Douglas Voet

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.

The Mutational Landscape of Head and Neck Squamous Cell Carcinoma

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

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.

Initial genome sequencing and analysis of multiple myeloma

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

The genomic complexity of primary human prostate cancer

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, ‘copy-neutral’) rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2–ERG, but inversely correlated with these regions in tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies and engage prostate tumorigenic mechanisms.

Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer

Prostate cancer is the second most common cancer in men worldwide and causes over 250,000 deaths each year. Overtreatment of indolent disease also results in significant morbidity. Common genetic alterations in prostate cancer include losses of NKX3.1 (8p21) and PTEN (10q23), gains of AR (the androgen receptor gene) and fusion of ETS family transcription factor genes with androgen-responsive promoters. Recurrent somatic base-pair substitutions are believed to be less contributory in prostate tumorigenesis but have not been systematically analyzed in large cohorts. Here, we sequenced the exomes of 112 prostate tumor and normal tissue pairs. New recurrent mutations were identified in multiple genes, including MED12 and FOXA1. SPOP was the most frequently mutated gene, with mutations involving the SPOP substrate-binding cleft in 6–15% of tumors across multiple independent cohorts. Prostate cancers with mutant SPOP lacked ETS family gene rearrangements and showed a distinct pattern of genomic alterations. Thus, SPOP mutations may define a new molecular subtype of prostate cancer.

Melanoma genome sequencing reveals frequent PREX2 mutations

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5–55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)—a PTEN-interacting protein and negative regulator of PTEN in breast cancer—as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

Somatic mutation of CDKN1B in small intestine neuroendocrine tumors

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and genome-sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NETs.

FireCloud, a scalable cloud-based platform for collaborative genome analysis: Strategies for reducing and controlling costs

FireCloud, one of three NCI Cloud Pilots, is a collaborative genome analysis platform built on a cloud computing infrastructure. FireCloud aims to solve the many challenges presented by the increasingly large data sets and computing requirements employed in cancer research. However, cost uncertainty associated with cloud computing’s pay-as-you-go model is proving to be a barrier to adoption of cloud computing. In this paper we present guidelines for optimizing workflows to minimize cost and reduce latency. Our guidelines include: (i) dynamic disk sizing to efficiently utilize virtual disks; (ii) tuned provisioning of virtual machines (VMs) using a performance monitoring tool; (iii) taking advantage of steep price discounts of preemptible VMs; and (iv) utilizing the optimal parallelization of a task’s workload.

Abstract 4017: Dissecting the clonal hierarchy of cancer-driving genomic lesions.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Characterizing the genomic evolution of cancer is critical to understanding disease progression and identifying potential therapeutic targets. By examining the clonal hierarchy of genomic lesions in common tumors, it would be possible to reconstruct the path of oncogenic events that drive carcinogenesis. Reliable assessment of such paths from high-throughput genome sequencing data is complicated by the admixture of normal DNA in tumor samples and by reduced data signal for highly subclonal events. We introduce an approach that exploits individuals’ genetic background by using the abundant germline SNP genotype data provided by whole genome sequence coverage to assess the clonality of genomic alterations, including copy number changes, rearrangements, and point mutations. We developed a novel algorithm, CLONET (CLONality Estimate in Tumors), which analyzes patient-specific heterozygous SNP loci (informative SNPs) and mono-allelic somatic deletions to assess levels of stromal DNA admixture and infer the clonal status of each aberration. For every mono allelic deletion, CLONET assesses the allelic fractions of informative SNPs to determine the apparent proportion of normal cells DNA. Next, through a conservative use of simulation-based error estimates, deletions with the lowest proportions of normal DNA reads are considered clonal. For point mutations, the tumor allelic fraction is corrected for stromal DNA admixture level and subclonality is inferred when it differs significantly from the expected value for clonal lesions. Similarly, the proportions of reads that span each side of a putative breakpoint involved in a rearrangement are matched against the expected values. CLONET also addresses tumor aneuploidy by searching for chromosomes with coverage and allelic fractions of informative SNPs not consistent with a diploid genome. CLONET was tested on 55 whole genome sequences from prostate cancers, a highly heterogeneous tumor type, to catalogue the accumulation of somatic alterations during oncogenesis and progression. In 98% of the cases CLONET made confident assessment of admixture and clonality. We observed consistent clonal lesions involving NKX3-1, the 3Mb region between TMPRSS2 and ERG and FOXP1, as well as early point mutations in SPOP and FOXA1. Overall, we observed a higher rate of subclonal protein-coding point mutation versus deletions (p-value < 10−7). We validated this approach by IHC and FISH for predicted clonal and sub-clonal events. A predicted subclonal homozygous deletion of CHD1 was confirmed by FISH that demonstrated the presence of both nuclei with homozygous and with hemizygous deletion of CHD1. Finally, to assess the general validity of CLONET, we analyzed data from 53 additional tumor genomes, including 25 melanomas and 28 lung adenocarcinomas. In summary, our results imply the existence of consensus paths of tumor carcinogenesis that favor dysregulation of cancer genes in a defined sequence. Citation Format: Davide Prandi, Sylvan C. Baca, Michael S. Lawrence, Juan Miguel Mosquera, Alessandro Romanel, Yotam Drier, Kyung Park, Naoki Kitabayashi, Theresa Y. MacDonald, Eliezer Van Allen, Gregory V. Kryukov, Jean-Philippe Theurillat, T. David Soong, Elizabeth Nickerson, Daniel Auclair, Ashutosh Tewari, Himisha Beltran, Robert C. Onofrio, Gunther Boysen, Candace Guiducci, Christopher E. Barbieri, Kristian Cibulskis, Andrey Sivachenko, Scott L. Carter, Gordon Saksena, Douglas Voet, Alex H. Ramos, Wendy Winckler, Michelle Cipicchio, Kristin Ardlie, Philip W. Kantoff, Michael F. Berger, Stacey B. Gabriel, Todd R. Golub, Matthew Meyerson, Eric S. Lander, Olivier Elemento, Gad Getz, Francesca Demichelis, Mark A. Rubin, Levi A. Garraway. Dissecting the clonal hierarchy of cancer-driving genomic lesions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2013-4017