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.

Insertions and Deletions Target Lineage-Defining Genes in Human Cancers.

Certain cell types function as factories, secreting large quantities of one or more proteins that are central to the physiology of the respective organ. Examples include surfactant proteins in lung alveoli, albumin in liver parenchyma, and lipase in the stomach lining. Whole-genome sequencing analysis of lung adenocarcinomas revealed noncoding somatic mutational hotspots near VMP1/MIR21 and indel hotspots in surfactant protein genes (SFTPA1, SFTPB, and SFTPC). Extrapolation to other solid cancers demonstrated highly recurrent and tumor-type-specific indel hotspots targeting the noncoding regions of highly expressed genes defining certain secretory cellular lineages: albumin (ALB) in liver carcinoma, gastric lipase (LIPF) in stomach carcinoma, and thyroglobulin (TG) in thyroid carcinoma. The sequence contexts of indels targeting lineage-defining genes were significantly enriched in the AATAATD DNA motif and specific chromatin contexts, including H3K27ac and H3K36me3. Our findings illuminate a prevalent and hitherto unrecognized mutational process linking cellular lineage and cancer.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Impact of Lineage Plasticity to and from a Neuroendocrine Phenotype on Progression and Response in Prostate and Lung Cancers.

Intratumoral heterogeneity can occur via phenotype transitions, often after chronic exposure to targeted anticancer agents. This process, termed lineage plasticity, is associated with acquired independence to an initial oncogenic driver, resulting in treatment failure. In non-small cell lung cancer (NSCLC) and prostate cancers, lineage plasticity manifests when the adenocarcinoma phenotype transforms into neuroendocrine (NE) disease. The exact molecular mechanisms involved in this NE transdifferentiation remain elusive. In small cell lung cancer (SCLC), plasticity from NE to nonNE phenotypes is driven by NOTCH signaling. Herein we review current understanding of NE lineage plasticity dynamics, exemplified by prostate cancer, NSCLC, and SCLC.

A landscape of driver mutations in melanoma.

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.

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.

Next-generation characterization of the Cancer Cell Line Encyclopedia.

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.

Integrated mutational landscape analysis of uterine leiomyosarcomas.

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

SvABA: genome-wide detection of structural variants and indels by local assembly.

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50-300 bp) SVs.

Mutational heterogeneity in cancer and the search for new cancer-associated genes.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour-normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour-normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Signatures of mutational processes in human cancer.

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, 'kataegis', is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

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.

Common genetic variants on 5p14.1 associate with autism spectrum disorders.

Autism spectrum disorders (ASDs) represent a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal communication, impairment of social interaction, and restricted and repetitive patterns of interests and behaviour. To identify common genetic risk factors underlying ASDs, here we present the results of genome-wide association studies on a cohort of 780 families (3,101 subjects) with affected children, and a second cohort of 1,204 affected subjects and 6,491 control subjects, all of whom were of European ancestry. Six single nucleotide polymorphisms between cadherin 10 (CDH10) and cadherin 9 (CDH9)-two genes encoding neuronal cell-adhesion molecules-revealed strong association signals, with the most significant SNP being rs4307059 (P = 3.4 x 10(-8), odds ratio = 1.19). These signals were replicated in two independent cohorts, with combined P values ranging from 7.4 x 10(-8) to 2.1 x 10(-10). Our results implicate neuronal cell-adhesion molecules in the pathogenesis of ASDs, and represent, to our knowledge, the first demonstration of genome-wide significant association of common variants with susceptibility to ASDs.

Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.

Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with approximately 550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 x 10(-3)). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 x 10(-3)). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 x 10(-6)). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.