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Chromosome 18 is frequently rearranged in carcinomas. We explored the distribution of breakpoints affecting chromosome 18 by mapping 56 breakpoints in 26 carcinoma cell lines by fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) and band paints. The distribution of breaks among 18 intervals of chromosome 18 was significantly nonrandom. The interval spanning the centromere contained the greatest number of breaks and had the highest average copy number of any interval. There was a high density of breaks close to the centromere as well as actually within the centromere. A cluster of breaks encompassing SMAD4 was associated with the minimum average copy number, consistent with SMAD4 being a tumor suppressor gene. There may be another cluster of breaks around 18q12. We offer two interpretations of the concentration of breaks near the centromere. It may reflect selection for an oncogene near the centromere, or there may be an underlying bias of breakage toward the centromere. We show that the latter is predicted by a simple model that invokes random breakage following anchorage of some random point on the chromosome, or selection of breaks proximal to one of several tumor suppressor genes.

Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas. We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization. All three lines had a breakpoint close to D8S505, proximal to HGL. Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other. The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity. Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment. ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL. This line also had an interstitial deletion several megabases more centromeric. The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements.

Expression of Wnt-4, a member of the Wnt gene family, is induced during early pregnancy in the mouse mammary gland. To investigate the function of Wnt-4, we used a recombinant retrovirus to constitutively express the gene in transplanted mammary epithelium grown in virgin animals. In fully grown glands, Wnt-4 expression resulted in ducts that were more highly branched than normal and caused some premature alveolar development. These changes resembled those seen during pregnancy, suggesting that endogenous Wnt-4 expression may regulate epithelial branching in early pregnancy. The modified growth pattern induced by Wnt-4 expression was similar to that induced by Wnt-1, one of the members of the Wnt gene family activated by mouse mammary tumour virus. As Wnt-1 is not normally expressed in the mammary gland, it may exert its effect on the mammary gland by activating a developmental pathway normally regulated by Wnt-4.

Introduction A series of clonal expansions are thought to underlie the progression of Barrett’s oesophagus (BE) to oesophageal adenocarcinoma (OAC). Each expansion carries with it somatic driver mutation (s) fixing it within a larger population and therefore increasing the likelihood of acquiring a second mutation. However, the precise order in which somatic variants occur remains unknown. Methods We performed whole genome sequencing in 25 cases of OAC and 3 matched cases of BE. Findings were validated in a larger cohort of OACs (n = 90), metaplastic never-dysplastic BE (NDBE, n = 66 with a median follow-up of 58 months) and high-grade dysplasia (n = 43) using amplicon resequencing. Mutational signatures and gene-centric somatic mutations were determined using an in-house pipeline incorporating standard statistical methods and the publically available EMu pipeline. Results There were 7 distinct mutational signatures present in both early (BE) and late disease (OAC). Fifteen genes were determined to be potential novel drivers of OAC development. Surprisingly in 53% of NDBE tissue samples we identified clonal expansion of cells (>10% mutant fraction) harbouring mutations in one or more of 13/15 of these putative driver genes. No difference in the frequency of mutation of these genes was observed between any of the disease stages studied. TP53 mutations clearly delineate between HGD/OAC and benign NDBE (p SMAD4 mutations are only observed in OAC (p Conclusion Mutagenic processes active in OAC are also active in the earliest stages of BE. Recurrent driver mutations identified in cancer may be acquired very early in the disease and may provide little or no progression advantage. Molecular diagnostic approaches must account for this. Disclosure of Interest None Declared.

randomize 90 patients per cohort, stratified by IL28B status and GT1 subtype (1a vs non– 1a), in a 2:1:2:1 fashion to 1 of 4 arms: simeprevir 150 mg once daily (QD) plus sofosbuvir 400 mg QD for 24 weeks with or without RBV, or simeprevir 150 mg QD plus sofosbuvir 400 mg QD for 12 weeks with or without RBV. For cohort 1, a safety and efficacy interim analysis was conductedwhen approximately 20% of the planned randomized patients reached the end of treatment (EOT) or permanently discontinued treatment early.Results: At the time of this interim analysis, 80 patients in cohort 1 have been randomized and treated; 19 patients have reached EOT and have post-treatment follow-up data available. Duration of treatment exposure ranges from 3 to 171 days; median is 63 days. One patient discontinued study drugs due to an adverse event (AE). Two patients discontinued treatment early for non-safety reasons. RBV dose was reduced in 4/51 patients due to decline in hemoglobin. No serious AEs were attributed to study drugs. Baseline characteristics and efficacy results are summarized. At the time of the presentation, results of an additional interim analysis will be presented, including SVR data in approximately 50 patients.Conclusions: Once-daily simeprevir + sofosbuvir with or without RBV for 12 or 24 weeks was generally well tolerated and resulted in high SVR4 rates in this initial group of study patients with null response to prior PegIFN/RBV therapy.

It has been recognised for some time that reciprocal chromosome translocation breakpoints are often complex. Most familiar are net deletions of material at the breakpoints, up to megabases in extent. We describe here the opposite phenomenondduplication of tens of kilobases of sequence so that the same sequence is present on both products of a translocation and the breakpoints overlap. Breakpoints from three breast cancer cell lines were determined by array painting. In array painting, chromosomes are isolated by flow cytometry and hybridized to DNA microarrays. This showed eighteen approximately balanced translocations. In six cases, all breakpoint junctions were mapped to 2-kb resolution using high-resolution array painting, FISH with fosmids and PCR on sorted chromosomes. A total of four translocations, in two of the cell lines, showed overlap of breakpoints, ranging in size from 46 kb to 200 kb. By SNP analysis, the duplicated sequences derived from the same chromosome. Of the remaining twelve translocations mapped to lower resolution, two reciprocal translocations showed large overlapping breakpoints of around 1.5 Mb. In most cases, knowledge of the overlap was necessary for correct interpretation of gene targets. Several of the overlapping breakpoints broke genes, and a fusion was formed between CTCF and SCUBE2. These overlaps would generally be missed by conventional FISH analysis. We propose that these duplicated sequences could arise through errors at replication, possibly by the production of stalled replication ‘bubbles’, rather than single replication forks. If translocations form during replication, the gains we have seen could be the counterpart of the previously described losses of sequence: of the two daughter cells formed, one would show gain and the other loss at the breakpoint(s). IDENTIFYING PROGNOSTIC MARKERS FOR HIGH GRADE NON-MUSCLE INVASIVE BLADDER CANCER