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Background: Folate is involved in multiple genetic, epigenetic, and metabolic processes, and inadequate folate intake has been associated with an increased risk of cancer., Objective: We examined whether folate intake is differentially associated with colorectal cancer (CRC) risk according to somatic mutations in genes linked to CRC using targeted sequencing., Design: Participants within 2 large CRC consortia with available information on dietary folate, supplemental folic acid, and total folate intake were included. Colorectal tumor samples from cases were sequenced for the presence of nonsilent mutations in 105 genes and 6 signaling pathways (IGF2/PI3K, MMR, RTK/RAS, TGF-β, WNT, and TP53/ATM). Multinomial logistic regression models were analyzed comparing mutated/nonmutated CRC cases to controls to compute multivariable-adjusted odds ratios (ORs) with 95% confidence interval (CI). Heterogeneity of associations of mutated compared with nonmutated CRC cases was tested in case-only analyses using logistic regression. Analyses were performed separately in hypermutated and nonhypermutated tumors, because they exhibit different clinical behaviors., Results: We included 4339 CRC cases (702 hypermutated tumors, 16.2%) and 11,767 controls. Total folate intake was inversely associated with CRC risk (OR = 0.93; 95% CI: 0.90, 0.96). Among hypermutated tumors, 12 genes (AXIN2, B2M, BCOR, CHD1, DOCK3, FBLN2, MAP3K21, POLD1, RYR1, TET2, UTP20, and ZNF521) showed nominal statistical significance (P < 0.05) for heterogeneity by mutation status, but none remained significant after multiple testing correction. Among these genetic subtypes, the associations between folate variables and CRC were mostly inverse or toward the null, except for tumors mutated for DOCK3 (supplemental folic acid), CHD1 (total folate), and ZNF521 (dietary folate) that showed positive associations. We did not observe differential associations in analyses among nonhypermutated tumors, or according to the signaling pathways., Conclusions: Folate intake was not differentially associated with CRC risk according to mutations in the genes explored. The nominally significant differential mutation effects observed in a few genes warrants further investigation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human aging is marked by the emergence of a tapestry of clonal expansions in dividing tissues, particularly evident in blood as clonal hematopoiesis (CH). CH, linked to cancer risk and aging-related phenotypes, often stems from somatic mutations in a set of established genes. However, the majority of clones lack known drivers. Here we infer gene-level positive selection in whole blood exomes from 200,618 individuals in UK Biobank. We identify 17 additional genes, ZBTB33, ZNF318, ZNF234, SPRED2, SH2B3, SRCAP, SIK3, SRSF1, CHEK2, CCDC115, CCL22, BAX, YLPM1, MYD88, MTA2, MAGEC3 and IGLL5, under positive selection at a population level, and validate this selection pattern in 10,837 whole genomes from single-cell-derived hematopoietic colonies. Clones with mutations in these genes grow in frequency and size with age, comparable to classical CH drivers. They correlate with heightened risk of infection, death and hematological malignancy, highlighting the significance of these additional genes in the aging process., (© 2024. The Author(s).)

Phylogenetic trees are a powerful means to display the evolutionary history of species, pathogens and, more recently, individual cells of the human body. Whole-genome sequencing of laser capture microdissections or expanded stem cells has allowed the discovery of somatic mutations in clones, which can be used as natural barcodes to reconstruct the developmental history of individual cells. Here we describe Sequoia, our pipeline to reconstruct lineage trees from clones of normal cells. Candidate somatic mutations are called against the human reference genome and filtered to exclude germline mutations and artifactual variants. These filtered somatic mutations form the basis for phylogeny reconstruction using a maximum parsimony framework. Lastly, we use a maximum likelihood framework to explicitly map mutations to branches in the phylogenetic tree. The resulting phylogenies can then serve as a basis for many subsequent analyses, including investigating embryonic development, tissue dynamics in health and disease, and mutational signatures. Sequoia can be readily applied to any clonal somatic mutation dataset, including single-cell DNA sequencing datasets, using the commands and scripts provided. Moreover, Sequoia is highly flexible and can be easily customized. Typically, the runtime of the core script ranges from minutes to an hour for datasets with a moderate number (50,000-150,000) of variants. Competent bioinformatic skills, including in-depth knowledge of the R programming language, are required. A high-performance computing cluster (one that is capable of running mutation-calling algorithms and other aspects of the analysis at scale) is also required, especially if handling large datasets., (© 2024. Springer Nature Limited.)

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation 1 , antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments., (© 2022. The Author(s).)

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited 1-3 . Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2 V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2 V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2 V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2 V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes 1-8 . Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease 9-13 than in normal liver 13-16 , which enables positive selection to shape the genomic landscape 9-13 . Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1 S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes 17-19 , and GPAM, which produces storage triacylglycerol from free fatty acids 20,21 , also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Starting from the zygote, all cells in the human body continuously acquire mutations. Mutations shared between different cells imply a common progenitor and are thus naturally occurring markers for lineage tracing 1,2 . Here we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissections. Reconstructed embryonic progenitors in the same generation of a phylogeny often contribute to different extents to the adult body. The degree of this asymmetry varies between individuals, with ratios between the two reconstructed daughter cells of the zygote ranging from 60:40 to 93:7. Asymmetries pervade subsequent generations and can differ between tissues in the same individual. The phylogenies resolve the spatial embryonic patterning of tissues, revealing contiguous patches of, on average, 301 crypts in the adult colonic epithelium derived from a most recent embryonic cell and also a spatial effect in brain development. Using data from ten additional men, we investigated the developmental split between soma and germline, with results suggesting an extraembryonic contribution to primordial germ cells. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations 1 . Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

Background: Smoking is associated with colorectal cancer (CRC) risk. Previous studies suggested this association may be restricted to certain molecular subtypes of CRC, but large-scale comprehensive analysis is lacking., Methods: A total of 9789 CRC cases and 11 231 controls of European ancestry from 11 observational studies were included. We harmonized smoking variables across studies and derived sex study-specific quartiles of pack-years of smoking for analysis. Four somatic colorectal tumor markers were assessed individually and in combination, including BRAF mutation, KRAS mutation, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. A multinomial logistic regression analysis was used to assess the association between smoking and risk of CRC subtypes by molecular characteristics, adjusting for age, sex, and study. All statistical tests were 2-sided and adjusted for Bonferroni correction., Results: Heavier smoking was associated with higher risk of CRC overall and stratified by individual markers ( P trend < .001). The associations differed statistically significantly between all molecular subtypes, which was the most statistically significant for CIMP and BRAF . Compared with never-smokers, smokers in the fourth quartile of pack-years had a 90% higher risk of CIMP-positive CRC (odds ratio = 1.90, 95% confidence interval = 1.60 to 2.26) but only 35% higher risk for CIMP-negative CRC (odds ratio = 1.35, 95% confidence interval = 1.22 to 1.49; P difference = 2.1 x 10 -6 ). The association was also stronger in tumors that were CIMP positive, MSI high, or KRAS wild type when combined ( P difference < .001)., Conclusion: Smoking was associated with differential risk of CRC subtypes defined by molecular characteristics. Heavier smokers had particularly higher risk of CRC subtypes that were CIMP positive and MSI high in combination, suggesting that smoking may be involved in the development of colorectal tumors via the serrated pathway., (© The Author(s) 2021. Published by Oxford University Press.)

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases 1,2 . Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis., Competing Interests: The authors have declared that no competing interests exist.

Placentas can exhibit chromosomal aberrations that are absent from the fetus 1 . The basis of this genetic segregation, which is known as confined placental mosaicism, remains unknown. Here we investigated the phylogeny of human placental cells as reconstructed from somatic mutations, using whole-genome sequencing of 86 bulk placental samples (with a median weight of 28 mg) and of 106 microdissections of placental tissue. We found that every bulk placental sample represents a clonal expansion that is genetically distinct, and exhibits a genomic landscape akin to that of childhood cancer in terms of mutation burden and mutational imprints. To our knowledge, unlike any other healthy human tissue studied so far, the placental genomes often contained changes in copy number. We reconstructed phylogenetic relationships between tissues from the same pregnancy, which revealed that developmental bottlenecks genetically isolate placental tissues by separating trophectodermal lineages from lineages derived from the inner cell mass. Notably, there were some cases with full segregation-within a few cell divisions of the zygote-of placental lineages and lineages derived from the inner cell mass. Such early embryonic bottlenecks may enable the normalization of zygotic aneuploidy. We observed direct evidence for this in a case of mosaic trisomic rescue. Our findings reveal extensive mutagenesis in placental tissues and suggest that mosaicism is a typical feature of placental development.

The rare benign giant cell tumour of bone (GCTB) is defined by an almost unique mutation in the H3.3 family of histone genes H3-3A or H3-3B; however, the same mutation is occasionally found in primary malignant bone tumours which share many features with the benign variant. Moreover, lung metastases can occur despite the absence of malignant histological features in either the primary or metastatic lesions. Herein we investigated the genetic events of 17 GCTBs including benign and malignant variants and the methylation profiles of 122 bone tumour samples including GCTBs. Benign GCTBs possessed few somatic alterations and no other known drivers besides the H3.3 mutation, whereas all malignant tumours harboured at least one additional driver mutation and exhibited genomic features resembling osteosarcomas, including high mutational burden, additional driver event(s), and a high degree of aneuploidy. The H3.3 mutation was found to predate the development of aneuploidy. In contrast to osteosarcomas, malignant H3.3-mutated tumours were enriched for a variety of alterations involving TERT, other than amplification, suggesting telomere dysfunction in the transformation of benign to malignant GCTB. DNA sequencing of the benign metastasising GCTB revealed no additional driver alterations; polyclonal seeding in the lung was identified, implying that the metastatic lesions represent an embolic event. Unsupervised clustering of DNA methylation profiles revealed that malignant H3.3-mutated tumours are distinct from their benign counterpart, and other bone tumours. Differential methylation analysis identified CCND1, encoding cyclin D1, as a plausible cancer driver gene in these tumours because hypermethylation of the CCND1 promoter was specific for GCTBs. We report here the genomic and methylation patterns underlying the rare clinical phenomena of benign metastasising and malignant transformation of GCTB and show how the combination of genomic and epigenomic findings could potentially distinguish benign from malignant GCTBs, thereby predicting aggressive behaviour in challenging diagnostic cases. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Epidemiological studies have identified many environmental agents that appear to significantly increase cancer risk in human populations. By analyzing tumor genomes from mice chronically exposed to 1 of 20 known or suspected human carcinogens, we reveal that most agents do not generate distinct mutational signatures or increase mutation burden, with most mutations, including driver mutations, resulting from tissue-specific endogenous processes. We identify signatures resulting from exposure to cobalt and vinylidene chloride and link distinct human signatures (SBS19 and SBS42) with 1,2,3-trichloropropane, a haloalkane and pollutant of drinking water, and find these and other signatures in human tumor genomes. We define the cross-species genomic landscape of tumors induced by an important compendium of agents with relevance to human health.

Somatic mutations in driver genes may ultimately lead to the development of cancer. Understanding how somatic mutations accumulate in cancer genomes and the underlying factors that generate somatic mutations is therefore crucial for developing novel therapeutic strategies. To understand the interplay between spatial genome organization and specific mutational processes, we studied 3,000 tumor-normal-pair whole-genome datasets from 42 different human cancer types. Our analyses reveal that the change in somatic mutational load in cancer genomes is co-localized with topologically-associating-domain boundaries. Domain boundaries constitute a better proxy to track mutational load change than replication timing measurements. We show that different mutational processes lead to distinct somatic mutation distributions where certain processes generate mutations in active domains, and others generate mutations in inactive domains. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation-rate variations observed in human cancers.

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDS as a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overt MDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement., (© 2020 by The American Society of Hematology.)

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium 1,2 . Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.

The discovery of drivers of cancer has traditionally focused on protein-coding genes 1-4 . Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers 6,7 , raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

Tobacco smoking causes lung cancer 1-3 , a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA 4,5 . The profound effects of tobacco on the genome of lung cancer cells are well-documented 6-10 , but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Cohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies., (© 2019 by The American Society of Hematology.)

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes 1-7 . Stem cells from normal livers have a low mutational burden and limited diversity of signatures 8 , which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer 1 . However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Risk stratification is critical in the care of patients with myelodysplastic syndromes (MDS). Approximately 10% have a complex karyotype (CK), defined as more than two cytogenetic abnormalities, which is a highly adverse prognostic marker. However, CK-MDS can carry a wide range of chromosomal abnormalities and somatic mutations. To refine risk stratification of CK-MDS patients, we examined data from 359 CK-MDS patients shared by the International Working Group for MDS. Mutations were underrepresented with the exception of TP53 mutations, identified in 55% of patients. TP53 mutated patients had even fewer co-mutated genes but were enriched for the del(5q) chromosomal abnormality (p < 0.005), monosomal karyotype (p < 0.001), and high complexity, defined as more than 4 cytogenetic abnormalities (p < 0.001). Monosomal karyotype, high complexity, and TP53 mutation were individually associated with shorter overall survival, but monosomal status was not significant in a multivariable model. Multivariable survival modeling identified severe anemia (hemoglobin < 8.0 g/dL), NRAS mutation, SF3B1 mutation, TP53 mutation, elevated blast percentage (>10%), abnormal 3q, abnormal 9, and monosomy 7 as having the greatest survival risk. The poor risk associated with CK-MDS is driven by its association with prognostically adverse TP53 mutations and can be refined by considering clinical and karyotype features.

The histological diagnosis of peripheral T-cell lymphoma (PTCL) can represent a challenge, particularly in the case of closely related entities such as angioimmunoblastic T-lymphoma (AITL), PTCL-not otherwise specified (PTCL-NOS), and ALK-negative anaplastic large-cell lymphoma (ALCL). Although gene expression profiling and next generations sequencing have been proven to define specific features recurrently associated with distinct entities, genomic-based stratifications have not yet led to definitive diagnostic criteria and/or entered into the routine clinical practice. Herein, to improve the current molecular classification between AITL and PTCL-NOS, we analyzed the transcriptional profiles from 503 PTCLs stratified according to their molecular configuration and integrated them with genomic data of recurrently mutated genes (RHOA G17V , TET2, IDH2 R172 , and DNMT3A) in 53 cases (39 AITLs and 14 PTCL-NOSs) included in the series. Our analysis unraveled that the mutational status of RHOA G17V , TET2, and DNMT3A poorly correlated, individually, with peculiar transcriptional fingerprints. Conversely, in IDH2 R172 samples a strong transcriptional signature was identified that could act as a surrogate for mutational status. The integrated analysis of clinical, mutational, and molecular data led to a simplified 19-gene signature that retains high accuracy in differentiating the main nodal PTCL entities. The expression levels of those genes were confirmed in an independent cohort profiled by RNA-sequencing., (© 2019 Wiley Periodicals, Inc.)

COSMIC, the Catalogue Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk) is the most detailed and comprehensive resource for exploring the effect of somatic mutations in human cancer. The latest release, COSMIC v86 (August 2018), includes almost 6 million coding mutations across 1.4 million tumour samples, curated from over 26 000 publications. In addition to coding mutations, COSMIC covers all the genetic mechanisms by which somatic mutations promote cancer, including non-coding mutations, gene fusions, copy-number variants and drug-resistance mutations. COSMIC is primarily hand-curated, ensuring quality, accuracy and descriptive data capture. Building on our manual curation processes, we are introducing new initiatives that allow us to prioritize key genes and diseases, and to react more quickly and comprehensively to new findings in the literature. Alongside improvements to the public website and data-download systems, new functionality in COSMIC-3D allows exploration of mutations within three-dimensional protein structures, their protein structural and functional impacts, and implications for druggability. In parallel with COSMIC's deep and broad variant coverage, the Cancer Gene Census (CGC) describes a curated catalogue of genes driving every form of human cancer. Currently describing 719 genes, the CGC has recently introduced functional descriptions of how each gene drives disease, summarized into the 10 cancer Hallmarks.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of BAP1 alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene VISTA in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options. SIGNIFICANCE: Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene VISTA in epithelioid MPM. See related commentary by Aggarwal and Albelda, p. 1508 . This article is highlighted in the In This Issue feature, p. 1494 ., (©2018 American Association for Cancer Research.)

Background: Myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment., Methods: We sequenced coding exons from 69 myeloid cancer genes in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort., Results: A total of 2035 patients were included in the analysis. A total of 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy., Conclusions: Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients' outcomes and may support the treatment of patients with myeloproliferative neoplasms. (Funded by the Wellcome Trust and others.).

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure 1 . The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion 2,3 . However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH) 4-8 . Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunit pole-4 Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of N C G > NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms . The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes., (© 2018 Meier et al.; Published by Cold Spring Harbor Laboratory Press.)

Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.

Chordoma is a malignant, often incurable bone tumour showing notochordal differentiation. Here, we defined the somatic driver landscape of 104 cases of sporadic chordoma. We reveal somatic duplications of the notochordal transcription factor brachyury (T) in up to 27% of cases. These variants recapitulate the rearrangement architecture of the pathogenic germline duplications of T that underlie familial chordoma. In addition, we find potentially clinically actionable PI3K signalling mutations in 16% of cases. Intriguingly, one of the most frequently altered genes, mutated exclusively by inactivating mutation, was LYST (10%), which may represent a novel cancer gene in chordoma.Chordoma is a rare often incurable malignant bone tumour. Here, the authors investigate driver mutations of sporadic chordoma in 104 cases, revealing duplications in notochordal transcription factor brachyury (T), PI3K signalling mutations, and mutations in LYST, a potential novel cancer gene in chordoma.

Background: The authors describe a family with a high penetrance of plasma cell dyscrasias, suggesting inheritance of an autosomal dominant risk allele., Methods: The authors performed whole-exome sequencing and reported on a combined approach aimed at the identification of causative variants and risk loci, using the wealth of data provided by this approach., Results: The authors identified gene mutations and single-nucleotide polymorphisms of potential significance, and pinpointed a known risk locus for myeloma as a potential area of transmissible risk in the family., Conclusions: To the authors' knowledge, the current study is the first to provide a whole-exome sequencing approach to such cases, and a framework analysis that could be applied to further understanding of the inherited risk of developing plasma cell dyscrasias. Cancer 2017;123:3701-3708. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Patterns of genomic evolution between primary and metastatic breast cancer have not been studied in large numbers, despite patients with metastatic breast cancer having dismal survival. We sequenced whole genomes or a panel of 365 genes on 299 samples from 170 patients with locally relapsed or metastatic breast cancer. Several lines of analysis indicate that clones seeding metastasis or relapse disseminate late from primary tumors, but continue to acquire mutations, mostly accessing the same mutational processes active in the primary tumor. Most distant metastases acquired driver mutations not seen in the primary tumor, drawing from a wider repertoire of cancer genes than early drivers. These include a number of clinically actionable alterations and mutations inactivating SWI-SNF and JAK2-STAT3 pathways., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation, we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. It may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.

Deregulated expression of the type I cytokine receptor, CRLF2, is observed in 5-15% of precursor B-cell acute lymphoblastic leukaemia (B-ALL). We aimed to determine the clinical and genetic landscape of those with IGH-CRLF2 or P2RY8-CRLF2 (CRLF2-r) using multiple genomic approaches. Clinical and demographic features of CRLF2-r patients were characteristic of B-ALL. Patients with IGH-CRLF2 were older (14 y vs. 4 y, P < .001), while the incidence of CRLF2-r among Down syndrome patients was high (50/161, 31%). CRLF2-r co-occurred with primary chromosomal rearrangements but the majority (111/161, 69%) had B-other ALL. Copy number alteration (CNA) profiles were similar to B-other ALL, although CRLF2-r patients harbored higher frequencies of IKZF1 (60/138, 43% vs. 77/1351, 24%) and BTG1 deletions (20/138, 15% vs. 3/1351, 1%). There were significant differences in CNA profiles between IGH-CRLF2 and P2RY8-CRLF2 patients: IKZF1 (25/35, 71% vs. 36/108, 33%, P < .001), BTG1 (11/35, 31% vs. 10/108, 9%, P =.004), and ADD3 deletions (9/19, 47% vs. 5/38, 13%, P =.008). A novel gene fusion, USP9X-DDX3X, was discovered in 10/54 (19%) of patients. Pathway analysis of the mutational profile revealed novel involvement for focal adhesion. Although the functional relevance of many of these abnormalities are unknown, they likely activate additional pathways, which may represent novel therapeutic targets., (© 2017 The Authors Genes, Chromosomes and Cancer Published byWiley Periodicals, Inc.)

Approximately 1-5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (∼1-5%) who could have selective therapeutic sensitivity to PARP inhibition.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. The transcriptomic consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.

COSMIC, the Catalogue of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk) is a high-resolution resource for exploring targets and trends in the genetics of human cancer. Currently the broadest database of mutations in cancer, the information in COSMIC is curated by expert scientists, primarily by scrutinizing large numbers of scientific publications. Over 4 million coding mutations are described in v78 (September 2016), combining genome-wide sequencing results from 28 366 tumours with complete manual curation of 23 489 individual publications focused on 186 key genes and 286 key fusion pairs across all cancers. Molecular profiling of large tumour numbers has also allowed the annotation of more than 13 million non-coding mutations, 18 029 gene fusions, 187 429 genome rearrangements, 1 271 436 abnormal copy number segments, 9 175 462 abnormal expression variants and 7 879 142 differentially methylated CpG dinucleotides. COSMIC now details the genetics of drug resistance, novel somatic gene mutations which allow a tumour to evade therapeutic cancer drugs. Focusing initially on highly characterized drugs and genes, COSMIC v78 contains wide resistance mutation profiles across 20 drugs, detailing the recurrence of 301 unique resistance alleles across 1934 drug-resistant tumours. All information from the COSMIC database is available freely on the COSMIC website., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types., (Copyright © 2016, American Association for the Advancement of Science.)

Disordered transcriptomes of cancer encompass direct effects of somatic mutation on transcription, coordinated secondary pathway alterations, and increased transcriptional noise. To catalog the rules governing how somatic mutation exerts direct transcriptional effects, we developed an exhaustive pipeline for analyzing RNA sequencing data, which we integrated with whole genomes from 23 breast cancers. Using X-inactivation analyses, we found that cancer cells are more transcriptionally active than intermixed stromal cells. This is especially true in estrogen receptor (ER)-negative tumors. Overall, 59% of substitutions were expressed. Nonsense mutations showed lower expression levels than expected, with patterns characteristic of nonsense-mediated decay. 14% of 4,234 rearrangements caused transcriptional abnormalities, including exon skips, exon reusage, fusions, and premature polyadenylation. We found productive, stable transcription from sense-to-antisense gene fusions and gene-to-intergenic rearrangements, suggesting that these mutation classes drive more transcriptional disruption than previously suspected. Systematic integration of transcriptome with genome data reveals the rules by which transcriptional machinery interprets somatic mutation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice., Methods: We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes., Results: We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials., Conclusions: The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

Somatic mutations in human cancers show unevenness in genomic distribution that correlate with aspects of genome structure and function. These mutations are, however, generated by multiple mutational processes operating through the cellular lineage between the fertilized egg and the cancer cell, each composed of specific DNA damage and repair components and leaving its own characteristic mutational signature on the genome. Using somatic mutation catalogues from 560 breast cancer whole-genome sequences, here we show that each of 12 base substitution, 2 insertion/deletion (indel) and 6 rearrangement mutational signatures present in breast tissue, exhibit distinct relationships with genomic features relating to transcription, DNA replication and chromatin organization. This signature-based approach permits visualization of the genomic distribution of mutational processes associated with APOBEC enzymes, mismatch repair deficiency and homologous recombinational repair deficiency, as well as mutational processes of unknown aetiology. Furthermore, it highlights mechanistic insights including a putative replication-dependent mechanism of APOBEC-related mutagenesis.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.