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Background: Many postmortem studies address the cardiovascular effects of COVID-19 and provide valuable information, but are limited by their small sample size. Objectives: The aim of this systematic review is to better understand the various aspects of the cardio-vascular complications of COVID-19 by pooling data from a large number of autopsy studies. Data sources: We searched the online databases Ovid EBM Reviews, Ovid Embase, Ovid Medline, Scopus, and Web of Science for concepts of autopsy or histopathology combined with COVID-19, published be-tween database inception and February 2021. We also searched for unpublished manuscripts using the medRxiv services operated by Cold Spring Harbor Laboratory. Study eligibility criteria: Articles were considered eligible for inclusion if they reported human post-mortem cardiovascular findings among individuals with a confirmed SARS coronavirus type 2 (CoV-2) infection. Participants: Confirmed COVID-19 patients with post-mortem cardiovascular findings. Interventions: None. Methods: Studies were individually assessed for risk of selection, detection, and reporting biases. The median prevalence of different autopsy findings with associated interquartile ranges (IQRs). Results: This review cohort contained 50 studies including 548 hearts. The median age of the deceased was 69 years. The most prevalent acute cardiovascular findings were myocardial necrosis (median: 100.0%; IQR, 20%-10 0%; number of studies = 9; number of patients = 64) and myocardial oedema (median: 55.5%; IQR, 19.5%-92.5%; number of studies = 4; number of patients = 46). The median re-ported prevalence of extensive, focal active, and multifocal myocarditis were all 0.0%. The most prevalent chronic changes were myocyte hypertrophy (median: 69.0%; IQR, 46.8%-92.1%) and fibrosis (median: 35.0%; IQR, 35.0%-90.5%). SARS-CoV-2 was detected in the myocardium with median prevalence of 60.8% (IQR 40.4-95.6%). Conclusions: Our systematic review confirmed the high prevalence of acute and chronic cardiac pathologies in COVID-19 and SARS-CoV-2 cardiac tropism, as well as the low prevalence of myocarditis in COVID-19. (C) 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma. Single-cell and spatial transcriptomic profiling of the human endometrium highlights pathways governing the proliferative and secretory phases of the menstrual cycle. Analyses of endometrial organoids show that WNT and NOTCH signaling modulate differentiation into the secretory and ciliated epithelial lineages, respectively. Correction in: Nature Genetics, 55, page 165 (2023)DOI: 10.1038/s41588-022-01287-6

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20–30 cancer genes1–8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9–13 than in normal liver13–16, which enables positive selection to shape the genomic landscape9–13. 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 FOXO1S22W 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 hepatocytes17–19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, 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.

Funder: Wellcome Clinical PhD fellowship, Funder: Wellcome PhD Studentship, Funder: Jean Shank/Pathological Society Intermediate Fellowship, 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.

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 tracing1,2. 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. Somatic mutations obtained from laser microdissected biopsies of human tissues are used to reconstruct the developmental phylogenies of these tissues back to the zygote.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. 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. Whole-genome sequencing of haematopoietic colonies from human fetuses reveals the somatic mutations acquired by individual progenitors, which are used as barcodes to construct a phylogenetic tree of blood development.

Gastric cancer is the third leading cause of cancer-related mortality globally and is often associated with infectious agents such as the bacterium Helicobacter pylori. However, the background mutational landscape in normal gastric epithelium and the first genomic steps towards the formation of gastric cancer remain poorly understood. Here, we use whole-genome sequencing of microdissected gastric glands (n=271) from 30 patients, 18 of whom had gastric cancer. We show that gastric glands are clonal structures and accrue approximately 27 single base substitutions per year. While the mutational signatures in most normal glands reflect age-related mutagenesis, gastric glands sampled close to a tumor showed exposure to a mutagenic process enriched in tumors (COSMIC reference mutational signature SBS17). Phylogenetic analysis shows that acquisition of SBS17 substitutions is closely linked to overt malignant transformation. We also observe widespread trisomies of specific chromosomes, which are recurrently and independently acquired in many gastric glands of the same patient. Mutations in genes encoding epigenetic modifiers and chromatin remodelers showed evidence of positive selection and were highly enriched in some patients. This was confirmed by targeted sequencing of cancer genes in a further 1008 gastric glands. Strikingly, glands that exhibit driver mutations, a recurrent trisomy or elevated mutation loads only overlap minimally, suggesting a highly variable and patient-specific mutation and selection landscape in the normal gastric epithelium. Taken together, these results give novel insights into the preclinical evolution of gastric malignancies. Citation Format: Tim H. Coorens, Grace Collord, Hyungchul Jung, Yichen Wang, Sonia Zumalave, Daniel Leongamornlert, Luiza Moore, Krishnaa Mahbubani, Kourosh Saeb-Parsy, Suet Yi Leung, Michael R. Stratton. Recurrent trisomies, variable selection and precancerous evolution in the normal gastric epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 226.

Subclonality is a universal feature of cancers yet how clones grow, are spatially organised, differ phenotypically or influence clinical outcome is unclear. To address this, we developed base specific in situ sequencing (BaSISS). In fixed tissues, transcripts harbouring clone-defining mutations are detected, converted into quantitative clone maps and characterised through multi-layered data integration. Applied to 8 samples from key stages of breast cancer progression BaSISS localised 1.42 million genotype informative transcripts across 4.9cm2 of tissue. Microscopic clonal topographies are shaped by resident tissue architectures. Distinct transcriptional, histological and immunological features distinguish coexistent genetic clones. Spatial lineage tracing temporally orders clone features associated with the emergence of aggressive clinical traits. These results highlight the pivotal role of spatial genomics in deciphering the mechanisms underlying cancer progression.

Mismatch repair (MMR) is a critical defence against mutation, but we lack quantification of its activity on different DNA lesions during human life. We performed whole-genome sequencing of normal and neoplastic tissues from individuals with constitutional MMR deficiency to establish the roles of MMR components, tissue type and disease state in somatic mutation rates. Mutational signatures varied extensively across genotypes, some coupled to leading-strand replication, some to lagging-strand replication and some independent of replication, implying that the various MMR components engage different forms of DNA damage. Loss of MSH2 or MSH6 (MutSα), but not MLH1 or PMS2 (MutLα), caused 5-methylcytosine-dependent hypermutation, indicating that MutSα is the pivotal complex for repairing spontaneous deamination of methylated cytosines in humans. Neoplastic change altered the distribution of mutational signatures, particularly accelerating replication-coupled indel signatures. Each component of MMR repairs 1-10 lesions/day per normal human cell, and many thousands of additional events during neoplastic transformation.HighlightsMMR repairs 1-10 lesions/day in every normal cell and thousands more in tumor cellsMMR patterns and rates are shaped by genotype, tissue type and malignant transformationMSH2 and MSH6 are pivotal for repairing spontaneous deamination of methylated cytosineReplication indels and substitutions vary by leading versus lagging strand and genotype

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated single-cell and spatial reference maps of the human uterus and 3D endometrial organoid cultures. We dissect the signalling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids highlights common pathways regulating the differentiation of secretory and ciliated lineage in vivo and in vitro. We show in vitro that downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. These mechanistic insights provide a platform for future development of treatments for a range of common endometrial disorders including endometriosis and carcinoma.

During the course of a lifetime normal human cells accumulate mutations. Here, using multiple samples from the same individuals we compared the mutational landscape in 29 anatomical structures from soma and the germline. 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, potentially reflecting oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The mutation rate was lowest in spermatogonia, the stem cell 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 mutation processes and may be partially attributable to a low cell division rate of basal spermatogonia. The results provide important insights into how mutational processes affect the soma and germline.

Starting from the zygote, all cells in the developing and adult human body continuously acquire mutations. A mutation shared between two different cells implies a shared progenitor cell and can thus be used as a naturally occurring marker for lineage tracing. Here, we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissected samples from multiple organs. Early embryonic progenitor cells inferred from the phylogenies often contribute in different proportions to the adult body and the extent of this asymmetry is variable between individuals, with ratios between the first two reconstructed cells ranging from 56:44 to 92:8. Asymmetries also pervade subsequent cell generations and can differ between tissues in the same individual. The phylogenies also resolve the spatial embryonic origins and patterning of tissues, revealing a spatial effect in the development of the human brain. Supplemented by data on eleven men, we timed the split between soma and germline, with the earliest observed segregation occurring at the first cell divisions. 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.

Over the course of an individual’s lifetime, normal human cells accumulate mutations1. 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 damage2, 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.

Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Rates of clonal expansion varied substantially (200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth trajectories drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.

Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 31 years (range 12-54 years). Rates of clonal expansion varied substantially (200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth and evolution drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.

Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy., Metastatic melanoma is associated with a poor prognosis and understanding the genetic features of metastases may enable better treatment strategies. Here, the authors analyse multiple metastases from individual patients finding high levels of heterogeneity in metastases from different organs.

Clonal dynamics and mutation burden in healthy human prostate epithelium are relevant to prostate cancer. We sequenced whole genomes from 409 microdissections of normal prostate epithelium across 8 donors, using phylogenetic reconstruction with spatial mapping in a 59-year-old man's prostate to reconstruct tissue dynamics across the lifespan. Somatic mutations accumulate steadily at ∼16 mutations/year/clone, with higher rates in peripheral than peri-urethral regions. The 24-30 independent glandular subunits are established as rudimentary ductal structures during fetal development by 5-10 embryonic cells each. Puberty induces formation of further side and terminal branches by local stem cells disseminated throughout the rudimentary ducts during development. During adult tissue maintenance, clonal expansions have limited geographic scope and minimal migration. Driver mutations are rare in aging prostate epithelium, but the one driver we did observe generated a sizable intraepithelial clonal expansion. Leveraging unbiased clock-like mutations, we define prostate stem cell dynamics through fetal development, puberty, and aging. ispartof: CELL STEM CELL vol:28 issue:7 pages:1262-+ ispartof: location:United States status: published

Here, we report the pathological findings of nine complete autopsies of individuals who died in community settings in the UK, three of which were positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), three tested negative for SARS-CoV-2 but are likely false negatives, and three died of other respiratory infections. Autopsy revealed firm, consolidated lungs or lobar pneumonia. Histology of the lungs showed changes of diffuse alveolar damage with fibrin membrane formation, thickened alveolar walls and interstitium with lymphocytic infiltrate, and type 2 pneumocyte hyperplasia with shedding into the alveolar space. This series is the first in the world to describe autopsy findings in individuals dying suddenly in the community, not previously known to have COVID-19 infection, and the first autopsy series in the UK. During a time when testing in the UK is currently primarily offered to patients in hospital or symptomatic key workers, with limited testing available in community settings, it highlights the importance of testing for COVID-19 at autopsy. Two deaths occurred in care homes where a diagnosis of COVID-19 allowed the health protection team to provide support in that ‘closed setting’ to reduce the risks of onward transmission. This work highlights the need for frequent COVID-19 testing in the management of patients in community settings. Comprehensive virology and microbiology assessment is pivotal to correctly identify the cause of death, including those due to COVID-19 infection, and to derive accurate death statistics.

Mutation accumulation over time in normal somatic cells contributes to cancer development and is proposed as a cause of ageing. DNA polymerases Pol ε and Pol δ replicate DNA with high fidelity during normal cell divisions. However, in some cancers defective proofreading due to acquired mutations in the exonuclease domains of POLE or POLD1 causes markedly elevated somatic mutation burdens with distinctive mutational signatures. POLE and POLD1 exonuclease domain mutations also cause familial cancer predisposition when inherited through the germline. Here, we sequenced normal tissue DNA from individuals with germline POLE or POLD1 exonuclease domain mutations. Increased mutation burdens with characteristic mutational signatures were found to varying extents in all normal adult somatic cell types examined, during early embryogenesis and in sperm. Mutation burdens were further markedly elevated in neoplasms from these individuals. Thus human physiology is able to tolerate ubiquitously elevated mutation burdens. Indeed, with the exception of early onset cancer, individuals with germline POLE and POLD1 exonuclease domain mutations are not reported to show abnormal phenotypic features, including those of premature ageing. The results, therefore, do not support a simple model in which all features of ageing are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.

To date, ontogeny of the human haematopoietic system during foetal development has been characterized mainly through careful microscopic observations. Here we used whole-genome sequencing (WGS) of 511 single-cell derived haematopoietic colonies from healthy human foetuses of 8 and 18 post-conception weeks (pcw) coupled with deep targeted sequencing of tissues of known embryonic origin to reconstruct a phylogenetic tree of blood development. We found that in healthy foetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 pcw. Using these mutations as barcodes, we 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 analysis has shown that ectoderm originates from a smaller set of blood antecedents compared to endoderm and mesoderm. Finally, our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations

Background In colorectal and breast cancer, the density and localisation of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations. Methods Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. Results Epithelial CD8 [p = 0.027, hazard ratio (HR) = 0.83], stromal CD68 (p = 3 × 10−4, HR = 0.44) and stromal CD45RO (p = 7 × 10−4, HR = 0.76) were positively associated with survival and remained so when averaged across the tumour and stromal compartments. Using principal component analysis, we identified optimised multiparameter survival models combining information from all immune markers (p = 0.016, HR = 0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components. Conclusions Combining measures of immune infiltration provided improved survival modelling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was associated with survival, underscoring the benefits evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

A childhood tumor—from the beginning Many adult cancers arise from clonal expansions of mutant cells in normal tissue. These premalignant expansions are defined by somatic mutations shared by the cancers. Whether pediatric cancers originate in a similar way is unknown. Coorens et al. studied Wilms tumor, a childhood kidney cancer. Phylogenetic analyses revealed large clones of mutant cells in histologically and functionally normal kidney tissue long before tumor development. Thus, like adult tumors, Wilms tumor appears to arise from a premalignant tissue bed. Science , this issue p. 1247

Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal that metastatic cells may depart the primary tumour very early in the disease course and follow a branched pattern of evolution. Truncal UV-induced mutations that often swamp downstream analyses of heterogeneity, were found to be replaced by APOBEC-associated mutations in the branches of the evolutionary tree. Multi-sample analyses from a further 7 patients confirmed that branched evolution was pervasive, representing an important mode of melanoma dissemination. Our analyses illustrate that combining cancer cell fraction estimates across multiple metastases provides higher resolution phylogenetic reconstructions relative to single sample analyses and highlights the limitations of accurately inferring inter-tumoural heterogeneity from a single biopsy.

Here we use deep transfer learning to quantify histopathological patterns across 17,396 H&E stained histopathology image slides from 28 cancer types and correlate these with underlying genomic and transcriptomic data. Pan-cancer computational histopathology (PC-CHiP) classifies the tissue origin across organ sites and provides highly accurate, spatially resolved tumor and normal distinction within a given slide. The learned computational histopathological features correlate with a large range of recurrent genetic aberrations, including whole genome duplications (WGDs), arm-level copy number gains and losses, focal amplifications and deletions as well as driver gene mutations within a range of cancer types. WGDs can be predicted in 25/27 cancer types (mean AUC=0.79) including those that were not part of model training. Similarly, we observe associations with 25% of mRNA transcript levels, which enables to learn and localise histopathological patterns of molecularly defined cell types on each slide. Lastly, we find that computational histopathology provides prognostic information augmenting histopathological subtyping and grading in the majority of cancers assessed, which pinpoints prognostically relevant areas such as necrosis or infiltrating lymphocytes on each tumour section. Taken together, these findings highlight the large potential of PC-CHiP to discover new molecular and prognostic associations, which can augment diagnostic workflows and lay out a rationale for integrating molecular and histopathological data. Key points Pan-cancer computational histopathology analysis with deep learning extracts histopathological patterns and accurately discriminates 28 cancer and 14 normal tissue types Computational histopathology predicts whole genome duplications, focal amplifications and deletions, as well as driver gene mutations Wide-spread correlations with gene expression indicative of immune infiltration and proliferation Prognostic information augments conventional grading and histopathology subtyping in the majority of cancers

Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, prognosis, and even treatment decisions. However, the field lacks a consensus on analysis and result interpretation. Using whole-genome sequencing of multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, we compare the performance of public signature analysis tools. We describe caveats and pitfalls of de novo signature extraction and fitting approaches, reporting on common inaccuracies: erroneous signature assignment, identification of localized hyper-mutational processes, overcalling of signatures. We provide reproducible solutions to solve these issues and use orthogonal approaches to validate our results. We show how a comprehensive mutational signature analysis may provide relevant biological insights, reporting evidence of c-AID activity among unmutated CLL cases or the absence of BRCA1/BRCA2-mediated homologous recombination deficiency in a MM cohort. Finally, we propose a general analysis framework to ensure production of accurate and reproducible mutational signature data.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. 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.

Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, prognosis, and even treatment decisions. However, the field lacks a consensus on analysis and result interpretation. Using whole-genome sequencing of multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, we compare the performance of public signature analysis tools. We describe caveats and pitfalls of de novo signature extraction and fitting approaches, reporting on common inaccuracies: erroneous signature assignment, identification of localized hyper-mutational processes, overcalling of signatures. We provide reproducible solutions to solve these issues and use orthogonal approaches to validate our results. We show how a comprehensive mutational signature analysis may provide relevant biological insights, reporting evidence of c-AID activity among unmutated CLL cases or the absence of BRCA1/BRCA2-mediated homologous recombination deficiency in a MM cohort. Finally, we propose a general analysis framework to ensure production of accurate and reproducible mutational signature data., Mutational signature analysis provides important information about the mutational processes underpinning different stages of tumorigenesis. Here, the authors compare publicly available signature extraction tools and suggest a framework for the generation of accurate and reproducible signature data.

All normal somatic cells are thought to acquire mutations. However, characterisation of the patterns and consequences of somatic mutation in normal tissues is limited. Uterine endometrium is a dynamic tissue that undergoes cyclical shedding and reconstitution and is lined by a gland-forming epithelium. Whole genome sequencing of normal endometrial glands showed that most are clonal cell populations derived from a recent common ancestor with mutation burdens differing from other normal cell types and manyfold lower than endometrial cancers. Mutational signatures found ubiquitously account for most mutations. Many, in some women potentially all, endometrial glands are colonised by cell clones carrying driver mutations in cancer genes, often with multiple drivers. Total and driver mutation burdens increase with age but are also influenced by other factors including body mass index and parity. Clones with drivers often originate during early decades of life. The somatic mutational landscapes of normal cells differ between cell types and are revealing the procession of neoplastic change leading to cancer.

Human endometrium is a highly dynamic tissue that undergoes numerous cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone. The marked regenerative capacity of the tissue’s epithelial compartment is maintained by the intra-glandular adult stem cells (ASCs) that are retained during menstruation. Although the endometrial epithelial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and arge intestine. In particular, the size of the stem cell pool within individual glands, clonal dynamics and genomic landscape are largely unknown. In this study, we laser-capture microdissected 257 morphologically normal endometrial glands from pre- and post-menopausal women (aged 19 to 81). In selected donors, we also obtained samples of normal endocervical and Fallopian tubular epithelium. Isolated cellular material was subjected to our modified library preparation protocol and subsequent whole genome sequencing (WGS). Analysis of the WGS data identified that the majority of the glands were clonal cell populations sharing a recent common ancestor. Somatic mutations accumulated at a rate of 29 substitutions per year. Total mutation burden in the normal endometrial epithelium is higher than in other normal cells and manyfold lower than in endometrial cancers. Overall, despite the heterogeneity in age, reproductive history and BMI, we found relatively homogenous mutational processes to be operative in this tissue with only occasional outliers. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Using a generalised linear mixed effect model, we found that age has a positive association with the accumulation of driver mutations, while parity has a negative association. Over time, the mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. Our data identify a distinct mutational landscape in normal endometrium that is in keeping with the presence of early positive selection in this highly regenerative tissue. Citation Format: Luiza Moore. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA007.

Background Recurrent mutations in cancer-associated genes drive tumour outgrowth, however, the timing of driver mutations and the dynamics of clonal expansion remain largely unknown. Philadelphia-negative myeloproliferative neoplasms (MPN) are unique cancers capturing the earliest stages of tumorigenesis through to disease evolution. Most patients harbor JAK2V617F, present as the only driver mutation or occurring in combination with driver mutations in genes such as DNMT3A or TET2. We aimed to identify the timing of driver mutations and clonal dynamics in adult MPN. Method We undertook whole-genome sequencing of individual single-cell derived hematopoietic colonies (n=952) together with targeted resequencing of longitudinal blood samples from 10 patients with MPN who presented with disease between ages 20 and 76 years. We identified 448,553 somatic mutations which were used to reconstruct phylogenetic trees of hematopoiesis, tracing blood cell lineages back to embryogenesis. We timed driver mutation acquisition, characterised the dynamics of tumour evolution and measured clonal expansion rates over the lifetime of patients. Resequencing of bulk blood samples corroborated clonal trajectories and provided population estimates. Results JAK2V617F was acquired in utero or childhood in all patients in whom JAK2V617F was the first or the only driver mutation. Earliest age estimates were within a few weeks post conception, and upper estimates of age of acquisition were between 4.1 months and 11.4 years, despite wide ranging ages of MPN presentation. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Subsequent driver mutation acquisition, including for JAK2V617F, was separated by decades. Disease latency following acquisition of JAK2V617F as a second driver event was still 12-27 years. DNMT3A mutations, commonly associated with age-related clonal hematopoiesis (CH), occurred as the first driver event, subsequent to mutated-JAK2, and as independent clones representing CH in MPN patients. DNMT3A mutations were also first acquired in utero or childhood, at the earliest 1.2 weeks post conception, and the latest 7.9 weeks of gestation to 7.8 years across 4 patients. A recurrent feature of the clonal landscape in MPN was the observation of similar genetic changes repeatedly occurring in unrelated clones within the same patient. Such 'parallel evolution' was observed for chr9p loss-of-heterozygosity, chr1q+ and mutations in myeloid cancer genes, suggesting that patient-specific factors flavour selective landscapes in MPN. Normal hematopoietic stem cells accumulated ~18 somatic mutations/year, however, mutant clones, particularly those with mutant-JAK2, acquired 1.5-5.5 excess mutations/ year and had shorter telomeres, reflecting increased cell divisions during clonal expansion. We modelled the rates of clonal expansion and found that they varied substantially, both across patients and within individuals. In one patient, an in utero acquired DNMT3A-mutated clone expanded slowly at 200%/year, doubling in size every 7 months. JAK2V617F as a single driver mutation also expanded variably across patients, highlighting that other factors, which may include germline, cytokine or stem cell differences between individuals, also influence selection for driver mutations. JAK2V617F associated clonal expansion rates in MPN were greater than that reported for JAK2-CH. Furthermore, rates of expansion in the cohort predicted time to clinical presentation, more so than age of mutation acquisition or tumour burden at diagnosis. This suggests that JAK2-mutant clonal expansion rates determine both if and when clinical manifestations occur. Driver mutations and rates of clonal expansion would have been detectable in blood one to four decades before clinical presentation. Conclusions MPN originate from driver mutation acquisition very early in life, even before birth, with life-long clonal expansion and evolution, establishing a new paradigm for blood cancer development. Early detection of mutant-JAK2 together with determination of clonal expansion rates could provide opportunities for early interventions aimed at minimising thrombotic risk and targeting the mutant clone in at risk individuals. Disclosures No relevant conflicts of interest to declare.

Background: In colorectal and breast cancer, the density and localization of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations. Methods and Findings: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. Epithelial CD8 (p=0.027, HR=0.83), stromal CD68 (p=3 × 10-4, HR=0.44) and stromal CD45RO (p=7 × 10-4, HR=0.76) were positively associated with survival and remained so when averaged across the tumor and stromal compartments.Using principal component analysis (PCA), we identified optimized multiparameter survival models that combined information from all immune markers (p=0.016, HR=0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components. Conclusions: Combining measures of immune infiltration provided improved survival modeling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was strongly associated with survival, underscoring the benefits that evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC. Citation Format: Anne Montfort, Steph J. Owen, Anna M. Piskorz, Anna Supernat, Luiza Moore, Sarwah Al-Khalidi, Steffen Boehm, Paul Pharoah, Jacqueline McDermott, Frances R. Balkwill, James D. Brenton. Combining measures of immune infiltration uncovers predictors of survival in high-grade serous ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4976.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited because of the difficulty of detecting somatic mutations in normal cells. Each colorectal crypt is a small clone of cells derived from a single recently-existing stem cell. Here, we whole genome sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during some phases of the cell lineage from zygote to adult cell. Likely driver mutations were present in ∼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.

BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.

Tumours with profound copy-number aberration elude molecular stratification due to their genomic complexity. By representing this complexity as a mixture of copy-number signatures, we provide molecular explanations for differing clinical outcomes. Here we present a method for copy-number signature identification, deriving eight signatures in 117 shallow whole-genome sequenced high-grade serous ovarian cancers (HGSOC), which validated on independent cohorts of 95 deep whole-genome sequenced, and 402 SNP array-profiled cases. Three copy-number signatures predicted longer overall survival, while the others predicted poorer outcome. We found evidence for the mutational processes giving rise to copy-number change for six of the eight signatures via correlations with other genomic features. Our results provide insights into the pathogenesis of HGSOC by uncovering multiple mutational processes that shape genomes following TP53 mutation. Importantly, our work shows that most HGSOC have a mixture of mutational processes suggesting that targeting a single mutator phenotype may be therapeutically suboptimal.

Human endometrium is a highly dynamic tissue that undergoes hundreds of cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone during female reproductive years. The marked regenerative capacity of this tissue’s epithelial compartment is maintained by intra-glandular adult stem cells (ASCs) that reside within the stratum basalis which is retained during menstruation. Although the endometrial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and the large intestines. In particular, the size of the stem cell pool within individual glands, the rates of their division, and mutational landscape are largely unknown. In this study, we isolated 215 morphologically normal endometrial glands from women aged 19 to 81 using laser capture microscopy. Analysis of whole-genome sequencing data identified that the overwhelming majority of the glands were clonal cell populations, and thus originating from a single ASC. Somatic mutations were found to accumulate at a linear rate during adult life. Elevated body mass index (BMI), a well-recognised risk factor for endometrial cancer, accelerated the rate of mutation acquisition. Surprisingly, despite the heterogeneity in age, reproductive history and BMI in our cohort, we find relatively homogenous mutational processes within normal endometrium. Comparison with cancer, shows lower somatic mutation burden and fewer operative signatures. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Over time, these mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. In older individuals, we observe a shift in the spectrum of acquired cancer-associated mutations, possibly reflecting post-menopausal changes in the levels of sex-steroid hormones and the resultant tissue microenvironment. Citation Format: Luiza Moore, Daniel Leongamornlert, Tim Coorens, Mathijs Sanders, Peter Ellis, Francesco Maura, Kevin Dawson, Simon F. Brunner, Jyoti Nangalia, Henry Lee-Six, Raheleh Rahbari, Patrick Tarpey, Yvette Hooks, Krishnaa Mahbubani, Christine A. Iacobuzio-Donahue, Jan J. Brosens, Inigo Martincorena, Kourosh Saeb-Parsy, Peter J. Campbell, Michael R. Stratton. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 970.

Summary The commonest causes of chronic liver disease are excess alcohol intake, viral hepatitis or non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation through cirrhosis to liver failure or hepatocellular carcinoma. The hepatocellular carcinoma genome exhibits diverse mutational signatures, resulting in recurrent mutations across >20-30 cancer genes1–7. Stem cells from normal livers have low mutation burden and limited diversity of signatures8, suggesting that the complexity of hepatocellular carcinoma arises during progression to chronic liver disease and subsequent malignant transformation. We sequenced whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers. Compared to normal liver, cirrhotic liver had higher mutation burden. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, both point mutations and structural variants, affected 1-5% clones. Clonal expansions millimetres in diameter occurred in cirrhosis, sequestered by bands of fibrosis engirdling regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCC; some were substantially more active in HCC than chronic liver disease; and others, arising from exogenous exposures, were present in a subset of patients. Up to 10-fold within-patient variation in activity of exogenous signatures existed between adjacent cirrhotic nodules, arising from clone-specific and microenvironmental forces. Synchronous hepatocellular carcinomas 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.