Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–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. Here, 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 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) 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 allow true cancer genes to rise to attention.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

We developed a computational method (ABSOLUTE) that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. ABSOLUTE can detect subclonal heterogeneity, somatic homozygosity, and calculate statistical sensitivity to detect specific aberrations. We used ABSOLUTE to analyze ovarian cancer data and identified pervasive subclonal somatic point mutations. In contrast, mutations occurring in key tumor suppressor genes, TP53 and NF1 were predominantly clonal and homozygous, as were mutations in a candidate tumor suppressor gene, CDK12. Analysis of absolute allelic copy-number profiles from 3,155 cancer specimens revealed that genome-doubling events are common in human cancer, and likely occur in already aneuploid cells. By correlating genome-doubling status with mutation data, we found that homozygous mutations in NF1 occurred predominantly in non-doubled samples. This finding suggests that genome doubling influences the pathways of tumor progression, with recessive inactivation being less common after genome doubling.

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., the most common primary bone tumor, is an aggressive cancer that affects children, adolescents, and young adults. In contrast to the improvements in 5-year overall survival for childhood cancers from 58% to 82% in the past three decades, the overall survival for pediatric OS has remained static over that same time period at 60% (1, 2).Predisposition to OS is associated with germline syndromes, including hereditary retinoblastoma and Li-Fraumeni syndrome (3, 4). OS is also seen in syndromes with mutations in RECQ helicases and SQSTM1 (5, 6). However, most cases of OS develop sporadically and are characterized by complex genomics. The first genome-wide association study conducted in OS only identified two susceptibility loci implicating one gene, GRM4, a glutamate receptor (7).Linkage with hereditary retinoblastoma and Li Fraumeni led to the recognition of recurrent somatic alterations in TP53, RB1, and genes interacting with TP53 and RB1 in OS (8, 9). Candidate-gene approaches demonstrated recurrent somatic mutations, deletions, and rearrangement affecting TP53 (9). Additional mechanisms of p53 inactivation described in OS are MDM2 and COPS3 amplification (8, 9). RB1 mutations are present in 6% and deletions or structural alterations are seen in 40% of cases (10,11). CDKN2A is deleted in 10-20% of OS (9, 12). Multiple other cancer-associated genes have been reported to be altered in OS [reviewed in Kansara and Thomas (5)]. Many of these studies SignificanceWe present, to our knowledge, the first comprehensive nextgeneration sequencing of osteosarcoma in combination with a functional genomic screen in a genetically defined mouse model of osteosarcoma. Our data provide a strong rationale for targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway in osteosarcoma and a foundation for rational clinical trial design. These findings present an immed...