Paw Edwards

Molecular cytogenetic analysis of breast cancer cell lines

The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish.

High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation.

The short arm of chromosome 8, 8p, is often rearranged in carcinomas, typically showing distal loss by unbalanced translocation. We analysed 8p rearrangements in 48 breast, pancreatic and colon cancer cell lines by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization, with a tiling path of 0.2 Mb resolution over 8p12 and 1 Mb resolution over chromosome 8. Selected breast lines (MDA-MB-134, MDA-MB-175, MDA-MB-361, T-47D and ZR-75-1) were analysed further. Most cell lines showed loss of 8p distal to a break that was between 31 Mb (5′ to NRG1) and the centromere, but the translocations were accompanied by variable amplifications, deletions and inversions proximal to this break. The 8p12 translocation in T-47D was flanked by an inversion of 4 Mb, with a 100 kb deletion at the proximal end. The dicentric t(8;11) in ZR-75-1 carries multiple rearrangements including interstitial deletions, a triplicated translocation junction between NRG1 and a fragment of 11q (unconnected to CCND1), and two separate amplifications, of FGFR1 and CCND1 . We conclude that if there is a tumour suppressor gene on 8p it may be near 31 Mb, for example WRN; but the complexity of 8p rearrangements suggests that they target various genes proximal to 31 Mb including NRG1 and the amplicon centred around ZNF703/FLJ14299.

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1–AHCY and RIF1–PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene.

Neuregulin-1 (NRG1) is both a candidate oncogene and a candidate tumour suppressor gene. It not only encodes the heregulins and other mitogenic ligands for the ERBB family, but also causes apoptosis in NRG1-expressing cells. We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than that in various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, whereas normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. Small interfering RNA (siRNA)-mediated depletion of NRG1 increased net proliferation in a normal breast cell line and a breast cancer cell line that expressed NRG1. The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is antiproliferative and is silenced in many breast cancers.

The NEUREGULIN1 gene and breast cancer

not available at time of printing.

PWE-171 Mobile element insertions are frequent and potentially mutagenic events in oesophageal carcinogenesis

Introduction Mobile elements (ME) are repeat sequences found throughout the genome that can move to, or insert copies of themselves at, other genomic loci. The most active generate new copies by retrotransposition: mRNA is transcribed then cDNA is synthesised by reverse transcription and inserted. Such events may also transduce small fragments of unique DNA sequence found adjacent to the ME, thus making them identifiable in sequencing data. ME retrotransposition has recently been described in several tumour types, but how frequent and widespread this is, and whether it contributes significantly to gene mutation, is not yet clear. This project aimed to explore whether ME retrotransposition was identifiable in oesophageal adenocarcinomas (OACs) and its mutagenic potential. Method Possible ME insertions were identified from whole genome sequencing data for 43 OACs by three methods. The first identified cases where the same short unique sequence from one chromosome, which would be a sequence transduced by a mobile element, was joined to multiple different loci within the same tumour. The second method isolated rearrangements adjacent to 90 retrotransposition-capable MEs. The third identified all poly (A) sequences >20 nucleotides long that appeared to be unique to the tumour. A subset of 71 potential insertions was tested by PCR using patient-matched normal DNA as a control. Results The first two approaches combined identified 649 candidate ME retrotransposition events. 42/45 (93%) inserts selected for PCR verification had tumour-unique products, with sequencing demonstrating target site duplication, a poly (A) tract and ME repeat sequence and/or unique transduced sequence, confirming these to be retrotransposition events. 40/43 (93%) tumours had at least one probable ME insertion with a mean of 15 per tumour (range 0–153). 193/649 (30%) inserts fell within genes, mainly intronic, however five were within exons. These two approaches identified retrotransposition events that transduced unique sequence downstream of MEs; however this is only a subset of ME insertions. A search for poly (A) tracts, a marker of all ME insertions, in tumour-unique loci was undertaken to estimate their overall frequency. In total, 5422 tumour-unique poly (A) tracts were identified in the 43 OACs, mean 126 (range 6–858) per tumour. 24/26 (92%) randomly selected inserts had a tumour-specific product on PCR validation. Conclusion ME retrotransposition is highly prevalent in OACs, and occurs frequently in each tumour. Since almost all ME insertions are capable of interrupting mRNA transcription, they could contribute substantially to gene mutation. Disclosure of interest None Declared.

OC-066 The Cancer Research UK (Cruk) Funded ICGC Oesophageal Adenocarcinoma Project: MRC Research Centre and Cruk Cambridge Research Institute

Introduction Esophageal adenocarcinoma (EAC) has one of the fastest rising incidences of any cancer in the western world. With a 5-year survival below 10% it is one of the most common causes of cancer death in US and UK. Currently little is understood about the genetic alterations that drive the development of OAC. Better understanding of these alterations may allow the development of novel therepeutic approaches Methods We have performed whole genome sequencing on 22 cases. Targeted amplicon resequencing of 27 recurrently mutated genes was performed on a validation cohort of 100 further oesophageal adenocarcinomas. Results In the discover set of 22 OACs we identified recurrent mutations (>3 tumours) in 31 genes including several implicated in tumorigenesis; TP53, CDKN2A, ARID1A. Strikingly in the validation cohort we observed that > 30% of EAC samples harbour mutation of one or both of the SWI/SNF complex members ARID1A and SMARCA4. In addition we identified highly recurrent mutations in several additional genes including TRIM58, SSTR4 and MYO18B. Conclusion Whole genome sequencing provides an unbiased screen of mutational architecture of OAC. This has allowed the identification of several recurrently mutated genes not previously implicated in this disease providing a unique insight to it’s pathogenesis Disclosure of Interest None Declared

Chromosome translocations may play a significant role in breast cancer

Chromosome translocations that form fusion transcripts and/or activate expression of genes by promoter insertion are key events in leukaemias and lymphomas, and mesenchymal tumours, but it has been fashionable to think they are irrelevant to the common epithelial cancers such as breast cancer. However, that view is now being challenged [1-4]; in particular, we have shown that NRG1 is translocated in breast cancers [3]. It seems likely that some translocations in breast cancers target specific genes at their breakpoints, and this is particularly likely for reciprocal translocations. We are cataloguing translocation breakpoints in breast cancer cell lines and tumours. We use array painting, in which individual chromosomes are purified in a cell sorter and their DNA hybridized to microarrays. We have analysed all the chromosomes of three breast cancer lines to 1 Mb resolution or better. A striking finding was that reciprocal and more complex balanced translocations are far more frequent than expected. Together the three lines had at least 14 balanced translocations, almost three times more than identified by cytogenetics — the cryptic ones involved small fragments, or were obscured by subsequent rearrangement. Furthermore, several translocation breaks were in genes, including known cancer-critical genes such as EP300/p300 and CTCF. This supports the emerging idea that chromosome rearrangement plays a major role in the gene changes that cause breast cancer.