High-throughput analysis of chromosome translocations and other genome rearrangements in epithelial cancers.

Genes that are broken or fused by structural changes to the genome are an important class of mutation in the leukemias and sarcomas but have been largely overlooked in the common epithelial cancers. Large-scale sequencing is changing our perceptions of the cancer genome, and it is now being applied to structural changes, using the 'paired end' strategy. This reveals more clearly than before the extent to which many cancer genomes are rearranged and how much these rearrangements contribute to the mutational burden of epithelial tumors. In particular, there are probably many fusion genes, analogous to those found in leukemias, to be found in common cancers, such as breast carcinoma, and some of these will prove to be important in cancer diagnosis and treatment.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Targeted next-generation sequencing for routine clinical screening of mutations

In many fields it is now desirable to sequence large panels of genes for mutation, to aid management of patients. The need for extensive sample preparation means that current approaches for assessing mutation status in the clinical setting are limited. A recent publication demonstrates a single-step, targeted, true single-molecule sequencing approach to assessing the mutational status of BRCA1. Fragmented DNA samples are loaded directly onto a flow cell and sequenced, thus detecting both small- and large-scale mutations with minimal sample preparation and high accuracy

The relative timing of mutations in a breast cancer genome.

Many tumors have highly rearranged genomes, but a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. Chromosome instability might arise early, be a late event contributing little to cancer development, or happen as a single catastrophic event. Another unknown is which of the point mutations and rearrangements are selected. To address these questions we show, using the breast cancer cell line HCC1187 as a model, that we can reconstruct the likely history of a breast cancer genome. We assembled probably the most complete map to date of a cancer genome, by combining molecular cytogenetic analysis with sequence data. In particular, we assigned most sequence-level mutations to individual chromosomes by sequencing of flow sorted chromosomes. The parent of origin of each chromosome was assigned from SNP arrays. We were then able to classify most of the mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome, endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided earlier and later, suggesting that genetic instability was relatively constant throughout the life of this tumor, and chromosome instability was not a late event. Mutations that caused chromosome instability would be in the earlier set. Strikingly, the great majority of inactivating mutations and in-frame gene fusions happened earlier. The non-random timing of some of the mutations may be evidence that they were selected

High-resolution array CGH clarifies events occurring on 8p in carcinogenesis.

BACKGROUND: Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb - 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. METHODS: We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. RESULTS: We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb - 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb - 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb - 23.4 Mb) in two lines and one of 98 tumours. CONCLUSION: The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene

Mobile element insertions are frequent in oesophageal adenocarcinomas and can mislead paired-end sequencing analysis.

BACKGROUND: Mobile elements are active in the human genome, both in the germline and cancers, where they can mutate driver genes. RESULTS: While analysing whole genome paired-end sequencing of oesophageal adenocarcinomas to find genomic rearrangements, we identified three ways in which new mobile element insertions appear in the data, resembling translocation or insertion junctions: inserts where unique sequence has been transduced by an L1 (Long interspersed element 1) mobile element; novel inserts that are confidently, but often incorrectly, mapped by alignment software to L1s or polyA tracts in the reference sequence; and a combination of these two ways, where different sequences within one insert are mapped to different loci. We identified nine unique sequences that were transduced by neighbouring L1s, both L1s in the reference genome and L1s not present in the reference. Many of the resulting inserts were small fragments that include little or no recognisable mobile element sequence. We found 6 loci in the reference genome to which sequence reads from inserts were frequently mapped, probably erroneously, by alignment software: these were either L1 sequence or particularly long polyA runs. Inserts identified from such apparent rearrangement junctions averaged 16 inserts/tumour, range 0-153 insertions in 43 tumours. However, many inserts would not be detected by mapping the sequences to the reference genome, because they do not include sufficient mappable sequence. To estimate total somatic inserts we searched for polyA sequences that were not present in the matched normal or other normals from the same tumour batch, and were not associated with known polymorphisms. Samples of these candidate inserts were verified by sequencing across them or manual inspection of surrounding reads: at least 85 % were somatic and resembled L1-mediated events, most including L1Hs sequence. Approximately 100 such inserts were detected per tumour on average (range zero to approximately 700). CONCLUSIONS: Somatic mobile elements insertions are abundant in these tumours, with over 75 % of cases having a number of novel inserts detected. The inserts create a variety of problems for the interpretation of paired-end sequencing data.Funding was primarily from Cancer Research UK program grants to RCF and ST (C14478/A15874 and C14303/A17197), with additional support awarded to RCF from UK Medical Research Council, NHS National Institute for Health Research (NIHR), the Experimental Cancer Medicine Centre Network and the NIHR Cambridge Biomedical Research Centre, and Cancer Research UK Project grant C1023/A14545 to PAWE. JMJW was funded by a Wellcome Trust Translational Medicine and Therapeutics grant

Single-molecule analysis of genome rearrangements in cancer.

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome

Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines-ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4-all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.This work was funded by an MRC Programme Grant to R.C.F. and a Cancer Research UK grant to PAWE. The pipeline for mutation calling is funded by Cancer Research UK as part of the International Cancer Genome Consortium. G.C. is a National Institute for Health Research Lecturer as part of a NIHR professorship grant to R.C.F. AGL is supported by a Cancer Research UK programme grant (C14303/A20406) to Simon Tavaré and the European Commission through the Horizon 2020 project SOUND (Grant Agreement no. 633974)

Active Trachoma and Ocular Chlamydia trachomatis Infection in Two Gambian Regions: On Course for Elimination by 2020?

Trachoma is the leading infectious cause of blindness worldwide, and is mainly found in tropical and poor countries. It is caused by infection of the eyes with the bacterium Chlamydia trachomatis. However, sometimes the clinical signs of disease can be present without infection being detected. Control efforts involve surgery, antibiotic treatment, face washing, and environmental improvement for better hygiene. Surveys of trachoma help countries to know whether and where they should implement control interventions. The Gambia is found in West Africa and has suffered from trachoma for decades. We conducted a survey of two Gambian regions to look at how much trachoma disease and C. trachomatis infection there is in the eyes. We found that although there was enough disease (≥10%) to warrant antibiotic treatment for everyone in the regions, there was nearly no infection (0.3%). This means that using clinical signs alone to make treatment decisions in low prevalence settings like The Gambia can lead to the waste of scarce resources. Our results also suggest that since less than 1% of children are infected with C. trachomatis, The Gambia is on course to achieve the World Health Organization's aim of eliminating blinding trachoma by the year 2020

The geographical distribution and burden of trachoma in Africa.

BACKGROUND: There remains a lack of epidemiological data on the geographical distribution of trachoma to support global mapping and scale up of interventions for the elimination of trachoma. The Global Atlas of Trachoma (GAT) was launched in 2011 to address these needs and provide standardised, updated and accessible maps. This paper uses data included in the GAT to describe the geographical distribution and burden of trachoma in Africa. METHODS: Data assembly used structured searches of published and unpublished literature to identify cross-sectional epidemiological data on the burden of trachoma since 1980. Survey data were abstracted into a standardised database and mapped using geographical information systems (GIS) software. The characteristics of all surveys were summarized by country according to data source, time period, and survey methodology. Estimates of the current population at risk were calculated for each country and stratified by endemicity class. RESULTS: At the time of writing, 1342 records are included in the database representing surveys conducted between 1985 and 2012. These data were provided by direct contact with national control programmes and academic researchers (67%), peer-reviewed publications (17%) and unpublished reports or theses (16%). Prevalence data on active trachoma are available in 29 of the 33 countries in Africa classified as endemic for trachoma, and 1095 (20.6%) districts have representative data collected through population-based prevalence surveys. The highest prevalence of active trachoma and trichiasis remains in the Sahel area of West Africa and Savannah areas of East and Central Africa and an estimated 129.4 million people live in areas of Africa confirmed to be trachoma endemic. CONCLUSION: The Global Atlas of Trachoma provides the most contemporary and comprehensive summary of the burden of trachoma within Africa. The GAT highlights where future mapping is required and provides an important planning tool for scale-up and surveillance of trachoma control