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PURPOSE Off-label use of vemurafenib (VMF) to treat BRAFV600E mutation–positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated. PATIENTS AND METHODS Fifty-four patients from 12 countries took VMF 20 mg/kg/d. They were classified according to risk organ involvement: liver, spleen, and/or blood cytopenia. The main evaluation criteria were adverse events (Common Terminology Criteria for Adverse Events [version 4.3]) and therapeutic responses according to Disease Activity Score. RESULTS LCH extent was distributed as follows: 44 with positive and 10 with negative risk organ involvement. Median age at diagnosis was 0.9 years (range, 0.1 to 6.5 years). Median age at VMF initiation was 1.8 years (range, 0.18 to 14 years), with a median follow-up of 22 months (range, 4.3 to 57 months), whereas median treatment duration was 13.9 months (for 855 patient-months). At 8 weeks, 38 complete responses and 16 partial responses had been achieved, with the median Disease Activity Score decreasing from 7 at diagnosis to 0 ( P < .001). Skin rash, the most frequent adverse event, affected 74% of patients. No secondary skin cancer was observed. Therapeutic plasma VMF concentrations (range, 10 to 20 mg/L) seemed to be safe and effective. VMF discontinuation for 30 patients led to 24 LCH reactivations. The blood BRAFV600E allele load, assessed as circulating cell-free DNA, decreased after starting VMF but remained positive (median, 3.6% at diagnosis, and 1.6% during VMF treatment; P < .001) and was associated with a higher risk of reactivation at VMF discontinuation. None of the various empirical therapies (hematopoietic stem-cell transplantation, cladribine and cytarabine, anti-MEK agent, vinblastine, etc) used for maintenance could eradicate the BRAFV600E clone. CONCLUSION VMF seemed safe and effective in children with refractory BRAFV600E-positive LCH. Additional studies are needed to find effective maintenance therapy approaches.

Rare Brain Tumor Registry., [Background] Embryonal tumours with multi-layered rosettes (ETMRs) are a newly recognised, rare paediatric brain tumour with alterations of the C19MC microRNA locus. Due to varied diagnostic practices and scarce clinical data, disease features and determinants of outcomes for these tumours are poorly defined. We did an integrated clinicopathological and molecular analysis of primary ETMRs to define clinical phenotypes, and to identify prognostic factors of survival and key treatment modalities for this orphan disease., [Methods] Paediatric patients with primary ETMRs and tissue available for analyses were identified from the Rare Brain Tumor Consortium global registry. The institutional histopathological diagnoses were centrally re-reviewed as per the current WHO CNS tumour guidelines, using histopathological and molecular assays. Only patients with complete clinical, treatment, and survival data on Nov 30, 2019, were included in clinicopathological analyses. Among patients who received primary multi-modal curative regimens, event-free survival and overall survival were determined using Cox proportional hazard and log-rank analyses. Univariate and multivariable Cox proportional hazard regression was used to estimate hazard ratios (HRs) with 95% CIs for clinical, molecular, or treatment-related prognostic factors., [Findings] 159 patients had a confirmed molecular diagnosis of primary ETMRs (median age at diagnosis 26 months, IQR 18–36) and were included in our clinicopathological analysis. ETMRs were predominantly non-metastatic (94 [73%] of 128 patients), arising from multiple sites; 84 (55%) of 154 were cerebral tumours and 70 (45%) of 154 arose at sites characteristic of other brain tumours. Hallmark C19MC alterations were seen in 144 (91%) of 159 patients; 15 (9%) were ETMR not otherwise specified. In patients treated with curative intent, event-free survival was 57% (95% CI 47–68) at 6 months and 31% (21–42) at 2 years; overall survival was 29% (20–38) at 2 years and 27% (18–37) at 4 years. Overall survival was associated with non-metastatic disease (HR 0·48, 95% CI 0·28–0·80; p=0·0057) and non-brainstem location (0·42 [0·22–0·81]; p=0·013) on univariate analysis, as well as with gross total resection (0·30, 0·16–0·58; p=0·0014), high-dose chemotherapy (0·35, 0·19–0·67; p=0·0020), and radiotherapy (0·21, 0·10–0·41; p, [Interpretation] Prompt molecular diagnosis and post-surgical treatment with intensive multi-modal therapy tailored to patient-specific risk features could improve ETMR survival., Canadian Institute of Health Research, Canada Research Chair Awards, Australian Lions Childhood Cancer Research Foundation, Spanish Society of Pediatrics, Consejería de Salud y Familias de la Junta de Andalucía, Miracle Marnie, Phoebe Rose Rocks, Tali's Funds, Garron Cancer Centre, Grace's Walk, Meagan's Hug, Brainchild, Nelina's Hope, and Jean Martel Foundation.

BackgroundETMRs are a newly recognized rare paediatric brain tumor with alterations of the C19MC microRNA locus. Due to varied diagnostic practices and limited clinical data, disease features and determinants of outcome are poorly defined. We performed an integrated clinico-pathologic and molecular analyses of 159 primary ETMRs to define clinical phenotypes, identify predictors of survival and critical treatment modalities for this orphan disease.MethodsPrimary ETMR patients were identified from the Rare Brain Tumor Consortium (rarebraintumorconsortium.ca) global registry using histopathologic and molecular assays. Event-Free (EFS) and Overall Survival (OS) for 108 patients treated with curative multi-modal regimens were determined using Cox proportional hazard and log rank analyses.FindingsETMRs were predominantly non-metastatic (73%) tumors arising from multiple sites; 55% were cerebral tumors, 45% arose at sites characteristic of other brain tumors. Hallmark C19MC alterations were seen in 91%; 9% were ETMR-NOS. Survival and hazard analyses showed a 6 month median EFS and 2-4yr OS of 27-29% with metastatic disease (HR=0.44, 95% CI 0.26-0.74; p=0.002) and brainstem location (HR=0.40, 95% CI 0.021-0.75; p=0.005) correlating with adverse OS. Gross total resection (GTR: HR=0.38, 95% CI 0.21-0.68; p=0.001), high dose chemotherapy (HDC: HR=0.55, 95% CI 0.31-0.97; p=0.04) and radiation (RT: HR=0.32, 95% CI 0.16-0.60; p=InterpretationPrompt molecular diagnosis and post-surgical treatment with multi-modal therapy tailored to patient-specific risk features improves ETMR survival.FundingThis work was supported by the Canadian Institute of Health Research Grant No. 137011, Canada Research Chair Awards to AH. Funds from Miracle Marnie, Phoebe Rose Rocks, Tali’s Funds, Garron Cancer Centre, Grace’s Walk, Meagan’s Walk, Nelina’s Hope and Jean Martel Foundation are gratefully acknowledged. SK and PS were respectively supported by the Australian Lions Children’s Cancer Foundation and the Spanish Society of Pediatrics, Consejería de Salud y Familias de la Junta de Andalucía Project EF-0451-2017.

ETMR, an aggressive disease characterised by C19MC alterations, were previously categorised as various histologic diagnoses. The clinical spectrum and impact of conventional multi-modal therapy on this new WHO diagnostic category remains poorly understood as a majority of ~200 cases reported to date lack molecular confirmation. We undertook comprehensive clinico-pathologic studies of a large molecularly confirmed cohort to improve disease recognition and treatment approaches. Amongst 623 CNS-PNETs patients enrolled in the RBTC registry, 159 primary ETMRs were confirmed based on a combination of FISH (125), methylation analysis (88), SNP and RNAseq (32) analyses; 91% had C19MC amplification/gains/fusions, 9% lacked C19MC alterations but had global methylation features of ETMR NOS. ETMRs arose in young patients (median age 26 months) predominantly as localized disease (M0-72%, M2-3 -18%) at multiple locations including cerebrum (60%) cerebellum (18%), midline structures (6%); notably 10% were brainstem primaries mimicking DIPG. Uni-and multivariate analyses of clinical and treatment details of curative regimens available for 110 patients identified metastatic disease (p=0.002), brainstem locations(p=0.005), extent of surgery, receipt of multi-modal therapy including high dose chemotherapy and radiation (P

Background Children with malignant bone tumors have average 5-year survival rates of 60% to 70% with current multimodality therapy. Local control modalities aimed at preserving function greatly influence the quality of life of long-term survivors. In developing countries, the limited availability of multidisciplinary care and limited expertise in specialized surgery and pediatric radiation therapy, as well as financial cost, all form barriers to achieving optimal outcomes in this population. Methods We describe the establishment of a collaborative pediatric bone tumor program among a group of pediatric oncologists in Lebanon and Syria. This program provides access to specialized local control at a tertiary children’s cancer center to pediatric patients with newly diagnosed bone tumors at participating sites. Central review of pathology, staging, and treatment planning is performed in a multidisciplinary tumor board setting. Patients receive chemotherapy at their respective centers on a unified treatment plan. Surgery and/or radiation therapy are performed centrally by specialized staff at the children’s cancer center. Cost barriers were resolved through a program development initiative led by St Jude Children’s Research Hospital. Once program feasibility was achieved, the Children’s Cancer Center of Lebanon Foundation, via fundraising efforts, provided continuation of program-directed funding. Results Findings over a 3-year period showed the feasibility of this project, with timely local control and protocol adherence at eight collaborating centers. We report success in providing standard-of-care multidisciplinary therapy to this patient population with complex needs and financially challenging surgical procedures. Conclusion This initiative can serve as a model, noting that facilitating access to specialized multidisciplinary care, resolution of financial barriers, and close administrative coordination all greatly contributed to the success of the program.

Background The presence of the EWS-WT1 gene fusion transcript (GFT) is characteristic of desmoplastic small round cell tumor (DSRCT), a rare and very aggressive disease for which the treatment has not yet been clearly standardized. Methods This was a retrospective national multicenter analysis of young patients

We analyzed risk factors influencing outcomes after related (R) human leukocyte antigen-identical cord blood transplantation (CBT) for 147 patients with malignancies reported to Eurocord–European Group for Blood and Marrow Transplantation. CBT has been performed since 1990; median follow-up was 6.7 years. Median patient age was 5 years. Acute leukemia was the most frequent diagnosis (74%). At CBT, 40 patients had early, 70 intermediate, and 37 advanced disease. CB grafts contained a median of 4.1 × 107/kg total nucleated cells (TNCs) after thawing. The cumulative incidence (CI) of neutrophil recovery was 90% at day +60. CIs of acute and chronic graft-versus-host disease (GVHD) were 12% and 10% at 2 years, respectively. At 5 years, CIs of nonrelapse mortality and relapse were 9% and 47%, respectively; the probability of disease-free survival (DFS) and overall survival were 44% and 55%, respectively. Among other factors, higher TNCs infused was associated with rapid neutrophil recovery and improved DFS. The use of methotrexate as GVHD prophylaxis decreased the CI of engraftment. Patients without advanced disease had improved DFS. These results support banking and use of CB units for RCBT. Cell dose, GVHD prophylaxis not including methotrexate, and disease status are important factors for outcomes after RCBT.

We retrospectively studied a series of 23 patients (median age 50 years, range 29-59 years) with multiple myeloma (MM), treated in first relapse by a sequential autologous-allogeneic tandem approach. Tandem transplantation (TT) consisted in high dose melphalan (HDT) and auto-SCT followed by an (allo-SCT) preceded by two gray TBI non-myeloablative conditioning. All patients received a first HDT as frontline treatment. At day 100 post allo-SCT, complete donor chimerism was detected in 22 patients (95%). Acute GVHD was observed in 19 patients (15 grade I-II (65%) and 4 grade III-IV (17%)). Ten patients (43%) developed an extensive chronic GVHD. The non-relapse mortality at 1 year was 17%. After TT, the overall response rate was 91% (17% partial response, 35% very good partial remission and 39% complete remission). At 2 years, OS was 61%. Median event-free survival and OS were 36.8 and 60 months, respectively. Based on the propensity score matching method, a significant survival advantage could be seen in patients treated with TT as compared with non-allografted patients. Thus, allo-SCT, in TT approach, provides a high response rate with low toxicity and may improve survival of patients with relapsing MM.

The use of umbilical or placental donor cord blood transplantation (CBT) in children with malignant and non-malignant diseases has witnessed important progress, mainly because of better cord blood donor choice and patient selection translating into better patient outcome. Approximately 2000 children with malignant diseases (about 75 % with acute leukemias) have been transplanted with a related (n=199) or unrelated CBT (UCBT, n=1663) and reported to Eurocord registry from 1990-2008. Disease-specific studies have been carried out after UCBT for acute lymphoblastic and myeloid leukemia and myelodysplastic syndromes in others to identify the risk factors that may improve outcomes. Outcomes after CBT have been compared with other alternative allogeneic hematopoietic SCT (HSCT) donors. Briefly, after CBT, myeloid engraftment is delayed, acute and chronic GVHD decreased and disease-free survival was not statistically different when compared with HLA identical and other alternative HSCT donor. Therefore, any physician has to carefully evaluate, for each single pediatric patient in need of an allograft, all the possible alternatives in order to choose the best hematopoietic stem cell donor, taking into account type of disease, urgency of transplantation, donor characteristics and center experience. This review will analyze the current results of CBT for pediatric patients with malignant diseases and the advantages and limitations of using this stem cell source.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children

Twenty-three children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) after ex vivo elimination of αβ(+) T cells and CD19(+) B cells. The median number of CD34(+), αβ(+)CD3(+), and B cells infused was 16.8 × 10(6), 40 × 10(3), and 40 × 10(3) cells/kg, respectively. No patient received any posttransplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD). All but 4 patients engrafted, these latter being rescued by a second allograft. Three patients experienced skin-only grade 1 to 2 acute GVHD. No patient developed visceral acute or chronic GVHD. Cumulative incidence of transplantation-related mortality was 9.3%. With a median follow-up of 18 months, 21 of 23 children are alive and disease-free, the 2-year probability of disease-free survival being 91.1%. Recovery of γδ(+) T cells was prompt, but αβ(+) T cells progressively ensued over time. Our data suggest that this novel graft manipulation strategy is safe and effective for haplo-HSCT. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

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.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients. "\"\\\"\\\\\\\"\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\"\\\\\\\"\\\"\""

Blood disorders caused by abnormal β-globin — β-thalassaemia and sickle cell disease — are the most prevalent inherited disorders worldwide, with patients often remaining dependent on blood transfusions throughout their lives. So a report of the successful use of gene therapy in a case of severe β-thalassaemia — using a lentiviral vector expressing the β-globin gene — is an eagerly awaited event. More than two years after gene transfer, the adult male patient has been transfusion-independent for 21 months. The therapeutic benefit seems to result from a dominant, myeloid-biased cell clone that may remain benign, although it could yet develop into leukaemia — a reminder that gene therapy is still at an early stage. Disorders caused by abnormal β-globin, such as β-thalassaemia, are the most prevalent inherited disorders worldwide. For treatment, many patients are dependent on blood transfusions; thus far the only cure has involved matched transplantation of haematopoietic stem cells. Here it is shown that lentiviral β-globin gene transfer can be an effective substitute for regular transfusions in a patient with severe β-thalassaemia. The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas1,2. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability1,2. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent1,2. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl−1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

e21020 Background: Osteosarcoma and Ewing sarcoma are the most common malignant bone tumors in pediatrics. Average 5-year survival rates of 60-70% are achieved with current multimodality therapy. L...

Introduction Hereditary spherocytosis (HS), the most common cause of inherited chronic hemolysis in Northern Europe and North America, is characterized by a wide variety of clinical features, ranging from an asymptomatic condition to a fulminant hemolytic anemia. Guidelines regarding the management and diagnosis of HS have been updated in 2012. Few reports exist on HS prevalence and related molecular abnormalities in Mediterranean populations. The prevalence and features of HS in Lebanon have not yet been reported. The aim of this study is to determine demographic, clinical, hematological and therapeutic features as well as outcome of a group of Lebanese patients with HS. Methods This is a retrospective hospital-based surveillance study conducted at 4 centers in Northern Lebanon and Central Beirut. Data was collected from archived files on 31 patients between May 2012 and May 2013 and included patients’ demographics, clinical presentation, diagnostic laboratory tests, treatment modalities and outcome. Analysis was conducted using SPSS 19, and t-test was used to compare two groups. We also conducted an Analysis of Variances where we included demographic variables that were different between the two groups as covariates (ANCOVA) in order to look for group effect. Statistical significance was set at p Results Thirty one patients, median age at diagnosis 60 months, age range 11 -.180 months were included in this study. 58.1% and 41.9% were females and males. 54.8% (17/31) had a family history for HS and 32.3% (10/31) had one parent with HS. The predominant clinical manifestations at diagnosis were pallor in 27/31 (87.1%), splenomegaly in 23/31 (74.2%) and anemia in 20/31 (64.5%) patients respectively. The mean size of the spleen at diagnosis was 5.51±2.91 cms below the costal margin. 9.7 % of patients had symptomatic gallstones. The mean hemoglobin, MCV, MCHC and reticulocyte count at time of diagnosis were 8.7 ± 2.1 g/dL, 73.0 ± 9.9 fL/red cell, 34.13±2 g/dl and 6.58±4.2% respectively. 13/17 (68.4%) had spherocytes on baseline blood smear. Osmotic fragility test performed in 27/31 (87.1%) was compatible with diagnosis of HS in 88.24 % patients. HPLC performed in 17/31 (54.8%) showed a mean HbF of 3.82 % and sickle cell trait in 4/17(2,35%) patients. 5/19 (26.3%) had concomitant G6PD deficiency. Folic acid supplementation was provided to patients with moderate to severe disease. 23/31 patients (74.2%) required intermittent or regular blood transfusions and 10/31 patients (32.3%) underwent splenectomy as part of their treatment. All splenectomies were done after 5 years of age except in one patient who had recurrent severe hemolysis necessitating splenectomy at 3 years. There were no correlations between the need for splenectomy with age at diagnosis (p-value 0.97), MCHC level (p-value 0.43) and blood type (p-value 0.17). There was a positive correlation between the need for splenectomy and the need for blood transfusion (r=0.671; p=0.006) and between G6PD status and the need for transfusion (r=0.778; p=0.002). 30/31(96%) patients are alive and doing well while 1 patient died from acute splenic sequestration. Of the 30 surviving patients, 3 (10%) had had a complicated disease course characterized by very severe and early hemolysis in 2 patients and significant iron overload in a third patient. Conclusions This is the first study from Lebanon describing the demographic, clinical, hematological and therapeutic features as well as outcome of patients with HS. Data generated from this study suggest underreporting of the disease and over utilization of the osmotic fragility test underscoring the need to initiate more awareness amongst Lebanese health providers about this disease and its new diagnostic guidelines. Clinical features, therapeutic modalities and excellent patients’ outcome in this Lebanese group appear to be similar to modern published reports. Major limitations of this study include its retrospective design and the small sample size. Prospective studies with long term follow up and enrolling a larger sample size are needed to better assess the utility of newer diagnostic tests and the long term complications associated with splenectomy. Disclosures: No relevant conflicts of interest to declare.

Abstract 532 Intensive and risk-adapted chemotherapy protocols may cure more than 80% of children with acute lymphoblastic leukemia (ALL). The detection of a positive minimal residual disease (MRD) in childhood ALL, is an independent prognostic factor of poor outcomes after chemotherapy treatment and has been used recently as a tool for risk stratification in most ALL treatment protocols. Its value before allogeneic related or unrelated hematopoietic stem cell transplantation (HSCT) has been described in small series of patients and it has been associated with outcomes. With the aim to investigate the prognostic significance of MRD before unrelated cord blood transplantation (UCBT) for ALL, we retrospectively analyzed children reported to the Eurocord registry. From 2000 to 2009, 316 children and adolescents with ALL in remission underwent a first single UCBT after a myeloablative conditioning regimen in 38 EBMT centers. Among these patients, 170 had an assessment of MRD before UCBT and were considered in this analysis. Of those, 72 patients underwent UCBT in first complete remission (CR), 77 in CR2 and 21 in CR3. The MRD assay was based on PCR (71%) or multiparameter flow cytometric analysis (FC) (29%). A positive MRD was defined according to treatment protocols of the various participating centers. MRD test before UCBT was positive (MRD+) in 74 patients (44%) and negative (MRD-) in 96 (56%). MRD was positive in 32 out of 72 patients in CR1, 31 out of 77 patients in CR2 and 11 out of 21 patients in CR3. The median interval from the last MRD test to UCBT was 18 days (12-42 days). The median follow-up was 46 months (4-104 months) and the median age at UCBT was 6.5 years (0.6-17 years). The most common immunephenotype was B-cell precursor ALL (83%). Of 72 patients transplanted in CR1, 56% had poor risk cytogenetics (t4;11 or t9;22). For patients in CR2 and CR3, the median interval between remission to relapse was 22 months (3-130 months). All patients received a TBI-based (63%) or Busulfan-based (37%), myeloablative conditioning regimen. GVHD prophylaxis consisted of cyclosporin (CSA) ± steroids in 75%. Sixty percent of cord blood units had 0–1 HLA mismatches with recipients and 40% had 2–3 mismatches (antigen level for HLA-A and B, allelic level for DRB1). Median infused TNC cell dose was 4.8 ×107/kg (1.30-18×107/kg) and median CD34+ was 1.8× 105/kg (1-10×105/kg). Cumulative incidence (CI) of day-60 neutrophil recovery (>500 mm3) and grade II-IV acute GVHD were 85±3% and 26±4%, respectively. Fifty-eight patients developed acute GVHD (grade 2, n=39, grade 3, n=14, grade 4, n= 5). The overall incidence of cGVHD was 17% at 4 years. Twenty-four patients had chronic GVHD (limited involvement, n=15, extensive disease, n=9). Overall CI of non-relapse mortality (NRM), relapse incidence (RI) and probability of leukemia free survival (LFS) at 4 years were 22±3%, 30±3%, and 44±4, respectively. In a univariate analysis, CI of NRM, RI and LFS at 4 years were 26±5%, 39±6%, and 29±5% in children with MRD+ and 20±4%, 24±3% and 54±5% in children with MRD- (p=0.08, p=0.05, p=0.006) respectively. In a multivariate analysis adjusted, a positive MRD before UCBT was an independent factor statistically associated with higher RI and decreased LFS (RI, HR=2.15, p=0.01, LFS, HR=1.97, p=0.003) (Figure 1). Also patients transplanted in more advanced status of disease at UCBT had higher RI and decreased LFS (RI, HR=4.25, p=0.002, LFS, HR=3.7, p= Estimated 4-year Leukemia free Survival according to MRD status before UCBT [(••• MRD negative) (— MRD positive)] Disclosures: No relevant conflicts of interest to declare.

Despite recent advances in the treatment including Autologous Stem Cell Transplantation (A-PBSCT) and new drugs, Multiple Myeloma (MM) is still an incurable disease and the outcome of relapsing patients is still extremely grim. The use of conventional allogeneic hematopoietic SCT is limited by a high transplantation-related mortality (TRM). Allografting with nonmyeloablative conditioning (NMA-C) has therefore been considered to improve survival. We retrospectively studied a series of 23 consecutive patients with relapsing MM who underwent allogeneic transplantation after NMA-C and compared their outcome with those of patients who relapsed but were not allografted. Owing to these non-randomized available data, there was a need to correct for potential recruitment bias. The propensity score (PS) methodology allows coping with the presence of such a bias (Rosenbaum PR, Rubin DB. The central role of the propensity score in observational studies for causal effects. Biometrika1983;70:41–55). The idea was to model, for each patient with MM alive at 6 months after relapse, the probability of receiving NMA-C transplantation, according to a set of baseline characteristics (namely, baseline age, serum beta2 microglobulin level (b2M), and time to progression (TTP) after the first A-PBSCT). This PS was estimated using logistic regression. It was then used to match 1:1 patients with similar propensity to receive allograft, based on nearest neighbor matching using calipers of width 0.2. Data of patients treated in the MAG-95 and MAG-2002 trials were used. Standardized differences in covariates between subjects in the matched sample were computed, with imbalances tested by paired t tests. A random effects model was finally fitted in order to account for the matched nature of the data. 23 patients with MM in first (n=21) or second (n=2) relapse were treated with high dose therapy (melphalan 200 mg/m2 followed by A-PBSCT) preceding allogeneic PBSCT with a 2 Gy TBI NMA-C. Donors were HLA-identical siblings in 13 (56 %) patients. Six patients (26%) had a high b2M (>3 mg/L). Median age at allograft was 50 years (29–59 years). Median follow-up after allograft was 27.4 months (1.8–61.8 months). Post-allograft response was CR in 9 patients, VGPR in 8, PR in 4 and progressive disease (PD) in 2. TRM at one year was 13 % (1 severe GvHD, 2 infections). Two patients died from PD at 2 and 9 months after allograft respectively. Acute GvHD occurred in 19 patients (15 grade I/II, 4 grade III/ IV) and 11 developed chronic GvHD. Among the 10 patients (41.6%) who relapsed with a median of 10,7 months, 6 are alive with a median survival of 38.3 months. So far, 8 patients (34,7%) including 5 in CR and 3 in VGPR are still alive without relapse with a median follow-up of 36.8 months. From the 23 allografted patients, 21 matched pairs were successfully constituted. Expectedly, the distribution of age (mean difference: −0.97, p= 0.54), b2M (p=1.00), and TTP (mean difference: 3.2 months, p= 0.37) were not different between treated by allograft and untreated subjects. Based on these matched pairs, the estimated hazard ratio of death was 0.35 (95% confidence interval: 0.14–0.88, p=0.027) for allografted patients compared with non allografted. In conclusion, the PS is a useful tool for the analysis of observational data. Applied to MM data, it suggests that NMA-C allograft in MM patients in first relapse provides a high and durable response rate with a low TRM. These promising results must be further evaluated in clinical trials.

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein TIRAP, mediates downstream signaling of TLR2 and TLR4. A leucine (Leu) substitution at position 180 serine (Ser) of TIRAP was shown to protect from infections. We hypothesized that the incidence of infections and outcome after bone marrow transplantation (BMT) could be influenced by the TIRAP gene polymorphisms. Genotyping for TIRAP at Ser180 was performed in 107 donor/recipients pairs: 64% of recipients and 63% of donors were homozygote wild type (wt) Ser180/Ser180, 20% and 19% were Ser180/Leu180 and 17% and 18% Leu180/Leu180. Severe bacterial (pneumonia, septicemia and septic shock), viral or invasive fungal infections during 180 days after BMT, TRM (transplant related mortality), acute GVHD and survival were studied retrospectively. An allogeneic HLA-identical BMT was performed in 107 patients (median age was 35 years), with acute (n=39) or chronic Leukemia (n=68). Conditioning regimen consisted of Busulfan+Cyclophosphamide±others (BU-CY) in 73 (68%) patients and TBI+CY in 15 (14%) or TBI+Melphalan in 17 (16%). GVHD prophylaxis consisted of CsA+MTX in 94%. Median follow-up time was 77 months (18–147). Cumulative incidence of neutrophil recovery was 94%. Acute GVHD (II-IV) was observed in 42%, and chronic GVHD in 50 out of 98 at risk. Estimate 5-year survival was 54%. The cumulative incidence of any first infection by day 180 was 56%, being 28% for at least one bacterial infection, 39% for viral infection and 12% for invasive fungal infection (8 invasive aspergillosis, 4 disseminated candidiosis and 1 disseminated Malassezia furfur fungal infection). TRM cumulative incidence at 1 year was 35%. Recipient’s TIRAP gene polymorphisms were not associated with outcome. However, patients transplanted from a mutant TIRAP donor (Leu/Ser or Leu/Leu) had a trend to lower incidence of fungal infections (p=0.08); lower incidence of acute GVHD (p=0.03), decreased TRM (p=0.02) and improved overall survival (p=0.02). In a multivariate analysis, adjusting for other prognostic factors, TIRAP donor polymorphisms were associated with decreased TRM (HR=0.49, p=0.02): TRM at 1 year was 43% when donors were wt (Ser/Ser) as compared to 20% when donors were mutants. There was also a trend to decreased incidence of acute GVHD in patients with a mutant TIRAP donor as compared to the others (30% versus 49%; HR 1.88 p=0.06) and better overall survival (67% versus 44%, respectively HR 1.82; p=0.07). Recipients of bone marrow (BM) from wt TIRAP donors seemed to die more frequently from infection (1 yr cumulative incidence 21% vs 8%, p=0.07). In conclusion, the presence of a Leu substitution at position 180 in the TIRAP gene of BM donors decreases the incidence of TRM in HLA-identical BMT, probably reflecting better immune reconstitution and protection against infections. This finding can help defining better surveillance and prophylaxis of bacterial infections in BMT recipients from donors with the wt TIRAP (Ser/Ser) genotype.

Between July 2004 and June 2006, 209 transplants were performed in our department. Only 3 patients (pts) developed candidemia (Cp n=2; Cg n=1) during that period. They were all receiving long-term C therapy. Pt 1 A 18 year-old male with severe aplastic anemia received a 1st unrelated HSCT in January 2004 without sustained engraftment. Two months later, he developed a definite lung invasive aspergillosis (IA) successfully treated with Voriconazole (V). He received a 2nd unrelated cord blood transplant on May 12, 2005. V was continued as secondary prophylaxis. Due to liver function test abnormalities V was switched to C on day (d) 7. On d 48, he was febrile, and a blood culture (BC) was positive (+) for Cp. C was stopped [total treatment duration (TTD)=47 d] and liposomal amphotericin B (LAB) was initiated. Due to the pt’s poor condition, the CVC was not removed. BCs remained + until d 53 (9 + BCs). He ultimately died from multi-organ failure and no engraftment on d 72. Skin and throat colonization (Co) with Cp was documented from d 39 on. Pt 2 A 46-year old male with ALL in 1st complete remission (CR) underwent a genoidentical HSCT on May 26, 2005. Antifungal prophylaxis consisted of fluconazole (F). C was empirically introduced on d 6. Granulocytes (G) recovery occurred on d 28. He further experienced a severe acute graft-versus-host disease (GvHD) treated with increased immunosuppression (IS), and a CMV infection. On d 58–60, although he was afebrile, 3 BCs were + for Cp. C was stopped [TTD=50 d], LAB and F were initiated, and the CVC was replaced. The BCs became negative. However, the pt ultimately died from GvHD and respiratory failure of unknown origin on d 86. Skin, GI and respiratory tracks Co with Cp was documented from d 33 on. Pt 3 A 32-year old male with lymphoblastic T cell lymphoma in 1st CR underwent an unrelated HSCT on November 10, 2005. Antifungal prophylaxis consisted of F. G recovery occurred on d 21. He experienced recurrent episodes of acute GvHD treated with increased IS and developed several episodes of CMV infections and bacteremias, including a septic shock on d 61 which lead to CVC replacement. Antifungal prophylaxis was switched to C on d 95. On d 118, he was febrile, and a single BC was + for Cg. Noteworthy, he developed a probable lung IA concomitantly. C was stopped [TTD=26 d], and V was initiated. The BCs became negative, and the IA resolved. He ultimately died from GvHD and respiratory failure on d 197. GI and respiratory racks Co with Cp was documented between d 116–123. [Table 1] The MICs of Cp and of Cg are known to be slightly higher than those of other Candida spp., which is in accordance with our results for the 2 Cp isolates. In deeply immunocompromised pts, the emergence of Candida known to have a decreased susceptibility to C could become an increasing problem. Persisting Co with such Candida spp may be an indication, in C treated pts, for switching to an alternative antifungal drug. Minimal Inhibitory Concentrations (MICs, μg/ml) of first blood isolates (EUCAST) C. parapsilosis (pt1) C. parapsilosis (pt2) C. guillermondii (pt3) Amphotericin B 0.03 0.03 ≤0.015 Fluorocytosine 0.5 ≤0.125 ≤0.125 Fluconazole 2 1 8 Itraconazole 0.06 0.06 0.5 Voriconazole 0.03 0.03 0.06 Caspofungin 0.5 0.125 0.06

Unrelated cord blood transplantation (UCBT) is an alternative option to treat children with haematological diseases without an HLA-identical donor. We have analyzed a total of 323 children with ALL receiving an UCBT, from 1994 to 2004 in 99 transplant centres in 24 countries, mostly in Europe. Cumulative incidence with competing risk and KM estimates were used to calculate outcomes. Seventy six children were transplanted in CR1, 136 in CR2 and 111 in more advanced phase of the disease. Among those children poor cytogenetics were observed in 89% of children in 1CR, 33% in 2CR and 42% in advanced phase. Twenty percent of children transplanted in advanced phase had been previously autografted. The median age was 6.5 years at UCBT, median cell dose infused was 4.1x107/kg and the median follow time was 22 months (3–96). The cord blood was HLA identical (6/6) in 12% of the cases, 5/6 in 46%, 4/6 in 39% and 3/6 in 3%. All children received myeloablative conditioning regimen (TBI in 66%) and the majority (67%) received CsA+corticoids as GVHD prophylaxis. Cumulative incidence of neutrophil recovery at day 60, platelets recovery (>20.000) at day 180, acute (grade II–IV) and chronic GVHD were 76±5%, 54±5%, 42±3%, 14±2%, respectively. Overall 2 year-LFS was 36±3%. In a multivariate analysis, only CR1 or CR2 were associated with better LFS (HR=1.8; p Outcomes CR1 (n=76) CR2 (n=136) Advanced (n=111) TRM at day 100 22+/−5% 25+/−4% 34+/−5% Relapse at 2 years 34+/−8% 37+/−5% 48+/−7% LFS at 2 years 42+/−6% 41+/−4% 24+/−4% For those patients transplanted with poor cytogenetics, LFS at 2 years was 32±6% and it was 37% for CR1, 43% for CR2 and 0% for advanced phase of the disease. In conclusion, in these large series of high risk ALL patients, these results show that UCBT should be proposed as alternative source of allogeneic transplantation for children lacking an HLA identical donor, in earlier status of the disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) with HLA geno-identical sibling donor is the treatment of choice for children and young adults with constitutional or acquired (SAA) bone marrow failure (BMF). However, results of unrelated HSCT for BMF have been poorer due to high transplant related mortality mainly related to rejection and GVHD. Generally, a myeloablative regimen HSCT is used for acquired and some constitutional BMF but not for Fanconi anemia (FA) patients for whom a low dose conditioning regimen is employed. We have driven the hypothesis that immunosuppressive reduced intensity conditioning regimen should decrease TRM, decreasing GVHD and allowing engraftment. In a Phase I-II trial, 20 patients (pts) with BMF were enrolled and transplanted between 2002 and 2004. Thirteen pts had a constitutional aplasia: FA n=11, congenital megakaryocytopenia (CMK) n=1, Rothmund-Thomson syndrome n=1 and 7 pts had SAA among those two had paroxystic nocturnal hemoglobinuria (PNH). There were 12 male and 8 female. Median age was 8 years for constitutional BMF and 26 years for SAA. The HSC source was bone marrow for 11 pts, PBSC for 1 pt and cord blood for 8 pts. Ten of the twelve BM or PBSC donors were HLA matched for 10 loci (A, B, C, DRB1, and DQB1) and eight cord blood donors were HLA mismatched with 2 generic differences and were used for FA. All pts received the same conditioning regimen consisting of Busulfan (3mg/kg x 2), cyclophosphamide (10mg/kg x 4), fludarabine (30mg/m2 x3) and ATG (2.5mg/kg x4). The mean of nucleated stem cells infused and CD34 + cells was 2.8x108/kg and 5.9x106/kg respectively for the 12 pts who received BM stem cells and PBSC and 6.4x107/kg and 4.6x106/kg respectively for the eight pts who received CB cells. Acute GVH disease prophylaxis consisted of ciclosporine A (CsA) for pts with constitutional BMF and CsA and short course methotrexate for 6 of the 7 pts with acquired BMF, (one received tacrolimus instead of CsA due to thrombotic pre-existing co-morbidity). One pt (with CMK) died on day 0 from cerebral haemorrhage. Eighteen pts out of 19 had WBC recovery with a median time of 23 days (11–42); one FA pt did never reach sustained engraftment and died at D+291 from adenovirus infection. Three others had late graft rejection: in a context of acute GvHD and EBV infection and pulmonary aspergillosis for two pts with SAA who received BM graft and with acute GvHD and adenovirus infection for one FA pt who received CB graft. The conditioning was well tolerated without severe mucositis even in FA patients, sixteen patients experienced transient liver abnormalities. Nine patients developed reversible haemorrhagic cystitis at a median of 47 days post-transplant. There were 3 bacterial, 10 viral and 5 fungal infections with a cumulative incidence of TRM at one year of 45 ±24%. The cumulative incidence of acute GVH (II–IV) was 50 ±23%. Overall survival (OS) at one year was 55±11 %. It was 86%± 13 for SAA and 38% ± 13 for constitutional BMF. In spite of the short follow-up and few patients included, reduced intensity conditioning regimen provides encouraging results for patients with SAA. For constitutional BMF, low toxicity was observed, however the overall results seem similar to those reported in the literature using other RIC regimen and are probably related to other factors than the conditioning regimen.