Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

Cancer vulnerabilities unveiled by genomic loss.

Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy number losses, we performed integrated analyses of genome-wide copy number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy number loss of that gene. These CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes are enriched for spliceosome, proteasome, and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.

Patterns of somatic structural variation in human cancer genomes.

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

The evolutionary history of 2,658 cancers.

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

Tangent normalization for somatic copy-number inference in cancer genome analysis.

MOTIVATION: Somatic copy-number alterations (SCNAs) play an important role in cancer development. Systematic noise in sequencing and array data present a significant challenge to the inference of SCNAs for cancer genome analyses. As part of The Cancer Genome Atlas, the Broad Institute Genome Characterization Center developed the Tangent normalization method to generate copy-number profiles using data from single-nucleotide polymorphism (SNP) arrays and whole-exome sequencing (WES) technologies for over 10 000 pairs of tumors and matched normal samples. Here, we describe the Tangent method, which uses a unique linear combination of normal samples as a reference for each tumor sample, to subtract systematic errors that vary across samples. We also describe a modification of Tangent, called Pseudo-Tangent, which enables denoising through comparisons between tumor profiles when few normal samples are available. RESULTS: Tangent normalization substantially increases signal-to-noise ratios (SNRs) compared to conventional normalization methods in both SNP array and WES analyses. Tangent and Pseudo-Tangent normalizations improve the SNR by reducing noise with minimal effect on signal and exceed the contribution of other steps in the analysis such as choice of segmentation algorithm. Tangent and Pseudo-Tangent are broadly applicable and enable more accurate inference of SCNAs from DNA sequencing and array data. AVAILABILITY AND IMPLEMENTATION: Tangent is available at https://github.com/broadinstitute/tangent and as a Docker image (https://hub.docker.com/r/broadinstitute/tangent). Tangent is also the normalization method for the copy-number pipeline in Genome Analysis Toolkit 4 (GATK4). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The whole-genome landscape of medulloblastoma subtypes.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

Genomic evolution and chemoresistance in germ-cell tumours.

Germ-cell tumours (GCTs) are derived from germ cells and occur most frequently in the testes. GCTs are histologically heterogeneous and distinctly curable with chemotherapy. Gains of chromosome arm 12p and aneuploidy are nearly universal in GCTs, but specific somatic genomic features driving tumour initiation, chemosensitivity and progression are incompletely characterized. Here, using clinical whole-exome and transcriptome sequencing of precursor, primary (testicular and mediastinal) and chemoresistant metastatic human GCTs, we show that the primary somatic feature of GCTs is highly recurrent chromosome arm level amplifications and reciprocal deletions (reciprocal loss of heterozygosity), variations that are significantly enriched in GCTs compared to 19 other cancer types. These tumours also acquire KRAS mutations during the development from precursor to primary disease, and primary testicular GCTs (TGCTs) are uniformly wild type for TP53. In addition, by functional measurement of apoptotic signalling (BH3 profiling) of fresh tumour and adjacent tissue, we find that primary TGCTs have high mitochondrial priming that facilitates chemotherapy-induced apoptosis. Finally, by phylogenetic analysis of serial TGCTs that emerge with chemotherapy resistance, we show how TGCTs gain additional reciprocal loss of heterozygosity and that this is associated with loss of pluripotency markers (NANOG and POU5F1) in chemoresistant teratomas or transformed carcinomas. Our results demonstrate the distinct genomic features underlying the origins of this disease and associated with the chemosensitivity phenotype, as well as the rare progression to chemoresistance. These results identify the convergence of cancer genomics, mitochondrial priming and GCT evolution, and may provide insights into chemosensitivity and resistance in other cancers.

Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset Cancers.

BACKGROUND & AIMS: Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients. METHODS: We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays. RESULTS: We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5-7.7). RHOA activity was reduced by an R5W substitution-the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells. CONCLUSIONS: In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

Exome sequencing of lymphomas from three dog breeds reveals somatic mutation patterns reflecting genetic background.

Lymphoma is the most common hematological malignancy in developed countries. Outcome is strongly determined by molecular subtype, reflecting a need for new and improved treatment options. Dogs spontaneously develop lymphoma, and the predisposition of certain breeds indicates genetic risk factors. Using the dog breed structure, we selected three lymphoma predisposed breeds developing primarily T-cell (boxer), primarily B-cell (cocker spaniel), and with equal distribution of B- and T-cell lymphoma (golden retriever), respectively. We investigated the somatic mutations in B- and T-cell lymphomas from these breeds by exome sequencing of tumor and normal pairs. Strong similarities were evident between B-cell lymphomas from golden retrievers and cocker spaniels, with recurrent mutations in TRAF3-MAP3K14 (28% of all cases), FBXW7 (25%), and POT1 (17%). The FBXW7 mutations recurrently occur in a specific codon; the corresponding codon is recurrently mutated in human cancer. In contrast, T-cell lymphomas from the predisposed breeds, boxers and golden retrievers, show little overlap in their mutation pattern, sharing only one of their 15 most recurrently mutated genes. Boxers, which develop aggressive T-cell lymphomas, are typically mutated in the PTEN-mTOR pathway. T-cell lymphomas in golden retrievers are often less aggressive, and their tumors typically showed mutations in genes involved in cellular metabolism. We identify genes with known involvement in human lymphoma and leukemia, genes implicated in other human cancers, as well as novel genes that could allow new therapeutic options.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

ATARiS: computational quantification of gene suppression phenotypes from multisample RNAi screens.

Genome-scale RNAi libraries enable the systematic interrogation of gene function. However, the interpretation of RNAi screens is complicated by the observation that RNAi reagents designed to suppress the mRNA transcripts of the same gene often produce a spectrum of phenotypic outcomes due to differential on-target gene suppression or perturbation of off-target transcripts. Here we present a computational method, Analytic Technique for Assessment of RNAi by Similarity (ATARiS), that takes advantage of patterns in RNAi data across multiple samples in order to enrich for RNAi reagents whose phenotypic effects relate to suppression of their intended targets. By summarizing only such reagent effects for each gene, ATARiS produces quantitative, gene-level phenotype values, which provide an intuitive measure of the effect of gene suppression in each sample. This method is robust for data sets that contain as few as 10 samples and can be used to analyze screens of any number of targeted genes. We used this analytic approach to interrogate RNAi data derived from screening more than 100 human cancer cell lines and identified HNF1B as a transforming oncogene required for the survival of cancer cells that harbor HNF1B amplifications. ATARiS is publicly available at http://broadinstitute.org/ataris.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Pan-cancer analysis of whole genomes identifies driver rearrangements promoted by LINE-1 retrotransposition.

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.

Genomic and Functional Approaches to Understanding Cancer Aneuploidy.

Aneuploidy, whole chromosome or chromosome arm imbalance, is a near-universal characteristic of human cancers. In 10,522 cancer genomes from The Cancer Genome Atlas, aneuploidy was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. Chromosome arm-level alterations show cancer-specific patterns, including loss of chromosome arm 3p in squamous cancers. We applied genome engineering to delete 3p in lung cells, causing decreased proliferation rescued in part by chromosome 3 duplication. This study defines genomic and phenotypic correlates of cancer aneuploidy and provides an experimental approach to study chromosome arm aneuploidy.

SETD2 Is Recurrently Mutated in Whole-Exome Sequenced Canine Osteosarcoma.

Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species.Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421-31. ©2018 AACR.

Author Correction: Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition.

In the Supplementary Information originally published with this article, a lane was missing in the ß-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.