Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

The repertoire of mutational signatures in human cancer.

Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature1. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identified 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses3-15, enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated-but distinct-DNA damage, repair and/or replication mechanisms. By estimating the contribution of each signature to the mutational catalogues of individual cancer genomes, we revealed associations of signatures to exogenous or endogenous exposures, as well as to defective DNA-maintenance processes. However, many signatures are of unknown cause. This analysis provides a systematic perspective on the repertoire of mutational processes that contribute to the development of human cancer.

Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans.

Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents.

Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress.

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Clusters of Multiple Mutations: Incidence and Molecular Mechanisms.

It has been long understood that mutation distribution is not completely random across genomic space and in time. Indeed, recent surprising discoveries identified multiple simultaneous mutations occurring in tiny regions within chromosomes while the rest of the genome remains relatively mutation-free. Mechanistic elucidation of these phenomena, called mutation showers, mutation clusters, or kataegis, in parallel with findings of abundant clustered mutagenesis in cancer genomes, is ongoing. So far, the combination of factors most important for clustered mutagenesis is the induction of DNA lesions within unusually long and persistent single-strand DNA intermediates. In addition to being a fascinating phenomenon, clustered mutagenesis also became an indispensable tool for identifying a previously unrecognized major source of mutation in cancer, APOBEC cytidine deaminases. Future research on clustered mutagenesis may shed light onto important mechanistic details of genome maintenance, with potentially profound implications for human health.

Correction: Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

[This corrects the article DOI: 10.1371/journal.pbio.3000464.].

Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases.

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

Mutational signatures of redox stress in yeast single-strand DNA and of aging in human mitochondrial DNA share a common feature.

Redox stress is a major hallmark of cancer. Analysis of thousands of sequenced cancer exomes and whole genomes revealed distinct mutational signatures that can be attributed to specific sources of DNA lesions. Clustered mutations discovered in several cancer genomes were linked to single-strand DNA (ssDNA) intermediates in various processes of DNA metabolism. Previously, only one clustered mutational signature had been clearly associated with a subclass of ssDNA-specific apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. Others remain to be elucidated. We report here deciphering of the mutational spectra and mutational signature of redox stress in ssDNA of budding yeast and the signature of aging in human mitochondrial DNA. We found that the predominance of C to T substitutions is a common feature of both signatures. Measurements of the frequencies of hydrogen peroxide-induced mutations in proofreading-defective yeast mutants supported the conclusion that hydrogen peroxide-induced mutagenesis is not the result of increased DNA polymerase misincorporation errors but rather is caused by direct damage to DNA. Proteins involved in modulation of chromatin status play a significant role in prevention of redox stress-induced mutagenesis, possibly by facilitating protection through modification of chromatin structure. These findings provide an opportunity for the search and identification of the mutational signature of redox stress in cancers and in other pathological conditions and could potentially be used for informing therapeutic decisions. In addition, the discovery of such signatures that may be present in related organisms should also advance our understanding of evolution.

Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells.

Mutation signatures specific to DNA alkylating agents in yeast and cancers.

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (SN1 and SN2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that SN1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of SN1-type agents was conserved across yeast, mice and human cancers. Conversely, SN2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to SN2-type alkylating chemicals. The estimates of mutation loads associated with the SN2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the SN2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers.

Tracing Lung Cancer Risk Factors Through Mutational Signatures in Never-Smokers.

Epidemiologic studies often rely on questionnaire data, exposure measurement tools, and/or biomarkers to identify risk factors and the underlying carcinogenic processes. An emerging and promising complementary approach to investigate cancer etiology is the study of somatic "mutational signatures" that endogenous and exogenous processes imprint on the cellular genome. These signatures can be identified from a complex web of somatic mutations thanks to advances in DNA sequencing technology and analytical algorithms. This approach is at the core of the Sherlock-Lung study (2018-ongoing), a retrospective case-only study of over 2,000 lung cancers in never-smokers (LCINS), using different patterns of mutations observed within LCINS tumors to trace back possible exposures or endogenous processes. Whole genome and transcriptome sequencing, genome-wide methylation, microbiome, and other analyses are integrated with data from histological and radiological imaging, lifestyle, demographic characteristics, environmental and occupational exposures, and medical records to classify LCINS into subtypes that could reveal distinct risk factors. To date, we have received samples and data from 1,370 LCINS cases from 17 study sites worldwide and whole-genome sequencing has been completed on 1,257 samples. Here, we present the Sherlock-Lung study design and analytical strategy, also illustrating some empirical challenges and the potential for this approach in future epidemiologic studies.

Infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies.

Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10-3 subs/site/year and 8.9 x 10-4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.

Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions.

Mutations are typically perceived as random, independent events. We describe here nonrandom clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of "strand-coordinated" changes of either cytosines or guanines in the same strand, mutation patterns, and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand DNA (ssDNA) formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hypermutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600­13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.

Division of labor at the eukaryotic replication fork.

DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many years. Here we investigate the contribution of Pol delta to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol delta allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T x dGTP) than for its complement (A x dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol delta replication is performed using the lagging strand template. When combined with recent evidence implicating Pol epsilon in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol epsilon and Pol delta, respectively.

Clustered and genome-wide transient mutagenesis in human cancers: Hypermutation without permanent mutators or loss of fitness.

The gain of a selective advantage in cancer as well as the establishment of complex traits during evolution require multiple genetic alterations, but how these mutations accumulate over time is currently unclear. There is increasing evidence that a mutator phenotype perpetuates the development of many human cancers. While in some cases the increased mutation rate is the result of a genetic disruption of DNA repair and replication or environmental exposures, other evidence suggests that endogenous DNA damage induced by AID/APOBEC cytidine deaminases can result in transient localized hypermutation generating simultaneous, closely spaced (i.e. "clustered") multiple mutations. Here, we discuss mechanisms that lead to mutation cluster formation, the biological consequences of their formation in cancer and evidence suggesting that APOBEC mutagenesis can also occur genome-wide. This raises the possibility that dysregulation of these enzymes may enable rapid malignant transformation by increasing mutation rates without the loss of fitness associated with permanent mutators.