[Show abstract][Hide abstract] ABSTRACT: Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole-genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional (“T-class”) asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations and replicative (“R-class”) asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of transcription-coupled damage (TCD) on the non-transcribed DNA strand and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair.
[Show abstract][Hide abstract] ABSTRACT: Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution.
Full-text · Article · Dec 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Summary There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these tumors fell into one of seven subtypes defined by specific gene fusions (ERG, ETV1/4, and FLI1) or mutations (SPOP, FOXA1, and IDH1). Epigenetic profiles showed substantial heterogeneity, including an IDH1 mutant subset with a methylator phenotype. Androgen receptor (AR) activity varied widely and in a subtype-specific manner, with SPOP and FOXA1 mutant tumors having the highest levels of AR-induced transcripts. 25% of the prostate cancers had a presumed actionable lesion in the PI3K or MAPK signaling pathways, and DNA repair genes were inactivated in 19%. Our analysis reveals molecular heterogeneity among primary prostate cancers, as well as potentially actionable molecular defects.
[Show abstract][Hide abstract] ABSTRACT: Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole-exome sequencing of 86 matched brain metastases, primary tumors, and normal tissue. In all clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases.
SIGNIFICANCE: Decisions for individualized therapies in patients with brain metastasis are often made from primary-tumor biopsies. We demonstrate that clinically actionable alterations present in brain metastases are frequently not detected in primary biopsies, suggesting that sequencing of primary biopsies alone may miss a substantial number of opportunities for targeted therapy.
[Show abstract][Hide abstract] ABSTRACT: Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these 'hotspot' sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.
No preview · Article · Sep 2015 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Large-scale tumor sequencing projects enabled the identification of many new cancer gene candidates through computational approaches. Here, we describe a general method to detect cancer genes based on significant 3D clustering of mutations relative to the structure of the encoded protein products. The approach can also be used to search for proteins with an enrichment of mutations at binding interfaces with a protein, nucleic acid, or small molecule partner. We applied this approach to systematically analyze the PanCancer compendium of somatic mutations from 4,742 tumors relative to all known 3D structures of human proteins in the Protein Data Bank. We detected significant 3D clustering of missense mutations in several previously known oncoproteins including HRAS, EGFR, and PIK3CA. Although clustering of missense mutations is often regarded as a hallmark of oncoproteins, we observed that a number of tumor suppressors, including FBXW7, VHL, and STK11, also showed such clustering. Beside these known cases, we also identified significant 3D clustering of missense mutations in NUF2, which encodes a component of the kinetochore, that could affect chromosome segregation and lead to aneuploidy. Analysis of interaction interfaces revealed enrichment of mutations in the interfaces between FBXW7-CCNE1, HRAS-RASA1, CUL4B-CAND1, OGT-HCFC1, PPP2R1A-PPP2R5C/PPP2R2A, DICER1-Mg(2+), MAX-DNA, SRSF2-RNA, and others. Together, our results indicate that systematic consideration of 3D structure can assist in the identification of cancer genes and in the understanding of the functional role of their mutations.
Full-text · Article · Sep 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Barrett's esophagus is thought to progress to esophageal adenocarcinoma (EAC) through a stepwise progression with loss of CDKN2A followed by TP53 inactivation and aneuploidy. Here we present whole-exome sequencing from 25 pairs of EAC and Barrett's esophagus and from 5 patients whose Barrett's esophagus and tumor were extensively sampled. Our analysis showed that oncogene amplification typically occurred as a late event and that TP53 mutations often occurred early in Barrett's esophagus progression, including in non-dysplastic epithelium. Reanalysis of additional EAC exome data showed that the majority (62.5%) of EACs emerged following genome doubling and that tumors with genomic doubling had different patterns of genomic alterations, with more frequent oncogenic amplification and less frequent inactivation of tumor suppressors, including CDKN2A. These data suggest that many EACs emerge not through the gradual accumulation of tumor-suppressor alterations but rather through a more direct path whereby a TP53-mutant cell undergoes genome doubling, followed by the acquisition of oncogenic amplifications.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND
Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas.
We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes.
Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma.
The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma.
(Funded by the National Institutes of Health)
Full-text · Article · Jun 2015 · New England Journal of Medicine
[Show abstract][Hide abstract] ABSTRACT: We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.
[Show abstract][Hide abstract] ABSTRACT: Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.
[Show abstract][Hide abstract] ABSTRACT: The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.
[Show abstract][Hide abstract] ABSTRACT: Pompe disease, an inherited deficiency of lysosomal acid alpha-glucosidase (GAA), is a metabolic myopathy with heterogeneous clinical presentations. Late-onset Pompe disease (LOPD) is a debilitating progressive muscle disorder that can occur anytime from early childhood to late adulthood. Enzyme replacement therapy (ERT) with recombinant human GAA is currently available for Pompe patients. Although ERT shows some benefits, the reversal of skeletal muscle pathology - lysosomal glycogen accumulation and autophagic buildup - remains a challenge. In this study, we examined the clinical status and muscle pathology of 22 LOPD patients and one atypical infantile patient on ERT to understand the reasons for muscle resistance to ERT.
The patients were divided into three groups for analysis, based on the age of onset and diagnosis: adult-onset patients, juvenile-onset patients, and those identified through newborn screening (NBS). The areas of autophagic buildup found in patients’ biopsies of all three groups, contained large autofluorescent inclusions which we show are made of lipofuscin, an indigestible intralysosomal material typically associated with ageing. These inclusions, analysed by staining, spectral analysis, time-resolved Fluorescence Lifetime Imaging (FLIM), and Second Harmonic Generation (SHG) imaging, were the major pathology remaining in many fibers after ERT. The best outcome of ERT both clinically and morphologically was observed in the NBS patients.
The muscle biopsy, in spite of its shortcomings, allowed us to recognize an underreported, ERT-resistant pathology in LOPD; numerous lysosomes and autolysosomes loaded with lipofuscin appear to be a hallmark of LOPD skeletal muscle. Lipofuscin accumulation - a result of inefficient lysosomal degradation - may in turn exacerbate both lysosomal and autophagic abnormalities.
Full-text · Article · Dec 2014 · Acta Neuropathologica
[Show abstract][Hide abstract] ABSTRACT: Cancers exhibit extensive mutational heterogeneity, and the resulting long-tail phenomenon complicates the discovery of genes and pathways that are significantly mutated in cancer. We perform a pan-cancer analysis of mutated networks in 3,281 samples from 12 cancer types from The Cancer Genome Atlas (TCGA) using HotNet2, a new algorithm to find mutated subnetworks that overcomes the limitations of existing single-gene, pathway and network approaches. We identify 16 significantly mutated subnetworks that comprise well-known cancer signaling pathways as well as subnetworks with less characterized roles in cancer, including cohesin, condensin and others. Many of these subnetworks exhibit co-occurring mutations across samples. These subnetworks contain dozens of genes with rare somatic mutations across multiple cancers; many of these genes have additional evidence supporting a role in cancer. By illuminating these rare combinations of mutations, pan-cancer network analyses provide a roadmap to investigate new diagnostic and therapeutic opportunities across cancer types.
[Show abstract][Hide abstract] ABSTRACT: 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.