Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.

The epigenetic landscape of cancer is regulated by many factors, but primarily it derives from the underlying genome sequence. Chromothripsis is a catastrophic localized genome shattering event that drives, and often initiates, cancer evolution. We characterized five esophageal adenocarcinoma organoids with chromothripsis using long-read sequencing and transcriptome and epigenome profiling. Complex structural variation and subclonal variants meant that haplotype-aware de novo methods were required to generate contiguous cancer genome assemblies. Chromosomes were assembled separately and scaffolded using haplotype-resolved Hi-C reads, producing accurate assemblies even with up to 900 structural rearrangements. There were widespread differences between the chromothriptic and wild-type copies of chromosomes in topologically associated domains, chromatin accessibility, histone modifications, and gene expression. Differential epigenome peaks were most enriched within 10 kb of chromothriptic structural variants. Alterations in transcriptome and higher-order chromosome organization frequently occurred near differential epigenetic marks. Overall, chromothripsis reshapes gene regulation, causing coordinated changes in epigenetic landscape, transcription, and chromosome conformation.

Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

The mutational landscape of human somatic and germline cells.

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.

Extensive heterogeneity in somatic mutation and selection in the human bladder.

The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted (n = 1914 microbiopsies), whole-exome (n = 655), and whole-genome (n = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.

Bionanomining: biotechnological synthesis of metal nanoparticles from mining waste-opportunity for sustainable management of mining environmental liabilities.

Mining is an important activity for many countries, especially some in development, such as Chile, where it is a pillar of its economy. However, it generates large impacts that are undesirable for the population such as the generation of polluting solid and effluents with a high content of heavy metals and metalloids, which are traditionally accumulated in deposits. In recent years, bionanomining emerged as a cutting-edge scientific-technological development associated with the application of micro- and macro-organisms to generate nanotechnological products by using mining and industrial wastes and wastewaters. Biomass of many species of bacteria, plants, algae and fungi have the ability to reduce or oxidise cations, which can physically be deposited as nanometric materials such as the nanoparticles. Nanoparticles are materials that are increasingly used, and therefore, their demand increase, based on the high surface area characteristics to improve thermal, electrical and optical properties of materials, and metallic ones have also antimicrobial activity. This review addresses the biosynthesis of metal nanoparticles, focusing on mining waste recovery strategies, which is an emerging reality in mining countries. Transformation of potentially hazardous waste into a valuable product through techniques that are eco-friendly is an opportunity to develop sustainably depressed or polluted sites.

Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected.

ascatNgs: Identifying Somatically Acquired Copy-Number Alterations from Whole-Genome Sequencing Data.

We have developed ascatNgs to aid researchers in carrying out Allele-Specific Copy number Analysis of Tumours (ASCAT). ASCAT is capable of detecting DNA copy number changes affecting a tumor genome when comparing to a matched normal sample. Additionally, the algorithm estimates the amount of tumor DNA in the sample, known as Aberrant Cell Fraction (ACF). ASCAT itself is an R-package which requires the generation of many file types. Here, we present a suite of tools to help handle this for the user. Our code is available on our GitHub site (https://github.com/cancerit). This unit describes both 'one-shot' execution and approaches more suitable for large-scale compute farms. © 2016 by John Wiley & Sons, Inc.

Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

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

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

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

Subclonal diversification of primary breast cancer revealed by multiregion sequencing.

The sequencing of cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient's tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole-genome and targeted sequencing to multiple samples from each of 50 patients' tumors (303 samples in total). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13 out of 50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resistance to chemotherapy and the acquisition of invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer.

Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin.

How somatic mutations accumulate in normal cells is central to understanding cancer development but is poorly understood. We performed ultradeep sequencing of 74 cancer genes in small (0.8 to 4.7 square millimeters) biopsies of normal skin. Across 234 biopsies of sun-exposed eyelid epidermis from four individuals, the burden of somatic mutations averaged two to six mutations per megabase per cell, similar to that seen in many cancers, and exhibited characteristic signatures of exposure to ultraviolet light. Remarkably, multiple cancer genes are under strong positive selection even in physiologically normal skin, including most of the key drivers of cutaneous squamous cell carcinomas. Positively selected mutations were found in 18 to 32% of normal skin cells at a density of ~140 driver mutations per square centimeter. We observed variability in the driver landscape among individuals and variability in the sizes of clonal expansions across genes. Thus, aged sun-exposed skin is a patchwork of thousands of evolving clones with over a quarter of cells carrying cancer-causing mutations while maintaining the physiological functions of epidermis.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

Polygenic in vivo validation of cancer mutations using transposons.

The in vivo validation of cancer mutations and genes identified in cancer genomics is resource-intensive because of the low throughput of animal experiments. We describe a mouse model that allows multiple cancer mutations to be validated in each animal line. Animal lines are generated with multiple candidate cancer mutations using transposons. The candidate cancer genes are tagged and randomly expressed in somatic cells, allowing easy identification of the cancer genes involved in the generated tumours. This system presents a useful, generalised and efficient means for animal validation of cancer genes.

Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.

Long interspersed nuclear element-1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3' transduction. Because 3' transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3' transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3' transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3' transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.

Processed pseudogenes acquired somatically during cancer development.

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5' truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia.

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

Whole exome sequencing of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a rare malignancy that can occur in multiple organ sites and is primarily found in the salivary gland. While the identification of recurrent fusions of the MYB-NFIB genes have begun to shed light on the molecular underpinnings, little else is known about the molecular genetics of this frequently fatal cancer. We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease. We identified multiple mutated genes that, combined, implicate chromatin deregulation in half of cases. Further, mutations were identified in known cancer genes, including PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, and CYLD. Mutations in NOTCH1/2 were identified in 3 cases, and we identify the negative NOTCH signaling regulator, SPEN, as a new cancer gene in ACC with mutations in 5 cases. Finally, the identification of 3 likely activating mutations in the tyrosine kinase receptor FGFR2, analogous to those reported in ovarian and endometrial carcinoma, point to potential therapeutic avenues for a subset of cases.