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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. Genome sequencing of hundreds of normal colonic crypts from 42 individuals sheds light on mutational processes and driver mutations in normal colorectal epithelial cells.

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. Whole-genome sequencing of liver microdissections from five healthy individuals and nine with cirrhosis demonstrates the effects of liver disease on the genome, including increased rates of mutation, complex structural variation and different mutational signatures.

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. Whole-genome sequencing of normal human endometrial glands shows that most are clonal cell populations and frequently carry cancer driver mutations that occur early in life, and that parity has a protective effect.

Over the course of an individual’s lifetime, normal human cells accumulate mutations 1 . Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage 2 , and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma. The authors report the mutational landscape of 29 cell types from microdissected biopsies from 19 organs and explore the mechanisms underlying mutation rates in normal tissues.

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. Whole-genome sequencing analysis of somatic mutations in liver samples from patients with chronic liver disease identifies driver mutations in metabolism-related genes such as FOXO1 , and shows that these variants frequently exhibit convergent evolution.

Mutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE / POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE / POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE / POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE / POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life. Healthy tissues from individuals with germline mutations in POLE or POLD1 show increased mutational burden, suggesting that normal cells are capable of tolerating high mutation rates.

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1 , widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX , RBL2 , and SP100 , providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of “BRCAness”, including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies. The molecular genetic landscape of leiomyosarcoma (LMS) is largely unknown. Here, the authors identify frequent DNA copy number alterations, whole-genome duplication, TP53 and RB1 inactivation, alternative telomere lengthening, and genomic imprints of defective DNA repair via homologous recombination as a potential therapeutic target in LMS patients.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time. This protocol describes a workflow that combines laser-capture microdissection with low-input genome sequencing, while circumventing the use of whole-genome amplification, for accurate detection of somatic mutations in non-neoplastic tissues.

Genome-wide methylome sequencing of serial samples obtained from patients with acute myeloid leukemia reveals that epigenetic alleles and genetic alleles follow independent courses during disease evolution. Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. DNA double-strand breaks are repaired by multiple pathways. The balance of these pathways depends on the local chromatin context, but the underlying mechanisms are poorly understood. Here the authors uncover a network of proteins that regulate pathway balance in a chromatin context-dependent manner.