Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

The Immune Landscape of Cancer.

We performed an extensive immunogenomic analysis of more than 10,000 tumors comprising 33 diverse cancer types by utilizing data compiled by TCGA. Across cancer types, we identified six immune subtypes-wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet, and TGF-ß dominant-characterized by differences in macrophage or lymphocyte signatures, Th1:Th2 cell ratio, extent of intratumoral heterogeneity, aneuploidy, extent of neoantigen load, overall cell proliferation, expression of immunomodulatory genes, and prognosis. Specific driver mutations correlated with lower (CTNNB1, NRAS, or IDH1) or higher (BRAF, TP53, or CASP8) leukocyte levels across all cancers. Multiple control modalities of the intracellular and extracellular networks (transcription, microRNAs, copy number, and epigenetic processes) were involved in tumor-immune cell interactions, both across and within immune subtypes. Our immunogenomics pipeline to characterize these heterogeneous tumors and the resulting data are intended to serve as a resource for future targeted studies to further advance the field.

Genomic basis for RNA alterations in cancer.

Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.

Targeting the oncogene LSF with either the small molecule inhibitor FQI1 or siRNA causes mitotic delays with unaligned chromosomes, resulting in cell death or senescence.

BACKGROUND: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. METHODS: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. RESULTS: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. CONCLUSIONS: These data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.

Landscape of genomic alterations in cervical carcinomas.

Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma-normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour-normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

GATK PathSeq: a customizable computational tool for the discovery and identification of microbial sequences in libraries from eukaryotic hosts.

Summary: We present an updated version of our computational pipeline, PathSeq, for the discovery and identification of microbial sequences in genomic and transcriptomic libraries from eukaryotic hosts. This pipeline is available in the Genome Analysis Toolkit (GATK) as a suite of configurable tools that can report the microbial composition of DNA or RNA short-read sequencing samples and identify unknown sequences for downstream assembly of novel organisms. GATK PathSeq enables sample analysis in minutes at low cost. In addition, these tools are built with the GATK engine and Apache Spark framework, providing robust, rapid parallelization of read quality filtering, host subtraction and microbial alignment in workstation, cluster and cloud environments. Availability and implementation: These tools are available as a part of the GATK at https://github.com/broadinstitute/gatk. Supplementary information: Supplementary data are available at Bioinformatics online.

The tumor virus landscape of AIDS-related lymphomas.

Immunodeficiency dramatically increases susceptibility to cancer as a result of reduced immune surveillance and enhanced opportunities for virus-mediated oncogenesis. Although AIDS-related lymphomas (ARLs) are frequently associated with known oncogenic viruses, many cases contain no known transforming virus. To discover novel transforming viruses, we profiled a set of ARL samples using whole transcriptome sequencing. We determined that Epstein-Barr virus (EBV) was the only virus detected in the tumor samples of this cohort, suggesting that if unidentified pathogens exist in this disease, they are present in <10% of cases or undetectable by our methods. To evaluate the role of EBV in ARL pathogenesis, we analyzed viral gene expression and found highly heterogeneous patterns of viral transcription across samples. We also found significant heterogeneity of viral antigen expression across a large cohort, with many patient samples presenting with restricted type I viral latency, indicating that EBV latency proteins are under increased immunosurveillance in the post-combined antiretroviral therapies era. Furthermore, EBV infection of lymphoma cells in HIV-positive individuals was associated with a distinct host gene expression program. These findings provide insight into the joint host-virus regulatory network of primary ARL tumor samples and expand our understanding of virus-associated oncogenesis. Our findings may also have therapeutic implications, as treatment may be personalized to target specific viral and virus-associated host processes that are only present in a subset of patients.

Genetic modifiers of EGFR dependence in non-small cell lung cancer.

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.

Characterization of HPV and host genome interactions in primary head and neck cancers.

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.

Complementary genomic approaches highlight the PI3K/mTOR pathway as a common vulnerability in osteosarcoma.

Osteosarcoma is the most common primary bone tumor, yet there have been no substantial advances in treatment or survival in three decades. We examined 59 tumor/normal pairs by whole-exome, whole-genome, and RNA-sequencing. Only the TP53 gene was mutated at significant frequency across all samples. The mean nonsilent somatic mutation rate was 1.2 mutations per megabase, and there was a median of 230 somatic rearrangements per tumor. Complex chains of rearrangements and localized hypermutation were detected in almost all cases. Given the intertumor heterogeneity, the extent of genomic instability, and the difficulty in acquiring a large sample size in a rare tumor, we used several methods to identify genomic events contributing to osteosarcoma survival. Pathway analysis, a heuristic analytic algorithm, a comparative oncology approach, and an shRNA screen converged on the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation in osteosarcoma. Osteosarcoma cell lines are responsive to pharmacologic and genetic inhibition of the PI3K/mTOR pathway both in vitro and in vivo.

Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome.

BACKGROUND: Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin. METHODS: We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls. RESULTS: DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease. CONCLUSIONS: We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen. (Funded by the National Cancer Institute and others.)

Validation and calibration of next-generation sequencing to identify Epstein-Barr virus-positive gastric cancer in The Cancer Genome Atlas.

The Epstein-Barr virus (EBV)-positive subtype of gastric adenocarcinoma is conventionally identified by in situ hybridization (ISH) for viral nucleic acids, but next-generation sequencing represents a potential alternative. We therefore determined normalized EBV read counts by whole-genome, whole-exome, mRNA and miRNA sequencing for 295 fresh-frozen gastric tumor samples. Formalin-fixed, paraffin-embedded tissue sections were retrieved for ISH confirmation of 13 high-EBV and 11 low-EBV cases. In pairwise comparisons, individual samples were either concordantly high or concordantly low by all genomic methods for which data were available. Empiric cutoffs of sequencing counts identified 26 (9 %) tumors as EBV positive. EBV positivity or negativity by molecular testing was confirmed by EBER-ISH in all but one tumor evaluated by both approaches (kappa = 0.91). EBV-positive gastric tumors can be accurately identified by quantifying viral sequences in genomic data. Simultaneous analyses of human and viral DNA, mRNA and miRNA could streamline tumor profiling for clinical care and research.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.

The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

DNA copy number analysis of metastatic urothelial carcinoma with comparison to primary tumors.

BACKGROUND: To date, there have been no reports characterizing the genome-wide somatic DNA chromosomal copy-number alteration landscape in metastatic urothelial carcinoma. We sought to characterize the DNA copy-number profile in a cohort of metastatic samples and compare them to a cohort of primary urothelial carcinoma samples in order to identify changes that are associated with progression from primary to metastatic disease. METHODS: Using molecular inversion probe array analysis we compared genome-wide chromosomal copy-number alterations between 30 metastatic and 29 primary UC samples. Whole transcriptome RNA-Seq analysis was also performed in primary and matched metastatic samples which was available for 9 patients. RESULTS: Based on a focused analysis of 32 genes in which alterations may be clinically actionable, there were significantly more amplifications/deletions in metastases (8.6% vs 4.5%, p < 0.001). In particular, there was a higher frequency of E2F3 amplification in metastases (30% vs 7%, p = 0.046). Paired primary and metastatic tissue was available for 11 patients and 3 of these had amplifications of potential clinical relevance in metastases that were not in the primary tumor including ERBB2, CDK4, CCND1, E2F3, and AKT1. The transcriptional activity of these amplifications was supported by RNA expression data. CONCLUSIONS: The discordance in alterations between primary and metastatic tissue may be of clinical relevance in the era of genomically directed precision cancer medicine.

Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

A comprehensive clinically informed map of dependencies in cancer cells and framework for target prioritization.

Genetic screens in cancer cell lines inform gene function and drug discovery. More comprehensive screen datasets with multi-omics data are needed to enhance opportunities to functionally map genetic vulnerabilities. Here, we construct a second-generation map of cancer dependencies by annotating 930 cancer cell lines with multi-omic data and analyze relationships between molecular markers and cancer dependencies derived from CRISPR-Cas9 screens. We identify dependency-associated gene expression markers beyond driver genes, and observe many gene addiction relationships driven by gain of function rather than synthetic lethal effects. By combining clinically informed dependency-marker associations with protein-protein interaction networks, we identify 370 anti-cancer priority targets for 27 cancer types, many of which have network-based evidence of a functional link with a marker in a cancer type. Mapping these targets to sequenced tumor cohorts identifies tractable targets in different cancer types. This target prioritization map enhances understanding of gene dependencies and identifies candidate anti-cancer targets for drug development.