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Demographic Information of Patient Cohort. A) Age category of patients. B) Cytogenetic category.

Patient Characteristics

List of all segment variations

Clonal cluster analysis. Mutations were clustered using DBSCAN based on the VAF and graphed according to timepoint. Panels A-T illustrate possible scenarios of how individual mutations may originate, evolve, and resolve based on VAF.

Clonal evolution of mutations from diagnosis to relapse. These graphs depict the Variant Allele Frequency (VAF) of each patient's mutations at diagnosis and relapse. Note: Mutations absent at diagnosis or relapse are depicted with VAF of zero. Gene names are noted to the left of each variant and are available in Supplemental Table 2.

List of all verified mutations

Purpose: The volume of data produced by the biomedical research community continues to grow rapidly. Efficient collection, curation, and sharing of these data are expected to enable researchers to synthesize larger datasets and apply novel methods to discover new patterns for diagnosis, treatment, and care of disease. Consequently, data platforms that support these functionalities have become an important instrument for the biomedical research community. The National Cancer Institute’s (NCI’s) Cancer Research Data Commons (CRDC) is a data platform for the cancer research community that provides cloud-based, secure storage and analytic tools for cancer data, including genomics, proteomics, imaging, and clinical trial data. CRDC has been funded by multiple sources, including the Beau Biden Cancer Moonshot program. Achieving long-term sustainability of the CRDC program is the focus of our study. Methods: We applied a Financial Operations (FinOps) framework to map all costs to functionalities provided by the CRDC, including processes such as data intake, storage, compute, user support, and project management. We established a comprehensive financial baseline for the operations of the CRDC, identified opportunities for optimization across multiple dimensions (people, processes, technology), projected future costs based on trends for cancer data, and evaluated sustainability under defined funding scenarios. Results: We identified 15 recommendations for optimization of the CRDC, including automation and centralization of core services (e.g., intake, curation, indexing), optimization of storage (e.g., compression, archiving), and harmonization of common services across the CRDC components (e.g., common architecture, governance). If implemented, we could demonstrate that these recommendations offer a path to long-term sustainability of the CRDC program under a variety of funding scenarios. Conclusion: Data platforms such as the CRDC will see rapid increases in operational costs due to the growing data volumes and demand from researchers. Optimizing governance and operational frameworks will result in efficiency gains that can ensure long-term sustainability. Taking these steps will be critical to provide the necessary infrastructure to advance the field of data-driven biomedical research. Citation Format: Juergen A. Klenk, Angela Maggio, Bhavani S. Singh, Erin N. Byrne, Erika Kim, Tanja M. Davidsen. A path to sustainability for the National Cancer Institute's Cancer Research Data Commons. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6582.

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children's Oncology Group (COG) AML trials. The COG-National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in2% of cases. In contrast, somatic structural variants, including new gene fusions and focal deletions of MBNL1, ZEB2 and ELF1, were disproportionately prevalent in young individuals as compared to adults. Conversely, mutations in DNMT3A and TP53, which were common in adults, were conspicuously absent from virtually all pediatric cases. New mutations in GATA2, FLT3 and CBL and recurrent mutations in MYC-ITD, NRAS, KRAS and WT1 were frequent in pediatric AML. Deletions, mutations and promoter DNA hypermethylation convergently impacted Wnt signaling, Polycomb repression, innate immune cell interactions and a cluster of zinc finger-encoding genes associated with KMT2A rearrangements. These results highlight the need for and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML.

BACKGROUND: Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS: We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS: Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS: The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.)

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

The mission of the National Cancer Institute’s (NCI) Office of Cancer Genomics (OCG) is to advance the molecular understanding of cancers in order to improve clinical outcomes through precision medicine. Although vast amounts of genomic data are available for many types of cancers, identifying genetic alterations in rare and pediatric cancers is still a challenge. Efficient bioinformatics tools to analyze, manage, store, and access data are also necessary for the research community. To develop effective and targeted treatments, clinically accurate genotypic and phenotypic research models are much needed. OCG’s programs focus on addressing these challenges through multidisciplinary, collaborative research efforts. The four initiatives of OCG support research on structural, functional, and translational genomics, as well as development of next-generation cancer models. The Cancer Genome Characterization Initiative (CGCI) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) programs use transcriptomic, genomic, and epigenomic approaches to examine genetic alterations between various tumors and matched normal tissues. CGCI projects focus on HIV-associated and rare cancers such as Burkitt lymphoma, while TARGET focuses on high-risk childhood cancers. The goals of these programs are to attain insights into key mutations that drive tumors and genetic abnormalities specific to cancer subtypes and to develop effective and less toxic therapies for patients. CGCI and TARGET data are available to the research community through data matrices on the OCG website. OCG also recently launched the Pediatric Genomic Data Inventory (PGDI) as a new resource for investigators to access molecular characterization data.The Cancer Target Discovery and Development (CTD2) Network advances cancer research by bridging the knowledge gap between cancer genomics and precision oncology. The Network aims to understand the cancer metastasis, tumor heterogeneity, and drug resistance to develop optimal combinations of small-molecules or immunotherapy with small molecules. As a community resource program, the CTD2 Network develops and provides access to data, tools, methods, and reagents through the Data Portal and the Dashboard. The Human Cancer Models Initiative (HCMI) is an international consortium that is generating novel human tumor-derived culture models from a wide variety of cancer types including rare and understudied cancers. The models, together with related clinical and genomic data, will be available as a resource to the world-wide research community. OCG’s policies on data usage, as well as guides to accessing data, are explained on the OCG website (https://ocg.cancer.gov/). Researchers, potential collaborators, and interested members of the public are encouraged to visit the OCG webpages or contact OCG at ocg@mail.nih.gov. Citation Format: Cindy W. Kyi, Pamela C. Birriel, Tanja M. Davidsen, Martin L. Ferguson, Patee Gesuwan, Nicholas B. Griner, Yiwen He, Subhashini Jagu, Eva Tonsing-Carter, Daniela S. Gerhard. NCI Office of Cancer Genomics: Promoting multidisciplinary research to translate findings into the clinic and advance precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4341.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Childhood cancers represent distinct clinical entities, often with unique genomic alterations and therapeutic responses that differ from cancers arising in adults. Pediatric acute myeloid

This report summarizes the proceedings of the 9th workshop of the Genomic Standards Consortium (GSC), held at the J. Craig Venter Institute, Rockville, MD, USA. It was the first GSC workshop to have open registration and attracted over 90 participants. This workshop featured sessions that provided overviews of the full range of ongoing GSC projects. It included sessions on Standards in Genomic Sciences, the open access journal of the GSC, building standards for genome annotation, the M5 platform for next-generation collaborative computational infrastructures, building ties with the biodiversity research community and two discussion panels with government and industry participants. Progress was made on all fronts, and major outcomes included the completion of the MIENS specification for publication and the formation of the Biodiversity working group.

The JCVI metagenomics analysis pipeline provides for the efficient and consistent annotation of shotgun metagenomics sequencing data for sampling communities of prokaryotic organisms. The process can be equally applied to individual sequence reads from traditional Sanger capillary electrophoresis sequences, newer technologies such as 454 pyrosequencing, or sequence assemblies derived from one or more of these data types. It includes the analysis of both coding and non-coding genes, whether full-length or, as is often the case for shotgun metagenomics, fragmentary. The system is designed to provide the best-supported conservative functional annotation based on a combination of trusted homology-based scientific evidence and computational assertions and an annotation value hierarchy established through extensive manual curation. The functional annotation attributes assigned by this system include gene name, gene symbol, GO terms [1], EC numbers [2], and JCVI functional role categories [3].

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria , the Cyanobacteria , and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N 2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

TIGRFAMs is a collection of protein family definitions built to aid in high-throughput annotation of specific protein functions. Each family is based on a hidden Markov model (HMM), where both cutoff scores and membership in the seed alignment are chosen so that the HMMs can classify numerous proteins according to their specific molecular functions. Most TIGRFAMs models describe 'equivalog' families, where both orthology and lateral gene transfer may be part of the evolutionary history, but where a single molecular function has been conserved. The Genome Properties system contains a queriable set of metabolic reconstructions, genome metrics and extractions of information from the scientific literature. Its genome-by-genome assertions of whether or not specific structures, pathways or systems are present provide high-level conceptual descriptions of genomic content. These assertions enable comparative genomics, provide a meaningful biological context to aid in manual annotation, support assignments of Gene Ontology (GO) biological process terms and help validate HMM-based predictions of protein function. The Genome Properties system is particularly useful as a generator of phylogenetic profiles, through which new protein family functions may be discovered. The TIGRFAMs and Genome Properties systems can be accessed at http://www.tigr.org/TIGRFAMs and http://www.tigr.org/Genome_Properties.

The genomic and clinical information used to develop and implement therapeutic approaches for acute myelogenous leukemia (AML) originated primarily from adult patients and has been generalized to patients with pediatric AML. However, age-specific molecular alterations are becoming more evident and may signify the need to age-stratify treatment regimens. The NCI/COG TARGET-AML initiative used whole exome capture sequencing (WXS) to interrogate the genomic landscape of matched trios representing specimens collected upon diagnosis, remission, and relapse from 20 cases of de novo childhood AML. One hundred forty-five somatic variants at diagnosis (median 6 mutations/patient) and 149 variants at relapse (median 6.5 mutations) were identified and verified by orthogonal methodologies. Recurrent somatic variants [in (greater than or equal to) 2 patients] were identified for 10 genes (FLT3, NRAS, PTPN11, WT1, TET2, DHX15, DHX30, KIT, ETV6, KRAS), with variable persistence at relapse. The variant allele fraction (VAF), used to measure the prevalence of somatic mutations, varied widely at diagnosis. Mutations that persisted from diagnosis to relapse had a significantly higher diagnostic VAF compared with those that resolved at relapse (median VAF 0.43 vs. 0.24, P < 0.001). Further analysis revealed that 90% of the diagnostic variants with VAF >0.4 persisted to relapse compared with 28% with VAF

The completion of the 5,373,180-bp genome sequence of the marine psychrophilic bacterium Colwellia psychrerythraea 34H, a model for the study of life in permanently cold environments, reveals capabilities important to carbon and nutrient cycling, bioremediation, production of secondary metabolites, and cold-adapted enzymes. From a genomic perspective, cold adaptation is suggested in several broad categories involving changes to the cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and strategies to overcome temperature-dependent barriers to carbon uptake. Modeling of three-dimensional protein homology from bacteria representing a range of optimal growth temperatures suggests changes to proteome composition that may enhance enzyme effectiveness at low temperatures. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition.

Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encoding enzymes for energy transduction, together with inferred cellular location of the enzymes, provides a basis for proposing an expansion to the 'hydrogen-cycling' model for increasing energy efficiency in this bacterium. Plasmid-encoded functions include modification of cell surface components, nitrogen fixation and a type-III protein secretion system. This genome sequence represents a substantial step toward the elucidation of pathways for reduction (and bioremediation) of pollutants such as uranium and chromium and offers a new starting point for defining this organism's complex anaerobic respiration.

We present the complete 2,843,201-bp genome sequence of Treponema denticola (ATCC 35405) an oral spirochete associated with periodontal disease. Analysis of the T. denticola genome reveals factors mediating coaggregation, cell signaling, stress protection, and other competitive and cooperative measures, consistent with its pathogenic nature and lifestyle within the mixed-species environment of subgingival dental plaque. Comparisons with previously sequenced spirochete genomes revealed specific factors contributing to differences and similarities in spirochete physiology as well as pathogenic potential. The T. denticola genome is considerably larger in size than the genome of the related syphilis-causing spirochete Treponema pallidum . The differences in gene content appear to be attributable to a combination of three phenomena: genome reduction, lineage-specific expansions, and horizontal gene transfer. Genes lost due to reductive evolution appear to be largely involved in metabolism and transport, whereas some of the genes that have arisen due to lineage-specific expansions are implicated in various pathogenic interactions, and genes acquired via horizontal gene transfer are largely phage-related or of unknown function.

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana . The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa , yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

The 1,995,275-bp genome ofCoxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies ofC. burnetiiresemble that ofRickettsiaeandChlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in theC. burnetiigenome. These analyses suggest that the obligate intracellular lifestyle ofC. burnetiimay be a relatively recent innovation.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Acute myeloid leukemia (AML) carries a poor prognosis across age groups. In children, AML has become the leading cause of leukemia mortality, with only 60% of cases securing long-term remission. In adults, outcomes are far worse, with 5 year survival approaching 24%. The mutational and transcriptional characterization of AML1has not yet translated into improved outcomes for most patients. The TARGET AML project is an effort of Children's Oncology Group (COG) and the National Cancer Institute to characterize molecular abnormalities in pediatric AML. 197 cases were selected for whole genome sequencing (WGS) of diagnostic specimens, 284 cases for mRNA sequencing, 289 cases for DNA methylation arrays, and 721 cases for targeted sequencing (182 assayed by WGS). Most patients (93%) were uniformly treated on COG study AAML0531 or AAML03P1. The Cancer Genome Atlas (TCGA) AML project1characterized 177 comparable adult AMLs with identical assays. DNA methylation changes radically during differentiation of blood cells2, and recurrent pre-leukemic mutations in adult AML3affect DNA methylation and chromatin modifiers. We thus investigated whether differences in cell-of-origin, immune signalling, and regulatory aberrations were captured by focal or regional differences in DNA methylation, within or between adult and pediatric AML patients. In cytogenetically similar TARGET and TCGA AML cases, striking differences in DNA methylation emerge (fig. 1). Pediatric FLT3-mutant AMLs dominate a cluster with normal-progenitor-like DNA methylation. Mutant DNMT3A, RUNX1, and TP53, which selectively favor preleukemic hematopoietic stem cells3,4,5 (HSCs), are common in adult AML, rare in pediatric AML, and tend towards HSC-like hypermethylation. Transcriptional & epigenetic signatures of the cell of origin persist even after leukemic transformation6. Thus we sought to identify the most likely cell of origin for each case. Previous studies of mRNA7 and DNA methylation8 differences in HSCs and progenitor cells (HSPCs), leukemic stem cells (LSCs), and AML blasts allowed us to model these differences in TCGA and TARGET AMLs. RNAseq results revealed many LSC-like cases with aberrant β-catenin signaling and TP53 regulation, distinct from blasts and normal HSPCs (fig. 2a). DNA methylation segregated cases resembling granulocyte/monocyte progenitors (GMPs) from those resembling other HSPC subsets (fig. 2b). DNMT3A mutants strongly associated with HSC/LSC-like mRNA expression, as did most MLL-rearranged AMLs. Nearly all TP53 and RUNX1 mutants presented LSC-like mRNA expression and retained HSC-like methylomes. These results suggest that decades of selective HSC attrition enable cooperating adult-specific mutations to initiate leukemia, while the timescales in pediatric AML favor fusion genes capable of transforming progenitors as well as HSCs. With matched mRNA expression & DNA methylation data from 256 TARGET cases and 156 TCGA cases, we found over 100 genes where DNA methylation accompanied loss of transcription (silencing) in AML but not in normal HSPCs (fig. 3a). Many such genes lie in regions affected by recurrent copy number aberrations, most notably chromosome arms 5q and 19q. Recurrently mutated or deleted genes such as DNMT3A, TET2, SPRY4, and CDKN2A/B are silenced, some mutually exclusively with mutations or CNV. Functional enrichment analyses of silenced genes with DAVID9revealed 4 clusters: NK-cell signaling, innate immune response regulation, transcriptional regulation, and (on chromosome 19q) zinc finger genes involved in Toll-like receptor signaling. Some silencing co-occurs with specific molecular features, but no event was perfectly predicted by any molecular or cytogenetic feature (fig. 3b). Drug-gene interaction mining with DGIDb10 suggests silencing may inform treatment. Silencing of the mitotic checkpoint gene CHFR may confer sensitivity to microtubule inhibitors11, silencing of MGMT suggests greater benefit from alkylating agents12, and demethylating agents may benefit cases with silenced immune response13. Biomarker driven clinical trials will be needed to evaluate these and other markers in pediatric and adult AML, but evidence of independent genetic and epigenetic evolution in AML14supports their continued investigation. This work is dedicated to the late Robert J. Arceci, without whom none of this would have been possible. Disclosures No relevant conflicts of interest to declare.

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.

Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

The age distribution of acute myeloid leukemia is unusual among malignancies, with onset spanning from early infancy until past the 9th decade. Despite similar histology, cytogenetic abnormalities and recent identification of somatic mutations (e.g., DNMT3A mutations) have highlighted differences in the events driving adult compared to childhood de novo AML. However, the full extent of these differences remains unknown and is likely to have relevance to treatment approaches. The TARGET AML initiative is an effort of the Children's Oncology Group (COG) and the National Cancer Institute to comprehensively characterize the molecular abnormalities of pediatric AML. The dataset comprises 1) whole genome sequencing (WGS) of AML and matched remission bone marrow in 197 cases, 2) mRNA transcriptome sequencing of 284 cases, 3) miRNA sequencing of 692 cases, 4) methylation array data on 289 cases, and 5) targeted capture sequencing of 174 candidate genes identified from WGS in 800 diagnostic samples, including 182 with WGS (Figure 1). The majority of patients (93%) studied were uniformly treated on COG study AAML0531 or its pilot safety precursor study, AAML03P1. Relapsed specimen data (not shown) are available for a subset of these cases. All patient samples were obtained by written consent upon enrollment in the clinical trial. Consistent with adult studies, we identified a relatively low mutational burden, with 2206 somatic tier 1 mutations resulting in a coding change in 1682 genes (median 6 per patient) from the WGS discovery data. We successfully verified 70-90% of variant calls by secondary methods. Also as with adult data, there were relatively few recurrently mutated genes, with fewer than 40 genes altered in >2% of samples. However, there were marked differences in somatic mutation frequencies in comparison to adult TCGA data, both by raw frequency and after adjustment for cytogenetic subtypes present among the two cohorts (Figure 2). Mutations in TP53, NPM1, IDH1, IDH2, TET2 and DNMT3A are more frequent in adult compared to pediatric disease; in contrast, mutations in NRAS, KRAS, WT1, FLT3, PTPN11, GATA2, ASXL2, MYC, SETD2, EZH2 and IKZF1 appear more common in pediatric AML. Mutations of several genes, including CEBPA, ASXL2 and KRAS are not only more common in pediatric AML, they show peak prevalence within specific pediatric age groups. In addition, several genes, including FLT3, WT1, and KIT show significant differences in mutational hotspots compared to adults. Pediatric-adult differences in AML were not limited to somatic gene mutations, but extended to focal and chromosomal copy number alterations (CNA), translocations, miRNA expression, and methylation-induced gene silencing. We identified recurrent focal CNAs in multiple regions not reported in adult AML including 15 heterozygous focal deletions impacting ELF1, an ETS-family transcriptional regulator of hematopoiesis and leukemia driver as well as deletions of the splicing regulator MBNL1 in 10 cases, 8 of which co-occurred with focal deletions of the hematopoietic transcriptional regulator, ZEB2. De novo assembly of mRNA sequencing data identified fusion transcripts in 63% of cases compared to 45% of TCGA LAML. In addition to cytogenetically evident fusions with well-described enrichment for MLL translocations in pediatrics, we identified 29 diagnostic samples (10%) with nucleoporin family fusions (NUP98 with NSD1, KDM5A, PHF23, HOXD13, HMGB3, BRWD3, and CLINT; NUP214 with DEK and SET), CBFA2T3-GLIS2 fusions in 5, and rare fusions of ETS transcription factor genes (FUS-FEV, ETV6-INO8D). Comparison of miRNA expression patterns between adult and pediatric specimens similarly showed marked differences in expression of key regulatory miRNAs including let-7 family members. Finally, analysis of mRNA expression and DNA methylation for the identification of epigenetically silenced genes suggested that, although specific events favor silencing in adults or children, an overall pattern of gene silencing was more prevalent in pediatric compared to adult cases. This work extends our understanding of the heterogeneity of AML, demonstrates fundamental differences in the biology of pediatric- and adult-onset disease, and suggests important age-related differences within "pediatric" AML. This rich dataset should provide a foundation for the establishment of biologically-guided treatment in children with AML. Disclosures No relevant conflicts of interest to declare.

In efforts to discover genes uniquely expressed in childhood AML, we performed transcriptomesequencing (RNA-Seq) in pediatric AML and contrasted the expression signature to that in normal marrow hematopoiesis. This effort led to the discovery of over 200 genes that lack expression in normal hematopoietic cells, but are variably expressed in pediatric AML cells. Mesothelin(MSLN) was discovered to be one of the most highly expressed genes in a subset of childhood AML cases (p In this study, RNA obtained from diagnostic bone marrow specimens from childhood AML (N=434) and normal marrow (N=20) was subjected to wholetranscriptomesequencing and MSLN expression was quantified and normalized and reported as reads perkilobaseof exon per million reads mapped (RPKM). Similar data was obtained from adult TCGA AML database. Quantitative RT-PCR (qRT-PCR) and multidimensional flowcytometry(MDF) was used for confirmation of the transcript and cell surface protein expression. TARGET AML methylation data was used for correlation with transcript expression. Of the 434 specimens analyzed, MSLN mRNA expression was variably expressed (RPKM range 0-618.8), with expression detected in 119 patients (27%). Confirmatory studies by qRT-PCR on specimens with and without MSLN expression (N=137) showed correlation between RNA-Seqand PCR data. Cell surface MSLN expression was assessed by MDF using a PE-conjugated MSLN antibody (PE-mesoAb) and verified expression of MSLN protein on the leukemic cell surface in every case with MSLN transcript expression (Figure 1A). Evaluation of CD34+/CD38- hematopoietic progenitor cells by PE-mesoAbdemonstrated lack of MSLN expression by MDF. Evaluation of matched diagnostic and relapse specimens from MSLN-expressing patients (n=27) confirmed that MSLN expression was largely stable (R2=0.87), thus substantiating its expression in the major AML clone. Comparison of MSLN expression in pediatric vs. adult AML demonstrated a higher prevalence in pediatric AML (TARGET: 27% vs. TCGA: 11%)(Figure 1B). Evaluation of the clinical and biologic features in MSLN expressing (MSLN+) and non-MSLN expressing (MSLN-) pediatric patients revealed that MSLN expression was rarely observed in patients with normal karyotype (p We further inquired about the mechanism by which MSLN expression might be regulated. Whole genome sequencing data failed to identify any genomic alterations in MSLN that could result in high expression. Therefore, we interrogated the possibility of epigenetic regulation of MSLN expression. Integration of the expression and methylation profiling cases with matching RNA-Seqand methylation data (N=246) demonstrated thathypomethylationof the MSLN promoter significantly correlated with high MSLN expression, implicating epigenetic regulation in the expression of MSLN in AML. Mesothelin, a therapeutic target in solid tumors, is highly expressed in biologically distinct subsets of childhood AML. High expression on leukemic blasts and lack of expression in normal hematopoiesis makes this antigen an ideal target for therapeutic intervention in AML. As a cell surface protein, this antigen avails itself for immune targeting by antibody drug conjugates, CAR-T cells, and T cell receptor mediated targeting. Figure 1 Mesothelin expression in childhood AML. Figure 1. Mesothelin expression in childhood AML. Disclosures Loken: Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment.

The Gemina system (http://gemina.tigr.org) developed at TIGR is a tool for identification of microbial and viral pathogens and their associated genomic sequences based on the associated epidemiological data. Gemina has been designed as a tool to identify epidemiological factors of disease incidence and to support the design of DNA-based diagnostics such as the development of DNA signature-based assays. The Gemina database contains the full complement of microbial and viral pathogens enumerated in the Microbial Rosetta Stone database (MRS) [1]. Initially, curation efforts in Gemina have focused on the NIAID category A, B, and C priority pathogens [2] identified to the level of strains. For the bacterial NIAID category A-C pathogens, for example, we have included 38 species and 769 strains in Gemina. Representative genomic sequences are selected for each pathogen from NCBI’s GenBank by a three tiered filtering system and incorporated into TIGR’s Panda DNA sequence database. A single representative sequence is selected for each pathogen firstly from complete genome sequences (Tier 1), secondly from whole genome shotgun (WGS) data from genome projects (Tier 2), or thirdly from genomic nucleotide sequences from genome projects (Tier3). The list of selected accessions is transferred to Insignia when new pathogens are added to Gemina, allowing Insignia’s Signature Pipeline [3] to be run for each pathogen identified in a Gemina query.

Abnormalities of epigenetic regulatory genes including DNMT3A, IDH1, IDH2 and TET2 are common in adults with AML. The prevalence of these abnormalities appears to increase with older age, but their impact in pediatric AML is less certain. The Children's Oncology Group (COG) has previously reported that mutations of DNMT3A, IDH1 and IDH2 are very rare in pediatric patients (Ho et al Leukemia 2010; Ho et al Pediatric Blood and Cancer 2011). In the current study we examined the prevalence and prognostic significance of TET2 gene mutations in a large cohort of pediatric patients with AML. We screened for genomic mutations in the TET2 gene using DNA extracted from diagnostic specimens of 949 pediatric de novo AML patients treated on the COG studies AAML03P1 (N=226) and AAML0531 (N=723). The COG trial AAML03P1 was a phase III pilot study with non-random assignment to gemtuzumab ozogamicin in combination with multi-agent chemotherapy. AAML0531 was a phase III trial with randomization to gemtuzumab ozogamicin. The entire coding sequence of TET2 and was amplified and sequenced. Mutational data was correlated with clinical characteristics and outcome data in both univariable and multivariable analyses. Mutations of the TET2 gene were found in 26 of 949 samples (2.7%). Mutations were found across TET2 gene exons 3-11 from amino acid 121 to 1920. There were 14 missense mutations, 8 nonsense mutations, 2 insertion/deletion (in/del) resulting in frame shift and 2 samples with multiple mutations (1 with missense mutation and in/del frameshift; 1 with nonsense mutation and in/del frameshift). There was no significant difference in the age, gender, race or ethnicity of patients with or without TET2 mutations. Patients with TET2 mutations had a higher prevalence of normal cytogenetics than those without a TET2 mutation (44% vs. 22%, P=0.011). There was also a significant association of TET2 mutations with core binding factor leukemia, although the direction of the association differed between t(8;21) and inv(16). Patients with TET2 mutations compared to those without these mutations were more likely to have t(8;21) (53% vs. 17%, P= 0.021) but less likely to have inv(16) (0% vs. 21%, P=0.039). Molecular mutations currently used in risk stratification of pediatric AML were evaluated. There was no association with FLT3/ITD or mutations of CEBPA, but there was a strong association with NPMc+ which is known to confer a favorable prognosis. TET2 mutant patients had a 32% prevalence of NPMc+ compared to only 7% NPMc+ among TET2 non-mutant (P= Comparing patients with and without TET2 mutations, there was no significant difference in overall survival (OS) (5 yr: 77% vs. 65%, P=0.194), event free survival (EFS) (5 yr: 58% vs. 50%, P=0.411) or relapse risk (40% vs. 37%, P=0.772). We performed multivariable analysis of OS and EFS for TET2 mutation status, risk grouping, NPMc+ status and treatment exposure to gemtuzumab ozogamicin. TET2 mutation status was not predictive of outcome in this multivariable analysis. However, genetic risk group was significantly associated with both OS and EFS and treatment with gemtuzumab ozogamicin was associated only with EFS (HR 0.824 for patients treated with gemtuzumab ozogamicin). Due to the strong association of TET2 mutation and NPMc+, we further evaluated Kaplan Meier estimates of EFS of the 4 groups of patients stratified by both TET2 mutation and NPMc+ status and determined that co-occurrence of TET2 mutation does not modify the clinical significance of NPMc+. (Figure 1) This study demonstrates that TET2 gene mutations are not common events in childhood AML but are highly associated with NPMc+ and may hint at potential cooperation of genetic and epigenetic factors that mediate myeloid leukemogenesis. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures Off Label Use: Arsenic Trioxide in pediatric patients with newly diagnosed APL. Gemtuzumab Ozogamicin in Pediatric AML..

Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention. Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G). Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation. Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse. We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. Disclosures No relevant conflicts of interest to declare.

Increasing use of high-throughput sequencing technologies has enabled the identification of novel somatic gene alterations in cancers, with important implications in understanding the mechanisms of transformation and potentially in improving therapeutic decision-making. In contrast to many solid tumors, the mutational spectrum of acute myeloid leukemia (AML) has proven bland, with relatively few recurrent somatic mutations per tumor. This result appears more pronounced in pediatric AML, where even mutation of genes commonly mutated in adult disease, such as IDH1/2 or DNMT3A, appear at much lower frequencies. Alterations of ASXL1, a regulator of the polycomb-repressive complex, have been identified in adults with myelodysplastic syndromes, myeloproliferative syndromes, and AML. Mutations in the related gene family member, ASXL2, were recently associated with t(8;21) AML in adults and children. In this study, we evaluated the prevalence and prognostic impact of mutations of these epigenetic regulator genes in pediatric AML patients treated on COG studies AAML03P1 and AAML0531. ASXL1 and ASXL2 (ASXL1/2) were sequenced from genomic DNA isolated by directed capture of the coding sequences of each gene followed by Illumina paired-end sequencing in a total of 769 diagnostic AML specimens. We identified ASXL1/2 mutations in 44/769 pediatric AML cases (5.7%), with ASXL2 altered more frequently (61% of mutated cases) than ASXL1. Consistent with prior reports, the majority of mutations (80%) were nonsense changes involving exons 11-12 of either gene. ASXL1 and ASXL2 mutations were mutually exclusive, with no case demonstrating concurrent mutation of both genes. In comparison of the clinical covariates of cases with ASXL1/2 mutation against those without, there were no differences in age, gender, or ethnicity; however we found an increased proportion of ASXL1/2 mutations in patients of Asian race, constituting 15% of those with mutated ASXL1/2 vs. 5% of those without (p=0.018). Patients possessing ASXL1/2 mutations tended to present with lower peripheral WBC and marrow blast counts (median 20K vs 30.8K, p=0.067 and 49% vs 70%, p=0.003, respectively). In agreement with recent reports, we identified a striking positive association of ASXL1/2 alterations and t(8;21), which was present in 68% of cases with ASXL1/2 vs 12% of those without (p We found no differences in the clinical outcomes of patients with ASXL1/2 mutations in comparison to those without. Complete remission rates were similar for both groups at the end of induction courses 1 and 2 (84 vs 75%, p=0.2 and 91 vs 89%, p=1.0, respectively). 5-year EFS and OS was 59 ± 15% vs 50 ± 4% (p=0.305) and 65 ± 15% vs. 66 ± 4% (p=0.851). Although prior reports have suggested the possibility of enhanced relapse risk with ASXL1/2 mutations, the 5-year RR for pediatric patients treated in these trials was similar at 31 ± 16% vs 40 ± 4% (p=0.407). Given the co-occurrence of ASXL1/2 mutations and t(8;21), we also evaluated outcomes of t(8;21) patients (n=113) with or without ASXL1/2 mutations, however no significant differences in 5-year OS, EFS, or RR were observed in this subset analysis. This work demonstrate that mutations of ASXL1 or ASXL2 are highly prevalent in childhood t(8;21) AML. Although there was no effect on treatment outcomes, this concomitance suggests the possibility of discrete cooperating lesions, even within the group of CBF-translocated AML, which may be amenable to specifically-directed therapy Disclosures No relevant conflicts of interest to declare.

Genetic alterations in the Nucleoporin (NUP) family of genes are involved in myeloid leukemogenesis and are associated with poor prognosis. We previously showed that NUP98-NSD1 is prevalent in acute myeloid leukemia (AML) and is highly associated with FLT3-ITD and dismal outcome. As genetic alterations in the NUP family are frequently cryptic by conventional karyotyping, their incidence has been underestimated. The COG/NCI TARGET AML initiative has performed comprehensive genome-wide characterization of diagnostic specimens from 200 pediatric AML cases in order to identify novel genetic lesions with prognostic and therapeutic significance. The interrogation of the whole genome and RNA sequencing data generated by this initiative identified numerous fusion transcripts involving the NUP family of genes, including NUP98-NSD1, NUP98-KDM5A, NUP98-HOXA9, NUP98-HMG3, NUP98-HOXD13, NUP98-PHF23, NUP98-BRWD3, CLINT-NUP98 and DEK-NUP214. All computationally identified NUP fusions were verified by orthogonal methodology and high-throughput screening assay was developed for frequency determination. The verified NUP fusions were screened in children treated on COG AAML0531 and AAML03P1 to define their prevalence, clinical characteristics and association with clinical outcome. The impact of NUP fusions was initially evaluated in patients with cytogenetically normal AML (CN-AML). NUP fusions were observed in 14.5% (35 of 242) patients: NUP98-NSD1 (N=21), DEK-NUP214 (N=3), NUP98-HMG3 (N=3), NUP98-HOXD13 (N=2), NUP98-PHF23 (N=2) and NUP98-KDM5A (N=4). The NUP fusions NUP98-BRWD3, NUP98-HOXA9 and CLINT-NUP98 were not found in CN-AML patients. Demographics and disease characteristics of CN-AML patients with and without NUP fusions were compared. Although patients of Asian descent comprised only 7% of the study population, they harbored significantly higher number of NUP fusions (29% vs 5%, P =0.002). Among those of Asian descent with CN-AML, 35% harbored a NUP fusion. We also noted an inverse association between NUP fusions and African-Americans where NUP fusions were not identified in any of African-American patients (P =0.031). NUP fusions were correlated with other common mutations in AML. NPM1 (9% vs 28%, P =0.007) and CEBPA (6% vs 19%, P =0.06) were rare in patients with NUP fusions, whereas FLT3/ITD (62% vs 34%, P =0.002) and WT1 (32% vs 8%, P Given the high co-occurrence of NUP fusions and FLT3-ITD, we investigated the prevalence and clinical correlation of NUP fusions in all FLT3-ITD-positive patients. The prevalence of NUP fusions in FLT3-ITD patients was 26% (43 of 164). The CR rate was lower in patients co-expressing the NUP fusion and FLT3-ITD (40% vs 71%, P In this study we report on the discovery, verification and frequency validation of NUP fusions, a new class of genetic alterations in AML. We demonstrate that NUP fusionsare common in pediatric patients and patients with CN-AML harboring NUP fusions have poor outcome and are more likely to have post-induction MRD than those without thesefusions. Furthermore, there is a high co-occurrence of FLT3-ITD and NUP fusions and patients harboring both genetic lesions have a lower CR rate and high post-induction MRD than those with FLT3-ITD alone. NUP fusions define a new subgroup of pediatric AML patients with an overall poor prognosis. AML harboring NUP fusions likely share similar mechanisms of leukemogenesis and targeting these genetic lesions will likely improve outcome in a significant subset of pediatric AML patients. Disclosures No relevant conflicts of interest to declare.

Activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) are present in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) Despite the high frequency of these mutations in CNL, they are quite rare in adult acute myeloid leukemia (AML), in which only a single CSF3R mutated case was found in the TCGA AML analysis (0.5%). We have previously demonstrated significant variation in genomic variants between pediatric and adult malignancies, thus prevalence of genomic variants identified in adults need be fully characterized in children. As part of COG/NCI TARGET AML initiative we interrogated the genomic makeup of 200 cases of childhood AML (discovery cohort) using whole genome sequencing and performed subsequent frequency determination of the variants in 787 unselected cases from COG AAML0531 (validation cohort). Somatic variants in CSF3R were initially found to be recurrent in the discovery cohort and underwent frequency determination in the validation cohort to establish their prevalence and correlations with clinical characteristics and outcome. Frequency determination of CSF3R mutation in 787 pediatric patients with available CSF3R data from AAML0531 identified 16 distinct CSF3R mutations in 28 patients (3.6%). Somatic mutations in CSF3R identified in pediatric AML included known oncogenic variants mutations such as T618I and T615A, previously identified in adult CNL studies as well as novel truncations of the CSF3R cytoplasmic domain (Q749X, Y767fs, Y787X and P819/820fs), and missense mutations (E149D, A208V, R223Q, E405K, A431V, and Q516K). Interestingly, although CSF3R truncations usually occur along with a T618I or T615A mutation in CNL/aCML, these two mutation categories were mutually exclusive in pediatric AML. Initial correlation of all CSF3R variants with demographic and clinical/laboratory parameters determined that CSF3R variants were less prevalent in younger patients (age 0-2, p=0.039), with significantly higher association with t(8;21) (32% vs. 14%, p=0.012) and CEBPA mutations (35% vs. 5%, p Compared to non-mutated cases, transforming CSF3R variants had a significant association with CEBPA mutations (44% vs. 5%, p CSF3R mutations define a distinct molecular subset of pediatric AML, which could be therapeutically targeted in the future using kinase inhibitors such as ruxolitinib. The oncogenic CSF3R mutations found in pediatric AML are either the same point mutations or similar truncation mutations as seen in CNL, suggesting that other cooperating genomic alterations may be important in directing these distinct diseases. Interestingly, we found that the majority of pediatric AML patients with CSF3R mutation have either a core binding factor alteration (such as t(8;21)) or a mutation in CEBPA. The enrichment of CEBPA mutations with CSF3R mutations is particularly striking, as CEBPA mutations are ~9 fold more frequent in patients with transforming CSF3R mutations than those without. Understanding the role of cooperating genomic alteration in CSF3R-driven myeloid malignancies will be the subject of future work. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures Radich: Novartis: Consultancy, Research Funding; Incyte: Consultancy; Gilliad: Consultancy; Ariad: Consultancy.

The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome. Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant. We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth. A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant. Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p Patients with CBL variants had a 100% clinical remission rate by end of induction II compared to 89% for CBL WT patients (p=0.014). Survival from study entry was similar between CBL mutant vs. WT patients (5 year OS 72% vs. 66%, p=0.24; 5 year EFS 61% vs. 50%, p=0.11). Due to the strong association of CBL mutation with core binding factor leukemia, we assessed whether CBL variant was prognostic of outcome within this favorable risk group, but there was no significant difference in outcomes. Variants of the CBL gene in pediatric AML include genetic mutations with and without whole exon deletions. These CBL variants are highly associated with low risk AML but do not provide independent risk prognosis. The cooperating events of CBL variants in core binding factor leukemia deserve greater study. Our initial analysis of the transcript variants in a cell line model suggest that these large exon 8+9 deletions represent important oncogenic events. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures No relevant conflicts of interest to declare.

Streptococcus pneumoniae(pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only twoS. pneumoniaestrains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae , the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

The complete genome sequence of Geobacter sulfurreducens , a δ-proteobacterium, reveals unsuspected capabilities, including evidence of aerobic metabolism, one-carbon and complex carbon metabolism, motility, and chemotactic behavior. These characteristics, coupled with the possession of many two-component sensors and many c-type cytochromes, reveal an ability to create alternative, redundant, electron transport networks and offer insights into the process of metal ion reduction in subsurface environments. As well as playing roles in the global cycling of metals and carbon, this organism clearly has the potential for use in bioremediation of radioactive metals and in the generation of electricity.

The PIM family of serine/threonine kinases (PIM1, PIM2, PIM3) are important regulators of signal transduction that phosphorylate proteins essential for cell proliferation, survival, and apoptosis. The PIM kinases are constitutively active and broadly expressed in multiple tissues and up-regulated in various malignancies. We report the discovery of a novel fusion transcript encoding the kinase domain of PIM3 fused to SCO2, a cytochrome c oxidase assembly protein. In transcript sequencing (RNA Seq) of 68 pediatric AML cases, PIM3-SCO2 fusion transcript was computationally identified and experimentally verified in index cases and studied in an independent cohort of pediatric patients with AML. RNA-Seq performed on the Illumina HiSeq in 68 diagnostic specimens from children with AML treated on COG clinical trials. Sequence reads were mapped to human genome using Novoalign. Four computational methods including Defuse, TophatFusion, FusionMap, and Snowshoes-FTD, were utilized to identify fusion transcripts and after filtering to eliminate false positives, fusions were selected based on observation in 2 or more fusion methods and presence in chimerDB. PIM3-SCO2 was identified as an in-frame fusion transcript in 3 cases with Inv(16) and subsequently verified by RT-PCR and Sanger sequencing. Frequency validation was performed by semi-quantitative expression analysis of PIM3-SCO2 expression levels in 235 AML diagnostic specimens as well as 6 normal bone marrow (NBM) controls. PIM3-SCO2 fusion protein was assessed by Western blot on whole cell lysates from cases with the fusion transcript. After verification of the fusion, available whole genome sequencing data in matching cases was interrogated and failed to demonstrate genomic counterpart to this fusion transcript, suggesting that this fusion may be the result of transcriptional read-through; also called transcription-induced chimera (TIC). Such fusion transcripts are generated when genes in proximity on a genome strand are spliced together to generate a chimeric product. Frequency validation studies in 235 diagnostic specimens from COG AAML0531 demonstrated that PIM3-SCO2 fusion transcript was highly prevalent in AML and expressed in 187 of the 235 cases of AML (80%) with variable prevalence across different cytogenetic cohorts, with prevalence of 87% in CBF, 56% in MLL, 79% in normal karyotype, and 70% in those with “other” karyotypes (p Our data show that a novel PIM3-SCO2 fusion transcript, which is likely a transcription-induced chimera of the two gene transcripts, may be involved in normal hematopoietic development whose expression is highly dysregulated in AML. Expression level of this chimeric product is highly variable in childhood AML, is associated with cytogenetic and molecular subsets, and may identify a potential target for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children’s Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ∼22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000. The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age>10 years or WBC>=50,000; p Supervised analysis of HELP profiles between paired relapse/diagnosis samples (n=67) revealed a markedly aberrant DNA methylation signature (1011 probesets, 888 genes, FDR25%, paired t-test), with 70% of the genes hyper- and 30% hypo-methylated in relapse samples. Using a Bayesian predictor and leave-one-out cross validation, this methylation signature could predict a sample as diagnosis or relapse with 95.3% accuracy. When comparing early (=36 months; n=18), we detected a profound hypermethylation signature in early relapse (96.6% of the 610 probesets, 544 genes, FDR25%). Finally, we identified 1800 probesets (1658 genes) as differentially methylated within all cytogenetic subtypes described above compared to the normal preB samples (Dunnett’s test with normal preB as reference, FDR25%). Again the majority (70%) of those genes were hypermethylated in relapse as compared to diagnostic and normal preB. The base-pair resolution and more comprehensive eRRBS methylation analysis for the eight pairs of samples identified 39,679 CpG sites as differentially methylated (dx >25%, FDR Taken together, our genome-wide high resolution DNA methylation analysis on a large cohort of relapsed childhood B-ALL from the COG trial identified unique methylation signatures that correlated with relapse and with specific genetic subsets. Those methylation signatures featured prevailing promoter hypermethylation and to a lesser extent, intrageneic hypomethylation. Epigenetically dysregulated gene networks in those relapse samples involved cell signaling, and embryonic and organismal development. Disclosures: No relevant conflicts of interest to declare.

Abstract 125 Despite identification of genomic alterations in AML, clinically actionable variants are limited. Further, mutations identified in adult AML are less frequent or not found in childhood AML. TARGET AML initiative, a collaboration between National Cancer Institute Office of Cancer Genomics (NCI/OCG) and Children's Oncology Group (COG) provided resources to perform multi-platform analysis of a large number of pediatric AML cases in order to identify therapeutic targets in childhood AML. As part of this effort, diagnostic, remission and relapse specimens from a large cohort of children with AML were subjected to whole genome sequencing (WGS). In addition, all specimens underwent characterization by expression profiling, SNP genotyping, methylation profiling and miRNA sequencing. Specimens from a subset of patients also underwent whole exome sequencing (WES) and whole transcript sequencing (RNA Seq). Here we present data from an interim analysis of the data that identified clinically significant, novel somatic mutations, deletions and translocations involving the ETV6 gene in pediatric patients with AML. Non-silent somatic mutations of ETV6, including missense, splice site mutations and indels were identified by WGS and WES. Large deletions involving ETV6 gene were identified by WGS and SNP/CGH arrays, and novel fusion transcripts (cryptic translocations) involving ETV6 were identified by WGS and RNA Seq. We present the prevalence and clinical significance of genomic alterations of ETV6 that were identified as part of TARGET AML study. WGS and WES data were available from 54 and 22 pediatric AML patients, respectively. In these 76 selected cases, somatic mutations in ETV6 were identified and verified in 3 patients. Frequency validation was performed by sequencing of the entire coding region of the ETV6 gene in diagnostic specimens from 180 children with AML. Somatic mutations of ETV6 gene were identified in 6% of patients, of which none had cytogenetic or molecular risk features. Those with somatic ETV6 mutations had an overall survival (OS) of 20% vs. 76% for those without mutations (p=0.002) with a corresponding relapse risk (RR) of 80% vs. 29% (p=0.004), demonstrating the potential impact of ETV6 mutations in pediatric AML. Deletions involving ETV6 gene (del ETV6) were detected in 3 patients by WGS and confirmed by SNP genotyping array and by fluorescent in situ hybridization (FISH). Subsequent interrogation of the SNP genotyping data identified deletions of 12p13 involving ETV6 in 19/242 patients (8%) ranging from 0.25 Mb to >20 Mb. Those with del ETV6 lacked cytogenetic (−7, or −5/del5q) or molecular (FLT3/ITD) high-risk features, but 50% of those with del ETV6 had core binding factor (CBF) AML. All patients with del ETV6 achieved a morphologic complete remission (CR) and had no evidence of minimal residual disease at the end of induction chemotherapy. Relapse risk for patients with and without del ETV6 was 63% vs. 45% (p=0.3) with a corresponding disease-free survival (DFS) of 32% vs. 53% (p=0.2). Due to the high prevalence of CBF AML in patients with del ETV6, we inquired whether this alteration might impact outcome in patients with CBF AML. In patients with CBF AML, del ETV6 was associated with adverse outcome, where CBF patients with and without del ETV6 had an event-free survival of 0% vs. 63%, respectively (p=0.002). Of the CBF AML patients who achieved an initial CR, those with compared to those without a del ETV6 had a RR of 88% (vs. 38%, p=0.08) with a DFS of 0% (vs. 61%, p=0.009) respectively. Cryptic ETV6 translocations involving at least two distinct translocation partners represented a third class of ETV6 alterations identified by WGS and RNA Seq. FISH evaluation of select patients was available and identified 16 patients with positive FISH for ETV6 variants. Those who had ETV6 alterations by FISH had a median age at diagnosis of 3.7 years. Evaluation of remission induction rate as well as post remission outcome demonstrated that all but 3 of these 16 patients either failed to achieve a CR (N=4) or relapsed after an initial remission (82% failure rate). These data identified varying genomic alterations of ETV6 by whole genome sequencing that identify a significant proportion of pediatric AML cases with adverse outcome who may be targeted for altered therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3519 Pediatric Acute Myeloid Leukemia (AML) offers a unique window into the genesis and progression of hematologic cancers because of its rapid progression and limited confounding factors. As part of the NCI TARGET AML initiative (target.cancer.gov), we report the initial integrative analysis of 58 pediatric AML whole-genome sequences [WGS, performed by Complete Genomics Inc. (CGI)] along with 225 Affymetrix microarrays, clinical cytogenetic and biomarker tests. WGS was performed on matched samples collected at diagnosis and remission from each patient. Candidate variants were identified by CGI and defined as being present only in tumor samples. To focus on the highest confidence variant calls, we developed stringent filters based on a set of 281 true-positive and 44 false-positive verified variants. We identified a total of 629 high-confidence variants in 58 cases (∼ 11 per patient). 99 of these variants occurred in 39 genes (2 to 8 per gene, up to 4 per sample). Analysis of the potential functional consequence of these variants identified 28 as deleterious impacting 17 genes in 21 of the 58 relapsed AMLs, including multiple mutations in AML-associated genes such as KIT, NRAS, PTPN11, and WT1, and several key hematopoietic genes (e.g. IKZF1, GATA2). Using microarray expression data, we identified 1,992 (1,051) genes that were differentially expressed in pediatric AML cases compared to 4 normal bone marrow samples [FDR-adjusted p-value = 0.05 (0.01)]. 249 differentially expressed genes were more than 6 standard deviations away from the control average in more than half of the samples (e.g. WT1, MYCN, miR155), suggesting the existence of a shared set of dysregulated processes across most pediatric AMLs. Network-oriented enrichment analysis using the Bioconductor (http://bioconductor.org) package DEGraph revealed differentially regulated interactions among 3,496 genes, including 1,437 cancer genes and highly enriched interactions involving growth factor/RTK signaling, down-regulated immune processes, and up-regulated transcription and translation. To pinpoint processes specific to AML subtypes, we used the Bioconductor package WGCNA to cluster the 225 microarray expression datasets and align them with clinically-identified cytogenetic and mutation data. We identified five distinct expression clusters. Three of these clusters corresponded to known cytogenetic abnormalities: MLL fusions, t(8;21), and Inv16. The other two clusters were cytogenetically normal, but all members of one cluster carry CEBPA mutations. The expression patterns of cases with t(8;21) and Inv16, while distinct, shared a number of features that distinguished them from samples with MLL abnormalities. For example, in both t(8;21) and Inv16 cases (but not in MLL cases) the fibroblast growth factor (FGF) receptor FGFR1 and the FGF ligand FGF11 were over-expressed compared to control samples. These differences may explain the higher rates of remission associated with Core Binding Factor (CBF) abnormalities. To identify potential relationships between sequence variants and differentially expressed genes, we used the Graphite Bioconductor package to search four publicly available databases (Reactome, NCI PID, KEGG, Biocarta) for known interactions of our candidate deleterious genes. Surprisingly, all but two of our candidate deleterious genes shared many interactors, suggesting they impacted shared processes. The core connected components of the mutation interaction network were all members of the set of dysregulated interactions that we identified by gene expression analysis and included multiple members of well-known pathways implicated in AML and other cancers (e.g. RTK/growth factor signaling, JAK/STAT signaling). Candidate mutations impacted both shared and distinct pathways. Furthermore, within shared pathways, the candidate mutations impacted shared and distinct targets. These findings have important implications for pathway-specific drug targeting. Comparing the interactions of candidate WGS mutations with those of clinically-identified chromosomal abnormalities, a further pattern emerges: CBF and MLL associated gene fusions appeared to impact a different set of genes and processes compared to WGS (presumably second-hit) variants, suggesting complementary roles. This finding has important implications for ongoing research and testing of targeted treatments. Disclosures: No relevant conflicts of interest to declare.

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis . Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.