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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., SCOPUS: er.j, info:eu-repo/semantics/published

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials. Postprint

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.

Normal skin's curiously abnormal genome Within every tumor, a battle is being waged. As individual tumor cells acquire new mutations that promote their survival and growth, they clonally expand at the expense of tumor cells that are “less fit.” Martincorena et al. sequenced 234 biopsies of sun-exposed but physiologically normal skin from four individuals (see the Perspective by Brash). They found a surprisingly high burden of mutations, higher than that of many tumors. Many of the mutations known to drive the growth of cutaneous squamous cell carcinomas were already under strong positive selection. More than a quarter of normal skin cells carried a driver mutation, and every square centimeter of skin contained hundreds of competing mutant clones. Science , this issue p. 880 ; see also p. 867

Technological and legislative safety solutions will only succeed if the social systems, attitudes and behaviours at work support them. Safety climate is one of the factors related to effective hazard management, including management of noise. Data on levels of noise, risk factors and compliance with the relevant Code of Practice were collected from 20 New Zealand small and medium workplaces with employee counts ranging from 2–48. One hundred and sixty-three (163) respondents from these workplaces also provided data on self-reported hearing protection use, safety climate and attitudes to controlling noise at work. Levels of compliance with New Zealand Approved Code of Practice requirements were low, even in workplaces with high levels of noise. Use of hearing protection devices (HPD) was highest in construction and manufacturing but no respondents in hospitality (cafes) or education (early childhood education) reported using hearing protection despite some high levels of noise.

It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

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.

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. ispartof: Nucleic acids research vol:41 issue:12 pages:6119-6138 ispartof: location:England status: published

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS-myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic "predestination," in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.

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

Item does not contain fulltext 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.

Summary Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis. PaperClip, Graphical Abstract Highlights ► Genome-wide analyses of mutations emerging through time in 21 breast cancers ► Minimal expansion of subclones occurs until thousands of mutations have accumulated ► Cancer-specific signatures of point mutations and genomic instability emerge late ► ERBB2 amplification begins early but continues to evolve over long molecular time, Newly developed algorithms allow the reconstruction of the genomic history of different breast cancers, tracing the temporal evolution of each tumor and the emergence of the dominant subclones that will eventually trigger diagnosis.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model., The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 249894 (SelSweep)

Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at www.openbiosystems.com). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.

© 2015 Nature America, Inc. All rights reserved.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.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

Aims and MethodThe ability of routine clinical practice to detect drug use at acute psychiatric admission was assessed by comparing the results of urinalysis with information on drug use extracted from patient records (n=112).ResultsUrinalysis detected drug use in 23% of the sample. Cannabis was the drug most frequently found. Of the cases of drug use positively identified by urinalysis, 54% were not identified in the notes. Some information on drug use proximal to admission was found in 40% of patient records. Patients asked about proximal drug use were younger than those not asked.Clinical ImplicationsAll patients should be asked about drug use.

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment., Multiple myeloma is a malignant plasma cell disorder with a complex molecular pathogenesis. Here, the authors perform whole-exome sequencing, copy-number profiling and cytogenetic analysis in 84 myeloma samples and highlight the diversity and evolution of the mutational profile underlying the disease.

As the evidence for the efficacy of treatment programmes for substance misuse and severe mental illness is very limited, a national three stage Delphi survey was conducted to try and achieve consensus of views amongst a panel of 154 experts in the UK. The panel reached consensus on the best ways to identify those with severe mental illness and substance misuse problems, on principles essential for a service and on training and research. There was also consensus on interventions/techniques considered the most important, and on those considered less important. There was no consensus on how services should be organised. The survey reflects the best evidence so far as to the views of those in the UK who are involved in this challenging area. The consensus items offer useful and realistic guidance for practitioners on the identification and treatment of those with severe mental illness and substance misuse problems.

Aims and MethodWe undertook a postal questionnaire survey of drug action teams in England and Wales with the aim of clarifying the nature of statutory specialist drug services.ResultsOf 159 drug action teams, 110 (69%) responded; 64 (58%) reported that mental health trusts exclusively provided their specialist drug services. Other providers were primary care and acute trusts, the non-statutory sector and social services. The majority of medical leads were psychiatrists (123 senior posts with 20% vacant/occupied by a locum), then general practitioners (GPs) (42) and other specialists (4).Clinical ImplicationsSpecialist drug services are offered by a range of treatment providers, with the medical lead being taken by GPs and other specialists in some areas. In view of the current difficulty in recruiting psychiatrists, we propose that alternative training pathways are considered for addiction specialists.

Item does not contain fulltext BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

A year-long survey, with samples collected weekly, was conducted to estimate long-term average concentrations of arsenic in treated and untreated drinking water for the city of Hamilton, New Zealand. The average concentration of arsenic in Waikato River water at Hamilton before processing was found to be (32.1 +/- 3.7) microg litre(-1), about three times above the new WHO limit of 10 microg litre(-1). However, full conventional water treatment effects a five-fold reduction in arsenic concentrations in drinking water to a level which meets the new standards of (6.2 +/- 0.8) microg litre(-1). The results of both this study and retrospective analysis of archived data suggest that total arsenic concentrations in the Waikato River are likely to follow a regular seasonal variation, being about 10-25 microg litre(-1) higher in the summer months. Changes in river flow rates have an almost negligible effect on the summer peak and winter trough; it is proposed that most of the variation in total arsenic concentrations may be due to the seasonal conversion of arsenic in the river sediments to more soluble forms.

SummaryAll cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed “kataegis,” was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed.PaperClip

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis(1), and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease.

Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium.-- et al., The myelodysplastic syndromes are a heterogeneous group of hematologic cancers characterized by low blood counts, most commonly anemia, and a risk of progression to acute myeloid leukemia.1 These disorders have increased in prevalence and are expected to continue to do so. Blood films and bone marrow¿biopsy specimens from patients with myelodysplastic syndromes show dysplastic changes in myeloid cells, with abnormal proliferation and differentiation of one or more lineages. Target genes of recurrent chromosomal aberrations have been mapped,2,3 and several genes have been identified as recurrently mutated in these disorders, including NRAS (encoding neuroblastoma RAS viral oncogene homologue), TP53 (encoding tumor protein p53), RUNX1 (encoding runt-related transcription factor 1), CBL (encoding Cas-Br-M ecotropic retroviral transforming sequence),4,5 TET2 (encoding tet oncogene family member 2),6,7 ASXL1 (encoding additional sex combs¿like protein 1),8,9 and EZH2 (encoding enhancer of zeste homologue 2).10 With the exception of TET2, most of these genes are mutated in no more than 5 to 15% of cases, and generally the mutation rates are lower in the more benign subtypes of the disease. The myelodysplastic syndromes can be divided into several categories on the basis of bone marrow and peripheral-blood morphologic characteristics and cytogenetic changes.11 In low-risk disease, such as refractory anemia, cytopenias are the major clinical challenge, whereas high-risk disease, such as refractory anemia with excess blasts, is characterized by both cytopenias and a high rate of transformation to acute myeloid leukemia. More than a quarter of patients with myelodysplastic syndromes have large numbers of ring sideroblasts in the bone marrow,12 a sufficiently distinctive morphologic abnormality to warrant a separate designation. Ring sideroblasts are characteristically seen on iron staining of bone marrow aspirates as differentiating erythroid cells with a complete or partial ring of iron-laden mitochondria surrounding the nucleus. Several genetic lesions underpinning inherited sideroblastic anemias have been identified,13 including loss-of-function mutations in the genes ALAS2 (encoding delta aminolevulinate synthase 2), ABCB7 (encoding ATP-binding cassette, subfamily B, member 7), and SLC25A38 (solute carrier family 25, member 38). The pathogenesis of ring sideroblasts in myelodysplastic syndromes, however, remains obscure, although gene-expression studies have revealed up-regulation of genes involved in heme synthesis (including ALAS2) and down-regulation of ABCB7.14,15 We reasoned that the identification of recurrently mutated cancer genes in low-grade myelodysplastic syndromes could prove useful for the diagnosis of these disorders and provide new insights into the molecular pathogenesis of these syndromes, Supported by grants from the Wellcome Trust (077012/Z/05/Z, for the overall study, as well as WT088340MA, to Dr. Campbell), the Kay Kendall Leukaemia Fund, Leukemia Lymphoma Research (for the overall study and to Drs. Boultwood, Green, Vyas, and Wainscoat), the Adenoid Cystic Carcinoma Research Foundation, the Medical Research Council (MRC) (to Dr. Warren), the Oxford National Institutes for Health Research Biomedical Research Centre (to Drs. Boultwood, Vyas, and Wainscoat), the Swedish Cancer Society (to Dr. Hellstrom-Lindburg), the International Human Frontier Science Program Organization (to Dr. Varela), the Department of Veterans Affairs and the National Institutes of Health (R01-124929, P01-155249, P50- 100007, and P01-78378, to Drs. Munshi and Anderson), the Association for International Cancer Research and the Leukemia Lymphoma Society (to Drs. Warren and Green), Associazione Italiana per la Ricerca sul Cancro (to the University of Pavia, the University of Milan Bicocca, and Dr. Cazzola), and Fondazione Cariplo (to the University of Pavia and the University of Milan Bicocca).

El pdf del artículo es el manuscrito de autor (PMCID: PMC3030920).-- et al., The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ~3,500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (also known as KDM6A)1, JARID1C (also known as KDM5C) and SETD2 (ref. 2). These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control3. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodelling complex gene PBRM1 (ref. 4) as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology., P.A.F. and M.R.S. would like to acknowledge the Wellcome Trust for support under grant reference 077012/Z/05/Z and A. Coffey, D. Turner and L. Mamanova for assistance with the exon capture. K.F., K.D. and B.T.T. acknowledge the support of the Van Andel Research Institute. B.T.T. would like to acknowledge support from the Lee Foundation. I.V. is supported by a fellowship from The International Human Frontier Science Program Organization. D.J.A. acknowledges the support of Cancer Research UK. D.A.T. and P.A.P.-M. acknowledge the support of the University of Cambridge, Cancer Research UK and Hutchison Whampo and thank W. Howatt, A. Hazelhurst and colleagues in the CRI core facilities for their support.

Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%¿3% of all cancers, across many subtypes, and is present in ~25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer., This work was supported by the Wellcome Trust (grant reference 077012/Z/ 05/Z) and the Chordoma Foundation. P.J.C. is personally funded through a Wellcome Trust Senior Clinical Research Fellowship (grant reference WT088340MA). I.V. is supported by a fellowship from The International Human Frontier Science Program Organization.

Background Xenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls. Results Here we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission. Conclusions We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

Christine Iacobuzio-Donahue and colleagues use whole-genome exome sequencing to analyse primary pancreatic cancers and one or more metastases from the same patients, and find that tumours are composed of distinct subclones. The authors also determine the evolutionary maps by which metastatic cancer clones have evolved within the primary tumour, and estimate the timescales of tumour progression. On the basis of these data, they estimate a mean period of 11.8 years between the initiation of pancreatic tumorigenesis and the formation of the parental, non-metastatic tumour, and a further 6.8 years for the index metastasis clone to arise. These data point to a potentially large window of opportunity during which it might be possible to detect the cancer in a relatively early form. Peter Campbell and colleagues use next-generation sequencing to detect chromosomal rearrangements in 13 patients with pancreatic cancer. The results reveal considerable inter-patient heterogeneity and indicate ongoing genomic instability and evolution during the development of metastases. But for most of the patients studied, more than half of the genetic rearrangements found were present in all metastases and the primary tumour, making them potential targets for therapeutic intervention at early and late stages of the disease. Pancreatic cancer is highly aggressive, usually because of widespread metastasis. Here, next-generation DNA sequencing has been used to detect genomic rearrangements in 13 patients with pancreatic cancer and to explore clonal relationships among metastases. The results reveal not only considerable inter-patient heterogeneity, but also ongoing genomic instability and evolution during the development of metastases. Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97–98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations1,2,3,4,5, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours6,7, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites7, and how the tumour disseminates. Here we harness advances in DNA sequencing8,9,10,11,12 to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2–M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

Within the setting of a regional secure unit, all doses of medication given p.r.n. over three months were ascertained and the details of each administration determined from prescription charts and a semistructured interview with the nursing staff involved. Thirty-two patients were resident for all or part of the study, all compulsorily detained. Only 15 were given medication p.r.n.) compared with the other patients they were significantly younger and more likely to be detained under civil orders than under the criminal provisions of the Mental Health Act 1983. Rarely was the medication the sole intervention in a crisis. In terms of effects on the overall treatment programme of the patient, medication given p.r.n. seemed to have an impact in only one small subgroup.

Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci ( l4Rn1 , l4Rn2 , and l4Rn3 ) and the fitness mutant brown-associated fitness ( baf ). We have also characterized misexpression of the basonuclin homologue, Bnc2 , associated with the inversion-mediated coat color mutant white-based brown ( B w ). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis.

Former consultant psychiatrist Torbay Hospital (b London 1951; q St Bartholomew's Hospital, London, 1977; MRCS, MRCPsych), died from postoperative sepsis on 2 June 2005 After higher training in Oxford Chris worked as a consultant psychiatrist with a special interest in rehabilitation for some 20 years in south Devon. He was instrumental in developing local addiction services, and at different times served as medical director and college tutor. With great expertise in drug therapy he was an early adopter of atypical antipsychotic medication. He had a real zest for life and outside medicine was a keen musician and cricketer, more latterly a cricket umpire. He leaves a wife, Joyce, and two children.

Patients who have had one oral cancer are at increased risk of developing a semi-malignant tumour. The detecting of oral cancer is made difficult (and is often delayed) by the unknown appearance of the early oral lesion. A technique that could reliably detect early cancers would be useful to the oral and dental health specialist. One possible technique is the use of a photosensitiser that may be preferentially taken up by cancerous cells. 5-aminolaevulinic acid (ALA) is one such drug that is converted to Protoporphyrin IX (PpIX) and fluoresces at 636nm when illuminated with light of wavelength 405nm. It has been hypothesized that cell inclined towards malignant change would have a higher metabolic rate, and thus convert more ALA into its metabolite PpIX. These drugs can then be detected using a technique called Photodynamic detection, through the analysis of their fluorescence spectra. We describe a pilot study that used a compact spectroscopic instrument designed to excite and measure fluorescence in the oral cavity. Some Inter-subject variation in PpIX time course characteristics may be evident in our volunteers, as has been reported by other researchers. The obtained data would suggest that this instrument may be a valuable tool for detecting early oral cancers. However, further studies are required, not least to ensure that these data are due to detection of ALA metabolite in cancer and not some other systemic effect.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

Corrigendum: Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

Nature 496, 498–503 (2013); doi:10.1038/nature12111 In this Letter, five authors were inadvertently omitted: Sharmin Begum and Christine Lloyd from the Wellcome Trust Sanger Institute, and Christa Lanz, Gunter Raddatz and Stephan C. Schuster from the Max Planck Institute for Developmental Biology. David Elliot was incorrectly listed as David Eliot, Beverley Mortimore was incorrectly listed as Beverly Mortimer, and James D.

BCR-ABL negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPLmutations remains obscure. Methods Exome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence. Results Exome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases. Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients. Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs. The mutation spectrum showed a predominance of C>T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers. Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student’s t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03). All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention. Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals. Conclusions We describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients. Disclosures: Bowen: Celgene: Honoraria. Harrison:S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Multiple myeloma (MM) is a malignancy of post-germinal centre B-cells whose pathogenesis is only partially understood. Chromosomal hyperdiploidy and recurrent immunoglobulin gene locus rearrangements are frequent, but are insufficient for malignant transformation, which is associated with additional events such as somatic mutations, epigenomic aberrations, and chromosomal copy-number changes. To investigate genomic event underlying MM pathogenesis and evolution, we used whole exome sequencing, copy number profiling and cytogenetics in 67 patients and 84 samples. For 15 patients, 2 or 3 serial samples (median 299 days apart) were available. Exome reads were used to call substitutions and indels. We used the Genome-Wide SNP Array 6.0 or exome reads to estimate the allele-specific copy number of the tumor. To cluster variants and estimate the clonal architecture of each sample and its evolution over time, we used the mutation burden, corrected for copy number and normal cell contamination. Analysis of the clonal structure of the tumors showed at least one subclone in 94% of patients at diagnosis, suggesting that myeloma is a heterogeneous disease at presentation. Interestingly, many mutations of known MM driver genes (KRAS, NRAS, BRAF, TP53, FAM46C) were subclonal at diagnosis. In 5/67 patients, BRAF and KRAS/NRAS mutations co-existed in the same sample, raising therapeutic implications given the paradoxical ERK-activating effect of BRAF inhibitors in RAS-mutated cells. Furthermore, only 3/10 BRAF variants were V600E, the current target of most inhibitors. Altogether, only the 5 previously known genes were significantly enriched in our cohort, highlighting marked heterogeneity of the spectrum of candidate driver gene mutations across MM patients. Nevertheless, we identified several new recurrent gene lesions: inactivating mutations of SP140 (7.5%), a gene previously linked to germline susceptibility to CLL, and in ROBO1 (7.5%), a gene recently implicated in pancreatic cancer; clustered missense substitutions in EGR1 (6%), a gene previously implicated in plasma cell apoptosis; clustered truncating mutations in LTB (4.5%), a TNF-family protein implicated in lymphoid development. The subclonal structure of the sample changed over time in 72% paired samples, highlighting genomic evolution at relapse. We described 4 different scenarios with striking concordance between mutations and chromosomal copy number changes: no change, linear evolution (a new clone appears in the later sample), differential clonal response (the relative proportions of the subclones change over time), and branching evolution (new clones emerge, while others decline in frequency or disappear). All subclonal variants in known driver myeloma genes increased their clonal fraction at the later time-point, consistent with the expected positive selection for the subclones harboring them. To investigate mutational processes responsible for the generation of the mutational repertoire in MM, we extracted the variant context and analyzed the mutational signatures. We found two signatures in our samples. The most represented one is enriched for spontaneous deamination of methylated cytosines, a common process in cancer and aged cells. The second signature was more represented in samples showing extremely high numbers of variants, sometimes clustered in small regions of ∼200 bp (kataegis). We hypothesize that it results from aberrant activity of the APOBEC family of cytosine deaminases, recently described in breast cancer. Interestingly, cases of extramedulary relapse were always associated with branching evolution and showed increased contribution from this APOBEC signature. In conclusion, in our cohort of MM samples we show: 1) evidence of tumor heterogeneity at the time of diagnosis; 2) discernable genetic changes and shifts in the clonal structure of disease at the time of progression; 3) different mutational processes responsible for an heterogeneous mutational repertoire across patients, and over time in the same patient; 4) a comprehensive list of recurrent variants, many of which are previously unreported. Our study provides new insights into the genomic architecture of MM, and will help identify molecular alterations associated with progression of disease and development of drug resistance. Disclosures: Tai: Onyx: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Moreau:Celgene: Honoraria, Speakers Bureau. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity’s Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder , Scientific Founder Other.

Background: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. Results: To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. Conclusion: This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

Background This paper reports on the second part of a major study in New Zealand to identify effective strategies for the prevention of noise induced hearing loss (NIHL). Aims/Objectives/Purpose The objective of the project was to evaluate existing work-related interventions to reduce NIHL, to identify critical factors in the development and implementation of such strategies, and to propose strategies/interventions where current interventions are considered ineffective. In addition, the research examined those aspects of workplace culture that affect decision-making around NIHL. Methods A systematic review of the research literature was completed specifically focussing on the effectiveness of interventions in the prevention of NIHL. Data collection involved surveys of specific industry sectors with ‘high, medium and low’ risk of NIHL. In addition to area noise measurements and personal dosimetry, assessments of the organisation9s conformance to current noise management standards and safety climate data were undertaken. Results/Outcome Five key effective strategies were identified from the literature. As anticipated, area and personal noise exposures were found to vary considerably within the ‘high risk’ (agriculture, manufacturing and construction; range: LAeq 8 h 80–90 dB), ‘moderate risk’ (cafes and restaurants) and ‘low risk’ sectors (pre-schools; range LAeq 8 h 70–80 dB). Most enterprises surveyed did not conform to the specific requirements of the noise management standard (Code of Practice). Safety climate was not the major factor in noise management within the enterprises. Significance/Contribution to the Field The research provided disappointing evidence on the implementation of noise management strategies in NZ businesses. As a consequence of the research, a comprehensive multi-level intervention strategy is proposed.

Background A selection of commercially available imported toys was assessed for acoustic safety by testing to the voluntary toy safety Standard. The results were compared to allowable noise levels for adults in a working environment. Aims/Objectives/Purpose The aim was to establish whether the revised toy Standard sets a voluntary acoustic level which offers sufficient hearing protection for children and identify changes to protect potential hearing damage in young children in future. Methods A number of commercially available toys were obtained and tested for compliance with the acoustic provisions of the 2002 and 2010 V of the Australian/New Zealand Standard AS/NZS ISO 8124.1, with consideration as to how they might be played with or used. Results/Outcome The examination revealed that many toys produced unacceptably high noise levels. It also highlighted that the revised Standard criteria exempted squeeze toys and rattles. No acoustical criteria were included for toys promoted as enhancing educational outcomes. No tolerance levels were included in the standard to account for interpretation of data and experimental error. This suggests a failure of the Standard to provide adequate hearing protection for young children. Amendments to the criteria have been recommended. Significance/Contribution to the Field Children may play with noisy toys for extended periods, or hold toys closer to the ear than expected by suppliers, which may be considered to be ‘reasonably foreseeable misuse’, as defined in AS/NZS ISO 8124.1. Failure to address this issue now may result in increasing incidence of early hearing impairment in future, raising questions of fiscal, educational and social cost. Conference topic Product safety Conference theme Safety design and sustainability oral presentation

Nature 469, 539–542 (2011) In this Letter, the accession number for the gene expression data in the Gene Expression Omnibus was wrongly published as GEO22316. The correct accession number is GSE22316. We thank P. Boutros for pointing this out. This has been corrected in the HTML and PDF versions online.

The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ∼3500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (KDM6A), JARID1C (KDM5C) and SETD2. These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodeling complex gene PBRM1 as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2011-2805

This short book, produced by the Clinical Governance Support Service at the Royal College of Psychiatrists' Research Unit, reports on 28 clinical audits carried out by a number of UK substance misuse services. Topic areas covered include patient assessment, aspects of clinical care such as shorter