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High-Resolution Analysis of Chromosome Rearrangements on 8P in Breast, Colon and Pancreatic Cancer Reveals a Complex Pattern of Loss, Gain and Translocation

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High-Resolution Analysis of Chromosome Rearrangements on 8P in Breast, Colon and Pancreatic Cancer Reveals a Complex Pattern of Loss, Gain and Translocation Oncogene (2006) 25, 5693–5706 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ONCOGENOMICS High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation JCM Pole1, C Courtay-Cahen1,2, MJ Garcia1, KA Blood1, SL Cooke1, AE Alsop1,3, DML Tse1,4, C Caldas1 and PAW Edwards1* 1Cancer Genomics Program, Departments of Pathology and Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK The short arm of chromosome 8, 8p, is often rearranged in Introduction carcinomas, typically showing distal loss by unbalanced translocation. We analysed 8p rearrangements in 48 Rearrangement of 8p, with loss of distal 8p, is one of the breast, pancreatic and colon cancer cell lines by most frequent genomic events in a range of common fluorescence in situ hybridization (FISH) and array epithelial cancers, but, in spite of much effort, their role comparative genomic hybridization, with a tiling path of in carcinogenesis is unclear (reviewed by Birnbaum 0.2 Mb resolution over 8p12 and 1 Mb resolution over et al., 2003). Classical cytogenetic analysis of several chromosome 8. Selected breast lines (MDA-MB-134, cancers, including breast, shows unbalanced transloca- MDA-MB-175, MDA-MB-361, T-47D and ZR-75-1) tions breaking around 8p12, with loss of distal 8p were analysed further. Most cell lines showed loss of 8p (Dutrillaux, 1995; also Teixeira et al., 2002). Compara- distal to a break that was between 31 Mb (50 to NRG1) tive genomic hybridization (CGH) also shows frequent and the centromere, but the translocations were accom- losses of distal 8p in breast, colorectal, prostate, bladder panied by variable amplifications, deletions and inversions and other cancers (e.g. Davidson et al., 2000; Abdel- proximal to this break. The 8p12 translocation in T-47D Rahman et al., 2001; Karan et al., 2003; Hurst et al., was flanked by an inversion of 4 Mb, with a 100 kb 2004; Hwang et al., 2004), and more recently array CGH deletion at the proximal end. The dicentric t(8;11) in ZR- has begun to provide more detail of these losses (e.g. 75-1 carries multiple rearrangements including interstitial Paris et al., 2003; Douglas et al., 2004; Hurst et al., 2004; deletions, a triplicated translocation junction between Nakao et al., 2004; Garcia et al., 2005). Distal 8p also NRG1 and a fragment of 11q (unconnected to CCND1), frequently shows loss of heterozygosity (LOH), which and two separate amplifications, of FGFR1 and CCND1 . would often reflect loss through unbalanced transloca- We conclude that if there is a tumour suppressor gene on tion (for references see Adams et al., 2005). 8p it may be near 31 Mb, for example WRN; but the There is also, often, amplification on 8p, proximal to complexity of 8p rearrangements suggests that they target the loss. In breast, for example, classical cytogenetic various genes proximal to 31 Mb including NRG1 and the banding shows hsrs (homogeneously staining regions, amplicon centred around ZNF703/FLJ14299. i.e. large-scale amplifications) attached to 8p12 (Bernar- Oncogene (2006) 25, 5693–5706. doi:10.1038/sj.onc.1209570; dino et al., 1998) and fluorescence in situ hybridization published online 24 April 2006 (FISH), CGH and Southern blotting show amplification generally in the region of 8p12 (Dib et al., 1995; Bautista Keywords: 8p12; breast cancer; array CGH; and Theillet, 1998; Davidson et al., 2000), which often, chromosome translocation; chromosome inversion; but not invariably, includes FGFR1, and may occasion- deletion ally extend as far distally as NRG1 (Ugolini et al., 1999). Recent array CGH studies that examine the 8p12 region ONCOGENOMICS in breast cancer have concentrated on the definition of the 8p12 amplicon (Ray et al., 2004; Garcia et al., 2005; *Correspondence: Dr PAW Edwards, Hutchison/MRC Research Prentice et al., 2005). The amplicon has been defined to Centre, Cambridge CB2 2XZ, UK. extend from 36.9 to 37.9 Mb (Garcia et al., 2005). The E-mail: [email protected] ZNF703/FLJ14299, SPFH2, BRF2 and RAB11FIP1 2Current address: The Animal Health Trust, Centre for Preventive Medicine, Lanwades Park, Kentford, Newmarket, CB8 7UU, UK. genes map to this region and their amplification 3Current address: Comparative Genomics Group, Research School of correlates with overexpression. This amplicon was Biological Sciences, The Australian National University, GPO Box present in 24% of primary breast tumours and found 475, Canberra, ACT 2601, Australia. to correlate with poor survival (Garcia et al., 2005; 4Current address: University of Cambridge Clinical School, Hills Road, Cambridge, UK. Prentice et al., 2005). The long-suspected amplification Received 11 November 2005; revised 23January 2006; accepted 28 target, FGFR1, was excluded from the current consensus February 2006; published online 24 April 2006 amplicon and detected as amplified in fewer, 10–15%, of 8p in carcinomas JCM Pole et al 5694 cases (Theillet et al., 1993; Ugolini et al., 1999; Prentice et al., 2005). Amplification of proximal 8p is also seen in other carcinomas (Veltman et al., 2003; Nakao et al., 2004). A further level of complexity is suggested by detailed analysis of the translocations that result in loss of distal 8p in cell lines. Firstly, in five breast and two pancreatic cell lines the breakpoints were clustered at the NRG1 gene (Adelaide et al., 2003, Huang et al., 2004). NRG1 encodes the heregulins/neuregulin 1 family of ligands, which bind to ErbB2, 3and 4. Our working hypothesis is that the translocations result in aberrant expression of normal or modified heregulins, which act in an autocrine loop on the cancer cells, as in at least one of the cell lines, MDA-MB-175, the translocation results in fusion of NRG1 and secretion of a fusion protein with soluble heregulin-like activity (Schaefer et al., 1997; Liu et al., 1999; Wang et al., 1999). We have subsequently shown that breakpoints in NRG1 are present in about 6% of uncultured breast tumours (Huang et al., 2004 also supported by Prentice et al., 2005). We had previously found another possible cluster of breaks about 2.5 Mb proximal to NRG1: three breast cancer cell lines, MDA-MB-361, T-47D, and ZR-75-1, had a break at about 34–35 Mb (Courtay-Cahen et al., 2000). This breakpoint cluster region is clearly distinct from the NRG1 cluster, as in two of the lines NRG1 is lost altogether. These translocations were accompanied by additional deletions, duplications and low-level amplifications (most of these amplifications are distinct Figure 1 Breaks on 8p defined by fluorescence in situ hybridiza- from the consensus amplicon described above), suggest- tion (FISH). On the left is an ideogram representing 8p. Breaks ing that 8p rearrangements in general might be complex. were mapped using bacterial artificial chromosome (BAC) clones and band-specific paints corresponding to the genes and bands We describe here a high-resolution analysis of marked. Rearranged copies of 8p in the cell lines are shown as rearrangements of 8p in carcinoma cell lines by FISH black bars, with the regions containing the breaks shown as open and array CGH. Our analysis shows a clear pattern of boxes. The width of the bars is proportional to the number of cell 8p loss extending, at least, to 30 Mb suggesting that the lines with that break pattern, to give an overall impression of the rearrangements could have multiple genetic targets, and distribution of breaks comparable to Figure 2. Grey boxes are used where there is amplification of a region immediately adjacent to the shows that the rearrangements are often highly complex. breakpoint, and the grey boxes represent the proximal boundaries of amplification. In MDA-MB-157 and MDA-MB-175, in addition to an unbalanced translocation of 8p, a separate, distal fragment is present, amplified in the case of MDA-MB-175. Some of the cell lines have two chromosomes with rearrangements of 8p. The cell Results lines in bold are breast; those highlighted in grey are colon; in italics, pancreatic; and other lines are in plain type. Rearrangements of 8p were identified in a set of breast, colon and pancreatic cancer cell lines, for most of which we had previously obtained SKY (spectral karyotyping) Array comparative genomic hybridization karyotypes (Davidson et al., 2000; Abdel-Rahman et al., To map rearrangements in more detail in a number of 2001; Sirivatanauksorn et al., 2001; available at http:// lines, and to discover additional examples of cell lines www.path.cam.ac.uk/Bpawefish). with a breakpoint in 8p12, we used array CGH Rearrangements were determined at the cytogenetic (Figure 2). Array CGH can detect breakpoints of level by metaphase FISH (Figure 1). Of 35 lines unbalanced chromosome rearrangements because a examined that had rearrangements of chromosome 8, copy number change occurs at the breakpoints. We 27 had breaks on 8p or at the centromere, as determined used a custom array that included near-tiling-path by 8q arm paint (ideograms of these chromosome 8 coverage of the 8p12 region, approximately from rearrangements are available at http://www.path.cam.a- WRN to FGFR1, together with roughly 1 Mb coverage c.uk/Bpawefish/). These were further analysed using of the rest of chromosome 8, and analysed 24 of the 27 8p11.2 and 8p22 band-specific paints, and BACs at the cell lines identified in the FISH screen that had 8p NRG1 and FGFR1 loci. Chromosomes with breaks rearrangements, and a further 24 breast cancer cell lines between NRG1 and 8p22 were then tested with a BAC that have been karyotyped by SKY or M-FISH containing WRN. This gave a low-resolution cytogenetic (Supplementary Table 1): a total of 48 cell lines, of classification of breakpoints on 8p (Figure 1).
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