American Lebanese Syrian Associated Charities Research Articles

Genome-Wide CRISPR/Cas9 Screen Identifies AraC-Daunorubicin-Etoposide (ADE) Response Modulators Associated with Clinical Outcomes in Pediatric AML

Cytarabine, daunorubicin, and etoposide (ADE) remain the standard backbone chemotherapy in pediatric AML despite newly approved drugs. However, many pediatric AML develop treatment resistance and relapse, resulting in poor survival. To overcome treatment resistance, it is crucial to understand the molecular mechanisms behind ADE treatment outcomes. In this study, we performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify ADE response modulators and integrated CRISPR-screen results with gene expression and clinical outcomes in pediatric AML treated with ADE. K-562 cells were transduced with the Brunello CRISPR/Cas9 library and puromycin selected before the transduced cell population was treated with DMSO, IC30 of cytarabine, daunorubicin, or etoposide (37uM, 0.1uM, and 0.85uM, respectively) for four days at 400x coverage. Genomic DNA was collected for all samples, sgRNAs were PCR amplified, and then sequenced on Illumina Hiseq 4000. The abundance of sgRNA was analyzed using MAGeCK-MLE to obtain normalized beta score per gene to identify treatment-related (treatment minus control beta values [diff-beta]) genes. Genes with diff-beta <2 standard deviations (SD) were associated with drug resistance (negative selection), and genes with diff-beta >2 SD were associated with increased drug sensitivity (positive selection). For translational impacts of CRISPR screen results with outcomes in ADE-treated AML patients, leukemic cell gene expression was evaluated for association with clinical endpoints such as event-free survival (EFS), overall survival (OS), minimal residual disease after induction 1 (MRD1) and relapsed/refractory disease (RR) in pediatric AML from two clinical cohorts, which multisite AML02 trial (n=163) and publicly available TARGET database (only patients treated with the ADE arm of AAML0531, n=213) were included. At the SD cut-off, 888, 114, and 878 are identified as drug sensitive genes, while 306, 1301, and 412 are resistant genes to cytarabine, daunorubicin, or etoposide, respectively (Figure 1). These significant genes are further tested for associations between expression levels and clinical endpoints in ADE-treated pediatric AML in two independent cohorts. Venn diagram in Figure 2 shows the overlapped genes between CRISPR hits and genes with significant clinical outcome associations in AML02 and TARGET-AAML0531. Of particular interest, five genes are significant CRISPR hits for all three drugs (all are sensitive to cytarabine and etoposide but resistant to daunorubicin), as well as for the clinical outcome associations. Among these, GABARAP, GYPA, and SUCLG2 are associated with better outcomes, while C11orf58 and MATR3 are associated with detrimental outcomes. Furthermore, Figure 2 also shows a heat map that illustrates the association between expression levels and clinical outcomes of these five genes with the available EFS, OS, MRD1, and RR (with and without risk adjustment) in two cohorts. Notably, GYPA (CD235a) is an erythroid cell membrane protein, and high leukemic cell GYPA expression is shown to be associated with favorable EFS and OS in AML02 (EFS HR=0.48, p=0.004 with risk-adjusted HR=0.45 p=0.002; OS HR=0.46, p=0.012 with risk-adjusted HR=0.43, p=0.006) and EFS in TARGET-AAML0531 (HR= 0.67, p=0.0038 with risk-adjusted HR=0.72 p=0.08). Another notable gene with detrimental outcome associations is MATR3, which has been shown to have a deleterious effect on cell fate transitions, chromatin structure, and cell differentiation (PMID: 34716321). Overall, our study coupled the results of a genome-wide CRISPR/Cas9 screen with leukemic cell gene expression and clinical outcomes identified clinically and functionally significant ADE genes. C11orf58, MATR3, GABARAP, GYPA, and SUCLG2 are potential novel biomarkers associated with prognosis in the context of pediatric AML treated with ADE. Our future work is focused on identifying the most optimal combination of induction regimens based on the transcriptomic signature of patients’ leukemic cells. Acknowledgments: NIH-R01-CA132946 (Lamba and Pounds), NIH-T32-HG008958 (UF PARADIGM Training Program), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC). We thank Dr. Roya Rafiee for CRISPR/Cas9 drug screen contribution; Drs. Campana, Coustan-Smith, and Susana Raimondi for MRD and cytogenetic data. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD19-CAR T Cells Develop Exhaustion Epigenetic Programs During a Clinical Response

Abstract The goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion in leukemia patients as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells during manufacturing and from peripheral blood mononuclear cells (PBMCs) of 15 patients enrolled on our institutional, autologous CD19-CAR T cell therapy study (NCT03573700). CAR T cell expansion and persistence were determined by measuring vector copy numbers in the PBMCs of treated patients. We had previously established novel exhaustion DNA methylation datasets that delineate between progenitor and fully exhausted T cells. These datasets served as a guide for stratifying our post-infusion CAR T cells along the exhaustion developmental trajectory. Our data show that CD19-CAR T cells lose repressive DNA methylation at effector loci (e.g. PRF1, TBET) while gaining methylation at genes associated with memory potential (e.g. LEF1, TCF7). We confirmed these epigenetic changes are coupled to endogenous human T cell effector and memory differentiation by cross-referencing our epigenetic data with publicly available transcriptional profiles for antigen-specific effector and long-lived memory CD8 T cells from individuals vaccinated for yellow fever. Furthermore, we show that CAR T cells were unable to mount an in vivo recall response after relapse of antigen-positive disease or recovery of endogenous B cells. These observations support the conclusion that CD19-CAR T cells acquire stable epigenetic exhaustion programs that limit their protective capacity. This work was supported by the National Institutes of Health (1R01AI114442 to BY and LRP to CCZ), the National Comprehensive Cancer Network Young Investigator Award (to CZ), Alex’s Lemonade Stand Foundation Young Investigator Grant (to CZ), Stand Up to Cancer- SU2C (to BY), the American Lebanese Syrian Associated Charities (ALSAC) to BY, and Assisi foundation to BY.

Impact of SAMHD1 Pharmacogenetics on Clinical Outcome in Pediatric AML

Acute myeloid leukemia (AML) is a heterogeneous disease characterized in part by genetic and epigenetic alterations. Cytarabine arabinoside (Ara-C) has been the cornerstone of chemotherapy treatment for patients diagnosed with AML for decades. Following cellular uptake, ara-C is phosphorylated into its active metabolite, ara-CTP, which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in ara-C activation/inactivation pathway (Fig 1A), can impact intracellular abundance of ara-CTP and thus its therapeutic benefit. Recently, deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) was shown to limit the efficacy of ara-C by intracellularly hydrolyzing its active metabolite (PMID: 27991919). Other nucleoside analogs, such as clofarabine, fludarabine, and gemcitabine have also been shown to be substrates of SAMHD1 (PMID:30305425). Among adult AML patients, higher SAMHD1 expression in the leukemic cells has been found to correlate with poor outcome (PMID: 30341277). However, whether genetic variation in SAMHD1 has any impact on the clinical outcomes in pediatric AML patients has not been evaluated in depth. In this study, we looked into 25 single nucleotide polymorphisms (SNPs) within SAMHD1 gene for their association with clinical outcome of newly diagnosed pediatric AML patients in 2 cohorts (multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=231] clinical trials). Briefly, genotypes for the 25 SNPs within SAMHD1 genes were obtained from Illumina 2.5 Omni data of AML patients. Association analysis was conducted using logistic regression models comparing minimal residual disease (MRD) positivity after treatment with chemotherapy using an unadjusted model and an adjusted model stratified by initial risk assignment of the patient determined at diagnosis (i.e. low, standard, and high). MRD positivity was defined as one or more leukemic cell per 1000 mononuclear bone-marrow cells. Cox proportional hazard models were used to examine the association of the SNPs with EFS and OS and considered three modes of inheritance. Cox regression models were performed with or without adjusting for provisional risk group assignment at time of diagnosis. Significance levels for association of SNP with clinical outcome were set at p < 0.05.Three top SNPs significantly associated with clinical outcomes were all localized in the 3'UTR region of SAMHD1(Fig 1B). Presence of the variant allele of rs7265241 was associated with a lower EFS and OS in both AML02 and AML08 cohorts. Figure 1C shows the results for OS in AML02 (HR = 2.24, 95% CI (1.05-3.55), p=0.02) and AML08 (HR = 1.52, 95% CI (1.04-1.99), p=0.01). Another 3'UTR SNP, rs1291128 was associated with poor OS in AML02 and in the clofarabine plus ara-C treatment arm in AML08 trial. For rs1291141, T allele was associated with higher MRD positivity (OR: 2.12, p=0.01) in AML02 and poor OS (HR = 1.55, 95% CI (1.12-2.14), p=0.008) in AML08(Fig 1D). Consistent with the clinical outcome results, rs1291141 T allele was also associated with higher SAMHD1 expression in the whole blood (p=8.1e-14) and several other tissues in the GTEx database. https://gtexportal.org/home/Our results suggest that genetic variation in SAMHD1 is associated with clinical outcomes in pediatric AML patients. Future studies are aimed at looking at haplotypes and SNP-SNP combinations to establish the impact SAMHD1 pharmacogenomics on its expression within leukemic cells and subsequent response to nucleoside analogs in AML patients. SAMHD1 has also been implicated as a tumor suppressor; thus, future studies will evaluate its role both as a proliferation regulator and in drug resistance.Acknowledgements: NIH-R01-CA132946 (Lamba and Pounds), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC), and American Society of Hematology Bridge funding (Lamba) funded the study. We thank Drs. Campana, Coustan-Smith, and Raimondi for MRD and cytogenetic data. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Global Impact of the COVID-19 Pandemic on Paediatric Cancer Care

Background: While mortality due to COVID-19 infection has been reportedly low among paediatric oncology patients, changes in healthcare services due to the pandemic have impacted cancer care delivery. This study assessed the effect of the COVID-19 pandemic on childhood cancer care worldwide.Methods: A cross-sectional survey was distributed to paediatric oncology providers from June to August 2020. The survey included questions to assess institution characteristics, the number of patients diagnosed with COVID-19, and disruptions and adaptations to cancer care. Responses were analysed at the institutional level.Findings: The survey was completed by 311 healthcare professionals at 213 institutions in 79 countries. Most centres had the capacity to test for SARS-CoV-2 (88%) and a median of two infections (range: 0-350) reported in children with cancer. Seven percent of centres reported complete closure of paediatric haematology-oncology services (median ten days). Overall, 43% of centres reported fewer than expected paediatric cancer diagnoses and 34% reported increased treatment abandonment. Institutions described reductions in clinical staff (66%) and decreased financial support (32%). Changes to cancer care delivery included: reduced surgical care (79%), blood product shortages (60%), chemotherapy modifications (55%), and interruptions to radiotherapy (40%). The decreased number of new cancer diagnoses did not vary based on country income status (p=0.1). However, lack of chemotherapy, treatment abandonment, and blood product shortages were more frequent in low- and middle-income countries (p=0.022; pInterpretation: The COVID-19 pandemic has considerably impacted the care of paediatric oncology patients worldwide, posing significant disruptions to cancer diagnosis and management. While paediatric cancer care has been affected globally, this impact is more pronounced in low-and middle-income countries.Funding Statement: This work was supported by the American Lebanese Syrian Associated Charities (ALSAC).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: This study was exempted by the institutional review board at St. Jude Children’s Research Hospital.

Pharmacogenomics of intracellular methotrexate polyglutamates in patients' leukemia cells in vivo.

BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).

Artificial Intelligence Assisted Prediction of Late Onset Cardiomyopathy Among Childhood Cancer Survivors

Objective: Early identification of childhood cancer survivors at high risk for treatment-related cardiomyopathy may improve outcomes by enabling intervention prior to development of heart failure. We implemented artificial intelligence methods using the Children’s Oncology Group (COG) guideline-recommended baseline electrocardiography (ECG) to predict cardiomyopathy. Materials and Methods: Seven artificial intelligence and signal processing methods were applied to 10-second 12-lead ECGs obtained on 1,217 adult survivors of childhood cancer prospectively followed in the St. Jude Lifetime Cohort (SJLIFE) study. Clinical and echocardiographic assessment of cardiac function was performed at initial and follow-up SJLIFE visits. Cardiomyopathy was defined as ejection fraction <50% or absolute drop from the baseline ≥10%. Genetic algorithm was used for feature selection and extreme gradient boosting (XGboost) was applied to predict cardiomyopathy during the follow-up period. Model performance was evaluated by five-fold stratified cross validation. Results: The median age at baseline SJLIFE evaluation was 31·7 years (range 18·4-66·4), and the time between baseline and follow-up evaluations was 5·2 years (0·5-9·5). Two-thirds (67·1%) of subjects were exposed to chest radiation and 76·6% to anthracycline chemotherapy. One hundred seventeen (9·6%) subjects developed cardiomyopathy during follow-up. In the model based solely on ECG features, the cross-validation AUC was 0·87 (CI 0·83-0·90), while the model based on clinical features had an AUC of 0·69 (CI 0·64-0·74). In the model based on ECG and clinical features, the cross-validation AUC was 0·89 (CI 0·86-0·91), with sensitivity 78% and specificity 81%. Conclusion: Artificial intelligence using ECG data may assist in the identification of childhood cancer survivors at increased risk for developing cardiomyopathy. Funding Statement: This study was supported by the National Cancer Institute (U01 CA195547, M. Hudson and L. Robison, Principal Investigators). Support to St. Jude Children’s Research Hospital was also provided by the Cancer Center Support (CORE) grant (CA21765, C. Roberts, Principal Investigator) and the American Lebanese Syrian Associated Charities (ALSAC). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Institutional Review Board and all survivors provided informed consent prior to participation.

The global burden of childhood and adolescent cancer in 2017: an analysis of the Global Burden of Disease Study 2017.

SummaryBackgroundAccurate childhood cancer burden data are crucial for resource planning and health policy prioritisation. Model-based estimates are necessary because cancer surveillance data are scarce or non-existent in many countries. Although global incidence and mortality estimates are available, there are no previous analyses of the global burden of childhood cancer represented in disability-adjusted life-years (DALYs).MethodsUsing the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 methodology, childhood (ages 0–19 years) cancer mortality was estimated by use of vital registration system data, verbal autopsy data, and population-based cancer registry incidence data, which were transformed to mortality estimates through modelled mortality-to-incidence ratios (MIRs). Childhood cancer incidence was estimated using the mortality estimates and corresponding MIRs. Prevalence estimates were calculated by using MIR to model survival and multiplied by disability weights to obtain years lived with disability (YLDs). Years of life lost (YLLs) were calculated by multiplying age-specific cancer deaths by the difference between the age of death and a reference life expectancy. DALYs were calculated as the sum of YLLs and YLDs. Final point estimates are reported with 95% uncertainty intervals.FindingsGlobally, in 2017, there were 11·5 million (95% uncertainty interval 10·6–12·3) DALYs due to childhood cancer, 97·3% (97·3–97·3) of which were attributable to YLLs and 2·7% (2·7–2·7) of which were attributable to YLDs. Childhood cancer was the sixth leading cause of total cancer burden globally and the ninth leading cause of childhood disease burden globally. 82·2% (82·1–82·2) of global childhood cancer DALYs occurred in low, low-middle, or middle Socio-demographic Index locations, whereas 50·3% (50·3–50·3) of adult cancer DALYs occurred in these same locations. Cancers that are uncategorised in the current GBD framework comprised 26·5% (26·5–26·5) of global childhood cancer DALYs.InterpretationThe GBD 2017 results call attention to the substantial burden of childhood cancer globally, which disproportionately affects populations in resource-limited settings. The use of DALY-based estimates is crucial in demonstrating that childhood cancer burden represents an important global cancer and child health concern.FundingBill & Melinda Gates Foundation, American Lebanese Syrian Associated Charities (ALSAC), and St. Baldrick's Foundation.

Temporal patterns in the risk of chronic health conditions in survivors of childhood cancer diagnosed 1970-99: a report from the Childhood Cancer Survivor Study cohort.

Treatments for childhood cancer have evolved over the past 50 years, with the goal of maximising the proportion of patients who achieve long-term survival, while minimising the adverse effects of therapy. We aimed to assess incidence patterns of serious chronic health conditions in long-term survivors of childhood cancer across three decades of diagnosis and treatment. We used data from the Childhood Cancer Survivor Study, a retrospective cohort with longitudinal follow-up of 5-year survivors of common childhood cancers (leukaemia, tumours of the CNS, Hodgkin lymphoma, non-Hodgkin lymphoma, Wilms tumour, neuroblastoma, soft tissue sarcoma, or bone tumours) who were diagnosed before the age of 21 years and from 1970 to 1999 in North America. We examined the cumulative incidence of severe to fatal chronic health conditions occurring up to 20 years post-diagnosis among survivors, compared by diagnosis decade. We used multivariable regression models to estimate hazard ratios per diagnosis decade, and we added treatment variables to assess whether treatment changes attenuated associations between diagnosis decade and chronic disease risk. Among 23 601 survivors with a median follow-up of 21 years (IQR 15-25), the 20-year cumulative incidence of at least one grade 3-5 chronic condition decreased significantly from 33·2% (95% CI 32·0-34·3) in those diagnosed 1970-79 to 29·3% (28·4-30·2; p<0·0001) in 1980-89, and 27·5% (26·4-28·6; p=0·012 vs 1980-89) in 1990-99. By comparison, the 20-year cumulative incidence of at least one grade 3-5 condition in 5051 siblings was 4·6% (95% CI 3·9-5·2). The 15-year cumulative incidence of at least one grade 3-5 condition was lower for survivors diagnosed 1990-99 compared with those diagnosed 1970-79 for Hodgkin lymphoma (17·7% [95% CI 15·0-20·5] vs 26·4% [23·8-29·1]; p<0·0001), non-Hodgkin lymphoma (16·9% [14·0-19·7] vs 23·8% [19·9-27·7]; p=0.0053), astrocytoma (30·5% [27·8-33·2] vs 47·3% [42·9-51·7]; p<0·0001), Wilms tumour (11·9% [9·5-14·3] vs 17·6% [14·3-20·8]; p=0·034), soft tissue sarcoma (28·3% [23·5-33·1] vs 36·5% [31·5-41·4]; p=0·021), and osteosarcoma (65·6% [60·6-70·6] vs 87·5% [84·1-91·0]; p<0·0001). By contrast, the 15-year cumulative incidence of at least one grade 3-5 condition was higher (1990-99 vs 1970-79) for medulloblastoma or primitive neuroectodermal tumour (58·9% [54·4-63·3] vs 42·9% [34·9-50·9]; p=0·00060), and neuroblastoma (25·0% [21·8-28·2] vs 18·0% [14·5-21·6]; p=0·0045). Results were consistent with changes in treatment as a significant mediator of the association between diagnosis decade and risk of grade 3-5 chronic conditions for astrocytoma (HR per decade without treatment in the model = 0·77, 95% CI 0·64-0·92; HR with treatment in the model=0·89, 95% CI 0·72-1·11; pmediation=0·0085) and Hodgkin lymphoma (HR without treatment=0·75, 95% CI 0·65-0·85; HR with treatment=0·91, 95% CI 0·73-1·12; pmediation=0·024). Temporal decreases in 15-year cumulative incidence comparing survivors diagnosed 1970-79 to survivors diagnosed 1990-99 were noted for endocrinopathies (5·9% [5·3-6·4] vs 2·8% [2·5-3·2]; p<0·0001), subsequent malignant neoplasms (2·7% [2·3-3·1] vs 1·9% [1·6-2·2]; p=0·0033), musculoskeletal conditions (5·8% [5·2-6·4] vs 3·3% [2·9-3·6]; p<0·0001), and gastrointestinal conditions (2·3% [2·0-2·7] vs 1·5% [1·3-1·8]; p=0·00037), while hearing loss increased (3·0% [2·6-3·5] vs 5·7% [5·2-6·1]; p<0·0001). Our results suggest that more recently treated survivors of childhood cancer had improvements in health outcomes, consistent with efforts over the same time period to modify childhood cancer treatment regimens to maximise overall survival, while reducing risk of long-term adverse events. Continuing advances in cancer therapy offer promise of further reducing the risk of long-term adverse events in childhood cancer survivors. However, achieving long-term survival for childhood cancer continues to come at a cost for many survivors, emphasising the importance of long-term follow-up care for this population. National Cancer Institute and the American Lebanese-Syrian Associated Charities.

The Transcriptional Landscape of B-Precursor Acute Lymphoblastic Leukemia: An International Collaborative Study of 1,223 Cases

Background: B-cell precursor acute lymphoblastic leukemia (BCP ALL) represents a heterogeneous group of hematological malignancies. Although most classes can be classifiend into known major gene expression subtypes, a substantial proportion of BCP ALL remaines poorly characterized with regard to unclear underlying genomic abnormalities. We therefore initiated a large-scale international collaborative study to comprehensively reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA-seq-driven genomic analyses. Methods: RNA-seq data were collected from five BCP ALL cohorts, LUH (Sweden), MaSpore (Singapore and Malaysia), JALSG (Japan), M-HOPES/SIH (China) and TARGET/COG (USA). We performed gene expression based analyses to identify mRNA features in different subgroups and their association with distinct gene fusions/driver mutations. We also evaluated the prognosis of subgroups with adequate number of patients treated. Findings: Fourteen BCP ALL gene expression subgroups (G1-G14) were identified. Apart from extending eight previously described subgroups (G1-G8 associated with MEF2D fusions, TCF3-PBX1 fusions, ETV6-RUNX1/-like, DUX4 fusions, ZNF384 fusions, BCR-ABL1/Ph-like, hyperdiploidy and KMT2A fusions), we additionally defined six gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11, respectively, with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH-CEBPE fusion or mutations in ZEB2 (p.H1038R), and G13 and G14, respectively, with TCF3/4-HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2-G5 and G7 (51-65/67 chromosomes) displayed a low risk, G7 (with ≤50 chromosomes) and G9 showed an intermediate risk, whereas G1, G6 and G8 defined high risk subgroups. In adult BCP ALL, G1, G2, G6 and G8 were associated with high risk, while G4, G5 and G7 had relatively favorable outcomes. Interpretation: Leukemogenic factors contributing to BCP ALL are highly heterogeneous. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete paths of BCP ALL, informing disease classification and prognostic stratification. Clinical Trial Information: Adult patients were enrolled in a SIH trial (Chinese Clinical Trial Registry, number ChiCTR-ONRC-14004968) which was basically a modification of the Vincristine, Daunorubicin, L‑ asparaginase, Cyclophosphamide and Prednisone (VDLCP) regimen. Pediatric patients in the Chinese cohort were enrolled in the Shanghai Children’s Medical Center ALL-2005 protocol (Chinese Clinical Trial Registry, number ONC-14005003) Funding Statement: This work was supported by Mega-Projects of Scientific Research for the 12th Five-Year Plan (2013ZX09303302); the National Natural Science Foundation of China (81570122, 81570122); Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161303); the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (QD2015005); the National Key Research and Development Program (Grant 2016YFC0902800); Fine classification and standardized treatment of children with acute leukemia of multi center clinical research (14411950600) Shanghai Municipal Science and Technology Commission; Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development; U.S. National Institutes of Health Grants CA21765, CA36401, and U01 GM115279, and American Lebanese Syrian Associated Charities (ALSAC); The Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Swedish Research Council, the Knut and Alice Wallenberg Foundation, and Governmental (ALF) Funding of Clinical Research within the Swedish National Health Service; NMRC/CSA/0053/2013; VIVA Foundation for Children with Cancer; Goh Foundation; Children's Cancer Foundation, Singapore Totalisator Board; the Samuel Waxman Cancer Research Foundation; the Center for HPC at Shanghai Jiao Tong University Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Ruijin hospital ethics committe gave approval for SIH trial.

MicroRNA‐124 Negatively Regulates Nuclear Receptor Nur77

The nuclear receptor Nur77 is commonly upregulated in adult cancers and has oncogenic functions. Nur77 is an immediate‐early response gene that acts as a transcription factor to promote proliferation and protect the cell from apoptosis. Conversely, Nur77 can translocate to the mitochondria and induce apoptosis upon treatment with various cytotoxic agents. Because Nur77 is upregulated in cancer and may have a role in cancer progression, it is of interest to understand the mechanism controlling its expression. MicroRNAs (miRNAs) are responsible for inhibiting translation of their target genes by binding to the 3′UTR and either degrading the mRNA or preventing it from being translated into protein, thereby making these non‐coding endogenous RNAs vital regulators of every cellular process. Several miRNAs have been predicted to target Nur77; however, strong evidence showing the regulation of Nur77 by any miRNA is lacking. In this study we used a luciferase reporter assay containing the 3′UTR of Nur77 to screen 296 miRNAs and found that miR‐124, the most abundant miRNA in the brain with a role in promoting neuronal differentiation, caused the greatest reduction in luciferase activity. Interestingly, we discovered an inverse relationship in Daoy medulloblastoma cells and undifferentiated granule neuron precursors (GNPs) where Nur77 is upregulated and miR‐124 is downregulated. Further elevating Nur77 levels in Daoy cells by exogenous expression increased proliferation and viability, but knocking down Nur77 via siRNA resulted in the opposite phenotype. Importantly, exogenous expression of miR‐124 reduced Nur77 expression, cell viability, proliferation, and tumor spheroid size in 3D culture. In all, we have discovered miR‐124 to be downregulated in instances of medulloblastoma in which Nur77 is upregulated, resulting in a proliferative state that abets cancer progression. This study provides evidence for increasing miR‐124 expression as a potential therapy for cancers with elevated levels of Nur77.Support or Funding InformationThis work was supported by the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's Research Hospital, and the National Institutes of Health [Grants RO1GM086415, RO1GM110034, &amp; P30‐CA21765].

Semisynthetic Natural Products for Antibacterial Drug Discovery

The pressing need for new treatments for multi‐drug resistant bacterial infections including MDR and XDR tuberculosis has led us to re‐explore the use of natural product antibiotics not normally used to treat target infections, especially those with unique mechanisms of action. Being the product of combinatorial evolution natural products have many advantages over purely synthetic antibacterial leads, but also some notable disadvantages often in regard to spectrum and human metabolism. This presentation will go over our efforts to advance under‐explored antibiotic scaffolds with semi‐synthetic approaches to tackle these disadvantages. Using spectinomycin as an example, synthetic modifications have been applied to overcome native bacterial efflux leading to potent leads with better than predicted in vivo efficacy in complex infection models. These observations have led us to examine active uptake and slow‐growing pathogen targeting hypotheses that are hard wired into many natural products, which are very hard to replicate by direct purely synthetic de novo antibacterial drug design.Support or Funding InformationThis work was supported by research grant R01AI090810 by the National Institutes of Health and the American Lebanese Syrian Associated Charities (ALSAC)

Pregnancy after chemotherapy in male and female survivors of childhood cancer treated between 1970 and 1999: a report from the Childhood Cancer Survivor Study cohort

The effect of many contemporary chemotherapeutic drugs on pregnancy and livebirth is not well established. We aimed to establish the effects of these drugs on pregnancy in male and female survivors of childhood cancer not exposed to pelvic or cranial radiotherapy. We used data from a subset of the Childhood Cancer Survivor Study cohort, which followed 5-year survivors of the most common types of childhood cancer who were diagnosed before age 21 years and treated at 27 institutions in the USA and Canada between 1970 and 1999. We extracted doses of 14 alkylating and similar DNA interstrand crosslinking drugs from medical records. We used sex-specific Cox models to establish the independent effects of each drug and the cumulative cyclophosphamide equivalent dose of all drugs in relation to pregnancies and livebirths occurring between ages 15 years and 44 years. We included siblings of survivors as a comparison group. We included 10 938 survivors and 3949 siblings. After a median follow-up of 8 years (IQR 4-12) from cohort entry or at age 15 years, whichever was later, 4149 (38%) survivors reported having or siring a pregnancy, of whom 3453 (83%) individuals reported at least one livebirth. After a median follow-up of 10 years (IQR 6-15), 2445 (62%) siblings reported having or siring a pregnancy, of whom 2201 (90%) individuals reported at least one livebirth. In multivariable analysis, survivors had a decreased likelihood of siring or having a pregnancy versus siblings (male survivors: hazard ratio [HR] 0·63, 95% CI 0·58-0·68; p<0·0001; female survivors: 0·87, 0·81-0·94; p<0·0001) or of having a livebirth (male survivors: 0·63, 0·58-0·69; p<0·0001; female survivors: 0·82, 0·76-0·89; p<0·0001). In male survivors, reduced likelihood of pregnancy was associated with upper tertile doses of cyclophosphamide (HR 0·60, 95% CI 0·51-0·71; p<0·0001), ifosfamide (0·42, 0·23-0·79; p=0·0069), procarbazine (0·30, 0·20-0·46; p<0·0001) and cisplatin (0·56, 0·39-0·82; p=0·0023). Cyclophosphamide equivalent dose in male survivors was significantly associated with a decreased likelihood of siring a pregnancy (per 5000 mg/m(2) increments: HR 0·82, 95% CI 0·79-0·86; p<0·0001). However, in female survivors, only busulfan (<450 mg/m(2) HR 0·22, 95% CI 0·06-0·79; p=0·020; ≥450 mg/m(2) 0·14, 0·03-0·55; p=0·0051) and doses of lomustine equal to or greater than 411 mg/m(2) (0·41, 0·17-0·98; p=0·046) were significantly associated with reduced pregnancy; cyclophosphamide equivalent dose was associated with risk only at the highest doses in analyses categorised by quartile (upper quartile vs no exposure: HR 0·85, 95% CI 0·74-0·98; p=0·023). Results for livebirth were similar to those for pregnancy. Greater doses of contemporary alkylating drugs and cisplatin were associated with a decreased likelihood of siring a pregnancy in male survivors of childhood cancer. However, our findings should provide reassurance to most female survivors treated with chemotherapy without radiotherapy to the pelvis or brain, given that chemotherapy-specific effects on pregnancy were generally few. Nevertheless, consideration of fertility preservation before cancer treatment remains important to maximise the reproductive potential of all adolescents newly diagnosed with cancer. National Cancer Institute, National Institutes of Health, and the American Lebanese-Syrian Associated Charities.

A Role For a Multi-Drug Resistance Protein (MRP4/ABCC4) In Pediatric Acute Myeloid Leukemia (AML)

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy comprising 15-20% of childhood acute leukemias and remains a challenging disease with cure rates of only 60% using conventional therapy. However, unlike ALL de novo AML is characterized by a very low burden of genomic alterations suggesting other genetic and/or epigenetic alterations occur within hematopoietic stem cells (HSC) or progenitors to affect their normal self-renewal, proliferation and/or differentiation in this disease. Multi-drug Resistance Protein 4 (MRP4/ABCC4) is the fourth member of the ABCC subfamily of ABC transporters (de novoAML, suggesting MRP4 has a role in myeloid leukemia. Moreover, gene expression profiling from 155 pediatric AML patients showed that MRP4 was differentially expressed among AML subtypes with M7 having the highest expression.To characterize the function of MRP4 in normal hematopoiesis and in the etiology of AML, we use a murine Mrp4 knockout model. Mrp4KO mice had a similar number of BM progenitors as WT mice did. However, hematopoietic stem cells of Mrp4KO appeared to have an intrinsic defect because short- and long-term repopulation was impaired in a competitive repopulation (CR) mouse model.To examine the role of MRP4 in AML etiology, we generated an NMYC-overexpressing AML model because NMYC overexpression is a frequent event in pediatric AML and is highly oncogenic in mouse myeloid cells (Kawagoe et al., 2007). In addition, NMYC expression was frequently associated with elevated MRP4 expression, a recently reported target of NMYC (Porro et al., 2010; Porro et al., 2011). We showed that NMYC-overexpressing WT BM cell had an increased progenitor population, which was absent in Mrp4KO cells. Because overexpression of MRP4 in lineage-depleted bone marrow cells increased the CD34+ population which correlated with increased expression of progenitor marker such as cKit, we tested whether exogenous MRP4 could rescue the progenitor population in Mrp4KO. We demonstrated that restoring MRP4 in Mrp4KO cells indeed rescued the progenitor populations. Furthermore, Mrp4-deficiency delayed in vivo NMYC-dependent leukemogenesis in an AML mouse model, possibly by decreasing the proliferation/survival of the tumor cells. Current studies are further elucidating pathways whereby MRP4 provides a biological advantage.In summary, Mrp4 is important for both normal and deregulated progenitor functions, especially during leukemogenesis when progenitor populations greatly increase. It is likely MRP4 alters survival and/or proliferation of progenitors. This work will further our understanding of ABC transporters in the normal biology and pathology of hematopoietic cells and has the potential to facilitate identification of new therapeutic targets for AML treatment.This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC).† Shang Li Lian, PhD shangli.lian@stjude.org, 901-595-2782, address: Chili's Care Center, Rm I5206, Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105. Disclosures:No relevant conflicts of interest to declare.

Obesity increases the severity of secondary bacterial coinfection following influenza virus infection

Obesity is a significant risk factor for influenza virus infection. However, little is known about obesity and secondary bacterial coinfections; the major cause of morbidity and mortality. Obesity increases inflammation, alters immune response and decreases wound repair in the lungs of obese mice infected with influenza virus, all risk factors for bacterial colonization. Therefore, we questioned the effect of obesity on secondary bacterial pneumonia. Infection of lean and obese mice with a nonlethal dose of influenza virus followed by a nonlethal dose of Streptococcus pneumoniae resulted in 100% mortality in obese mice by day 3 post bacterial challenge with no loss of lean controls. No differences were observed in viral titers between groups; however, lung bacterial titers were significantly increased in obese animals. Histological analysis revealed widespread damage to the lungs of obese mice. Obesity increases the severity and mortality from influenza‐associated complications and secondary bacterial pneumonia may be greatly enhanced in an increasingly obese human population. Further study is necessary to determine the mechanisms of increased susceptibility to influenza, secondary bacterial infection and possible therapeutic strategies in this expanding risk group.Grant Funding Source: NIH NIAID contract number HHSN266200700005C and the American Lebanese Syrian Associated Charities (ALSAC)

Fat flu: the obese host in influenza virus evolution

During the 2009 influenza pandemic, obesity was identified as an independent risk factor for increased disease severity and mortality. These epidemiological findings have been confirmed using obese mouse models. Previous studies in mice have demonstrated that nutritional status can affect virulence of RNA viruses including influenza. The global epidemic of obesity combined with increased disease severity in obese subjects prompted us to question whether obesity could influence influenza virus evolution. To determine if the obese lung microenvironment could differentially affect viral adaptation, A/California/04/2009 influenza virus was passaged five times in either lean or diet‐induced obese (DIO), C57Bl/6 male mice. Lean and DIO mice were then infected with parental and passaged viruses and monitored for morbidity and mortality. Interestingly, virus passaged in DIO mice induced greater morbidity, pathology and mortality in both lean and DIO animals compared to lean‐passaged virus. Studies are underway to define the molecular determinants associated with the enhanced pathogenicity; however, the obese virus retained increased pathogenicity following egg passage suggesting genetic changes to the virus itself. These studies suggest that the expanding obese population may not only be more susceptible to disease, but may differentially regulate viral evolution towards more virulent viral strains.Grant Funding Source: NIH NIAID contract number HHSN266200700005C and the American Lebanese Syrian Associated Charities (ALSAC)

The Abcc4 Knockout Reveals An Important Role for Abcc4 in Platelet Aggregation

Abstract 1141Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides.We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels.Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10.This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures:No relevant conflicts of interest to declare.

Combination chemotherapy with clofarabine, cyclophosphamide, and etoposide in children with refractory or relapsed haematological malignancies

Combination chemotherapy with clofarabine, cyclophosphamide, and etoposide in children with refractory or relapsed haematological malignancies

273 Erythropoietin plus danazole, prednisone, B12 and folate in refractory cytopenia with multilineage dysplasia (RCMD). Monocentric prospective study

About a fifth of children with acute T-lymphoblastic leukaemia (T-ALL) succumb to the disease, suggesting an unrecognised biological heterogeneity that might contribute to drug resistance. We postulated that T-ALL originating from early T-cell precursors (ETPs), a recently defined subset of thymocytes that retain stem-cell-like features, would respond poorly to lymphoid-cell-directed therapy. We studied leukaemic cells, collected at diagnosis, to identify cases with ETP features and determine their clinical outcome.Leukaemic cells from 239 patients with T-ALL enrolled at St Jude Children's Research Hospital (n=139) and in the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-2000 (n=100) were assessed by gene-expression profiling, flow cytometry, and single nucleotide polymorphism array analysis. Probabilities of survival and treatment failure were calculated for subgroups considered to have ETP-ALL or typical T-ALL.30 patients (12·6%) had leukaemic lymphoblasts with an ETP-related gene-expression signature or its associated distinctive immunophenotype (CD1a−, CD8−, CD5weak with stem-cell or myeloid markers). Cases of ETP-ALL showed increased genomic instability, in terms of number and size of gene lesions, compared with those with typical T-ALL. Patients with this form of leukaemia had high risk of remission failure or haematological relapse (72% [95% CI 40–100] at 10 years vs 10% [4–16] at 10 years for patients with typical T-ALL treated at St Jude Children's Research Hospital; and 57% [25–89] at 2 years vs 14% [6–22] at 2 years for patients treated in the AIEOP trial).ETP-ALL is a distinct, previously unrecognised, pathobiological entity that confers a poor prognosis with use of standard intensive chemotherapy. Its early recognition, by use of the gene expression and immunophenotypic criteria outlined here, is essential for the development of an effective clinical management strategy.US National Cancer Institute, Cariplo Foundation, Citta della Speranza Foundation, Italian Association for Cancer Research (AIRC), Italian Ministry for University and Research, and American Lebanese Syrian Associated Charities (ALSAC).

Minimal residual disease-directed therapy for childhood acute myeloid leukaemia: results of the AML02 multicentre trial

We sought to improve outcome in patients with childhood acute myeloid leukaemia (AML) by applying risk-directed therapy that was based on genetic abnormalities of the leukaemic cells and measurements of minimal residual disease (MRD) done by flow cytometry during treatment. From Oct 13, 2002, to June 19, 2008, 232 patients with de-novo AML (n=206), therapy-related or myelodysplasia-related AML (n=12), or mixed-lineage leukaemia (n=14) were enrolled at eight centres. 230 patients were assigned by block, non-blinded randomisation, stratified by cytogenetic or morphological subtype, to high-dose (18 g/m(2), n=113) or low-dose (2 g/m(2), n=117) cytarabine given with daunorubicin and etoposide (ADE; induction 1). The primary aim of the study was to compare the incidence of MRD positivity of the high-dose group and the low-dose group at day 22 of induction 1. Induction 2 consisted of ADE with or without gemtuzumab ozogamicin (GO anti-CD33 monoclonal antibody); consolidation therapy included three additional courses of chemotherapy or haematopoietic stem-cell transplantation (HSCT). Levels of MRD were used to allocate GO and to determine the timing of induction 2. Both MRD and genetic abnormalities at diagnosis were used to determine the final risk classification. Low-risk patients (n=68) received five courses of chemotherapy, whereas high-risk patients (n=79), and standard-risk patients (n=69) with matched sibling donors, were eligible for HSCT (done for 48 high-risk and eight standard-risk patients). All 230 randomised patients were analysed for the primary endpoint. Other analyses were limited to the 216 patients with AML, excluding those with mixed-lineage leukaemia. This trial is closed to accrual and is registered with ClinicalTrials.gov, number NCT00136084. Complete remission was achieved in 80% (173 of 216 patients) after induction 1 and 94% (203 of 216) after induction 2. Induction failures included two deaths from toxic effects and ten cases of resistant leukaemia. The introduction of high-dose versus low-dose cytarabine did not significantly lower the rate of MRD-positivity after induction 1 (34%vs 42%, p=0.17). The 6-month cumulative incidence of grade 3 or higher infection was 79.3% (SE 4.0) for patients in the high-dose group and 75.5% (4.2) for the low-dose group. 3-year event-free survival and overall survival were 63.0% (SE 4.1) and 71.1% (3.8), respectively. 80% (155 of 193) of patients achieved MRD of less than 0.1% after induction 2, and the cumulative incidence of relapse for this group was 17% (SE 3). MRD of 1% or higher after induction 1 was the only significant independent adverse prognostic factor for both event-free (hazard ratio 2.41, 95% CI 1.36-4.26; p=0.003) and overall survival (2.11, 1.09-4.11; p=0.028). Our findings suggest that the use of targeted chemotherapy and HSCT, in the context of a comprehensive risk-stratification strategy based on genetic features and MRD findings, can improve outcome in patients with childhood AML. National Institutes of Health and American Lebanese Syrian Associated Charities (ALSAC).

Conformal radiotherapy after surgery for paediatric ependymoma: a prospective study

Therapy for ependymoma includes aggressive surgical intervention and radiotherapy administered by use of methods that keep the risk of side-effects to a minimum. We extended this treatment approach to include children under the age of 3 years with the aim of improving tumour control. Between July 11, 1997, and Nov 18, 2007, 153 paediatric patients (median age 2.9 years [range 0.9-22.9 months]) with localised ependymoma were treated. 85 patients had anaplastic ependymoma; the tumours of 122 were located in the infratentorial region, and 35 had received previous chemotherapy. Patients received conformal radiotherapy after definitive surgery (125 patients had undergone gross total, 17 near total, and 11 subtotal resection). Doses of 59.4 Gy (n=131) or 54.0 Gy (n=22) were prescribed to a 10 mm margin around the target volume. Disease control, patterns of failure, and complications were recorded for patients followed over 10 years. Overall survival, event-free survival (EFS), cumulative incidence of local recurrences, and cumulative incidence of distant recurrences were assessed. Variables considered included tumour grade, tumour location, ethnic origin, sex, age when undergoing conformal radiotherapy, total radiotherapy dose, number of surgical procedures, surgery extent, and preradiotherapy chemotherapy. After a median follow-up of 5.3 years (range 0.4-10.4), 23 patients had died and tumour progression noted in 36, including local (n=14), distant (n=15), and combined failure (n=7). 7-year local control, EFS, and overall survival were 87.3% (95% CI 77.5-97.1), 69.1% (56.9-81.3), and 81.0% (71.0-91.0), respectively. The cumulative incidences of local and distant failure were 16.3% (9.6-23.0) and 11.5% (5.9-17.1), respectively. In the 107 patients treated with immediate postoperative conformal radiotherapy (without delay or chemotherapy), 7-year local control, EFS, and overall survival were 88.7% (77.9-99.5), 76.9% (63.4-90.4), and 85.0% (74.2-95.8), respectively; the cumulative incidence of local and distant failure were 12.6% (5.1-20.1), and 8.6% (2.8-14.3), respectively. The incidence of secondary malignant brain tumour at 7 years was 2.3% (0-5.6) and brainstem necrosis 1.6% (0-4.0). Overall survival was affected by tumour grade (anaplastic vs differentiated: HR 3.98 [95% CI 1.51-10.48]; p=0.0052), extent of resection (gross total vs near total or subtotal: 0.16 [0.07-0.37]; p<0.0001), and ethnic origin (non-white vs white: 3.0 [1.21-7.44]; p=0.018). EFS was affected by tumour grade (anaplastic vs differentiated: 2.52 [1.2705.01]; p=0.008), extent of resection (gross total vs near total or subtotal: 0.20 [0.11-0.39]; p<0.0001]), and sex (male vs female: 2.19 [1.03-4.66]; p=0.042). Local failure was affected by extent of resection (gross total vs near total or subtotal: 0.16 [0.067-0.38]; p<0.0001), sex (male vs female: 3.85 [1.10-13.52]; p=0.035), and age (<3 years vs >/=3 years: 3.25 [1.30-8.16]; p=0.012). Distant recurrence was only affected by tumour grade (anaplastic vs differentiated: 4.1 [1.2-14.0]; p=0.017). Treatment of ependymoma should include surgery with the aim of gross-total resection and conformal, high-dose, postoperative irradiation. Future trials might consider treatment stratification based on sex and age.