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To discover driver fusions beyond canonical exon-to-exon chimeric transcripts, we develop CICERO, a local assembly-based algorithm that integrates RNA-seq read support with extensive annotation for candidate ranking. CICERO outperforms commonly used methods, achieving a 95% detection rate for 184 independently validated driver fusions including internal tandem duplications and other non-canonical events in 170 pediatric cancer transcriptomes. Re-analysis of TCGA glioblastoma RNA-seq unveils previously unreported kinase fusions (KLHL7-BRAF) and a 13% prevalence of EGFR C-terminal truncation. Accessible via standard or cloud-based implementation, CICERO enhances driver fusion detection for research and precision oncology. The CICERO source code is available at https://github.com/stjude/Cicero.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood cancer-related death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories, and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking, and xenografting to formally define clonal structure, we identified 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. CREBBP, NOTCH1, and RAS signaling mutations arose from diagnosis subclones, whereas variants in NCOR2, USH2A, and NT5C2 were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%), or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using droplet digital PCR at levels comparable with orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones. Significance: This study defines the landscape of mutations that preexist and arise after commencement of ALL therapy and shows that relapse may be propagated from ancestral, major, or minor clones at initial diagnosis. A subset of cases exhibits hypermutation that results in expression of neoepitopes that may be substrates for immunotherapeutic intervention. See related video: https://vimeo.com/442838617 See related commentary by Ogawa, p. 21. See related article by S. Dobson et al . This article is highlighted in the In This Issue feature, p. 5

Background RNA editing leads to post-transcriptional variation in protein sequences and has important biological implications. We sought to elucidate the landscape of RNA editing events across pediatric cancers. Methods Using RNA-Seq data mapped by a pipeline designed to minimize mapping ambiguity, we investigated RNA editing in 711 pediatric cancers from the St. Jude/Washington University Pediatric Cancer Genome Project focusing on coding variants which can potentially increase protein sequence diversity. We combined de novo detection using paired tumor DNA-RNA data with analysis of known RNA editing sites. Results We identified 722 unique RNA editing sites in coding regions across pediatric cancers, 70% of which were nonsynonymous recoding variants. Nearly all editing sites represented the canonical A-to-I (n = 706) or C-to-U sites (n = 14). RNA editing was enriched in brain tumors compared to other cancers, including editing of glutamate receptors and ion channels involved in neurotransmitter signaling. RNA editing profiles of each pediatric cancer subtype resembled those of the corresponding normal tissue profiled by the Genotype-Tissue Expression (GTEx) project. Conclusions In this first comprehensive analysis of RNA editing events in pediatric cancer, we found that the RNA editing profile of each cancer subtype is similar to its normal tissue of origin. Tumor-specific RNA editing events were not identified indicating that successful immunotherapeutic targeting of RNA-edited peptides in pediatric cancer should rely on increased antigen presentation on tumor cells compared to normal but not on tumor-specific RNA editing per se.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors, and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor–normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. Significance: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers. This article is highlighted in the In This Issue feature, p. 2945

Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes. To better understand neuroblastoma pathogenesis, here we analyze whole-genome, whole-exome and/or transcriptome data from 702 neuroblastoma samples. Forty percent of samples harbor at least one recurrent driver gene alteration and most aberrations, including MYCN, ATRX, and TERT alterations, differ in frequency by age. MYCN alterations occur at median 2.3 years of age, TERT at 3.8 years, and ATRX at 5.6 years. COSMIC mutational signature 18, previously associated with reactive oxygen species, is the most common cause of driver point mutations in neuroblastoma, including most ALK and Ras-activating variants. Signature 18 appears early and is continuous throughout disease evolution. Signature 18 is enriched in neuroblastomas with MYCN amplification, 17q gain, and increased expression of mitochondrial ribosome and electron transport-associated genes. Recurrent FGFR1 variants in six patients, and ALK N-terminal structural alterations in five samples, identify additional patients potentially amenable to precision therapy., Genomic analysis of neuroblastoma has revealed important disease etiology. In this study, the authors assembled whole genome, exome and transcriptome data from over 700 neuroblastomas and identified molecular signatures correlated with age, and rare, potentially targetable variants overlooked in smaller cohorts.

Cancer genomics has illuminated a wide spectrum of genes and core molecular processes contributing to human malignancy. Still, the genetic and molecular basis of many cancers remains only partially explained. Genetic predisposition accounts for 5-10% of cancer diagnoses(1,2) and genetic events cooperating with known somatic driver events are poorly understood. Analyzing established cancer predisposition genes in medulloblastoma (MB), a malignant childhood brain tumor, we recently identified pathogenic germline variants that account for 5% of all MB patients(3). Here, by extending our previous analysis to include all protein-coding genes, we discovered and replicated rare germline loss-of-function (LoF) variants across Elongator Complex Protein 1 (ELP1) on 9q31.3 in 15% of pediatric MB(SHH) cases, thus implicating ELP1 as the most common MB predisposition gene and increasing genetic predisposition to 40% for pediatric MB(SHH). Inheritance was verified based on parent-offspring and pedigree analysis, which identified two families with a history of pediatric MB. ELP1-associated MBs were restricted to the molecular SHHα subtype(4) and were characterized by universal biallelic inactivation of ELP1 due to somatic loss of chromosome 9q. The majority of ELP1-associated MBs exhibited co-occurring somatic PTCH1 (9q22.32) alterations, suggesting that ELP1-deficiency predisposes to tumor development in combination with constitutive activation of SHH signaling. ELP1 is an essential subunit of the evolutionary conserved Elongator complex, whose primary function is to enable efficient translational elongation through tRNA modifications at the wobble (U(34)) position(5,6). Biochemical, transcriptional, and proteomic analyses revealed that ELP1-associated MB(SHH) are characterized by a destabilized core Elongator complex, loss of Elongator-dependent tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response (UPR), consistent with deregulation of protein homeostasis due to Elongator-deficiency in model systems(7–9). Our findings suggest that genetic predisposition to proteome instability is a previously underappreciated determinant in the pathogenesis of pediatric brain cancer. These results provide a strong rationale for further investigating the role of protein homeostasis in other cancer types and potential opportunities for novel therapeutic interference.

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing., Clinical oncology is rapidly adopting next-generation sequencing technology for nucleotide variant and indel detection. Here the authors present a three-platform approach (whole-genome, whole-exome, and whole-transcriptome) in pediatric patients for the detection of diverse types of germline and somatic variants.

Motivation Reliable identification of expressed somatic insertions/deletions (indels) is an unmet need due to artifacts generated in PCR-based RNA-Seq library preparation and the lack of normal RNA-Seq data, presenting analytical challenges for discovery of somatic indels in tumor transcriptome. Results We present RNAIndel, a tool for predicting somatic, germline and artifact indels from tumor RNA-Seq data. RNAIndel leverages features derived from indel sequence context and biological effect in a machine-learning framework. Except for tumor samples with microsatellite instability, RNAIndel robustly predicts 88–100% of somatic indels in five diverse test datasets of pediatric and adult cancers, even recovering subclonal (VAF range 0.01–0.15) driver indels missed by targeted deep-sequencing, outperforming the current best-practice for RNA-Seq variant calling which had 57% sensitivity but with 14 times more false positives. Availability and implementation RNAIndel is freely available at https://github.com/stjude/RNAIndel. Supplementary information Supplementary data are available at Bioinformatics online.

The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.

Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult1,2,3,4 but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues5. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.

Context: The genomic characterization of acute myeloid leukemia (AML) in adults, both at diagnosis and relapse, has been extensively reported. Previous work has shown a clear difference between AML in adults and children, yet the genetic changes associated with relapsed disease in children have yet to be fully described. Objective: To elucidate the genetic background of pediatric AML at relapse and identify gene alterations related to poor prognosis. Design: Transcriptomic and genetic analyses were performed on relapsed AML from multiple clinical studies held in St. Jude Children’s Research Hospital from 2002 to 2020. No blinding was performed in this study. Patients or Other Participants: RNA sequencing was performed from 136 patients (median age of 9.2 years) with relapsed pediatric AML. Ninety-one of these samples were also studied by tumor–normal-paired whole-genome sequencing. Additional transcriptomic data from pediatric AML at diagnosis (n=417) were also obtained from previous reports to define the molecular alterations common in relapsed pediatric AML. The clinical outcome of each molecular category is confirmed using clinical data of the diagnosis cohort of the TARGET pediatric AML study. Results: Genomic profiling of relapsed pediatric AML demonstrated an increase in KMT2A (n=36, 26.5%) and NUP98 rearrangements (n=18, 13.2%) when compared to de novo pediatric AML, as well as an increase in WT1 mutations (n=33, 24.3%). Comprehensive characterization of pediatric AML with extension RNA transcriptome data revealed 18 molecular categories with unique expression patterns and mutually exclusive gene alteration patterns. These molecular categories included rare categories with structural variants involving oncogenes of MECOM (n=3, 2.2%) or BCL11B (n=2, 1.5%), which led to the upregulation and allele-specific expression (ASE) of each gene. The entire molecular categories showed unique distribution in the relapse cohort compared to the TARGET cohort (p Conclusions: Integrated genomic profiling of pediatric AML identified unique molecular categories associated with relapsed disease and poor outcome.

Genetic alterations that activate NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors, are hallmarks of T-lineage acute lymphoblastic leukemia (T-ALL), but detailed genome-wide sequencing of large T-ALL cohorts has not been carried out. Using integrated genomic analysis of 264 T-ALL cases, we identified 106 putative driver genes, half of which had not previously been described in childhood T-ALL (for example, CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We describe new mechanisms of coding and noncoding alteration and identify ten recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOXA1 deregulated ALL, PTPN2 mutations in TLX1 deregulated T-ALL, and PIK3R1/PTEN mutations in TAL1 deregulated ALL, which suggests that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. Significance: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse. See related video: https://vimeo.com/442838617 See related article by E. Waanders et al .

We developed cis-X, a computational method for discovering regulatory noncoding variants in cancer by integrating whole-genome and transcriptome sequencing data from a single cancer sample. cis-X first finds aberrantly cis-activated genes that exhibit allele-specific expression accompanied by an elevated outlier expression. It then searches for causal noncoding variants that may introduce aberrant transcription factor binding motifs or enhancer hijacking by structural variations. Analysis of 13 T-lineage acute lymphoblastic leukemias identified a recurrent intronic variant predicted to cis-activate the TAL1 oncogene, a finding validated in vivo by chromatin immunoprecipitation sequencing of a patient-derived xenograft. Candidate oncogenes include the prolactin receptor PRLR activated by a focal deletion that removes a CTCF-insulated neighborhood boundary. cis-X may be applied to pediatric and adult solid tumors that are aneuploid and heterogeneous. In contrast to existing approaches, which require large sample cohorts, cis-X enables the discovery of regulatory noncoding variants in individual cancer genomes.

Reliable identification of expressed somatic insertion/deletion (indels) is an unmet demand due to artifacts generated in PCR-based RNA-Seq library preparation and the lack of normal RNA-Seq data, presenting analytical challenges for discovery of somatic indels in tumor trasncriptome.By implementing features characterized by PCR-free whole-genome and whole-exome sequencing into a machine-learning framework, we present RNAIndel, a tool for predicting somatic, germline and artifact indels from tumor RNA-Seq data alone. RNAIndel robustly predicts 87□93% of somatic indels from 235 samples with heterogeneous conditions, even recovering subclonal (VAF range 0.01–0.15) driver indels missed by targeted deep-sequencing, outperforming the current best-practice for RNA-Seq variant calling which had 57% sensitivity but with 12 times more false positives.RNAIndel is freely available at https://github.com/stjude/RNAIndelContactjinghui.zhang@stjude.org

Variant interpretation in the era of massively parallel sequencing is challenging. Although many resources and guidelines are available to assist with this task, few integrated end-to-end tools exist. Here, we present the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE), a web- and cloud-based platform for annotation, identification, and classification of variations in known or putative disease genes. Starting from a set of variants in variant call format (VCF), variants are annotated, ranked by putative pathogenicity, and presented for formal classification using a decision-support interface based on published guidelines from the American College of Medical Genetics and Genomics (ACMG). The system can accept files containing millions of variants and handle single-nucleotide variants (SNVs), simple insertions/deletions (indels), multiple-nucleotide variants (MNVs), and complex substitutions. PeCanPIE has been applied to classify variant pathogenicity in cancer predisposition genes in two large-scale investigations involving >4000 pediatric cancer patients and serves as a repository for the expert-reviewed results. PeCanPIE was originally developed for pediatric cancer but can be easily extended for use for nonpediatric cancers and noncancer genetic diseases. Although PeCanPIE's web-based interface was designed to be accessible to non-bioinformaticians, its back-end pipelines may also be run independently on the cloud, facilitating direct integration and broader adoption. PeCanPIE is publicly available and free for research use.

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosisin the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis andTP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition.This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia. Refereed/Peer-reviewed

The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.

GenomePaint (https://genomepaint.stjude.cloud/) is an interactive visualization platform for whole-genome, whole-exome, transcriptome, and epigenomic data of tumor samples. Its design captures the inter-relatedness between DNA variations and RNA expression, supporting in-depth exploration of both individual cancer genomes and full cohorts. Regulatory non-coding variants can be inspected and analyzed along with coding variants, and their functional impact further explored by examining 3D genome data from cancer cell lines. Further, GenomePaint correlates mutation and expression patterns with patient outcomes, and supports custom data upload. We used GenomePaint to unveil aberrant splicing that disrupts the RING domain of CREBBP, discover cis-activation of the MYC oncogene by duplication of the NOTCH1-MYC enhancer in B-lineage acute lymphoblastic leukemia, and explore the inter- and intra-tumor heterogeneity at EGFR in adult glioblastomas. These examples demonstrate that deep multi-omics exploration of individual cancer genomes enabled by GenomePaint can lead to biological insights for follow-up validation.

Gene fusions are important biomarkers for cancer diagnosis, subtype classification and therapeutic decision-making. While fusion detection using RNA-seq data has become a standard practice, existing computational methods primarily focus on identifying canonical exon-to-exon fusions. However, more complex events such as multi-partner fusions, truncations, enhancer hijacking and internal tandem duplications (ITD) can also lead to abnormal function or aberrant transcription of cancer driver genes. To aid discovery of complex and diverse driver fusions, we developed CICERO (CICERO Is Clipping Extended for RNA Optimization), a local assembly-based algorithm that integrates RNA-seq reads bearing aberrant mapping signatures with extensive annotation for ranking candidate fusions. Our benchmark data set, designed to support the main application of RNA-seq fusion analysis, consists of 184 driver fusions from 170 pediatric leukemia, solid tumor and brain tumor detected by paired tumor-normal WGS and orthogonally validated by capture sequencing, RT-PCR and/or FISH. CICERO detected 95% of these fusions with an average ranking of 1.9, whereas ChimeraScan, deFuse, FusionCatcher and STAR-Fusion detected only 63%, 66%, 77% and 63% with an average ranking of 37.0, 9.0, 18.1 and 4.4, respectively. Notably, events such as ITD and rearrangements involving the highly repetitive IGH locus were detected almost exclusively by CICERO. Our re-analysis of 167 RNA-seq data from the TCGA Glioblastoma Multiforme (GBM) cohort unveiled 158 fusions of cancer genes that were not reported previously. These include kinase fusions (KLHL7-BRAF), ITD of EGFR kinase domain and a 13% prevalence of EGFR C-terminal truncation compared to the 6% reported by the TCGA Network. CICERO has greatly improved our ability to discover non-canonical fusions which are overlooked by existing fusion detection methods, and has been used to analyze >2,000 RNA-seq samples generated by the two largest pediatric cancer genomics initiatives: the St. Jude/Washington University Pediatric Cancer Genome Project (PCGP) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project. We anticipate that CICERO will also improve fusion analysis for adult cancer RNA-seq data, as demonstrated through our re-analysis of TCGA-GBM and our recent discovery of MAP3K8 C-terminal truncation fusion in 2% of TCGA melanoma samples. CICERO is accessible via standard (https://github.com/stjude/Cicero) or cloud-based (https://platform.stjude.cloud/tools/rapid_rna-seq) implementation. To further improve accuracy, fusions predicted by CICERO can be curated by FusionEditor (https://proteinpaint.stjude.org/FusionEditor/), an interactive viewer allowing inspection of protein domains involved in the fusion and the gene expression status of fusion-positive samples. Citation Format: Liqing Tian, Yongjin Li, Michael N. Edmonson, Xin Zhou, Scott Newman, Clay McLeod, Yu Liu, Bo Tang, Michael C. Rusch, John Easton, Jing Ma, Austyn Trull, J. Robert Michael, Andrew Thrasher, Charles Mullighan, Suzanne J. Baker, James R. Downing, David W. Ellison, Jinghui Zhang. CICERO: An accurate method for detecting complex and diverse driver fusions using cancer transcriptome sequencing (RNA-seq) data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5478.

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

Purpose Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB). Patients and Methods Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available. Results A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH–MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses. Conclusion Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH–MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.

Ewing sarcoma is a primary bone tumor initiated by EWSR1–ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. Significance: Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers. Cancer Discov; 4(11); 1342–53. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1243

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathologic including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI, and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2 which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain of function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor (MEP) gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathologic drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

Pediatric high-grade glioma (HGG) is a devastating disease with a less than 20% survival rate 2 years after diagnosis. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs), by whole-genome, whole-exome and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPGs (32%), in addition to previously reported frequent somatic mutations in histone H3 genes, TP53 and ATRX, in both DIPGs and NBS-HGGs. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, NTRK2 and NTRK3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase-RAS-PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59% of pediatric HGGs, respectively, including in DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

SummaryRhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring. Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. Disclosures Mullighan: Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.

Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include: 1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples. 2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD. 3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care. 4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD. Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24-31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32-39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving subunits of the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n=87) and adult (n=78) samples, including cases with RUNX1-RUNX1T1 (n=85) or CBFB-MYH11 (n=80) rearrangements, by whole-genome or whole-exome sequencing. In addition to previously reported somatic mutations in the Ras signaling pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a recurrent and previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated a remarkably different spectrum of cooperating mutations as RUNX1-RUNX1T1 cases harbored recurrent somatic mutations in DHX15 and ZBTB7A, as well as an enrichment of somatic mutations in epigenetic regulators, including ASXL2, and in components of the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscape of cooperating mutations between these related AML subtypes.

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n=22), diffuse oligodendroglial tumors (d-OTs; n=20), diffuse astrocytomas (DAs; n=17), angiocentric gliomas (n=15), and gangliogliomas (n=17). Most LGNTs (84%) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82% of DNETs and 40% of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87%), and MYB fusion genes were the most common genetic alteration in DAs (41%). A BRAF:p.V600E mutation was present in 35% of gangliogliomas and 18% of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78% of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

BackgroundThe prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. MethodsIn 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data...

Summary Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

ERIC, the Enteropathogen Resource Integration Center (www.ericbrc.org), is a new web portal serving as a rich source of information about enterobacteria on the NIAID established list of Select Agents related to biodefense - diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Yersinia enterocolitica and Yersinia pestis. More than 30 genomes have been completely sequenced, many more exist in draft form and additional projects are underway. These organisms are increasingly the focus of studies using high-throughput experimental technologies and computational approaches. This wealth of data provides unprecedented opportunities for understanding the workings of basic biological systems and discovery of novel targets for development of vaccines, diagnostics and therapeutics. ERIC brings information together from disparate sources and supports data comparison across different organisms, analysis of varying data types and visualization of analyses in human and computer-readable formats. © 2007 The Author(s).

There is incomplete understanding of genetic heterogeneity and clonal evolution during cancer progression. Here we use deep whole-exome sequencing to describe the clonal architecture and evolution of 20 pediatric B-acute lymphoblastic leukaemias from diagnosis to relapse. We show that clonal diversity is comparable at diagnosis and relapse and clonal survival from diagnosis to relapse is not associated with mutation burden. Six pathways were frequently mutated, with NT5C2, CREBBP, WHSC1, TP53, USH2A, NRAS and IKZF1 mutations enriched at relapse. Half of the leukaemias had multiple subclonal mutations in a pathway or gene at diagnosis, but mostly with only one, usually minor clone, surviving therapy to acquire additional mutations and become the relapse founder clone. Relapse-specific mutations in NT5C2 were found in nine cases, with mutations in four cases being in descendants of the relapse founder clone. These results provide important insights into the genetic basis of treatment failure in ALL and have implications for the early detection of mutations driving relapse., Genetic heterogeneity and clonal evolution contribute to cancer progression. Here Ma et al. use deep whole-exome sequencing to identify recurrently mutated pathways and clonal architecture in pediatric acute lymphoblastic leukaemia, shedding light on the evolutionary trajectory from diagnosis to relapse

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

ASAP is a comprehensive web-based system for community genome annotation and analysis. ASAP is being used for a large-scale effort to augment and curate annotations for genomes of enterobacterial pathogens and for additional genome sequences. New tools, such as the genome alignment program Mauve, have been incorporated into ASAP in order to improve display and analysis of related genomes. Recent improvements to the database and challenges for future development of the system are discussed. ASAP is available on the web at https://asap.ahabs. wisc.edu/asap/logon.php.