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Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1− B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non–risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

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. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Hyperleukocytosis is observed in 5% to 20% of patients with newly diagnosed acute myeloid leukemia (AML) and is associated with an increased risk of early complications and mortality. While being used frequently in patients with AML and hyperleukocytosis, the clinical utility of leukapheresis has not been conclusive. Low-dose chemotherapy has also been used recently as a cytoreduction method in these patients, but the data are limited. Objectives: To describe and compare the clinical and laboratory characteristics, early adverse events, and outcomes of children with newly diagnosed AML and hyperleukocytosis according to cytoreductive methods; leukapheresis, low dose chemotherapy (cytarabine), or no intervention. Methods: We studied patients with newly diagnosed AML treated on three multi-institutional St. Jude protocols, AML97, AML02, and AML08, between 1997 and 2017. Hyperleukocytosis was defined as white blood cell (WBC) counts of 100 x 10 9/L or higher at diagnosis. The decision of cytoreductive treatment was made as the discretion of the treating physician. Leukoreduction was used in the AML97 and AML02 studies, and cytarabine (100mg/m 2/dose every 12 hours) was the first choice for AML08 study. We reviewed baseline clinical characteristics and laboratory data (complete blood cell counts [CBC], chemistries, coagulation) and adverse effects (grade 3 or higher on neurologic, renal, respiratory, and hemorrhagic complications based on Common Terminology Criteria for Adverse Events) from diagnosis to day 14 of protocol-based chemotherapy. Cairo-Bishop criteria was used for laboratory/clinical tumor lysis syndrome. The time from the first CBC to administration of protocol-based chemotherapy was calculated. Results: A total of 49 patients were identified: 8 patients in AML97, 19 in AML02, and 22 in AML08) (Table). The age at diagnosis was 10.8 years with a median initial WBC count of 157.6 x 10 9/L; CNS (CNS 2, 3 or traumatic lumbar puncture with blasts) was seen in 29 (59.2%) cases. FAB M4 or M5 subtype was found in 30 patients (61.2%), 11q23 abnormalities in 15 (30.6%) and inv(16) in 8 (16.3%). In regards to leukoreduction method, 16 patients received leukapheresis (14 patients in AML97/02 and 2 in AML08), 18 cytarabine (all in AML08) and 1 hydroxyurea (in AML08); 14 did not receive leukoreduction (13 patients in AML97/02 and 1 in AML08). Leukapheresis was used more often in patients with higher diagnostic WBC counts (218.7 x 10 9/L) than those treated with cytarabine (152.9 x 10 9/L) or without intervention (127.3 x 10 9/L) (P Conclusion: Low-dose cytarabine treatment appears to be a safe and effective mean of cytoreduction for patients with AML and hyperleukocytosis. Further studies are needed to determine if this approach is preferable among patients treated with contemporary treatment. Figure 1 Figure 1. Disclosures Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee.

CG Mullighan and T Haferlach: are co-senior authors Introduction: Recent genomic sequencing studies have advanced our understanding of the pathogenesis of myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and improved classification of specific subgroups. Unfortunately, these studies have mostly analyzed specific subtypes and/or used targeted DNA-sequencing, thus limiting discovery of novel mutational patterns and gene expression clusters. Here, we performed an integrated genome-wide mutational/transcriptomic analysis of a large cohort of adult AML and MDS samples to accurately define subtypes of diagnostic, prognostic and therapeutic relevance. Methods: We performed unbiased whole genome (WGS) and transcriptome sequencing (RNA-seq) of 1,304 adult individuals (598 AML and 706 MDS; Fig. 1A), incorporating analysis of somatic and presumed germline sequence mutations, chimeric fusions and structural complex variations. Transcriptomic gene expression data were processed by a rigorous bootstrap procedure to define gene expression subgroups in an unsupervised manner. Associations between genetic variants, gene expression groups and outcome were examined. Results: Genomic/transcriptome sequencing confirmed diagnosis according to WHO 2016 of AML with recurrent genetic abnormalities in 10.9% of cases. These cases had a distinct gene expression profile (Fig. 1A), good prognosis (Fig. 1B) and a combination of mutations in the following genes: KIT, ZBTB7A, ASXL2, RAD21, CSF3R and DNM2 in RUNX1-RUNXT1 leukemia; FLT3, DDX54, WT1 and CALR in PML-RARA promyelocytic leukemia; KIT and BCORL1 in CBFB-rearranged leukemia. In addition, 9% of cases showed rearrangements of KMT2A, with known (e.g. MLLT3) and non-canonical partners (e.g. ACACA, and NCBP1) and poor outcome. Although common targets of mutations have been previously described for myeloid malignancies, the heterogeneity and complexity of mutational patterns, their expression signature and outcome here described are novel. Gene expression analysis identified groups of AML and/or MDS lacking recurrent cytogenetic abnormalities (87%). The spectrum of the most frequently mutated genes (>10 cases) and associated gene expression subtypes is summarized in Figure 1A. TET2 (more frequent in MDS than AML, p=0.0011) and DNMT3A (more frequent in AML than MDS, p Conclusions: the integration of mutational and expression data from a large cohort of adult pan myeloid leukemia cases enabled the definition of subtypes and constellations of mutations and have prognostic significance that transcends prior gene panel-based classification schema. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction: Venetoclax (VEN) is a potent and selective inhibitor of BCL-2. It has demonstrated activity in adults with acute myeloid leukemia (AML) in combination with low-dose cytarabine ( Methods: VEN was given daily for 28 days and chemotherapy was started on day 8, or earlier in cases of disease progression. Dosages of VEN and chemotherapy were escalated separately using a rolling-six design. Response to the VEN window was determined using total peripheral blood blast count or, in a subset of patients, bone marrow blast percentage as determined by flow-cytometry based minimal residual disease (MRD). Pharmacokinetics of VEN both as a single agent and in combination with chemotherapy were measured in a subset of patients treated at the maximum tolerated combination dose of VEN. Response to therapy was determined using bone marrow evaluation between days 29-50 of therapy. All patients received antimicrobial prophylaxis, typically with levofloxacin and micafungin. Azoles were prohibited during VEN administration. Results: Thirty-six patients aged 2-22 years were enrolled. All dose levels were tolerated. The recommended phase 2 dose of VEN in combination with high-dose cytarabine with or without idarubicin was 360 mg/m2 daily (max 600mg). One patient (treated with 240mg/m2 of VEN and intermediate-dose cytarabine) experienced a dose-limiting toxicity due to delayed count recovery and one patient died of recurrent colitis (at dose level 3) which first occurred during prior therapy and was deemed unrelated to VEN. Patients experienced a mean of 2.4 non-hematologic grade 3+ toxicities, with infections including culture-negative febrile neutropenia, sepsis, and colitis the most common toxicities. Patient-reported quality of life was similar at study entry and at the completion of cycle 1 and was within normal limits in most patients. Among 22 patients receiving VEN with high-dose cytarabine ± idarubicin, 14 (64%) achieved a complete response (CR) or complete response with incomplete count recovery (CRi). Response to the VEN window was associated with end of cycle 1 response; 13/15 (87%) patients with a greater than 80% reduction in peripheral blasts achieved a partial response (PR; 3) or CR/CRi (10). In contrast, only 8/15 (53%) patients with less than an 80% reduction in blasts responded to combination therapy (7 CR, 1 PR). Window response to VEN was associated with BH3 dependence as determined by cytochrome c release from leukemia cells in a flow-cytometry based assay. 5 of the 6 (83%) patients with primary BCL-2 dependence had a >80% reduction in blasts; the single patient with a poor response had a change to BCL-XL dependence at the end of cycle 1. In contrast, 4 of the 6 (66%) patients with primary BCL-XL dependence had a 90% reduction had secondary BCL-2 dependence. None of the 4 patients with FLT3-ITD or point mutations responded as determined by end of cycle 1 marrow. VEN levels were consistent across weights and ages and similar to levels seen in adults. The levels were similar between patients who did and did not receive idarubicin (mean AUC24 38.3 ± 32.7 vs. 47.3 ± 22.9 μg•h/mL). Conclusion: VEN combined with high-dose cytarabine or high-dose cytarabine and idarubicin was well tolerated and effective in children and young adults with relapsed or refractory AML. Enrollment continues to refine estimates of response rate. VEN window response is associated with BH3 dependence and end of cycle 1 response rates. Targeting BCL-XL or FLT3 may improve response to combination therapy. Table Disclosures Karol: Abbvie: Other: Unrelated to this study, St. Jude has received a charitable contribution from AbbVie, Inc. The charitable contribution is not being used for clinical or research activities, including any activities related to this study.. Alexander:AbbVie: Other: travel funding. Salem:AbbVie: Employment, Other: Stock/stock options. Palenski:Abbvie: Employment, Other: Stock/ stock options. Opferman:AbbVie: Research Funding. Rubnitz:AbbVie: Research Funding.

Introduction: Cytarabine, daunorubicin and etoposide (ADE) are commonly used for remission and intensification of pediatric acute myeloid leukemia (AML). However, development of drug resistance is a major cause of treatment failure. In this study, we performed a comprehensive evaluation of expression levels of genes of pharmacological significance (pharmacokinetic /pharmacodynamic) to ADE and derived a drug response score predictive of treatment outcomes in pediatric AML patients. Methods: This study included 163 cases (median age=8.79 year, range= (0.013-21.1)) with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression and clinical data available. We used a penalized LASSO regression algorithm (glmnet R-package) to fit a cox regression model on diagnostic leukemic cell gene expression levels of 66 genes of pharmacological significance to ADE. We performed 1000 bootstraps of LASSO regression with event free survival (EFS) as the outcome variable and the five genes represented in at least 95% of the models were included to build an ADE-Response Score (ADE-RS) equation. Patients were classified into low or high score groups using recursive portioning implemented in Rpart-R package and evaluated for association with minimal residual disease after induction I (MRD1), EFS and overall survival (OS). ADE response score equation was further validated using RNA-Seq gene-expression data obtained from diagnostic samples of 432 pediatric AML patients enrolled in Children's Oncology Group (COG) AAML0531 and AAML03P1 treatment protocols. Results: After applying LASSO regression, we defined the equation: ADE-RS = (0.128 x DCTD) - (0.0993 x TOP2A) + (0.212 x ABCC1) - (0.113 x MPO) - (0.126 x CBR1) to develop ADE-response score (ADE-RS), followed by classifying patients into low (60%; 98 patients) or high (40%; 65 patients) score groups. Patients in the high ADE-RS group had significantly worse EFS (HR=4.07(2.43-6.84), P < 0.0001; Figure 1A) and OS (HR= 4.54(2.42-8.49), P In a multivariable cox-regression models that included pLSC6-ADE response score groups, MRD1 status, risk groups, WBC at diagnosis and age in AML02 cohort, high pLSC6-ADE score group was found significantly associated with poor EFS (HR=6.02(2.71-13.2), P Discussion: In this study, we defined a pharmacological response score focused on key genes of PK/PD significance to ADE. We further integrated LSC score with the ADE response score to improve our ability to predict treatment outcome in AML patients across different clinical trials. ADE-RS was composed of five genes: DCTD, which is a deaminase, involved in ara-C inactivation; CBR1, a carbonyl reductase involved in inactivation of daunorubicin (DNR); MPO, myeloperoxidase, an etoposide activator; ABCC1, an efflux transporter of DNR and etoposide; and TOP2A, DNA topoisomerase II alpha, which is a target for DNR and etoposide. Integrated pLSC6 and ADE-RS has a potential to predict treatment outcomes using diagnostic gene expression levels and accordingly develop treatment strategies to improve treatment outcome. Figure 1 Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding.

Acute myeloid leukemia (AML) comprises a heterogeneous group of malignancies that are linked by the presence of blasts displaying morphologic and immunophenotypic features of myeloid cell differentiation. With the development of genome-wide gene expression profiling (GEP), array-base comparative genomic hybridization methodologies, and next generation sequencing technologies, the field has gained a greater understanding of the molecular features of this malignancy. Several pathologic lesions have been found to have prognostic implications contributing to a continuous refinement of risk stratification in the context of modern therapy. Recently, the Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative molecularly characterized 993 pediatric AML cases including 197 specimens that underwent comprehensive whole genome sequencing. Of these, 94 carried one of three oncogenic fusions known to be strong drivers of leukemogenesis: RUNX1-RUNX1T1, CBFB-MYH11 and KMT2A rearrangements (KMT2Ar). Among all the alterations detected only ten occurred in more than 5% of subjects, all of which had been previously described. This suggested that low-frequency molecular subsets may exist that require larger cohorts to fully elucidate. To address this limitation, we selected 122 pediatric AML specimens that lacked RUNX1-RUNX1T1, CBFB-MYH11 and KMT2Ar by clinical testing for whole genome (WGS), exome (WES) and RNA (RNAseq) sequencing to enrich for cases that carry low-frequency events. GEP coupled with somatic mutation calls and outcome data were utilized to identify distinct molecular subtypes with prognostic implications. Structural variations, copy number alterations, single nucleotide variations and indels were determined by our established pipelines, as well as an evaluation for regulatory rearrangements driving oncogene overexpression through enhancer hijacking. In addition to known AML somatic mutations and rearrangements in genes such as CEBPA, GATA2, NPM1, WT1, FLT3, NRAS, KRAS, ETV6, Cohesin, NUP98 and KAT6A, we identified rare novel events in known oncogenic drivers. These include a GATA2-ITD as well as the repositioning of a distal MYC enhancer to ectopically activate either the MECOM or BCL11B loci. Interestingly, several AML cases carrying loss of function mutations in polycomb repressive complex 2 (PRC2) genes were found to resemble an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) GEP by gene set enrichment analysis. ETP-ALL exhibits aberrant expression of stem cell and myeloid markers and has been shown to have a GEP consistent with transformation of a stem cell progenitor. Further, mixed phenotype acute leukemias (MPAL) with T and myeloid lineage characteristics have been previously suggested to be in this spectrum of immature leukemias. We therefore hypothesized that these PRC2-mutated AML cases represented the myeloid end of this continuum. To provide global transcriptional context to these ETP-like AMLs and evaluate a comprehensive cohort encompassing a range of pediatric myeloid malignancies, we integrated results from previously published AML, MPAL, acute megakaryoblastic Leukemia (AMKL), and ETP-ALL datasets that had RNAseq and either WES or WGS available for a total of 436 cases. t-SNE visualization using a 381 gene list derived from the top 100 most variably expressed transcripts within each cohort revealed a clear molecular classifier identifying groups that had consistent mutational compositions and disease outcomes but were agnostic of immunophenotype. This approach allowed the distinction of 63 ETP-like cases comprising a mixture of AML, MPAL, and ETP-ALL leukemias which fell into two subgroups distinguished by FLT3-ITD and PRC2 alterations. Irrespective of treatment approach, FLT3-ITD positive ETP-like leukemias enjoyed a favorable outcome whereas those with PRC2 mutations had a poor prognosis. Our data support a refined classification of pediatric myeloid malignancies based on molecular determinants that can be used for risk stratification in therapeutic trials. Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding. Reinhardt:Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Novartis: Other: Participation in Advisory Boards; Roche: Research Funding. Locatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zwaan:Roche: Consultancy; Servier: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Incyte: Consultancy.

Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Acute myeloid leukemia (AML) treatment response remains poorly understood. Although multiple studies have focused on understanding the transcriptomic and epigenetic landscape of AML, a genome-wide analysis of SNPs in pediatric AML has not yet been investigated in depth. Thus, we sought to identify genetic variants predictive of AML response, relapse, and survival in pediatric AML patients. For this study, we generated genome-wide SNP data patients (n=160) treated on the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) using Infinium Omni 2.5M Exome Beadchip. Standard GWAS QC procedure was followed in order to remove SNPs with call rate < 95%, monomorphic SNPs, SNPs with MAF Acknowledgments: We are thankful for funding from NIH R01-CA139246 and ALSAC. Disclosures No relevant conflicts of interest to declare.

Introduction: Resistance and relapse remain major obstacles in the treatment of acute myeloid leukemia (AML). Pre-existence and persistence of drug resistant leukemia stem cells (LSCs) is considered one of the major causes of relapse. A previous study (Ng et al., 2016) reported a prognostic signature of 17 genes (LSC17 score) differentially expressed in LSC+ compared to LSC- cell fractions that predicted outcome in patients with AML thereby classifying patients into high and low risk groups. The goal of this study is to determine the validity of LSC17 score in pediatric AML patients and to enhance its clinical utility by exploring a new score with limited number of stem cell genes. Methods: 150 pediatric patients with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression data and clinical data were included in the study. Since only 14 of the 17 genes were represented on the Affymetrix U133A gene chip we tested the validity of the LSC14 score using the previously defined equation (Ng et al, 2016) with multiple clinical endpoints such as minimum residual disease (MRD), event free survival (EFS) and overall survival (OS). To reduce the model complexity, we applied a penalized regression algorithm called the least absolute shrinkage and selection operator (LASSO) implemented in the glmnet R-package using event free survival (EFS) as an outcome variable. Score of the new equation, which included three genes, was designated as pediatric-LSC3 (pLSC3). pLSC3 was tested in the AML02 cohort for association of high or low pLSC3 (based on the median value) with clinical endpoints mentioned above. pLSC3 score equation was validated using publically available gene-expression data from 117 pediatric relapse enriched AML patient cohort enrolled in Children's Oncology Group (COG) protocol (TARGET database). COX-proportional hazard models and Log rank test were used for survival data analysis. Results: AML02 cohort: Patients with high LSC14 scores (greater than median), had significantly worse MRD (p In a multivariate COX regression model, pLSC3 score groups was the only significant covariate (table 1). It explained 13.1% of variability in EFS and 11.6% of variability in OS, while other prognostic factors such as risk groups, FLT3 status, treatment arm and age collectively explained 15.1 and 12.1 % of variability. Discussion: In summary, our results show validity of a previously defined LSC14 score in a pediatric AML population from the multicenter AML02 clinical trial. To enhance the clinical utility, score equation was further simplified and the final score (pLSC3) was derived from three genes: DNMT3B, which encodes for DNA methyltransferase; CD34, an important cell surface marker for early-undifferentiated LSCs; and GPR56, a G protein coupled receptor of significance in AML. Given that there is need to refine classification of a highly heterogeneous group of patients with standard risk AML, we show that differentiating standard risk patients based on pLSC3 score should be considered in the future. We show the relevance of pLSC3 in two independent cohorts, opening up opportunities to improve treatment outcomes of pediatric patients with AML. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a clinically challenging disease with high interpatient variability in response to chemotherapy. Despite continuing advances in treatment options, current 5-year survival rates for pediatric AML are suboptimal at ~60%. The heterogeneous nature of AML contributes significantly to the variability in treatment response and survival outcomes. Several known genetic lesions and cytogenetic features contribute to disease progression. However, our understanding of how molecular mechanisms contribute to variation in treatment outcomes is still limited. Previous metabolomics studies have successfully identified significant metabolic alterations in hematological malignancies, but very few metabolomics studies have been conducted for the pediatric AML patient population. In this study, we used global and targeted metabolomics to identify differential metabolite abundance associated with chemosensitivity and treatment outcomes in pediatric AML patients. Serum metabolomics profiles were generated with serum samples obtained at diagnosis from patients treated in the multicenter AML02 study (n=94, NCT00136084). Clinical outcomes tested for association included half-maximal inhibitory concentration (IC50) of cytarabine, minimal residual disease (MRD), relapse free survival (RFS), and overall survival (OS). Global metabolomics profiling was performed using liquid chromatography/mass spectrometry (LC/MS). Targeted metabolomics profiling was generated for a select group of organic acids and acylcarnitines. The organic acid panel included eight metabolites related to the tricarboxylic acid cycle and glycolysis. The acylcarnitine panel featured 20 varieties of acylcarnitines detectable in human serum. Statistical analyses were performed using MetaboAnalyst and various R packages. A total of 3205 features were detected in the global metabolome, with 124 known metabolites and 3081 unknown features. All metabolites were used for association analysis, while annotated metabolites were used in pathway analyses. Association analysis of clinical endpoints vs. metabolome identified 10 known metabolites significantly associated with IC50 values, 17 associated with MRD, 7 associated with RFS, and 7 associated with OS (p Pathway enrichment analysis identified 11 metabolic pathways showing significant association with IC50 values and 12 pathways associated with MRD (FDR This study identifies several metabolites and metabolic pathways significantly associated with chemosensitivity and clinical endpoints in pediatric AML patients. These results help expand on previously conducted AML pilot studies, and metabolomics studies on other cancer types, to further clarify the metabolic differences associated with interpatient variability in chemotherapy response for AML patients. Continued metabolic profiling of AML patient populations can help establish targetable pathways that can be used to improve treatment efficiency for AML. In addition, in vitro functional modeling to validate results of the metabolomics study are currently underway. Disclosures No relevant conflicts of interest to declare.

Introduction B progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor. Methods To identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing. Results Using integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in "double/triple hit" lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion. We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL. Conclusions These results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. Disclosures McKay: ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

To characterize the biology and optimal therapy of acute mixed-lineage leukemia in children, we reviewed the pathologic and clinical features, including response to therapy, of 35 patients with mixed-lineage leukemia. The majority of cases (91%) had blasts cells that simultaneously expressed either T-lineage plus myeloid markers (T/myeloid, n = 20) or B-lineage plus myeloid markers (B/myeloid, n = 12). Overall survival rates for the B/myeloid and T/myeloid subgroups were not significantly different from each other or from the rate for acute myeloid leukemia (AML) but were inferior to the outcome in children with acute lymphoblastic leukemia (ALL). Patients who failed to achieve complete remission with AML-directed therapy could often be induced with a regimen of prednisone, vincristine, and L-asparaginase. Analysis of gene-expression patterns identified a subset of biphenotypic leukemias that did not cluster with T-cell ALL, B-progenitor ALL, or AML. We propose that treatment for biphenotypic leukemia begin with one course of AML-type induction therapy, with a provision for a switch to lymphoid-type induction therapy with a glucocorticoid, vincristine, and L-asparaginase if the patient responds poorly. We also suggest that hematopoietic stem cell transplantation is often not required for cure of these patients.

Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARα], t(8;21)[AML1-ETO], inv16 [CBFβ-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation.

Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.

Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES). At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio >0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11). We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML. In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. Disclosures Inaba: Arog: Research Funding.

Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

Cytarabine is the backbone of modern AML chemotherapy. However, extensive inter-patient variation in treatment response, development of resistance, and severe toxicity remain as major hurdles to effective cytarabine chemotherapy. Although the genes involved in cytarabine’s activation/inactivation and transport are well defined, we still lack in understanding of the PD genes of relevance. So far several efforts have been made to identify cytarabine response genes have some of these utilized transcriptional profiling to identify gene expression signatures that differentiate sensitive and resistant cell lines; others have utilized gene expression profiles from diagnostic specimens, to identify transcripts predictive of therapeutic outcome. To identify genes that show a biologically meaningful pattern of association with multiple pharmacologic and clinical variables, we have recently developed and applied the innovative PROMISE (PRojection Onto the Most Interesting Statistical Evidence) statistical analysis procedure. In our preliminary study using PROMISE, we identified transcriptional signatures (consisting of 60 genes) that were predictive of therapeutically beneficial (n=46 genes) or detrimental (n=14 genes) patterns of association with multiple clinical parameters. We then used this information, combined with genes reported in literature as being important to ara-C pharmacology and/or identified in relevant genome-wide screens, to define a high-priority list of 300 candidate genes, which we subjected to a multi-phased, high throughput siRNA/ cytarabine modifier screening campaign in human AML cells. The first screening phase used 3 individual siRNAs to target each of the 300 selected candidate genes in THP1 cells, tested alongside standard transfection controls in 384 well plates. Post siRNA transfection, cells were treated with different concentrations of cytarabine (0µM; 0.1µM-IC10; 0.8µM-IC50; and 10µM-IC90) followed by multi-parametric nuclear morphometry assays using automated microscopy to document the individual and combined phenotypic effects of siRNA gene silencing and cytarabine on cell growth and proliferation. Genes were classified as suppressors if their siRNA knockdown inhibited the drug response (i.e increased resistance) and enhancers if the knockdown enhanced the drug response (i.e increased drug sensitivity). This analysis yielded 72 candidate cytarabine modifier genes, which were then subjected to further technical and biological validation tests to document experimental reproducibility, siRNA targeting specificity and cell line specificity of observed phenotypes in THP1 (0, 0.8 and 10µM) and Kasumi cells (0, 0.05 and 1µM for IC50 and IC90 concentrations for Kasumi). Targeting specificity was assessed by comparing microscopy phenotypes to target knock-down as measured by RT-qPCR for candidate hit siRNAs and matched non-cleaving “C911” control siRNAs. Our results yielded clear validation of ara-C modifier effects for several genes with known functions in ara-C-relevant pathways, including DNA damage repair response factors and deoxynucleotide metabolism/catabolism enzymes. As such, these results confirm the patho-physiological relevance of our screening campaign in strengthening the predictive value of markers previously identified by transcriptional profiling analyses from AML patient samples. They also confirm our screen design’s potential for identifying novel modulators of cytarabine -induced phenotypes. Indeed, even from this relatively small screen of only 300 high-priority genes, our validation data supported ara-C suppressor effects from knocking down APOBEC3G, ANXA5, (DCK: involved in activation of cytarabine), KPNA2, NCF1, PLEKHM1, REPIN1 and XRCC1, as well as enhancer effects from knocking down CFLAR, CHEK1, DERA, EIF4F2, EXO1, GNB5, GRPEL1, IER3IP1, IQGAP1, MAPK11, NFKB2, NKX2, NUBP1, RPL31. These genes represent valuable biomarker candidates whose expression levels in AML patients may predict ara-C responsiveness. The identified enhancer genes also represent novel therapeutic target candidates for developing more effective ara-C combination treatments for AML. Disclosures Echeverri: Cenix BioScience Inc: Employment, Equity Ownership. Korn:Cenix BioScience Inc: Employment. Kochar:Cenix BioScience Inc: Employment.

Abstract 2470 Acute myeloid leukemia (AML) is the second most common form of childhood leukemia and has the one of the worst prognoses of all major childhood cancers. Development of resistance to the nucleoside analog cytarabine (ara-C) is one of the biggest challenges that clinicians face. Studies of diagnostic gene expression profiles have attempted to determine the molecular mechanisms that underlie drug resistance. In the present study, we evaluated cytarabine-induced gene expression changes in n=24 AML patients by using the Affymetrix U133A array. We measured gene expression in bone marrow samples obtained at diagnosis and 24 hours after initiation of cytarabine infusion. We identified genes with significant change in expression post cytarabine treatment (after adjusting for arm) and explored the association of cytarabine induced expression changes with minimal residual disease after one course (MRD), and event-free survival. We found significant change in the expression level of 11 genes at p≤0.001 (6 genes down-regulated and 5 up-regulated) and 146 with p≤0.01 (89 genes down-regulated and 57 up-regulated). Pathway analysis tool (IPA) identified Cancer, Tumor Morphology, Cellular Movement (score 32) and Cell Morphology, Nervous System Development and Function, Tissue Morphology (score 29) as top networks. The most significant pathways influenced by ara-C treatment included NRF2-mediated oxidative stress signaling, Hif 1a signaling, Aryl hydrocarbon receptor and acute phase signaling. The top genes with ∼ 3 fold increase in expression included VAX2, PMP22, GMFG, PTP4A1, TIMP3, CHMP2A, GPR56, NFIB and KLRB1, and top genes with ∼ 3 fold decrease in expression included CER1, PREB, CDK13 and CDK10, UVRAG, FGF17, ZNF768, YLPM1, SIK3, PRICKLE3, MAP3K7. We also identified 34 genes whose expression change was significantly associated (p Overall our results provide insight into cytarabine-induced gene expression differences in AML patients and the association of these expression differences with clinical outcome. Understanding the pathways that are altered in patients once treatment is initiated can provide opportunities to enhance our understanding of drug action and further improve treatment outcome by adjusting the combination therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 1073 Background: Aberrant receptor tyrosine kinase (RTK) signaling arising from genetic abnormalities, such as FLT3-internal tandem duplications (FLT3-ITD), is an important mechanism in the development and growth of acute myeloid leukemia (AML) and is often associated with a poor outcome. Hence, inhibition of RTK signaling is an attractive novel treatment option, particularly for disease that is resistant to conventional chemotherapy. We evaluated the clinical activity of the multikinase inhibitor sorafenib in children with de novo FLT3-ITD–positive AML or relapsed/refractory AML. Methods: Fourteen patients were treated. Six patients with newly diagnosed FLT3- ITD–positive AML (aged 9–16 years; median, 12 years) received 2 cycles of remission induction therapy and then started sorafenib (200 mg/m2 twice daily for 20 days) the day after completing induction II (low-dose cytarabine, daunorubicin, and etoposide). Nine patients (aged 6–17 years; median, 9 years) with relapsed AML (including one treated on the above regimen) received sorafenib alone (2 dose levels; 200 and 150 mg/m2) twice daily for the first week of therapy, concurrently with clofarabine and cytarabine on days 8–12, and then alone from days 13 to 28. Sorafenib pharmacokinetics were analyzed at steady-state on day 8 of sorafenib in patients with newly diagnosed AML and on day 7 in patients with relapsed AML. In patients with relapsed AML, the effect of sorafenib on signaling pathways in AML cells was assessed by flow cytometry. Results: All 6 newly diagnosed patients, including 2 whose AML was refractory to induction I, achieved a complete remission (CR) after induction II; 5 had negative minimal residual disease (MRD; 50% decrease in blast percentage and/or bone marrow cellularity after 1 week of sorafenib. After concurrent sorafenib and chemotherapy, 5 of the 9 patients with relapsed AML achieved CR (2 had negative MRD) and 2 achieved a partial remission (PR; 5%-25% AML cells in bone marrow); all 4 patients with FLT3-ITD had a CR or PR. After sorafenib treatment, 6 patients underwent HSCT while 2 with FLT3-ITD who could not receive HSCT were treated with single-agent sorafenib and have maintained CR for up to 8 months. Hand-foot skin reaction (HFSR) or rash occurred in all patients and improved with cessation of sorafenib. Dose-limiting toxicity (DLT, grade 3 HFSR and/or rash) was observed in 3 of the 6 patients with relapsed AML treated with 200 mg/m2 of sorafenib; no DLT was observed at 150 mg/m2. The effect of sorafenib on downstream RTK signaling was tested in the leukemic cells of 4 patients: in most samples, phosphorylation of S6 ribosomal protein and 4E-BP1 was inhibited. The mean (± SD) steady-state concentration (Css) of sorafenib was 3.3 ± 1.2 mg/L in the newly diagnosed group and 6.5 ± 3.6 mg/L (200 mg/m2) and 7.3 ± 3.6 mg/L (150 mg/m2) in those with relapsed AML. In both groups, the mean conversion of sorafenib to sorafenib N-oxide was 27%-35% (approximately 3 times greater than previously reported), and mean sorafenib N-oxide Css was 1.0–3.2 mg/L (2.1-6.7 μM). In a 442-kinase screen, the inhibitory profiles of sorafenib N-oxide and sorafenib were similar, and FLT3-ITD phosphorylation was potently inhibited by both forms (sorafenib N-oxide Kd = 0.070 μM; sorafenib Kd = 0.094 μM). Sorafenib N-oxide inhibited the growth of an AML cell line with FLT3-ITD (IC50 = 0.026 μM) and 4 AML cell lines with wild-type FLT3 (IC50 = 3.9–13.3 μM) at approximately half the potency of sorafenib. Conclusion: In children with de novo FLT3-ITD and relapsed/refractory AML, sorafenib given alone or with chemotherapy induced dramatic responses and inhibited aberrant RTK signaling in leukemic cells. Sorafenib and its active metabolite (sorafenib N-oxide) likely contribute to both efficacy and toxicity. These results warrant the incorporation of sorafenib into future pediatric AML trials. Disclosures: Inaba: Bayer/Onyx: Research Funding. Off Label Use: Sorafenib and clofarabine: both used for treatment of pediatric acute myeloid leukemia.

Abstract 849 Core-binding-factor (CBF) acute myeloid leukemia (AML) defined by the presence of t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is associated with favorable outcome. However, about 30–40% of patients are not cured by current treatment approaches. Secondary genetic changes are believed to cause clinical heterogeneity. To identify new secondary genetic lesions, we performed high-resolution, genome-wide analysis of copy number aberrations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 300 adult and pediatric CBF AMLs; t(8;21), n=157 (adult, n=114; pediatric, n=43); and inv(16), n=143 (adult, n=104; pediatric, n=39). Germline control DNA from remission bone marrow or peripheral blood was available for paired analysis in 175 patients. In addition, for 42 patients matched relapse samples were analyzed. Data were processed using reference alignment, dChipSNP and circular binary segmentation. Paired analysis revealed a median of 1.28 somatic CNAs per case [t(8;21): 1.14, range: 0–5; inv(16): 1.45, range: 0–9], with deletions more common than gains [t(8;21): 0.94 losses/case vs. 0.2 gains/case; inv(16): 0.95 vs. 0.5]. Recurrent deletions were detected at chromosomal bands 7q36.1 (n=23), 9q21.13 (n=15), 11p13 (n=7), 17q11.2 (n=6), 10q24.32 (n=2), and at the chromosomal breakpoints of t(8;21) and inv(16) on 8q21.3 (n=9), 21q22 (n=16), 16p13.11 (n=28), and 16q22.11 (n=21). Deletions at 7q, 11p and 17q were validated using FISH analysis. Minimally deleted regions (MDR) less than 1.5 Mb were identified at 7q36.1 (647 Kb, 4 genes), 9q21.13 (1125 Kb, 9 genes), 11p13 (130 Kb, 1 gene), and 17q11.2 (902 Kb, 11 genes), with each region containing a putative tumor suppressor (e.g., MLL3 on 7q, FRMD3 on 9q, WT1 on 11p, and NF1 on 17q). Sequence analysis of MLL3 in 23 cases with del(7q), 1 case with 7q CN-LOH, and 23 randomly selected cases identified a MLL3 truncating mutation leading to a premature stop codon in a case that lacked a 7q alteration. The del(11p13) contained only WT1 and primarily affected inv(16)-cases (5 of 7). Sequence analysis of WT1 in four cases with del(11p) revealed an additional frame shift mutation in the remaining allele in one case. Sequence analysis of WT1 in an additional 103 inv(16)-containing cases revealed mutations in 10 (9%) of the cases. The MDR at 17q11.2 was exclusively identified in inv(16)-containing cases (n=6) and included the tumor supressor NF1. Sequence analysis of all coding exons in NF1 in 4 additional inv(16)-containing AMLs revealed no additional mutation. Recurrent gains were identified at 22q11.21-q13.33 (n=20; 32 Mb), 8q24.21 (n=14; 138 Kb), 13q21.1-q34 (n=6; 14 Mb), and 11q25 (n=5; 368 Kb). The smallest gains were identified at 8q24.21 and 11q25, both containing only a single non-coding RNA gene (CCDC26 and LOC283177, respectively). Somatic CN-LOH were uncommon and only found at 1p36.33-p12 (n=2), 4q (n=2), and 19p (n=2). In a comparative analysis of paired diagnostic and relapse samples, novel CNAs at the time of relapse were identified at 3q13.31 (n=5), 5q (n=2), 17p (n=2), and 17q (n=2). The MDR at 3q was only 46 kb in size and contains a single transcript that has been connected to LSAMP, a putative tumor suppressor located 404 Kb upstream of the deletion. In summary, our data provide a comprehensive profiling of copy number alterations in pediatric and adult CBF AML. These data demonstrate a very low number of CNAs, with no significant differences noted between pediatric and adult cases. Interestingly, a number of novel recurrent secondary genetic alterations are identified. Exploring the biological role of these lesions in leukemogenesis and drug resistance should provide important insights into the CBF leukemias. Disclosures: No relevant conflicts of interest to declare.

Abstract 16 With improvements in risk-directed therapy and supportive care, cure rates for children with acute lymphoblastic leukemia are now approaching 90%. In contrast, little progress has been made for children with acute myeloid leukemia (AML). We designed a multicenter treatment protocol for children with newly diagnosed AML that used sequential minimal residual disease (MRD) assessment for risk assignment. Patients (n = 230) with AML were randomized to receive daunorubicin and etoposide plus high-dose cytarabine (HD-ADE, n = 113) or low-dose cytarabine (LD-ADE, n = 117) as Induction I. Induction II consisted of LD-ADE with or without gemtuzumab ozogamicin (GO), depending on the level of MRD after Induction I. The remaining treatment was based on presenting features and MRD levels, and included hematopoietic stem cell transplantation (HSCT) for high-risk patients. The protocol underwent 2 treatment changes. First, LD-ADE + GO was initially given only to patients with MRD more than 25%, but was later extended to patients with MRD more than 1% because the combination was found to be safe and effective at reducing MRD. Of the 30 patients who received LD-ADE + GO, 27 (90%) had a reduction in MRD and 13 (43%) became MRD negative, with a median overall MRD reduction of 98%. CNS-directed therapy initially consisted of intrathecal (IT) cytarabine alone. However, because 3 of the first 33 patients enrolled on AML02 developed CNS relapse, subsequent patients were treated with triple IT therapy consisting of methotrexate, hydrocortisone, and cytarabine. The cumulative incidence of CNS relapse decreased from 9% to 2% after this treatment change (P = 0.004). Of the 212 (92%) evaluable patients, 80% became MRD negative and 95% achieved clinical complete remission (CR) after 2 courses of therapy; overall 3-year event-free survival (EFS) and overall survival (OS) rates were 60% ± 5% and 70% ± 4%, respectively. Patients on the HD-ADE and LD-ADE treatment arms had similar MRD-negative rates after 1 (66% vs. 58%, P = 0.17) and 2 courses of therapy, as well as similar CR, EFS (59% vs. 61%, P = 0.79), and OS rates (68% vs. 73%, P = 0.41). Multipredictor logistic regression modeling indicated that the odds of having detectable MRD after 1 course of therapy was significantly lower for patients with core binding factor (CBF) leukemia [t(8;21) or inv(16)] (P In conclusion, risk-adapted therapy, incorporation of GO, and excellent supportive care contributed to a CR rate of 95%, low mortality, and an overall 3-year OS rate of 70% for children with AML enrolled on AML02. Although this is substantially higher than rates reported in our previous studies, it should be possible to further increase cure rates by additional refinements in risk assignment, as well as by introducing novel agents that can bypass drug resistance. To this end, our current AML08 protocol has incorporated newly discovered molecular predictors, highly sensitive MRD tests, and natural killer cell therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 593 Cytarabine (ara-C) is one of the most widely used chemotherapeutic agents in the treatment of acute myeloid leukemia (AML). Inter-patient variability in clinical response following ara-C treatment has been correlated with leukemic cell concentration of ara-C triphosphate (ara-CTP), the active metabolite of the drug. NT5C2 is an inactivating enzyme in the activation pathway of ara-C, and catalyzes conversion of ara-C monophosphate to ara-C. We identified genetic variations in NT5C2 and investigated the association of NT5C2 genetic variations with its mRNA expression and with intracellular ara-CTP accumulation. The coding exons of NT5C2 were resequenced using genomic DNA from EBV-transformed B-lymphoblastoid HapMap cell lines derived from 90 CEPH (representing 30 trios with European ancestry) and 90 YRI (30 trios with African ancestry) sample. Thirty-nine genetic variants (one insertion-deletion and 38 SNPs), including three nonsynonymous SNPs (Thr3Ala, Lys47Arg, Gln136Arg), were identified. The strongly linked SNPs (R2 > 0.8) in CEPH and YRI were grouped into ten distinct groups. NT5C2 mRNA expression levels were determined in all samples and was significantly associated with ethnicity (p = 8.5 × 10-6), with subjects of African ancestry having significantly higher NT5C2 mRNA expression as compared to the subjects with European ancestry (Figure A). Several NT5C2 SNPs were associated with its mRNA expression in both the ethnic groups. To determine the clinical implication of NT5C2 SNPs, we investigated selected germline NT5C2 SNPs in pediatric AML patients treated on the St Jude AML 97 protocol (n=55). Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hrs after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher when given as short daily infusion, as compared to continuous infusion (0.56 ±0.5 vs. 0.33± 0.31 nmol ara-CTP/2×107leukemic cells; p = 0.014). The inter-patient variability for blast ara-CTP concentration was 40-fold in the short infusion arm and 101-fold in continuous infusion arm. Karyotypes, gender, age, and ethnicity had no significant influence on the leukemic ara-CTP levels. NT5C2 Group 1 SNPs were found to be significantly associated with intracellular ara-CTP levels in leukemic blasts from ara-C-treated Pediatric AML Patients (Figure B). These results suggest that genetic variation in NT5C2 could influence its activity and/or expression and may predict the variability observed in intracellular levels of the ara-C active metabolite, ara-CTP, which in turn can influence treatment response. Disclosures: No relevant conflicts of interest to declare.

Abstract 114 To identify genes that influence responses to cytarabine (ara-C) treatment, we explored the association of gene expression in leukemic cells at diagnosis with multiple pharmacological and clinical end-points in children with acute myeloid leukemia (AML) treated with ara-C on the St. Jude AML97 clinical trial. We applied a novel statistical procedure, PRojection Onto the Most Interesting Statistical Evidence (PROMISE; PR), to identify genes with expression levels associated with clinical and pharmacological endpoints. To do this, we first defined the following values of the clinical and pharmacological variables as “therapeutically beneficial” :higher leukemic cell ara-C triphosphate levels, lower DNA synthesis values on days 1 and 2 of treatment relative to baseline, decreases in leukocyte counts on day 2 of treatment, improved response and decreased risk of relapse, death, or second malignancy. We considered a gene to show a therapeutically beneficial pattern of association if its expression was positively correlated with ara-CTP levels, negatively correlated with DNA synthesis levels, negatively correlated with decrease in leukocyte counts on day 2, positively correlated with better treatment response, and negativelycorrelated with the risk of relapse or death. A gene showed a therapeutically detrimental pattern of association if its expression had the opposite correlations with the clinical and pharmacological variables. We performed five variable (PR5 using early pharmacologically interesting phenotype measures) or seven variable (PR7 all the above indicated phenotypes) PROMISE analyses. PR5 identified 275 beneficial probe sets and 69 detrimental probe sets (p ≤ 0.005). PR7 analysis identified 112 beneficial probe sets and 115 detrimental probe sets (p ≤ 0.005). To confirm these results, we performed a PROMISE for a cohort of patients treated with ara-C and other agents on the AML02 protocol. Gene expression in leukemic cells at diagnosis was analyzed for a beneficial or detrimental pattern of association with three phenotypes (PR-3); diagnostic blast ara-C cytotoxicity, minimal residual disease (MRD) and event-free survival (EFS). Eighty-one probe sets identified by PR5 or PR7 analyses in the initial cohort were confirmed in the PR-3 analysis of AML02 data. Genes identified in the present study may serve as predictive markers of response and candidates for future drug development. Disclosures: No relevant conflicts of interest to declare.

Cytarabine (ara-C) is one of the most effective agents for the treatment of AML; however development of drug resistance remains a major limitation. To identify genes that impact ara-C therapy, we explored the association of leukemic blast gene expression (measured using the Affymetrix U133A microarray) with intracellular ara-CTP pharmacokinetic and pharmacodynamic variables (DNA synthesis relative to baseline measured in leukemia cells, peripheral blast clearance) measured in leukemia cells from 42 patients treated under St. Jude AML97 protocol. In this study, patients received either a daily short infusion of ara-C or a continuous infusion of ara-C in combination with cladribine (CdA). Probe sets with mRNA expression levels (at diagnosis) that positively correlate with leukemic blast ara-CTP levels at day 1 (after ara-C alone) and day 2 (after ara-C and CdA), negatively correlate with extent of inhibition in days 1 and day 2 DNA synthesis relative to baseline, and negatively correlate with absolute blast count at 48 hours relative to baseline were considered to exhibit a therapeutically beneficial pattern of association. Probe sets with correlations opposite to those mentioned above were considered to exhibit a therapeutically detrimental pattern of association. An association pattern test found statistically significant evidence (p ≤ 0.005, FDR ≈ 0.30) that the expression of 304 probe sets associates with a beneficial pattern of association and of 61 probe sets associates with a detrimental pattern of association. These results include 34 cell cycle genes with beneficial association patterns and 2 cell cycle genes with detrimental association patterns (PPP1R13B and CUL4B). Also, 2 tumor necrosis factor genes (TNFRSF25 and FASLG) showed significant association with beneficial association patterns. The significant probe sets included RAS oncogene pathway genes, RAP2A and RAB9A with a detrimental pattern of association and the RAS-like gene RIT2 with a beneficial pattern of association. It has been recently shown that AML patients with mutations in RAS genebenefit from high ara-C doses1. The erythroblastic leukemia viral oncogene homologs EGFR and THRB demonstrated a statistically significant beneficial pattern of association. The T-cell leukemia homeobox gene also showed a statistically significant beneficial pattern of association. We further analyzed these 365 genes using Ingenuity Pathway Analysis tools and identified 16 networks with scores greater than 11 (p value < 10−11). In addition to the apoptotic genes, genes in the metabolic pathway of ara-C as well as few previously identified genes were also found to be associated with the pharmacokinetic and/or pharmacodynamic measures of ara-C in circulating blasts. In conclusion, our study has identified molecular signatures (in leukemic blasts at diagnosis) associated with the ara-CTP pharmacokinetics and pharmacodynamics. Validation of these signatures will help in better understanding of ara-C response as well as the mechanism underlying drug resistance. We are concentrating on these candidate genes and pathways for functional validation studies.

To define the complement of genetic lesions in pediatric AML, we performed high resolution genome-wide analysis on bone marrow blasts from 111 cases using Affymetrix 100K and 500K SNP microarrays and genomic resequencing. Cases included t(8;21) (N=20), inv(16) (N=16), t(15;17) (N=7), MLL rearranged (N=16), FAB-M7 (N=9), miscellaneous cytogenetic abnormalities (N=24) and normal karyotype (N=19). Germline DNA was available for 67 of the cases. The mean number of somatic copy number abnormalities (CNA) was 2.38 (range 0–45) with 1.32 gains (range 0–41) and 1.06 losses (range 0–12). Focal CNAs were detected at the breakpoints of known chromosomal translocations, most commonly in inv(16) cases (7/16) and t(8;21) cases (4/20). Based on this observation, we examined focal CNAs that affected the 5’ or 3’ regions of genes for their possible involvement in cryptic translocations. CNAs involving NUP98 and MLL led to the identification of two cases with t(5;11)[ NUP98-NSD1 ], and two with t(6;11)[ MLL-MLLT4 ] not evident on cytogenetic analysis. RT-PCR for these fusions identified two additional NUP98-NSD1 cases. Identification of focal CNAs are thus useful in identifying clinically significant translocations in AML. Other recurring somatic CNAs were uncommon. Twelve regions containing a single gene were identified, including amplification of EBF , PRDM5 , CCDC26* , GDF10 , ABCC4*, and deletion of FAM20C* , TUSC1 , GPC5 , A2BP1 , INSR, BCOR* ( * involved in N>2 cases). The most frequent abnormality was amplification of CCDC26 at 8q24.21 ( N =15; focal in 4 cases, broad in 11) which encodes a putative mediator of retinoic acid receptor signaling. The focal amplifications are predicted to abolish expression of normal transcripts. Seventeen additional recurring CNAs involving 2 to 25 genes were identified, many of which contain known or putative tumor suppressor genes or oncogenes, including deletions of regions at 7q36, 9p21 ( CDKN2A/B ), 11p14-p12 ( WT1 , WIT1 ), and amplification at 19p13 ( JUNB, LYL1 ). These regions showed significant correlation between copy number and local gene expression in an integrated analysis of CNA and Affymetrix U133A gene expression data. Copy-neutral LOH was uncommon and recurrent only with LOH of chromosome 13 in 3 cases. Sequence analysis has been completed for several genes involved in recurring CNAs ( CCDC26, TUSC1, FBXW7 ) and for genes known to be mutated in AML ( N/KRAS, PTPN11 , BRAF, SOS1 , FLT3 , CEPBA , NPM1, AML1, CKIT , GATA1 ). No mutations were identified in CCDC26 , TUSC1 , FBXW7 , BRAF , or SOS1 . Marked differences in the combination of CNA and sequence mutation were identified across the different genetic subtypes of AML. FAB M7 cases had the highest frequency of CNA (mean 9.3 lesions/case) but had sequence mutations limited to GATA1 . Mutations of FLT3 , CEPBA and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, where as RAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 30% of the cases with recurring translocations had no other sequence or numerical abnormalities. These data demonstrated that, in contrast to pediatric ALL, AML is characterized by relatively few recurring CNAs, and that spectrum of CNA and sequence mutation is significantly associated with disease subtype.

Cytarabine (Ara-C) is a prodrug requiring conversion to its 5′-triphosphate (ara-CTP) form for antileukemic effect. Alteration of deoxycytidine kinase (DCK), a rate-limiting enzyme in this conversion, is associated with ara-C resistance. We sought to identify and determine the functional and clinical significance of DCK genetic variants. Sixty-four genetic polymorphisms were identified, including 3 coding changes (Ile24Val, Ala119Gly, and Pro122Ser amino acid substitutions), in subjects of European or African ancestry. The activity of recombinant DCK-24Val, DCK-119Gly, and DCK-122Ser was 85±5%, 66±3%, and 43±4%, respectively, that of wild-type (WT) DCK. These 3 DCK mutants also demonstrated altered substrate kinetics. Lymphoblast cell lines derived from subjects heterozygous for these coding changes had DCK activity significantly lower than that of homozygous-WT lymphoblasts (176.7±15 vs. 340.8±19 pmol CdA-MP/min/mg protein; p=0.0004). Moreover, for a 3′UTR 35708T>C SNP, the C allele was significantly associated with lower DCK mRNA expression in lymphoblast cell lines (p=0.02). To determine the clinical implications of DCK SNPs, we screened pediatric AML patients treated on the St Jude AML 97 protocol (n=55) for the 3 coding variants and the 3UTR SNP. Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hours after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher in patients given a short daily infusion than in those given a continuous infusion (0.56±0.5 vs. 0.33±0.31 nmol ara-CTP/2x107 leukemia cells; p = 0.014). The inter-patient variability of blast ara-CTP concentration was 40-fold (0.06–2.43 nmol/2x107 cells) when ara-C was administered as short infusion and 101-fold (0.012–1.22 nmol/2x107 cells) when given as a continuous infusion. Karyotype, sex, age, and ethnicity were not significantly associated with leukemia-cell ara-CTP level. An Ala119Gly or Pro122Ser non-synonymous polymorphism was present in 1 patient each; the patient heterozygous for Ala119Gly (continuous infusion arm) had the lowest intracellular concentration of ara-CTP (0.012 nmol/2x107 cells; Figure). Both of these patients had relapses, despite the presence of favorable cytogenetic abnormalities [inv16 and t(8;21)]. The 3′UTR 35708 T>C SNP was significantly associated with lower intracellular ara-CTP concentration in patients receiving ara-C as a continuous infusion (35708T allele 0.5±0.3 vs. 35708C allele 0.22±0.17 nmol/2x107 cells; p= 0.04; Figure). These results suggest that genetic polymorphism of DCK influences its activity and expression and may underlie interpatient variability in intracellular ara-CTP concentration, which in turn can influence treatment response. Influence of DCK SNPs on leukemic blast ara-CTP levels in AML patients receiving ara C-as continuous infusion Influence of DCK SNPs on leukemic blast ara-CTP levels in AML patients receiving ara C-as continuous infusion

To obtain a comprehensive registry of oncogenic lesions in pediatric acute lymphoblastic leukemia (ALL), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of heterozygosity (LOH) in leukemic blasts and matched remission samples from 250 ALLs. We studied B-progenitor ALLs with high hyperdiploidy, n=39; ETV6-RUNX1, n=47; MLL rearranged, n=11; TCF3-PBX1, n=17; BCR-ABL1, n=9; low hyperdiploidy, n=23; hypodiploidy, n=10; unclassified cases, n=42; and 50 T-lineage ALLs. Four arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 loci at a mean inter-marker distance of 4.8 kb. Data was analyzed using dChipSNP and a modified array normalization algorithm using only SNPs from regions known to be diploid by routine karyotyping. Copy number abnormalities were confirmed by FISH and genomic quantitative PCR. Complementary methylation analysis and sequencing of candidate genes was performed. 84% of B-ALLs and 96% of T-ALLs had at least one region of somatic deletion, and excluding cases with high hyperdiploidy, 68% of B-ALLs and 50% of T-ALLs had at least one region of somatic amplification. These included previously identified abnormalities including chromosomal duplications in hyperdiploid B-ALL; 1q duplication in TCF3-PBX1 ALL; and deletions of 9p21 (harboring CDKN2A/B, 70% of T-ALLs, 34.5% of B-ALL), 12p13 (ETV6; 25.5% of B-ALLs, 10% of T-ALLs), 6q16 (22 cases) and 11q (15 cases). The resolution of the arrays enabled precise mapping of the minimal regions of deletion at 9p21 to CDKN2A, and at 12p13 to ETV6. Combined LOH and copy number analysis identified several patterns of 9p21 abnormality: focal hemizygous deletion with corresponding LOH; focal homozygous and flanking hemizygous loss with corresponding LOH, indicating two focal deletional events; and focal homozygous loss with LOH of all of 9p or chromosome 9, indicating loss of the normal 9 or 9p and duplication of the chromosome or chromosomal arm containing the focal deletion. Copy-neutral LOH without any focal deletion in the affected region was uncommon. Deletions involving other genes with potential roles in leukemogenesis were identified including BTG1 (17 cases), ERG (10), FHIT (14), mir-16/-15a (19), MYB (5), NF1 (9), the glucocorticoid receptor NR3C1 (11), PTEN (4), and RB1 (20). Furthermore, deletions, translocations, amplifications, and point mutations of genes that regulate B-cell development and differentiation, including EBF, PAX5, Ikaros and Aiolos, were identified in 40% of B-ALL. For each of the listed genes, cases were identified that contained focal deletions limited to the specific gene. Overall, 73.6% B-ALL and 88% T-ALLs harbored deletions of one of the common lesions listed above, with 48% of B-ALLs and 48.5% of T-ALLs having multiple common lesions. The average number of deletions per case was 3.8 and 5.7 for B and T-lineage ALLs respectively. By contrast, when hyperdiploid cases were excluded, it was rare to find more than 2 regions of amplification in a single case, and the majority of cases contained no amplifications. These findings show the power of high-resolution copy number analysis for the identification of new genetic lesions in cancer, and demonstrate that multiple genetic abnormalities contribute to leukemogenesis in pediatric ALL.

Patients with acute myeloid leukemia (AML) who receive intensive chemotherapy regimens containing high-dose cytarabine are at high risk for bacterial sepsis due to prolonged neutropenia and severe mucositis. To determine if the use of prophylactic antibiotics during periods of neutropenia reduced bacterial infections and viridans streptococcal infections (VSI) in particular, we reviewed the charts of 66 AML patients who were treated on our front line protocol (AML02) from October 2002 to June 2006. During this time, several consecutive prophylactic antibiotic strategies were implemented for use after intensive myelosuppressive therapy. All patients also received prophylactic antifungal therapy. Overall, patients who received any prophylactic antibiotic regimen had an 86% (95% CI: 66–95%) reduction in VSI (P

To identify a comprehensive registry of oncogenic lesions in pediatric acute myeloblastic leukemia (AML), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of-heterozygosity (LOH) in leukemic blasts from 112 cases of pediatric AML and corresponding remission samples from 63 of these cases. The analyzed cases included t(8;21)[AML1-ETO] (n=20), inv(16)[CBFβ-MYH11](n=16), t(15;17)[PML-RARα] (n=7), MLL rearranged (n=17), FAB M7 (n=9) and normal cytogenetics or miscellaneous cytogenetic abnormalities (n=43). Four SNP arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 markers at a mean inter-marker distance of 4.8 kb. Combined data were analyzed using dChipSNP and a modified array normalization algorithm using only those SNPs from regions known to be diploid by routine karyotyping. These analyses not only detected known whole or partial chromosomal losses or gains, but also detected numerous copy number abnormalities that were not evident by conventional cytogenetics. Somatic DNA copy number abnormalities were identified in 102 (91.1%) cases. The mean number of lesions per patient was 3.2 (range 1–12), with a mean of 2.13 deletions/whole chromosome losses and 1.02 amplifications/whole chromosome gains per patient. Deletions were detected in the leukemic blasts from over 90% of patients, whereas amplifications were only seen in the leukemic blasts from 54% of patients. The vast majority of deletions were focal (

Chromosomal aberrations are a hallmark of acute lymphoblastic leukemia (ALL) but alone fail to induce leukemia. To identify cooperating oncogenic lesions, we performed genome-wide analysis of leukemic blasts from 242 pediatric ALL patients using high-resolution 100K and 250K Affymetrix single nucleotide polymorphism arrays, and genomic DNA sequencing. Remarkably, our analyses identified deletion, amplification, point mutation and structural rearrangement in genes encoding regulators of B lymphocyte development in over 40% of B-progenitor ALL. PAX5, which encodes a transcription factor critical for B cell commitment and differentiation, was the most frequent target of somatic mutation, being altered in 31.7% of cases. The most frequent PAX5 mutations were copy number alterations with mono-allelic loss in 53 cases, biallelic loss in 3 cases, and an internal amplification in 1 case. PAX5 deletions and the amplification were confirmed by FISH and/or RT-PCR, and were present in over 90% of blasts. Twenty-five of the mono-allelic deletions were confined to PAX5, with the majority deleting only a subset of PAX5 exons. Thus, of the 57 cases with PAX5 copy number changes, the majority had haploinsufficiency of PAX5 (n=30), or generated hypomorphic alleles that produce proteins that lack the DNA-binding domain (n=20) or the transcriptional activation domain (n=7). Four cases contained cryptic PAX5 translocations: PAX5-ETV6 (n=2), PAX5-FOXP1 and PAX5-ZNF521 (1 case each). In addition to the structural alterations, sequencing identified 14 B-ALLs with PAX5 point mutations. Mutations were identified in the DNA-binding paired domain, homeodomain and transactivation domains. The mutations were somatically acquired, and present in a dominant clone. Gel-shift and transcriptional reporter assays demonstrated reduced DNA binding and/or transcriptional activity for each of the identified PAX5 fusion proteins and point mutants. In addition to the PAX5 mutations, deletions were also detected in the B cell regulatory genes Ikaros (20 cases), Aiolos (3), EBF (8), E2A (1), LEF1 (3), and RAG1/2 (11). Importantly, genomic sequencing of Ikaros and EBF revealed no point mutations. Although the high frequency of mutations in genes regulating B-cell development was unexpected, even more surprising was the marked difference in the frequency and type of mutations among the various genetic subtypes of ALL. Hypodiploid ALLs had alterations in B-cell development genes in 100% of cases, with broad deletions of one PAX5 allele, mutation of the other PAX5 allele in 50% of the cases, and mono-allelic deletion of other regulators of B-cell development, frequently with multiple genes affected within a single case. In contrast ETV6-RUNX1 cases exhibited a much more restricted pattern of mutations, with 27% showing focal PAX5 deletion without alterations of the retained allele. At the other end of the spectrum were hyperdiploid ALLs, with only rare cases harboring a deletion of one of these genes. These data demonstrate that disruption of pathways controlling B cell development and differentiation contributes to the pathogenesis of ALL. Moreover, the approach used provides a rational roadmap for the application of genome-wide approaches to the identification of new molecular lesions in cancer.

Patients with acute myeloid leukemia (AML) and hyperleukocytosis are at increased risk of early death from neurological and pulmonary complications due to leukostasis. Although leukapheresis is often used for rapid cytoreduction in these patients, a recent adult study showed no difference in the rate of early mortality between patients who underwent leukapheresis and historical controls. The scarcity of information about leukapheresis in childhood AML led us review our experience of 106 children with AML and hyperleukocytosis (i.e., initial leukocyte count ≥ 100 × 109/L) treated between 1968 and 2002. The presenting clinical features, early complications, and clinical outcomes during the first 2 weeks of remission induction therapy were analyzed according to two treatment eras: early (1968–1982) vs. recent (1983–2002), when leukapheresis was available. The entire cohort had a median age of 9.7 years (range, 0–19.9 years); initial leukocyte count of 161 × 109/L (100 to 1,600); hemoglobin concentration of 8.2 g/dL (2.9–15.4); and platelet count of 38.5 × 109/L (0 to 300). The presenting features were comparable between patients treated in the two eras with the exception of higher hemoglobin concentrations among those treated in the recent era (p=0.03). Platelet transfusion prior to chemotherapy was used more often in the recent era (p=0.001). Half of the cases (53 of 106) had FAB M4 or M5 subtypes. Twenty-one patients (19.8%) had grade 3 or 4 neurological, respiratory, and/or renal complications (according to NCI common criteria) at initial presentation, and 35 patients (33%) had one or more of these complications during the first 2 weeks after diagnosis (17 neurological; 20 respiratory; 16 renal). The frequency of the complications did not differ significantly between patients treated in the two eras. Patients with FAB M4/M5 AML were significantly more prone than others to respiratory (p=0.005) and renal (p=0.0002) complications during the first two weeks of therapy. Seventeen patients (16%) died during the first 2 weeks after diagnosis. The rate of early death was significantly higher in the early era than in the recent era (16/70 vs. 1/36, p=0.01). The time between admission and initiation of chemotherapy was significantly shorter (20.2 vs. 33.6 hours, p

Therapy-related leukemia (t-ML) is a rare but devastating complication of anticancer treatment, including treatment of acute lymphoblastic leukemia (ALL). Although a number of treatment factors have been identified that contribute to the development of t-ML, little is known about the biology of the disease or the genetic risk factors. To identify possible genetic risk factors for and characteristics of t-ML among ALL patients, we used the Affymetrix GeneChip® Human Mapping 100K set to genotype over 100,000 single nucleotide polymorphisms (SNPs) in germline DNA from 13 cases with t-ML and from 13 matched controls, as well as DNA of the t-ML blast samples of the 13 cases. Controls were matched for treatment protocol, ALL risk classification, irradiation, prior use of G-CSF, and race. Germline allele frequencies differed (at the p < 0.01 level) for 309 SNPs (203 genes) between t-ML cases and controls. Cytogenetically, most of the cases had translocations or inversions of 11q23; however, three cytogenetically-detected regions of chromosomal loss were also detected (on chromosome 5, 7, and 9, each found only once among the cases). These chromosomal deletions were all detected as regions of loss of heterozygosity (LOH) and decreased copy number by paired intra-patient germline vs t-ML blast analyses of SNP calls. However, the paired SNP data indicated 216 regions of LOH (FDR = 2.5%) corresponding to a total of 81 non-contiguous regions (Figure), most of which were not detected as losses cytogenetically, and 38 shared (among pairs) regions of LOH. As many as 45 noncontiguous and 12 contiguous LOH regions were found among those cases with no cytogenetically defined regions of loss, including patients with simple balanced translocations. In addition to cytogenetically-defined cases, using SNP analyses we detected additional t-ML cases with regions of LOH on 5q (n=1), on chromosome 7 (n=4), and on chromosome 9 (n=2). In fact, only one patient, whose cytogenetics showed a balanced translocation involving 11q23, had no extended regions of LOH detected by SNP analysis. Altogether, 5 of the 13 cases demonstrated LOH on chromosome 7. Using a change-point model, we detected 18 regions of copy number increase and 21 regions of copy number decrease in the blasts compared to paired germline DNA (p Figure: 81 regions of inferred LOH in case t-ML blast DNA compared to germline DNA indicated losses involving multiple autosomes. Figure:. 81 regions of inferred LOH in case t-ML blast DNA compared to germline DNA indicated losses involving multiple autosomes.

We reviewed the records of 408 patients who were less than 21 years old at the time of diagnosis of acute myeloid leukemia (excluding patients with Down syndrome or acute promyelocytic leukemia) treated on five consecutive institution protocols from 1980–2002 to investigate prognostic factors for attainment of second complete remission (CR2) and overall survival (OS) after first relapse. Of the 320 (78%) patients who achieved CR, 158 patients suffered hematologic relapses at a median of 11.9 months (range, 2.9–119.9 months) from the time of diagnosis. Forty-one patients relapsed on therapy and 117 relapsed after the completion of all planned therapy. For patients who relapsed off therapy, 38 were diagnosed with relapse because of symptoms suggestive of recurrent leukemia and 78 were diagnosed at the time of a routine follow up (information not available for one patient). After relapse, 20 patients received palliative care, 82 received chemotherapy alone, 36 received chemotherapy followed by hematopoietic stem cell transplant (SCT), and 20 proceeded directly to SCT. Eighty-five (54%) patients attained CR2. In univariate analyses, factors associated with the achievement of CR2 include initial therapy (chemotherapy alone, 56%; autologous SCT, 71%; allogeneic SCT, 20%; p=0.008) and time from the diagnosis of AML to relapse (≤ 1 year, 44%; > 1 year, 65%; p=0.010). Logistic regression analysis demonstrated that patients with male gender (odds ratio [OR], 2.46; 95% confidence interval [CI], 1.14–5.28; p=0.021) and greater time from diagnosis to relapse (OR, 1.05; CI, 1.01–1.09; p=0.016) were more likely to achieve CR2, whereas patients with M7 morphology (OR, 0.11; CI, 0.01–1.04; p=0.054) and allogeneic SCT in first remission (OR, 0.17; CI, 0.03–0.85; p=0.031) were less likely to achieve CR2. At the time of last follow up, 19 patients were alive, 115 died of progressive disease, and 24 died of regimen-related toxicity. The 2-year OS estimate ± SE for the entire cohort of 158 patients was 15.8% ± 2.8%. For patients who relapsed off therapy, there was no significant difference in outcome between those whose relapse was diagnosed at the time of a routine follow up and those diagnosed because of symptoms. Cox proportional-hazards regression modeling indicated that M7 morphology (hazard ratio [HR], 3.06; CI, 1.44–6.51; p=0.004) and allogeneic SCT in first remission (HR, 2.17; CI, 1.22–3.87; p=0.008) were associated with significantly worse OS after relapse. In fact, there were no survivors in these two groups of patients. In contrast, age 1–10 years (HR, 0.53; CI, 0.36–0.77; p=0.001), M2 morphology (HR, 0.57; CI, 0.39–0.85; p=0.006), allogeneic SCT after relapse (HR, 0.34; CI, 0.22–0.53; p

The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine (A), daunorubicin (D), and etoposide (E) as Induction I; all subsequent therapy is risk-adapted. Pharmacokinetic, pharmacodynamic, and gene expression studies are performed after exposure to high-dose or low-dose cytarabine. Patients with no response (NR, ≥ 25% bone marrow blasts) to Induction I receive low-dose ADE + gemtuzumab ozogamicin (GO) as Induction II, whereas all others receive ADE. Minimal residual disease (MRD) is monitored by flow cytometry and is defined as ≥ 0.1% cells with leukemia-associated immunophenotypes among bone marrow mononuclear cells. Patients who are MRD+ after induction therapy receive GO as a single agent before consolidation chemotherapy or stem cell transplantation. Among the 112 patients enrolled since October 2002, 37 had -7, FAB M6 or M7, FLT3 internal tandem duplication (ITD), or MDS or treatment-related AML and were classified as high-risk (HR); 32 had AML1-ETO, CBFβ-MYH11, or MLL-AF9 (low-risk; LR); the remaining 43 were considered as standard-risk (SR). The rate of complete response (CR