismatches allowed between each CB unit and the pts). Pts with isolated bone marrow cytogenetic abnormality (absence of morphologic evidence of myelodysplastic syndrome) were also eligible. The conditioning regimen consisted of fludarabine 30mg/m2 (D-6 to D-3), cyclophosphamide 30mg/kg (D-6 to D-3), ATG (thymoglobulin) 2.5mg/kg at D-3 and D-2 (5mg/Kg total dose) and total body irradiation (2 Gray) on D-2. All pts received one injection of anti-CD20 (150 mg/m2) to prevent EBV reactivation (D+5). CsA was given alone as prophylaxis for graft versus host disease (GVHD). The trial used the Fleming's Single Stage Phase II Design, with sample size computed to demonstrate an improved overall survival (OS) rate at one year from 20% up to 50%, with type I and type II error rates set at 0.05. A minimum sample of 25 pts was required, with a minimum number of 9 pts alive at one year needed to indicate treatment efficacy. Secondary endpoints included cumulative incidences (CumI) of engraftment, acute and chronic GVHD (aGVHD and cGVHD), infections, relapse and late cause of death. Results: 29 pts were included between June 2011 and October 2015. One pt was diagnosed with dyskeratosis congenita and a matched unrelated donor was identified for another pt between inclusion and anticipated date of CBT (exclusion criteria). One pt died before CBT due to a septic shock (D+4 after inclusion). Analyses were conducted in the remaining 26 pts. Median time between SAA diagnosis and CBT was 12 months (interquartile range (IQR) 8.7 to 17.8). All pts received at least one course of IST before CBT (2 courses, n=8). Twenty-three pts received 20 or more transfusions before CBT. BM karyotyping showed 2 pts respectively with monosomy 7 in 4 and 15 mitoses as well as one pt with trisomy 8 in 4 mitoses before CBT. Eight pts (31%) have a PNH clone (median 4.5% [IQR: 3.1-9.7]). Median age at time of CBT was 16 years (IQR 8.25 to 23) and median follow-up is 13.2 months (IQR 4.1 - 23.5). Three out of 26 pts incorrectly received twice the dose of ATG (10mg/Kg total dose) due to protocol violation. Sixteen pts received one CB and 10 received two CB units. Median number of nucleated cells infused was 3.7 x 107/Kg (IQR, 3-4.9) and CD34+ cells infused was 1.4 x 105/Kg (IQR, 1.0-1.9). Myeloid engraftment occurred in 23 pts, with a 60 day-CumI of neutrophil engraftment of 88.5% with full chimerism for all of them. Three pts did not engraft (2 deaths at D71 and D92 and one successful second HSCT). The 100 day-CumI of grade II-IV acute GVHD was 40% (95% CI, 20-60) (8 grade II; 0 grade III; 2 grade IV). Among the 23 pts alive at day 100, five developed cGVHD leading to a one-year CumI of cGVHD at 27% (95% CI, 7-47) (severe cGvHD in 2 pts). During follow-up, 8 pts experienced a total of 18 grade III infections with a 6-month CumI of 32% (95% CI, 13-51). None of the pts presented post-transplant EBV-related lymphoproliferative disorder. Immune reconstitution is shown in Figure 1. Three pts died before 1 year due to non-engraftment (n=2) and infection (n=1, one of those who received twice the dose of ATG). With 23 pts alive at 1 year, the primary objective of the study was thus reached. The one-year treatment related mortality (TRM) was 15% (95%CI, 4-35). One additional pt who had also received twice the dose of ATG died (severe cGvHD) after one year (at 13.6 months), resulting in a 14-month OS of 81% (95%CI, 66-100) (Figure 2). Conclusion: CBT with at least 4 x 107/Kg frozen nucleated cells/Kg after a FLU-CY-TBI-ATG conditioning regimen gave rise to very encouraging results in this particular high risk SAA refractory population. Pediatric and young adult SAA pts who are refractory to first-line IST should now be considered either way for matched unrelated donor or CBT. Disclosures Peffault de Latour: Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Introduction: Fertility preservation is crucial in the setting of hematopoietic stem cell transplantation (HSCT) due to the severe impact of infertility on quality of life in long term survivors. Many chemotherapy agents as well as CNS or pelvic irradiation can affect gonads leading to impairment of pubertal development and/or infertility. In the last years different fertility preservation guidelines especially for cancer patients have been published. Despite these recommendations options for fertility preservation are not well established in children, it's discussed only in about 42% (Terenziani et al. PBC 2014) of all eligible patients. We present here a survey conducted by the Pediatric WP of the EBMT. Aim: to analyze and compare the different fertility preservation practices for children and adolescents across the EBMT centers and to identify hence the unmet needs in the field. Material and Methods: The survey was conducted between September 2014 and December 2015. 177 pediatric centers reporting to the EBMT were asked to fill-out a form with 24 questions related to fertility preservation strategies. Questions focused on institutional availability of a fertility preservation program and its options, number of patients included in the program, payers and suggestions for improvement. Results: 38 (21%) centers from 16 countries replied. In 2013 the number of HSCT per center ranges from 2 to 60. Totally, 834 children were transplanted in the 38 centers, 22% of the reported HSCT in children (3789) in 2013 from the EBMT centers. Of the 834 patients 612 (73%) were prepubertal and 203 (25%) postpubertal. 322/834 (39%) patients received a fertility preservation counselling and 243 (29%) of them had a fertility preservation procedure. Half of the 38 centers reported to have a fertility preservation program with standard operating procedure (SOP) for counselling. Centers with SOP counselled patients significantly more often than those without (Figure 1A). Regarding the number of applied procedures, no significant difference was found between centers with and without a SOP (Figure 1B). Centers with SOP counselled earlier (at the beginning of treatment/diagnosis; 61% versus 29%), and an interdisciplinary counselling team was largely used (60%). This team involved hematologists, gynecologists and /or reproductive medicine specialists. The most frequently cited reasons for missing counselling were lack of time (59%), refusal by parents (35%) and expected poor prognosis (29%). Reasons explaining parents/patients refusal were psychological distress (52%) and overwhelming feeling (50%) in this life-threatening moment. All responding centers considered the topic of fertility preservation as very relevant. Better education, more resources and financial funding emerged as pivotal factors to support and implement such programs. Conclusion: This EBMT survey showed the great interest for fertility preservation in participating centers. The collected data indicated that less than half of pediatric and adolescent patients undergoing HSCT in 2013 received a counselling on fertility preservation and/ or underwent fertility preservation procedures. This emphasizes the need for enhanced education of the transplant team and active multidisciplinary collaboration in the field. Fertility preservation becomes a standard of care; this data represents the basis to further efforts toward fertility preservation standardization in the EBMT community. Figure 1 Upper figures: Percentage of consulted patients in centers with and without SOP. Lower figures: Percentage of treated patients in centers with and without SOP Figure 1. Upper figures: Percentage of consulted patients in centers with and without SOP. Lower figures: Percentage of treated patients in centers with and without SOP Disclosures Bader: Servier: Consultancy, Honoraria; Neovii Biotech: Research Funding; Riemser: Research Funding; Medac: Consultancy, Research Funding; Novartis: Consultancy, Honoraria.

Objectives: A small number of previous studies have demonstrated prognostic significance of MRD detection before and after allogeneic transplantation in children ALL. A major challenge of studies has been low patient numbers that were insufficient for multivariate analysis and assessment of specific post-transplant time points. To address this, the Westhafen Intercontinental Group (WIG) performed a retrospective analysis to investigate the impact of pre- and post-transplant MRD assessment in children with ALL. Patients and Methods: Patients (pts) treated in Europe (France, Germany, Italy, and Netherlands), North America (COG/PBMTC, Seattle, Minnesota, and Johns Hopkins) and Australia were included. All pts were registered either in local or national trials. Pts were transplantations between 09/99 and 05/16; analysis was performed in 06/16. Altogether 747 pts were included on whom 2960 MRD measurements were performed in bone marrow. MRD prior to transplant was available in 650 pts. MRD was assessed in bone marrow prior to transplant and on days +30, +60, +100, +180, +365 and beyond day +365 post-transplant either by flow cytometry or by RQ-PCR of TCR and IG gene rearrangements. Of these 747 pts, 466 (62%) were male and 281 (38%) were female. Pts received their transplant in CR1 (n=275, 37% ), CR2 (n=410, 55%), >CR3 (n=55, 7%), or non-remission (NR) (n=7, 1%). 586 patients (78%) had pre-B ALL, 145 pts (20%) had T-cell phenotype and 16 pts (2%) had bi-lineage or bi-phenotype AL. Grafts were matched sibling donors (MSD; n=227, 30%), matched unrelated donors (MUD; n=314, 42%), from mismatched donors (MMD, n=75, 10%), cord blood (n=128, 17%) or no data (n=3, 1%). 31 patients (4%) were younger than 2, 365 pts (49%) were ≥2 and ≤ 10, and 351 pts (47%) were older than 10 years at the time of transplant. After MRD assessment, pts were grouped for analysis according to the level of MRD in three groups: pts in whom no MRD was detectable were categorized "MRD negative", pts with MRD Results: The total group of pts showed a 4y-pEFS and a 4y-pOS of 55.0% and 60%; cumulative incidence (CI) of relapse (CIR) and CI of treatment related mortality (CI TRM) were 31% and 13%. The following pre transplant factors did not influence outcome: age, gender, relapse site, cytogenetics, donor, and stem cell source. MRD prior to transplant was available in 648 pts. MRD high, low and negative, patients showed a 4y-pEFS of 37%, 67%, and 62% (p MRD values post-transplant were analysed as time-dependent covariates. 4y-pEFS at day +30 for MRD high, MRD low and MRD negative pts were: 43%, 65%, and 59%, respectively (p=0.002). At Day +60 for MRD high, low and MRD negative pts: 36%, 47%, and 64%, respectively (p There was an interaction between MRD and aGVHD at all time-points investigated after transplantation. At day +30, patients with high MRD had a 2.54 fold increased risk to develop an event which is defined as relapse or TRM, where the risk for high MRD pts with aGVHD was 0.64. This was due to a lower relapse risk compared to MRD positive patients without aGVHD. Summary and Conclusion: This world wide study confirmed in the largest patient population investigated so far, MRD prior to transplant is a powerful predictor for survival after transplant. When analysed either for the different time points or for the highest MRD value post- transplant, MRD results were always significantly associated with pEFS. All pts who showed an MRD load of >10E-4 (0.01% by flow) at any time after transplant had an increased risk. Occurrence of aGVHD in MRD positive pts reduced the risk of relapse. Serial MRD detection after transplantation allows for the prediction of relapse. Disclosures Bader: Medac: Consultancy, Research Funding; Servier: Consultancy, Honoraria; Neovii Biotech: Research Funding; Riemser: Research Funding; Novartis: Consultancy, Honoraria. Verneris:Bimogen: Other: Advisory Board. Borowitz:Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding; BD Biosciences: Research Funding; HTG Molecular: Consultancy. Peters:Medac: Consultancy; Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Pulsipher:Novartis: Consultancy, Other: Study Steering Committee; Jazz Pharmaceutical: Consultancy; Chimerix: Consultancy; Medac: Other: Housing support for conference.

Purpose The long-term impact of treatment in infants with acute leukemia (AL) is poorly understood and of concern. Patients and methods We analyzed the incidence, in the L.E.A. cohort, of and risk factors for late sequelae in the survivors of infant AL diagnosed before the age of one year, according to group: type of AL, transplantation, sex, and age ≤ or > 6 months. Of 113 survivors, 55 had had lymphoblastic AL and 58 myeloblastic AL. The median follow-up was 11.7 years [1.9-34.7]. Sixty-three were treated with chemotherapy only (and four with central nervous system irradiation) and 50 received transplantation (with a chemotherapy-based regimen except for three). Results The most frequently observed sequelae included problems of height, body mass index, and gonadal or thyroid function (Figure 1). We found a mean of 1.3 +/-0.1 sequelae per patient. In multivariate analysis, treatment after the age of six months significantly decreased the incidence of major growth failure (OR 0.38, p = 0.04) and low weight (OR 0.3, p = 0.02) (Figure 2). Transplants performed in first remission did not affect the occurrence of late effects (p = 0.2) (Figure 3). Parent-reported child quality of life (QoL) scores were similar among groups and to the normative population. Adults had diminished QoL as measured by psychosocial subscales relative to French norms but with no difference between groups. Conclusion Altogether, these results highlight the need for comprehensive follow-up with hormone replacement therapy and psychological intervention. Our results in a large cohort with very limited use of irradiation, did not show an increased risk of late sequelae relative to older children. We conclude, that transplantation should be considered when warranted, as there is no increased risk of late effects. Disclosures No relevant conflicts of interest to declare.

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.

Fatal diffuse capillaritis after hematopoietic stem-cell transplantation for dyskeratosis congenita despite low-intensity conditioning regimen

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples ( n = 92) and 78% from stool samples ( n = 100). No adenovirus was detected in samples from noninfected patients ( n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml ( r = 0.82 and 0.78, respectively) were correlated up to 6 log 10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus ( n = 41), CMV ( n = 30), EBV ( n = 26), JC virus ( n = 9), and HSV-1 ( n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.

We analyzed the outcomes of 485 patients with thalassemia major (TM) or sickle cell disease (SCD) receiving HLA-identical sibling cord blood transplantation (CBT, n = 96) or bone marrow transplantation (BMT, n = 389). Compared with patients given BMT, CBT recipients were significantly younger (median age 6 vs 8 years, P = .02), and were treated more recently (median year 2001 vs 1999, P < .01). A higher proportion of patients with TM belonging to classes II-III of the Pesaro classification received BMT (44%) compared with CBT (39%, P < .01). In comparison with patients receiving BMT (n = 259, TM; n = 130, SCD), those given CBT (n = 66, TM; n = 30, SCD) had slower neutrophil recovery, less acute graft-versus-host disease (GVHD) and none had extensive chronic GVHD. With a median follow-up of 70 months, the 6-year overall survival was 95% and 97% after BMT and CBT, respectively (P = .92). The 6-year disease-free survival (DFS) was 86% and 80% in TM patients after BMT and CBT, respectively, whereas DFS in SCD patients was 92% and 90%, respectively. The cell dose infused did not influence outcome of patients given CBT. In multivariate analysis, DFS did not differ between CBT and BMT recipients. Patients with TM or SCD have excellent outcomes after both HLA-identical sibling CBT and BMT.

We report French prospective experience with reduced-intensity conditioning-allo-SCT in 46 patients (median age: 15.5 years, 4.8-20.2) presenting high-risk AL (n=11), Hodgkin's lymphoma (n=15) or solid tumors (n=20). Graft sources were BM (n=21), PBSC (n=20) and cord blood (CB; n=5) from related (n=20) or unrelated (n=26) donors. For CB grafts, only one patient out of five achieved sustained engraftment. For PBSC/BM grafts, engraftment rate was 95%, hematopoietic recovery times were not significantly different between BM, PBSC, sibling or unrelated grafts, day+100. Full donor chimerism was achieved in 94% of patients, and incidences of primary acute GVHD and chronic GVHD were 49% and 14%, respectively. Underlying disease was fatal in 39% of patients. TRM was 6.9%. Three-year OS was 49.15%. OS and EFS were not significantly different between patients transplanted with different grafts and with or without primary GVHD. Patients with solid tumor or measurable disease at transplant had poorer outcomes. Three-year EFS: 33.3% for ALL, 75.0% for AML, 51.8% for Hodgkin's lymphoma, 28.6% for neuroblastoma and 22.2% for sarcoma patients. This multicentre study concluded that Bu/fludarabine/anti-thymocyte globulin conditioning with PB or BM, related or unrelated grafts in patients with various malignancies at high-risk for transplantation toxicity results in high engraftment rates, low TRM and acceptable survival.

Intravenous (IV) busulfan (Bu) dosing approved in Europe based on 5 body weight (BW) strata has been validated for targeting Bu exposures in children undergoing hematopoietic stem-cell transplantation and with mostly malignant diseases. The authors conducted an observational study aiming to investigate the behavior and ontogeny of IV Bu pharmacokinetic (PK) disposition, and to reevaluate the consistency of the BW-based dosing in very young children with rare diseases.The observational study comprised 115 patients, mostly infants with immunodeficiencies and metabolic inherited disorders and with altered liver function and/or iron overload. Additional data (90 children, mostly malignant diseases) were pooled with the first data set. The overall data (205 children aged from 10 days to 15 years) were analyzed using population PK modeling.The BW remained the main determinant of IV Bu PK, and no further covariate effect was identified. Bu clearance (CL) variability was best described by BW allometric functions. Increase of drug CL with the child's growth was faster in younger children. This pattern is likely related to the maturation of GSTA1 enzymes during infancy and was accounted for in the model by estimating a higher BW allometric exponent in children9 kg compared with that in children ≥9 kg. IV Bu PK was not modified in children with altered liver function and/or iron overload, and no disease specific difference was observed. Bu dosing either adjusted according to the final model or with the approved EU labeling yields similar targeting performances. For both dosing strategies, the percent of patients achieving the therapeutic area under the curve window (900-1500 μmole·min/L were 60% and 70%-90% in children9 and ≥9 kg, respectively.A population PK model accounting for the highest Bu CL in the youngest patients was validated on training and evaluation data sets. The BW-based dosing strategy recommended in Europe proved to be consistent on a large paediatric cohort representative of the population heterogeneity observed in hematopoietic stem-cell transplantation.

We investigated prospectively factors influencing the safety of hematopoietic stem cell (HSC) collection in 453 pediatric donors. The children in the study donated either BM or peripheral blood stem cells (PBSCs) according to center policy. A large variability in approach to donor issues was observed between the participating centers. Significant differences were observed between BM and PBSC donors regarding pain, blood allotransfusion, duration of hospital stay, and iron supplementation; however, differences between the groups undergoing BM vs PBSC donation preclude direct risk comparisons between the 2 procedures. The most common adverse event was pain, reported mainly by older children after BM harvest, but also observed after central venous catheter (CVC) placement for PBSC collection. With regard to severe adverse events, one patient (0.7%) developed a pneumothorax with hydrothorax after CVC placement for PBSC collection. The risk of allotransfusion after BM harvest was associated with a donor age of < 4 years and a BM harvest volume of > 20 mL/kg. Children < 4 years were at higher risk than older children for allotransfusion after BM harvest and there was a higher risk of complications from CVC placement before apheresis. We conclude that PBSC and BM collection are safe procedures in children. (Blood. 2012;119(12):2935-2942) We investigated prospectively factors influencing the safety of hematopoietic stem cell (HSC) collection in 453 pediatric donors. The children in the study donated either BM or peripheral blood stem cells (PBSCs) according to center policy. A large variability in approach to donor issues was observed between the participating centers. Significant differences were observed between BM and PBSC donors regarding pain, blood allotransfusion, duration of hospital stay, and iron supplementation; however, differences between the groups undergoing BM vs PBSC donation preclude direct risk comparisons between the 2 procedures. The most common adverse event was pain, reported mainly by older children after BM harvest, but also observed after central venous catheter (CVC) placement for PBSC collection. With regard to severe adverse events, one patient (0.7%) developed a pneumothorax with hydrothorax after CVC placement for PBSC collection. The risk of allotransfusion after BM harvest was associated with a donor age of < 4 years and a BM harvest volume of > 20 mL/kg. Children < 4 years were at higher risk than older children for allotransfusion after BM harvest and there was a higher risk of complications from CVC placement before apheresis.We conclude that PBSC and BM collection are safe procedures in children. © 2012 by The American Society of Hematology.

To determine the optimal serotherapy regimen in idiopathic aplastic anaemia stem cell transplantation, we analysed 1837 patients who underwent either in vivo T cell depletion with either ATG (n=1283) or Alemtuzumab (n=261) or no serotherapy (n=293) as part of their conditioning regimen. All patients had either undergone a matched sibling or matched unrelated donor stem cell transplant (at least 6 out of 6). Data was obtained retrospectively from the EBMT SAA database, between the periods 2000-2013. The major endpoints were graft versus host disease, overall survival and event free survival. Events were classified as graft failure, secondary malignancy, relapse and requirement for second transplant. The median follow up was 34 months in the ATG group, 30.9 months in the Alemtuzumab group and 47.9 months in the no serotherapy group. Rate of grade 2-4 acute GVHD was 19.1% without serotherapy; this was higher than that observed with both ATG (13.3%, p Figure 1. Figure 1. Disclosures Snowden: Celgene: Other: Educational support, Speakers Bureau; Janssen: Other: Educational support, Speakers Bureau; MSD: Consultancy, Other: Educational support, Speakers Bureau; Sanofi: Consultancy. Dufour:Pfizer: Consultancy. Risitano:Pfizer: Consultancy; Novartis: Research Funding; Rapharma: Consultancy, Research Funding; Alnylam: Consultancy, Research Funding; Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: this paper includes discussion of the use of alentuzumab for GVHD prophylaxis, which is currently off-label.

Bone marrow failure (BMF) syndrome is a group of rare hereditary or idiopathic disorders occurring at all ages. For BMF patients under 40 years of age and having an HLA-identical related donor, allogeneic bone marrow transplantation (HSCT) is indicated. The use of cord blood transplantation (CBT) from an HLA-identical sibling donor is of interest in non-malignant disease due to the low risk of graft-versus-host disease (GVHD) associated with this type of stem cell source and the absence of risk to the donor. We analyzed outcomes of 122 children and young adults with inherited or acquired BMF syndrome, who received a CBT from an HLA-identical related donor. Patients were transplanted in EBMT centers between 1988 and 2014 and reported to Eurocord and EBMT. Ninety-six patients had an inherited and 26 patients had an acquired BMF. The specific diagnosis were: Fanconi anemia (n=48), Diamond Blackfan anemia (n=25), Amegakariocytic thrombocytopenia (n=6), Kostmann syndrome (n=4), Dyskeratosis congenital (n=3), Schwachman-Diamond syndrome (n=2), and unclassified inherited BMF (n=8) for the inherited group and severe aplastic anemia (n=25), pure red cell aplasia (n=1) for the acquired group. Eighty-nine patients received a single cord blood (CB) unit, whereas 33 patients received a combination of CB and bone marrow (BM) cells from the same sibling donor. The main reason for adding BM was due to the low cell dose of the single CB unit alone. Of patients receiving CB+BM, 6 had an acquired and 24 had an inherited BMF. Median age at CBT was 6.7 (range 1-16) years (5.6 years for patients with acquired and 6.9 years for those with inherited BMF). Median interval between diagnosis and CBT was 21 (range 2-114) months for the acquired and 55 (range 2-160) months for the inherited group, respectively. Fifty-nine patients (53%) received a reduced intensity conditioning (RIC) and 52 (47%) were treated with myeloablative regimens (MAC) (data available for 111 of 122 patients). Fludarabine-based regimens were used in 54 patients (44%). For patients receiving a RIC the most common regimen was cyclophosphamide and fludarabine (56%), and for MAC, cyclophosphamide and busulfan (46%). Total body irradiation was used in 8 patients (both in RIC and MAC settings). GVHD prophylaxis consisted mainly of cyclosporine alone (CSA) in 53 patients (43%), CSA + micophenolate mofetil in 11 patients (9%) and CSA + methotrexate in 23 patients (19%). The median number of total nucleated cells (TNC) infused was 6.2x107/Kg (1-24.2x107/Kg) for patients receiving a single CB unit and 26.8x107/Kg (5.3-41x107/Kg) for those receiving CB+BM (median TNC was 3x107/Kg in CB, and 22x107/Kg in BM graft). Median follow up is 5 (range 0.6-25) years. The cumulative incidence (CI) of neutrophil recovery at day 60 was 91%+9%, and the median time to engraftment was 21 (range 7-100) days. The CI of platelets engraftment at day 180 was 89+3% with a median time of 35 (range 7-138) days. Thirteen patients experienced primary graft failure (PFG) (12 patients after a single CB and one after a CB+BM graft). For those patients with available information, treatment of graft failure was second BM-HSCT (n=6), and growth factors (n=1). Of the 13 patients with graft failure, 11 died within in a median time of 1.9 (0.3 to 6) months after CBT. Two patients are alive after the second HSCT. The 100-day CI of grade II-IV acute GVHD was 11+3% (15 patients: grade II, n=8; grade III, n=6; grade VI, n=1), and the 1-year CI of chronic GVHD was 12+4%. The 1-year CI of transplant-related mortality was 13+3%, 17 patients died: 11 due to PGF (infections (n=6) hemorrhagic syndrome (n=1) and other causes (n=4)) and the remaining 6 of acute respiratory distress syndrome (n=1), and infections (n=5). Five-year overall survival (OS) for the whole population was 87+3%. OS was 80+8% for patients with acquired BMF, 87+3% for Fanconi anemia patients, 95+4% for those with Diamond Blackfan, and 86+7% for other inherited BMFs. In pediatric and young adult patients with either inherited or acquired BMF syndrome, CBT from an HLA-identical sibling donor is associated with excellent long-term outcomes with particular low rates of both acute and chronic GVHD, this latter observation being of particular importance for patients with non-malignant disorders. In case of inherited BMF, collecting cord blood unit at the birth of a new sibling is strongly recommended. Disclosures Dufour: Pfizer: Consultancy.

Introduction: Hematopoietic stem cell transplantation (HSCT) is, to date, the only curative therapy for sickle cell disease (SCD). However, HSCT is offered to relatively few patients with SCD for a number of reasons including lack of a suitable HLA-matched donor, lack of consensus on indications for HSCT, the potential for trading one chronic condition (i.e., SCD) for another, such as chronic graft-versus-host disease (GVHD), and the mortality associated with the procedure. To-date, most HSCTs for SCD have utilized matched siblings as donors and are performed in children and adolescents. We report outcomes after HLA-matched sibling HSCT of patients reported to the Eurocord-Monacord/European Group for Blood and Marrow Transplantation (EBMT) and Center for International Blood and Marrow Transplant Research (CIBMTR). Material and methods: One thousandpatients with SCD received HLA identical sibling HSCT between 1991 and 2013; n=439 from CIBMTR and n=561 from EBMT centers. HSCTs were performed in 90 centers in 23 countries. Results: Median age at HSCT was 9 years (range 1-54y); 85% of patients were aged The cumulative incidence (CI) of neutrophil engraftment at day+60 was 98% (96.6% for CB, 98.3% for BM and 95.2% for PB) with a median time to recovery of 19 days, while that for platelet engraftment was 98 % (96±2% for CB, 99±1% for BM and 98±9% for PBSC) with a median time to recovery of 25 days. Twenty-six patients experienced primary and 47 patients secondary graft failure; 67 patients died mainly due to GVH or infection. The 3-year probabilities of overall (OS) and event-free survival (EFS, alive with engraftment) were 94% (95% CI 92-95) and 90% (95% CI 68-82), respectively. According to stem cell source, 3-year OS was 99% after CB, 94% after BM and 80% after PBS (p Conclusion: This large registry based international study shows that HLA identical sibling transplant is successful more than 90% of the patients with severe SCD with limited transplant related complications (rejection, GVHD). Strategies aimed at lowering graft failure and GVHD are desirable to further optimize the observed 3-year event-free survival. Importantly, these data should increase awareness to early referral to HSCT of patients with severe SCD. Disclosures Walters: ViaCord and AllCells, Inc: Other: Medical director. Bertrand:ERYTECH Pharma: Consultancy. Peters:Medac: Research Funding; Fresenius: Research Funding; Amgen: Research Funding; Jazz: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Sanovi: Research Funding; Pierre-Fabre: Research Funding.

INTRODUCTION: Infertility is a major long term side effect after hematopoietic stem cell transplantation (HSCT) when using myeloablative conditioning regimen based either on high dose chemotherapy or radiotherapy and chemotherapy association. These conditioning regimens are associated with high rates of gonadal failure and endocrine defects. These side effects strongly impact on survivor's quality of life. Then, several options for fertility preservation have been developed and are currently available for children i.e. sperm cryopreservation, gonadal tissue cryopreservation, oocyte cryopreservation but some technics are still experimental. AIMS: To analyse the acceptability of a fertility preservation program in pediatric HSCT (boys and girls). To evaluate the ovarian function several years after HSCT and to determine which patients are at risk to develop premature ovarian failure. PATIENTS AND METHODS: All children treated by HSCT since the initiation of the fertility program (1998 for girls and 2009 for boys) in our center were included in the study. Clinical and biological data were collected until the 30 April 2015 and were retrospectively analysed. Evaluation of ovarian function was made for girls older than 13 years. Gonadal function was determined by clinical data (pubertal development, menstruations status, substitutive hormonal therapy) and by biological data including follicle stimulating hormone (FSH), luteinizing hormone (LH), antimüllerian hormone (AMH) and oestradiol serum levels. Premature ovarian insufficiency was defined by amenorrhoea and a FSH serum level greater than 25UI/L. RESULTS: From May 1998 to December 2014, 445 children (187 boys, 258 girls) received a first allogeneic HSCT at a median age of 7.4 years (range: 0.4-18.8). 70% patients had been treated for a malignant pathology and 30% for a non-malignant disease. The conditioning regimen was administered according to the pathology and has been classified as myeloablative conditionning regimen (MAC) with or without total body irradiation (TBI) and reduced intensity conditioning (RIC). A fertility preservation was proposed by haematologist to 237 children (53%). Twenty five families declined the proposal. 208 families met the physician from fertility preservation program and after counselling, 28 (13%) refused the procedure and 23 (11%) presented with a medical contra indication to the surgery. Finally, 161 children had a cryopreservation: 4 postpubertal boys had a sperm cryopreservation, 64 boys underwent testicular biopsy and 93 girls an unilateral ovariectomy at a median age of 8.8 years (0.8 - 16) and 7.2 (0.6 - 16.5) for boys and girls, respectively. No grade 3-4 adverse effects of the surgery were reported. 120 out of 445 patients (40 boys, 80 girls) died after HSCT (from either TRM or underlying disease) during follow up (overall survival: 73%). Regarding females, assessment of ovarian function was available for 69 post pubertal girls with the median time of follow up after HSCT of 5.2 years (1-14, 7). The median age at HSCT for these 69 girls was 10.4 years (1.2-17.9). 62% were treated for a malignant disease and 38% for a non-malignant disease. Conditioning regimen was MAC for 56 girls (81%) with TBI for 18 (26%), and RIC for 13 girls (17%). 28/69 (40%) underwent ovarian cryopreservation before HSCT. 42 girls had developed premature ovarian insufficiency at a median age of 15.3 years (13-19, 5). From them, 11 (26%) were older than 13y at time of HSCT, 38 (90%) received MAC and 26 (62%) had an ovarian cryopreservation. Up to now, none of them used cryopreserved ovarian samples, including those transplanted for non malignant diseases. In multivariate analysis, both HSCT after 13 years and ovarian cryopreservation appeared as significant negative impact factors for ovarian failure (p=0.002 and p=0.001, respectively). CONCLUSION: Fertility preservation is a major concern for both physicians and patients in pediatric HSCT. Gonadal tissue cryopreservation is the only available technic at time. This technic is well accepted by patients and families. However, in girls this procedure may impair fertility by itself. Pediatric transplanters and fertility physicians have to conduct prospective programs to develop new technics and increase results. Disclosures Baruchel: Jazz pharmaceuticals: Consultancy; Novartis: Consultancy; SigmaTau: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Dalle:Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Macopharma: Membership on an entity's Board of Directors or advisory committees.

Background: "Drepagreffe" is a French national prospective trial involving 67 sickle cell anemia (SCA)-children with a history of abnormal cerebral arterial velocities by TCD, and comparing for the first time the outcome of cerebral vasculopathy following transfusion program (TP) or transplantation (HSCT). Inclusion criteria at enrollment were children with SCA (SS/Sb0), younger than 15 years, with a history of abnormal cerebral arterial velocities (TAMMX ≥ 200 cm/sec) and placed on long-term TP, with at least one non-SCA sibling, and with parents accepting HLA-typing and HSCT if a genoidentical donor was available. The 2 arms (TP/HSCT) were defined by the random-availability of a genoidentical donor. Seven of the 67 patients had a history of stroke. Transplanted patients (n=32) received a conditioning regimen of Busilvex-CY 200 mg/kg and 20 mg/kg rabbit Thymoglobulin, with CSA and a short course of MTX or MMF for GvHD prophylaxis. In the TP arm (n=35), HbS% was maintained at < 30%, with an Hb at 9-11g/dL. At enrollment and 12 months post-enrollment, blood screening, Doppler, and cerebral MRI/MRA were performed along with cognitive performance testing, the latter being done in parallel in the non-SCA siblings. Preliminary findings on cerebral velocities as the primary endpoint were reported at the last ASH meeting (abstract 67237), and demonstrated that all patients were alive at one year and that the 32 transplanted patients had no chronic GVHD and the same hemoglobin profile as their donor. Velocities were significantly lower post-HSCT than under TP (p Patients and Methods: We report here the cerebral imaging (MRI/MRA) and cognitive performance data performed at enrollment and after 12 months. The scoring applied for MRI was: 3 = territorial, 2 = borderzone (cortical and subcortical), 1 = white matter or basal ganglia infarcts, 0 to 3 = atrophia, and for MRA: 1 = mild stenosis (25-49%), 2 = moderate stenosis (50-74%), 3 = severe stenosis (75-99%), 4 = occlusion for each artery and 0 to 2 for Moya presence. Cognitive testing using the WPPSI-3 (3-6 yr), WISC-4 (7-16 yr) or WAIS-3 (>16 yr) scales, depending on the age, was performed in patients and in siblings when possible. Results: MRI/MRA data were available in 66/67 patients. At enrollment (M0), ischemic lesions and stenoses were present in 25 and 35/66 patients, respectively. Cognitive testing was obtained in 64 patients and 56 siblings. Paired analysis with siblings (Table 1) showed significant differences in Verbal Comprehension Index (VCI) with a mean difference of 7.6±14.5(p =0.0004), Processing Speed Index (PSI) 6.3±20.5 (p =0.04), and Full Scale IQ (FSIQ) 7.3±15.0 (p =0.01). After exclusion of the 7 patients with stroke history, significant differences were still observed in VCI (p =0.013) and FSIQ (p =0.019). Patient cognitive performance indexes were correlated negatively and significantly with the MRI and MRA scores (Table 2). At post-enrollment (M12),ischemic lesions and stenoses were present in 26/66 patients. The mean variation in MRI and MRA scores between M12 and M0 was not significantly different between the 2 arms (Table 3). The cognitive tests were performed at M12 in 60 patients (Table 4) and the performance indexes were improved in the TP compared to the HSCT arm, but only significantly for FSIQ. Conclusion This first prospective trial initially showed that HSCT reduces more significantly the cerebral velocities at M12 and in a higher proportion of patients than TP. Here, we show that patients with a history of abnormal cerebral velocities had significantly lower cognitive performances than their siblings, even in the absence of stroke history; however, there was no significant difference between the 2 arms for the outcomes of ischemic lesions and stenosis at M0 and M12. The fact that cognitive performances were improved in the TP compared to the HSCT arm might be explained by the stress of the HSCT procedure and the lack of schooling during this period. Despite the higher ability of HSCT to decrease velocities at M12 compared to TP, a longer follow-up will be required to demonstrate its effect on stenosis and cognitive performances; therefore, patients will be reassessed at 3 years post-HSCT. Disclosures Bernaudin: Novartis: Research Funding.

BACKGROUND. Allogeneic hematopoietic stem cell transplantation (HSCT) is of benefit in pediatric patients with high-risk acute lymphoblastic leukemia (ALL) in first or further remission. Whether transplantation from unrelated donors could yield similar results to transplantation from HLA identical siblings is still to be assessed within countries running different frontline and relapse protocols. AIM OF THE STUDY. A prospective study was initiated within the International BFM Study Group, in order to assess whether the outcome of HSCT from a 9 or 10 out of 10 HLA allelic matched compatible donor (MD) was inferior to the outcome of HSCT from a matched sibling donor (MSD) in children or young adults with ALL carrying very high risk eligibility criteria for transplantation. Primary endpoint was event-free survival (EFS) and secondary endpoints were non-relapse mortality (NRM) and incidence of acute and chronic graft-versus-host disease (aGVHD, cGVHD). PATIENTS AND METHODS. Between 2007 and 2013, 10 countries (Czech Republic, Denmark, France, Israel, Italy, The Netherlands, Poland, Sweden, Slovakia, Turkey) participated into the ALL SCT I-BFM Study, coordinated by Peters in Vienna. 298 consecutive patients, 18 years old or younger (70% male, median age 9 years), affected with VHR ALL in complete remission (CR), were enrolled in the core arm of the Study (MD vs MSD). Of 298, 107 patients were transplanted from a MSD (50% in CR1, 47% in CR2, 4% >CR2) and 191 from a MD (44% in CR1, 48% CR2, 9% >CR2), either related (5%) or unrelated (95%), at a median of 189 and 197 days, respectively, after diagnosis/relapse. As per protocol, conditioning regimen consisted of TBI-etoposide, in patients older than 2 years, or busulfan-cyclophosphamide-melphalan, in patients 2 years or younger, and GVHD-prophylaxis consisted of cyclosporine (CSA) only for MSD and CSA/methotrexate/ATG for MD recipients. Median follow-up for alive patients was 3.1 years. RESULTS. Three-year EFS was 67% (SE 5%) for MSD vs 61% (SE 4%) for MD recipients (p-value 0.254), overall survival (OS) 76% (SE 4%) vs 69% (SE 4%) (p-value 0.207), cumulative incidence of relapse (CIR) 24% (SE 4%) vs 22% (SE 3%) (p-value 0.935) and NRM 8% (SE 3%) vs 16% (SE 3%) (p-value 0.094), respectively. There was a trend for a higher risk NRM for MD patients, although no statistical significance was reached (HR 1.94, CI 0.85-4.41; p=0.114), after adjusting for risk profile and donor type. Being transplanted in CR2 was associated with lower EFS and higher NRM (p=0.001). Grade II-IV acute GVHD occurred in 37% and grade III-IV in 16% of MSD vs 42% and 15% of MD recipients; 39% of the evaluable MSD and 19% of the MD recipients experienced chronic GVHD, which was severe in 24% and 10%, respectively. Cumulative incidence of developing chronic GVHD was 39% (SE 5%) and 17% (SE 3%), for patients grafted from MSD and MD, respectively (p=0.001). Being transplanted from a MD, compared with a MSD, was significantly associated with a reduced risk of developing chronic GVHD (HR 0.31, CI 0.18-0.54, p CONCLUSIONS. Outcome of transplantation of MD pediatric recipients with VHR ALL in CR was not inferior to the outcome of MSD recipients in terms of EFS, OS, and CIR, with probability of chronic GVHD being lower for MD versus MSD recipients, which suggests the crucial role of serotherapy, and severe acute GVHD being associated with increased NRM and lower OS but similar CIR. Disclosures Dalle: Gilead: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Macopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Peters:Medac: Research Funding; Fresenius: Research Funding; Amgen: Research Funding; Jazz: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Sanovi: Research Funding; Pierre-Fabre: Research Funding.

Better understanding of the antitumor effect of allogeneic transplant and the need to reduce the toxicity of the procedure, particularly in elderly patients have spurred the development of reduced-intensity conditioning regimens (RIC). These regimens allow fast engraftment with very low chemotherapy-induced toxicity. They are widely used in adults and there are numerous studies to demonstrate their feasibility and efficiency, but in pediatrics, the place of RIC remains to be determined. They can be proposed in two pediatric populations. First, solid tumors or hematological malignancies remaining unresponsive to the reference strategies according to best practices in pediatrics. Second, in children presenting malignancies for which allografting is the only recognized curative indication but is contraindicated with myeloablative conditioning regimens. More than 100 pediatrics cases have been reported in various pathologies, including blood diseases, acute leukemia, Hodgkin's lymphoma and solid tumors, and promising results published recently underline how RIC warrants further investigation in prospective comparative multicentric trials. The use of new post-graft treatment modalities is expected to pave the way to the development of RIC in pediatric patients.

The uncommon C77G polymorphism of the Protein-Tyrosine Phosphatase (PTPRC) gene (PTPRC; previously termed CD45) could confer an increased risk of immunopathology. This study compared the outcome of children following human leucocyte antigen-matched unrelated haematopoïetic-stem cell transplantations (HSCT) from donors carrying (C77G cases: n = 8) or not (controls: n = 36) the PTPRC C77G polymorphism. Transmission of the PTPRC C77G polymorphism through the graft was suggested by unusual CD45RA phenotype in the donors and/or in the recipients after, but not before HSCT. Restriction-Fragment Length Polymorphism and sequencing confirmed the polymorphism. Overall survival rates were similar in C77G cases and controls (63% vs. 61%). Acute leukaemia relapse tended to be less frequent in C77G cases (0% vs. 32%; P = 0·09). Among recipients surviving ≥ 30 d, acute GVHD (aGVHD) ≥ grade 2 tended to be more frequent (100% vs. 58%; P = 0·07) and the rate of steroid-refractory or -dependant aGVHD higher (67% vs. 28%) in C77G cases. Finally, extensive chronic GVHD tended to occur more frequently (40% vs. 9%) in C77G cases. Recovery of lymphocyte subsets and virus-specific CD4 was similar in C77G cases and controls while interleukin 2 (IL2)-responses through CD3 stimulation were higher in C77G cases (P = 0·004). In conclusion, HSCT from PTPRC C77G donors could increase GVHD risk without compromising overall survival. Altered IL2-responses could be involved in this process.

Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), −7/7q (17.2%), and 11q− (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

In unrelated hematopoietic stem cell transplantation (HSCT), the prediction of donor search outcome at the time of search initiation is of great value for the physicians to delineate the strategy of patient care. The probability of finding an unrelated donor is high for patients who carry at least 1 of the 10 most common HLA haplotypes in Caucasians. As only 10% to 20% patients respond to this criterion, here we aimed at finding additional common haplotypes to improve the prediction of a successful search. HLA broad HLA-A/B/DRB1 haplotypes that were observed with frequencies ≥0.19% in patient families of European origin and that split into ≤2 predominant 4-digit HLA-A/B/C/DRB1/DQB1 haplotypes were considered as common. Carriage of at least 1 of those in 168 patients of various geographic areas with no family donor was confronted to the chance of finding ≥9/10 HLA-matched unrelated donors. Fifty common 4-digit haplotypes were identified. A higher (P < 5 × 10−6) chance of finding a suitable donor was found for 55 of 170 (32%) recipients that carried at least 1 of these common haplotypes. Up to now, estimates classified patients into ≥3 groups of probability with ≥1 intermediate group of poor utility for the clinicians. Considering carriage of these common haplotypes together with the frequencies of alleles and of B/C and DRB1/DQB1 associations, which are carried by patient HLA haplotypes, we could classify the patients into 2 groups of probability with a 98% and 26% chance of finding a donor, respectively. Prediction of search outcome could be improved by including the 50 most common HLA haplotypes in the current approaches.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.