(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 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 , 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 , 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 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 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 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 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). which 39 show some type of rearrangement on 8p.

Oncogene 8p in carcinomas JCM Pole et al 5695

Figure 2 (a) Array comparative genomic hybridization (CGH) of 8p in 48 cell lines, represented conventionally as gains and losses, relative to average ploidy of the whole genome. Gains above ratio of 1.2 are green, gains greater than a ratio of 2 are shown in yellow, losses below ratio of 0.8 are red. The blocks of colour represent consecutive data points (BACs) reaching the threshold. Regions between the thresholds are shown as black with thin red or green lines representing isolated data points crossing the thresholds. Each column is a separate cell line. White horizontal lines delimit the extent of the higher resolution 8p region of the array shown in Figure 2b. N and F indicate the approximate locations of genes NRG1 and FGFR1, respectively. BT549, MT3, SW620, SUM159, SKBR3, LS411, HCC1395, MCF7, and Vaco4A had no detected copy number shifts within 8p (HCC70 has a further deletion within the 8p loss that does not show on Figure 2a). (b) Array CGH of the interval from RP11-263C6 (29.01 Mb) to RP11-503E24 (42.50 Mb), including the region of 8p12 covered at near-tiling path resolution on the array. Each row represents a single clone on the array, with the approximate position on the genomic scale shown. Each column is a separate cell line. Log2 ratios are shown on a colour scale, shown to the right, where green represents gain and red loss. Grey boxes correspond to data rejected after quality tests for signal intensity and replicate reproducibility. Cell lines are shown ranked according to the approximate position of the most distal break (copy number step) that resulted in loss of distal sequences. Oncogene 8p in carcinomas JCM Pole et al 5696 The ability of the array CGH to detect copy number visible and the breakpoint could generally be placed to changes and identify breakpoints was remarkable. In the nearest BAC. As shown in Figure 3, in several lines most cell lines clear step changes in copy number were we were able to compare the array CGH to the detailed

Oncogene 8p in carcinomas JCM Pole et al 5697 FISH mapping of the chromosomes that is described small amplicon substantially proximal to the main below. For example, in T-47D, FISH had shown that break, and two BACs spanning the FGFR1 are there was a breakpoint within BAC RP11-419I6 and amplified in HCC1599. Several lines show a small region a deletion of most of BAC RP11-90P5: both are of relative loss within the retained region proximal to clearly visible in the array CGH. In MDA-MB-361 the break (C70, MDA-MB-361, HCC1569, ZR-75-1 and ZR-75-1, where FISH showed multiple copy (more than one), HCC1806, SW837, UACC812), and number changes in this region, the copy changes one line, HCC70, has a small region of additional loss corresponded clearly to the array CGH (Figure 3). distal to the main break at the centromere. These regions of loss could be interstitial deletions, but they may also be gaps between regions that are present in The pattern of rearrangement that results in loss of distal extra copies, as shown below for MDA-MB-361 and 8p ZR-75-1, or deletions at inversion junctions, as shown Taken together, at low resolution, Figures 1 and 2a below for T-47D. MDA-MB-175 is an extreme example, show that the majority of lines show the typical pattern with a small fragment around NRG1 present in several of loss of distal 8p, to form unbalanced translocations, copies inserted into chromosome 11 material (Figure 3), terminal deletions or isoderivatives. In Figure 2a, the and the remainder of 8p lost. cell lines have been approximately ranked according to Several of the breast lines also showed substantial the main breakpoint, to illustrate the distribution of amplifications between 37 and 40 Mb, as we have breaks. This is necessarily crude, as in several lines the described previously for breast tumour tissues and some transition from gain to loss occurs in more than one step cell lines (Garcia et al., 2005). Several cell lines not in (e.g. SW403has a break at the centromere and in our previous study that showed amplification or gain in proximal 8p12), and some lines show steps both up and this region included Cama-1, HCC1599, HCC38, down in copy number. UACC812, ZR-75-1, and the colon line SW837 However, Figure 2a shows clearly that most lines have (Figure 2b). lost distal 8p and the break(s) between gain and loss are In summary, the overall pattern is very variable but mostly in the interval NRG1 to FGFR1 or at the there is loss of distal 8p; the region immediately centromere. The WRN to FGFR1 region is covered at proximal to the loss is often amplified (or at least higher resolution by our array (average 1 BAC per duplicated), and in several cases a region proximal to 0.16 Mb) and Figure 2b shows 31 cell lines that show this may actually show loss. In some cases there is a copy number changes in this region as a heat map. The complex combination of losses and duplications/ampli- lines have been ordered slightly differently from fications, as analysed in more detail below in specific Figure 2a, roughly according to the most distal fall in cases. copy number. Several lines show a single step in copy number (Figure 2b), with loss of distal sequences, usually as a Retention of distal 8p result of unbalanced translocation (e.g. PaTuI, and, at Two lines showed an additional rearrangement: reten- this resolution, T-47D), or sometimes as an isoderivative tion of a fragment of 8p extending from 8pter to a break (e.g. MDA-MB-453). However, several lines have more around 8p22. Metaphase FISH detected this in PMC42 than one copy number change in the region, showing (Figure 1). Array CGH detected a further example in that the low-resolution picture in Figure 2a, and the HCC1806, and mapped both breaks to between 12 and cytogenetic picture, oversimplifies the pattern of breaks. 14 Mb. (Although in Figure 2a Colo357 and UACC812 Several (Cama-1, HCC1500, MDA-MB-134, MDA- appear similar, this is due to small copy number MB-175, MDA-MB-361, SUIT-2, SUM44, SUM52 fluctuations about the threshold and is not convincing). and ZR-75-1) show at least low-level amplification of In MCF7 and SKBR3, FISH showed that fragments a fragment immediately proximal to the main break. All corresponding to the whole of 8p were present of these, except Cama-1, have been reported before (Figure 1). (Lemieux et al., 1996; Courtay-Cahen et al., 2000; Adelaide et al., 2003; Ray et al., 2004; Garcia et al., Detailed fluorescence in situ hybridization analysis of 2005) and all, except those in Cama-1 and the SUM individual cell lines lines, have been verified by us using FISH with BACs The rearrangements of chromosome 8 in certain cell and chromosome 8 paint. HCC38 and UACC812 have a lines were mapped in more detail by FISH. Breaks were

Figure 3 Comparison of fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (CGH) data for selected lines, ZR-75-1, T-47D, MDA-MB-361, MDA-MB-175 and MDA-MB-134. For each line, ideograms representing all normal and rearranged chromosome 8 material are shown on the left; a key for the ideograms is shown bottom left. For each cell line, array CGH of the region 30–40 Mb is shown, expressed as log2 ratio on the y-axis, with the average chromosome copy number, equivalent to log2 ratio ¼ 0, shown to the right of each plot (modal chromosome number obtained from the spectral karyotyping (SKY) karyotype/ 23). Below array CGH plots, on the same scale, are the numbers of FISH signals from the same bacterial artificial chromosomes (BACs) on the various copies of chromosome 8, as detailed in Supplementary Table 2. Norm, normal copies; Trans, translocated or rearranged copies. þ , þþ, þþþ, represent 1, 2 and 3copies detected by FISH. *represents a cluster of several signals. W, weak signal compared to untranslocated copy. Vertical lines approximately separate regions of different copy number. The BAC on the breakpoint in T-47D is RP11-419I6.

Oncogene 8p in carcinomas JCM Pole et al 5698

Figure 4 Map showing 8p11–12 region, 30–40 Mb, and bacterial artificial chromosome (BAC) array, with breakpoints in selected lines mapped by fluorescence in situ hybridization (FISH), displayed using the UCSC Browser. Horizontal scale: genome position. Above: BACs on the in-house array. Horizontal bars represent regions of chromosome 8 present in translocated chromosomes in the five cell lines ZR-75-1, T-47D, MDA-MB-361, MDA-MB-175 and MDA-MB-134, with copy number determined by FISH shown as þ , þþetc. as in Figure 3. For simplicity, the second rearranged 8 chromosome in ZR-75-1 is not shown, but is detailed in Figure 3. Underneath: cytogenetic bands, and genes within this region. Vertical lines are at copy number changes determined by FISH.

mapped by FISH to the nearest BAC in a near- (Figure 5c). We then confirmed the predicted junctions contiguous set that covers the region, as summarized by fibre-FISH: the proposed junction of the chromo- in Figure 4, and detailed in Figure 3and Supplementary some 14 break with the proximal end of the inverted Table 2. fragment of chromosome 8 was detected as contiguous signals from BACs that span the breaks, respectively, RP11-893D14 and CTD-2277D22 (Figure 5e). The T-47D junction created by the inversion, between chromosome In the breast cancer cell line T-47D we found that the 8p 8 sequences, was similarly confirmed using BACs RP11- translocation was accompanied by cryptic inversion and 90P5 and RP11-419I6 (Figure 5f). deletion. This line has a der(8)t(8;14)(p12;q12), which is The extent of the deletion of chromosome 8 sequence present in two copies with two apparently normal at the proximal end of the inversion, in RP11-90P5, was chromosome 8 s (Morris et al., 1997; Courtay-Cahen estimated to be between 80 and 150 kb using fosmid et al., 2000, Figure 3). clones (Figure 5g) and BAC metaphase and fibre- We first mapped the expected breakpoints on FISH (Figure 5e and f). The distal end was between chromosomes 8 and 14, but found they were not joined. 38.07–38.08 Mb and the proximal end was between Conventional FISH and array CGH both positioned the 38.170–38.215 Mb. break on 8p within BAC RP11-419I6 (Figure 4). Fibre- In conclusion, the segment from about 34.50 (RP11- FISH showed loss of about one-third of this BAC, 419I6) to about 38.15 Mb (RP11-90P5) has been placing the break at approximately 34.5 Mb (not inverted, with loss of sequence contained within RP11- shown). On chromosome 14, following FISH with a 90P5, and the inverted piece is joined to chromosome 14 series of BACs on metaphase chromosomes (Supple- (Figure 5). Genes that might be affected include ASH2L mentary Table 2), fibre-FISH placed the break in BAC to BAG4, in the deletion, and genes that flank the new RP11-893D14 at 24.4 Mb (Figure 5a). In expectation junctions including EIF4EBP1, BAG4 or DDHD2, that the BAC RP11-419I6 from chromosome 8 and UNC5D, and, on chromosome 14, STXBP6 (Map, RP11-893D14 from chromosome 14 would be joined, we Figure 4). hybridized the two BACs together on T-47D fibres. We were unable to find any joined copies. To explain this, we hypothesized that sequences on 8p ZR-75-1 adjacent to the break were inverted (Figure 5b), as The breast cancer cell line ZR-75-1 has an extraordina- inversions could explain the complex 8p rearrangements rily complex rearrangement of 8p. At cytogenetic in ZR-75-1 and MDA-MB-361. The site of the proximal resolution, it has a dicentric unbalanced translocation inversion break was suggested by the partial deletion of of chromosomes 8 and 11, dic(8;11)(p12;q12), and three BAC RP11-90P5: this deletion had been detected as a apparently normal chromosome 8 s. One of the ‘normal’ weak RP11-90P5 signal on the translocated 8;14 8 chromosomes proves to have some of the changes chromosomes, and in the array CGH (Figures 3and 4). present on the dic(8;11) (Figures 3and 4) (Courtay- To test the hypothesis, we first used interphase FISH Cahen et al., 2000). to show that the relative distances between BACs at A reconstruction of the dicentric chromosome is each end of the inversion changed as predicted shown in Figure 6a based on the work described below,

Oncogene 8p in carcinomas JCM Pole et al 5699

Figure 5 Translocation der(8)t(8;14) with inversion in T-47D. (a) The position of the chromosome 14 breakpoint shown by fibre- FISH. Bacterial artificial chromosomes (BACs) RP11-893D14 (red) and RP11-725L2 (green), which overlap on chromosome 14. Two kinds of signal were found, in equal numbers, corresponding to normal copies (upper image) and translocated copies (lower image) of chromosome 14. The break is approximately at the centre of the overlap of the two BACs. (b) Inversion hypothesis. Postulated inversion of segment adjacent to translocation, illustrating consequences for the relative positions of BACs shown as blue (RP11- 893D14), red (RP11-701H6 or CTD-2277D22), open circles (RP11-419I6) and green (RP11-90P5, or more proximally, RP11-350N15). The proximal breakpoint of the inversion is postulated to be at the small deletion observed at BAC RP11-90P5. (c) Example of interphase FISH showing changed relative positions of BACs in normal and translocated copies of chromosome 8, as postulated in (b). Blue, marked by blue arrows, BAC at break on chromosome 14, RP11-893D14. It appears in this half interphase as a single isolated signal (an untranslocated copy) and two signals almost superimposed on red signals (two translocated copies). Red and green, respectively BACs RP11-701H6 and RP11-90P5, are almost superimposed in one copy, as expected for a normal 8 chromosome, and are separated in two copies, with the red signals instead superimposed on blue signals, as predicted in (b) for the translocated 8;14 chromosome. (d) Measurements of the distances between BACs by interphase FISH, in normal and translocated copies of chromosome 8. Distances between BACs in images such as shown in (c) were measured and the average and standard error of the mean are shown as vertical bars for various combinations of BACs. Translocated copies were distinguished from untranslocated by proximity of a chromosome 14 BAC. Note for example that RP11-419I6 moves close to RP11-90P5 in the translocated chromosome, while RP11- 90P5 is separated from RP11-701H6. (e) Confirmation of predicted junction between chromosomes 8 and 14 by fibre-FISH: in green, BAC RP11-893D14, which spans the chromosome 14 break; in red, BAC CTD-2277D22, which spans the distal border of the deletion on chromosome 8, leaving a small fragment of the BAC hybridizing to the translocated chromosome at the 8;14 junction. Magnification is larger than in (a). (f) Confirmation of predicted 8;8 junction created by the inversion and deletion: in red, BAC RP11- 419I6, which spans the distal break in chromosome 8; in green, RP11-90P5, which spans the proximal border of the deletion. (g) Detailed mapping of the deletion at the proximal inversion break, at BAC RP11-90P5. Horizontal scale, distance in Mb on normal 8p. Grey bars are annotated genes in the region. Black bars are BACs CTD-2277D22 and RP11-90P5: both were positive on the translocated chromosome, but weak and their signals were separated from each other in interphases, implying that the region of their overlap was deleted. Critical fosmids G248P85539D6, G248P8244D11, G248P88726F3 G248P81454D2, G248P89521H4 are shown, respectively, in orange and blue for positive and negative. and our earlier less detailed work (Courtay-Cahen et al., separate amplification of the CCND1 region. This may 2000; Adelaide et al., 2003). The main features are as not be complete as, for example, we would not have follows: a translocation of the NRG1 gene to chromo- detected inversions. some 11 (Adelaide et al., 2003; Huang et al., 2004); The following is illustrated in Figures 3, 4 and 6, and triplication of a complex fragment that includes this 8;11 details of the BAC FISH are in Supplementary Table 2. translocation junction and other neighbouring segments of chromosome 8 and 11 (previously reported as a Chromosome 11- NRG1 junction. We identified a duplication Courtay-Cahen et al., 2000); an interstitial fragment of 11q12 that was present in the dicentric deletion proximal to NRG1; a separate amplification of and joined to the breakpoint on 8p in NRG1 (we had not the FGFR1 region, which is also present on one of the previously investigated what the break in NRG1 was untranslocated copies of chromosome 8; a second joined to (Adelaide et al., 2003)). Fluorescence in situ interstitial deletion proximal to FGFR1, which is hybridization on extended metaphase chromosomes, associated with both copies of the FGFR1 amplification followed by fibre-FISH (Figure 6f, Supplementary Table (Courtay-Cahen et al., 2000); and involvement of at 2), showed that an approximately 300 kb fragment of least two fragments of chromosome 11q, including a 11q12, extending from approximately 62.8 (the

Oncogene 8p in carcinomas JCM Pole et al 5700

Figure 6 Structure of the Complex dic (8;11) of ZR-75-1. (a) A schematic representation of the dic(8;11). The middle diagram is the suggested form of the complex translocated 8;11 chromosome, above and below this are the pieces of chromosome 11 and 8 that contribute to the dic(8;11); a key is shown. (b) Triplication of chromosome 11 and 8 material in metaphase. Green, BAC RP11-669B22 (spans NRG1 breakpoint); red, BAC RP11-996J9 (one end of triplicated chromosome 11 fragment). Red arrows indicate the three signals on the translocated chromosome. (c) Triplication in interphase. Green, BAC RP11-669B22 (spans NRG1 breakpoint); red, BAC RP11-996J9 (one end of triplicated chromosome 11 fragment); blue, BAC RP11-838I13 (other end of the triplicated chromosome 11 fragment see part d). Blue signals are arrowed; vertical arrows mark the three triplicated copies, each with an almost-superimposed green and red signal, the horizontal arrow indicates a normal copy of chromosome 11. The distance between the chromosome 8 BAC RP11-669B22 and each of the chromosome 11 BACs was measured and is reported in (e). (d) The 11q12.3region that is triplicated on the dic(8;11). The region, mapped by FISH, is from 62.68 to 63.18 Mb and its ends are spanned by BACs RP11-838I13 and RP11- 996J9. (e) A summary of measured distances between BAC signals in multiple interphase images like (c). The signal from the NRG1, BAC RP11-669B22, is nearer to the BAC RP11-996J9 signal than the BAC RP11-838I13 signal in the triplicated copies. (f and g) Fibre-FISH showing the break on chromosome 11 spanned by RP11-996J9 joined to the NRG1 break, RP11-669B22. (f) The break on 11. The upper image shows the intact copy with overlapping signals for the two adjacent BACs, the lower image shows the broken copy. An arrowhead marks estimated position of break on RP11-996J9. (g) The junction between the break on 11, spanned by BAC RP11-996J9, and break in NRG1, spanned by BAC RP11-669B22. (h and i) Amplification of FGFR1 and CCND1 in metaphase and interphase. Red, CCND1 locus, BAC RP11-300I6; green, FGFR1 locus, BAC RP11-350N15. (h) The dicentric 8;11 chromosome is shown. There is a cluster of several red (CCND1) signals and there also appears to be a single signal between the green (FGFR1) signals. The FGFR1 BAC (green) hybridizes in multiple copies in a single location on the dicentric (shown more clearly on (i)) there is also at least one green signal between the two red (CCND1) signals. (i) The signals shown on an interphase nucleus. The multiple red signals for CCND1 are shown by the red arrow, the two proposed amplified signals for FGFR1, one on the dicentric chromosome, and one on the otherwise normal chromosome 8 are indicated by the green arrows.

midpoint of BAC RP11-996J9) to 63.1 Mb, was present 512I24 was present in a single copy (Figure 6h and i; in the dicentric and triplicated in the same locations as Supplementary Table 2). RP11-300I6 also hybridized in NRG1 (Figures 6b–d and Supplementary Table 2). The a separate location between the amplified and single proximal end of this fragment was shown by interphase copy of FGFR1 (Figure 6g). Other regions of 11q were measurements then fibre-FISH to be fused to the break absent by BAC FISH (Supplementary Table 2), and by in NRG1 (Figure 6g). hybridization of amplified, flow-sorted copies of the dicentric to the 1 Mb genomic array of Fiegler et al., 2003(KAB, unpublished observations). Other 11q sequences present on the dic(8;11). The only other 11q sequences we detected were a fragment of 4– 7 Mb spanning CCND1 (maximally 64.7 to 71.9 Mb). Details of chromosome 8 fragments. Different parts of Bacterial artificial chromosomes (BACs) RP11-126P21, the region between NRG1 and FGFR1 were present in RP11-300I6 (CCND1) showed amplification and RP11- anything from zero to three copies, as shown by FISH

Oncogene 8p in carcinomas JCM Pole et al 5701 with BACs from 8p12, confirmed by array CGH CGH placed the proximal limit of the duplicated (Figures 3and 4; Supplementary Table 2). Three segment at approximately 39 Mb. separate fragments were triplicated together with the triplicated fragment of 11q. Additionally there were two regions present in two copies, two regions present in MDA-MB-134 single copies, and a deletion proximal to NRG1. We MDA-MB-134 is known to have a high copy number propose that this is due to the duplication of some parts amplification of sequences from 8p12, including FGFR1, and then reduplication of part of the already duplicated forming an hsr with sequences from 8q and chromo- region. It appears that the second and third copies are some 11 (Figure 3) (Lemieux et al., 1996; Bautista and successively placed further from the 8 centromere, Theillet, 1998). The distal end of the 8p amplification because successively fewer of the chromosome 8 frag- was mapped to within BAC RP11-155H15, at 34.7 Mb ments are present. As the regions duplicated and by FISH and array CGH (Figure 3; Supplementary triplicated were not originally contiguous, deletions Table 2). The array CGH shows the amplification and/or inversions must have occurred before or during continuing proximally, beyond FGFR1, to about 42 Mb. the duplications. The proximal boundary of the The amplified region shows varying levels of gain, triplication is within BAC RP11-90P5 (38.1 Mb), just suggesting that the amplification may be the result of distal to the FGFR1 BAC RP11-350N15. repeated joining and re-breaking with potentially multi- ple boundaries.

Amplifications of FGFR1 and CCND1. The FGFR1 locus (detected in this study by BAC RP11-350N15), MDA-MB-175 although not in the triplication, was present as MDA-MB-175 has a translocation of 8p12 at NRG1 a small amplification, in at least three copies clustered that creates a fusion with the DOC4 gene on chromo- together, on both the translocated and one some 11 (Wang et al., 1999; Liu et al., 1999; Adelaide untranslocated chromosome (Figure 6h and i) as et al., 2003). The fragment containing the junction is previously described (Courtay-Cahen et al., 2000). amplified (Figure 3). We mapped the boundaries of the The hybridization signals were tightly clustered and amplified fragment of 8p using array CGH and FISH, separate from the triplication just described, hence it and it extends approximately from 32.4 to 33.2 Mb seems to be an independent event. Some sequences, (Figures 3, 4 and Supplementary Table 2). The copies hybridizing to BACs CTD-2343B20 and RP11-90P5, are present in a small cluster inserted into chromosome just distal to RP11-350N15, are also amplified on 11 material, remote from other parts of chromosome 8 the untranslocated chromosome 8, although not (Figure 3). on the dic(8;11). On the dicentric chromosome this region (CTD-2343B20 and RP11-90P5) is included in Clusters of breakpoints at NRG1 and UNC5D the triplication. We have previously reported that seven cell lines had We confirmed that, in the dic(8;11) chromosome, the breaks within or immediately 50 to NRG1, (Adelaide FGFR1 amplification was also well separated from the et al., 2003). The array CGH detected two more cluster of CCND1 signals (Figures 6h and i). examples, in HCC1569 and HCC1806, which were Centromeric to FGFR1 there is a second interstitial confirmed by FISH: the breaks were respectively at deletion, present on both the dicentric and the ‘normal about 32.1–31.5 Mb (between BACs RP11-22F19 and 8’ that has amplified FGFR1. This was previously RP11-275E10 and between BACs RP11-212N14 and detected by a YAC (Courtay-Cahen et al., 2000). The RP11-566H8). The known NRG1 breaks in MDA-MB- array CGH suggests that the deletion extends roughly 175, PaTuI, SUM52, SUIT-2, UACC812 and ZR-75-1 from 38.6 to 40.4 Mb. The array CGH also suggests the were also detected. The break in HCC1937 is reciprocal region proximal to this deletion and before the and so was not detected. centromere may also be amplified, but this was not Interestingly, in two cell lines, HCC38 and MDA- investigated further. This region may include the ANK1 MB-157, there is a break just proximal to NRG1 thus and PLAT genes which can be amplified separately removing this gene in the translocated chromosomes (Ugolini et al., 1999). (Figure 2b). Several cell lines have breaks 50 to UNC5D at 35.2– MDA-MB-361 35.8 Mb. We previously found three breaks near MDA-MB-361 has two complex 8p translocations that 34.5 Mb which appeared to be a cluster, in the breast appear both to have evolved from the same 8;17 cell lines MDA-MB-361, T-47D and ZR-75-1 (Courtay- translocation (Figure 3) (Morris et al., 1997; Courtay- Cahen et al., 2000). These were mapped precisely by Cahen et al., 2000). Both have duplicated or triplicated FISH and array CGH, to 34.2, 34.5 and 34.9 Mb, fragments of 8p from the region proximal to the respectively (Figure 4). At least five more cell lines had breakpoint, and the extra copies are inserted into the breaks 50 to UNC5D by array CGH: CF-PAC, C70 and adjacent chromosome 17 material (Courtay-Cahen HCC1569 at 34.1–34.4 Mb, MDA-MB-134 at 34.7 Mb, et al., 2000). We mapped the distal breakpoints of the and MDA-MB-453at 35.5Mb. VP229 and BT20 may translocation and extra copies to the nearest BAC by also have breaks nearby, but they were not precisely FISH (Figures 3and 4; Supplementary Table 2). Array located.

Oncogene 8p in carcinomas JCM Pole et al 5702 Discussion obvious support to this region as it is substantially distal to the breaks we have detected. However, if breaks are Our main observations and interpretation are summar- concentrated in regions of relative fragility, this argu- ized in Figure 7. At cytogenetic resolution, the overall ment would not hold. pattern of 8p rearrangement is simple (Figure 1 and 2a): the great majority of cases show loss of distal 8p, with Alternative theories of 8p breakage breakpoints scattered between WRN (31 Mb) and the Among alternative explanations for 8p breakage is that centromere (44–47 Mb), mostly between NRG1 and distal loss occurs simply in order for amplification of the FGFR1 (32 and 38.4 Mb). This would be compatible region around FGFR1 to occur by breakage-fusion- with the ‘traditional’ view that there is a tumour bridge cycles, and breaks may be clustered if there are suppressor gene on 8p of general importance in sites of preferential breakage (Birnbaum et al., 2003). carcinomas (reviewed by Birnbaum et al., 2003). This idea does not fit the data particularly well: some However, at higher resolution, there seems to be a great lines, including HCC70, MCF7, and SKBR3, have no deal more complexity than this simple idea would 8p material retained; others have 8p breaks but no predict. detectable amplification – HCC1143, MDA-MB-453, BT20, T-47D, C70, for example; and there are cases Is there a tumour suppressor? where loss occurs proximal to amplification – MDA- If there is a tumour suppressor gene on 8p, we would MB-175 and ZR-75-1, for example. While amplification expect the breaks to be scattered between the centromere often occurs immediately proximal to a break, we and the gene: Figure 2 shows breaks scattered between favour the interpretation that oncogenic fusions occur the centromere and the distal end of NRG1 (32 Mb), at the break that are subsequently amplified by a making genes immediately distal to NRG1, including mechanism independent of the original break. A WRN (31 Mb), obvious candidates. Most of the possible variation on this idea would be that the breaks candidates proposed previously – none of which are permit inversion. particularly convincing – are in the region 3–24 Mb (e.g. DLC1, DBC2, FEZ1/LZTS1 and TUSC3; for references Clusters of breakpoints at NRG1 and UNC5D see Birnbaum et al., 2003; Toomes et al., 2003; Asatiani A completely different interpretation is that some of the et al., 2005) and our array CGH (Figure 2) gives no 8p translocations break at specific genes and activate or

Figure 7 Summary of selected results and interpretation. The distribution of the majority of breakpoints found is shown schematically, together with the main targets of amplification and examples (in T-47D and ZR-75-1) of the complexity of rearrangements associated with 8p breaks. The distribution of breakpoints suggests that if there is a tumour suppressor gene it may be close to the most distal of these, for example, WRN. (Previously proposed tumour suppressor genes are shown except KIAA1456, which is adjacent to DLC1; note that DBC2 is RHOBTB2, FEZ1 is LZTS1). The group of breaks at NRG1 and possible cluster of breaks 50 of UNC5D are indicated. The most frequent core amplicon (defined previously, Garcia et al., 2005; Prentice et al., 2005) includes ZNF703, previously named FLJ14299, and may extend to RAB11FIP1. The inversion and deletion accompanying the translocation in T-47D illustrates that the distal break may not be the rearrangement that is selected for. The ZR-75-1 rearrangement shows that several apparently separate events have occurred: there is translocation of NRG1 with triplication of proximal sequences including the ZNF703 amplicon; and there is also physically separate amplification of FGFR1, showing that this is an independent amplicon.

Oncogene 8p in carcinomas JCM Pole et al 5703 inactivate them. Although 8p breakpoints are spread, Amplifications some are clustered in the NRG1 gene and there may be a The overall pattern of amplifications is complex, but we second cluster at UNC5D. suggest they can be divided into at least three separate We recently reported that seven breast or pancreatic categories. cancer cell lines have breaks at or within the 50 end of First, there is the consensus amplicon around the NRG1 that could activate or fuse NRG1, and this genes ZNF703/FLJ14299 to RAB11FIP1at about study adds two more lines. Similar breaks occur in 36.9–37.9 Mb. FGFR1, at 38.4 Mb, although originally fresh tumours (Adelaide et al., 2003; Huang et al., proposed as the principle target for amplification in this 2004). These translocations seem to specifically target region, is not included (Ray et al., 2004; Garcia et al., NRG1 and to be oncogenic: one of the NRG1 2005; Prentice et al., 2005). This amplicon we defined translocations, in HCC1937 is reciprocal; in at least using the same array on breast tumour tissues (Garcia one case, MDA-MB-175, the result is secretion et al., 2005), and it was independently identified by of an Nrg1 fusion protein with biological activity Prentice et al. (2005). In the present study Cama-1 (Schaefer et al., 1997); in several cases the trans- provided another example of high-level amplification of location junction is amplified; and there is a strong the ZNF703/FLJ14299 region with only slight gain of correlation between translocation and expression FGFR1. of NRG1 transcripts (Y-L Chua and PAWE, Secondly, our data make it clear that FGFR1 is unpublished). Nrg1 are ligands for the ERBB sometimes a distinct target of amplification, though less receptor family and overactivity of NRG1 promotes common. FGFR1 is amplified or gained without the mammary tumour development in transgenic mice ZNF703/FLJ14299-RAB11FIP1 region in UACC812, (Krane and Leder, 1996). At present, these transloca- and probably in HCC1599 and SW837 (colon), though tions appear to be distinct events from others affecting these cases may include the proximal part of the 8p – certainly in the two lines examined in detail by consensus amplicon. Most clearly, in ZR-75-1, FGFR1 FISH, MDA-MB-175 and ZR-75-1, the NRG1 junction amplification occurs in a physically separate location behaves as a separate rearrangement to other events on from triplication of the region that includes ZNF703/ 8p12. On the other hand, most translocations of 8p FLJ14299- RAB11FIP1, and so cannot simply be a result in complete loss of a copy of NRG1, so passenger. translocation of NRG1 is not a general explanation for A third class of low-level amplification, sometimes 8p rearrangement. only duplication or triplication, was seen immediately UNC5D, at 35.2–35.8 Mb, is also a candidate proximal to breaks in many cases, suggesting either that target gene, its 50-end lying proximal to a the mechanism of translocation often results in their number of breaks between 34.1 and 35.5 Mb, in at formation or that the translocation junction is onco- least eight cell lines (HCC1569, MDA-MB-134, genic and so tends to be amplified during subsequent MDA-MB-361, MDA-MB-453, T-47D, ZR-75-1, evolution. For example, several of the breaks at CF-PAC and C70). We originally reported three of NRG1are amplified – in MDA-MB-175, SUM52, ZR- these which appeared to be a cluster (Courtay-Cahen 75-1 and Suit2 – as are the breaks in MDA-MB-134 et al., 2000). We had previously found four (34.7 Mb), and MDA-MB-361 (34.2 Mb). Other exam- breast tumours with breaks at about 35.5 Mb ples extend into the ZNF703/FLJ14299 and FGFR1 (Garcia et al., 2005). regions. UNC5D could well be a critical gene, as it is one of the four human homologues of Caenorhabditis Inversions elegans unc5, which are receptors for Netrin. The We suggest that inversions may be frequent in these Netrin system, including the Netrin receptors tumours. In T-47D we demonstrated an inversion UNC5A-D, DCC and neogenin, were originally impli- accompanying the 8p translocation (Figure 5), and cated in axon guidance, but are also active in inversions, before or during translocation, would survival signalling, and are involved in morphogenesis explain the alternating regions of deletion and duplica- of the mammary gland (Hinck, 2004). The UNC5s tion seen in ZR-75-1. Inversions are difficult to detect have been proposed as tumour suppressors (Arakawa, but are an attractive explanation for the amplification of 2004), but could equally be activated, which would be apparently distinct regions of one chromosome (e.g. the more likely consequence of the translocations Orsetti et al., 2004): inversion, creating an oncogenic reported here. junction, would be followed by amplification of one At present, however, there is not much evidence inversion junction, manifest as two apparent regions of that UNC5D is targeted. The breaks are mostly amplification by array CGH, in the same way as quite a long way 50 of the gene, and with more breaks amplifications on distinct chromosomes can be coam- mapped in this region (Figure 2b), it is not plifications (Guan et al., 1994; Bautista and Theillet, clear that they form a distinct cluster. Furthermore, 1998; Volik et al., 2003). the complexity of the rearrangements associated with these breaks, particularly our finding that the adjacent segment is inverted in T-47D, may Rearrangements are complex well mean that these breaks are not in themselves In all cases where we investigated rearrangement in significant. detail, the translocations are accompanied by complex

Oncogene 8p in carcinomas JCM Pole et al 5704 additional events, including inversion, deletion Materials and methods proximal to the main translocation junction, and multiple independent duplications or amplifications. Cell culture, metaphase preparation and fluorescence in situ In ZR-75-1, for example (Figure 6), on the single hybridization dic(8;11) there is translocation of NRG1, triplication of Cell lines are listed in Supplementary Table 1. Metaphase spreads and FISH were carried out using standard methods as the translocation together with the region around the detailed in Alsop et al. (2006). For metaphase FISH, ZNF703/FLJ14299 amplicon, and physically separate chromosome paints kindly provided by Professor MA amplification of CCND1 and FGFR1. This complexity Ferguson-Smith were labelled using nick translation either cannot be dismissed as an in vitro artefact, as each of the directly with spectrum orange-dUTP (Vysis/Abbot Labora- events is common in uncultured breast tumours. In T- tories, IL, USA) or indirectly with biotin-dUTPs (Roche 47D, an apparently simple 8;14 translocation also has an Diagnostics, Basel, Switzerland). Bacterial artificial chromo- inversion and deletion of sequences at the inversion somes were labelled indirectly, using nick translation with junction. digoxigenin-, estradiol- and biotin-labelled dUTP (Roche There are now many examples, where translocations Diagnostics). Band-specific paints for 8p11.2, and 8p22 were are accompanied by additional rearrangements on a from Research Genetics/Invitrogen (Paisley, UK). Chromo- some 8q arm paint pre-labelled with digoxigenin was from scale of hundreds of kilobases or larger. For example, Cambio (Cambridge, UK). Detection of labelled, incorporated the translocation-plus-inversion structure that we found dUTPs was performed using the appropriate antibody or in T-47D probably occurs in some examples of the avidin conjugates (sheep anti-digoxigenin-FITC (Roche Diag- recurrent translocations ETV6-ABL1 (Van Limbergen nostics), avidin-Cy3, or avidin-Cy5 (GE Healthcare, Buck- et al., 2001) and MLL-AF10 (Klaus et al., 2003and inghamshire, UK) and biotinylated anti-avidin (Vector references therein). In both cases the inversion is Laboratories Inc., Burlingame, CA, USA). Images were necessary for the two participating genes to be in the captured on a Nikon Eclipse 800 microscope using Cytovision same orientation. Other translocations may be accom- software (Applied Imaging, Carlsbad, CA, USA). panied by deletions (e.g. Rousseau-Merck et al., 1999; Metaphase spreads with more extended chromosomes were Sinclair et al., 2000). Similarly, constitutional transloca- made from ZR-75-1 by culturing the cells with 40 mg/ml bromodeoxyuridine for 16 h, addition of 5 mg/ml ethidium tions are often accompanied by complex cryptic bromide, and harvesting an hour later without colcemid rearrangements (Gribble et al., 2005), and as these are treatment. likely to be single events, they suggest that the The BAC and PAC clones used in FISH experiments are mechanism of translocation often produces complex detailed in Supplementary Table 2. Clone positions are given rearrangements. according to Human genome sequence NCBI Build 35. Bacterial artificial chromosome clones were from the RPCI- 11 (RP11) library, the GS1 library and Caltech whole genome 8p12 rearrangements in real tumours libraries (CTA, CTC and CTD). They were identified from the Many of the rearrangements that we have reported in emerging genome assembly or BAC end sequence data cell lines affecting 8p12 have also been detected in real displayed on the UCSC Genome Browser website (http:// tumours. The recurrent breakpoint effecting NRG1 genome.ucsc.edu), or the Washington University Fingerprint (Huang et al., 2004), the ZNF703/FLJ14299-RAB11- maps. All BACs had been checked for chimerism and location FIP1 amplicon and a recurrent breakpoint at UNC5D in on 8p using conventional FISH on normal metaphases. They breast cancers (Garcia et al., 2005) have been previously were obtained from the Wellcome Trust Sanger Institute reported. In our previous publication we compare array (Hinxton, UK), Research Genetics/Invitrogen, or in some CGH results from breast cancer cell lines and breast cases from the laboratory that had sequenced them. Fosmids were selected from the UCSC genome browser tumours and the overall pattern of rearrangement is (http://genome.ucsc.edu) and obtained from the Sanger very similar, showing loss of distal 8p, gain of 8q and the Institute. They were also checked by hybridization to normal most frequent site of copy number change occurring at metaphase chromosomes. Isolated fosmid DNA was amplified 8p12, often with amplification (Garcia et al., 2005). This using Templiphi (GE Healthcare), and labelled by nick comparison suggests that the events we describe here in translation. cell lines are relevant to real carcinomas. Fibre-FISH was based on the method described by Mann et al. (1997). Briefly, 10 ml of fixed metaphase preparation was smeared across the width of a polylysine-coated slide (Poly- prep, Sigma, Dorset, UK) approximately 1 cm from the top of Conclusion the usable slide surface and allowed to dry for a minute before being submerged vertically in lysis buffer (0.5% (w/v) SDS, In conclusion, at present there is no grand unifying 50 mM EDTA, 0.2 M Tris-HCl pH7.4) in a coplin jar and left to theory of 8p rearrangement. If it is primarily driven by stand with the cell preparation just below the buffer surface. loss of a distal tumour suppressor gene, we have After 5 min 94% ethanol was slowly run on to the buffer evidence that the gene is close to NRG1. However, the surface to form an upper layer until the entire slide was covered. After incubating for 10 min, the slide was pulled out tumour suppressor hypothesis does not account well for of the solutions slowly and steadily at a 301 angle, submerged the details and complexity of rearrangements upstream, in 70% ethanol, incubated for 30 min, and dehydrated in an such as NRG1 translocations and amplification of more ethanol series. Slides were hybridized as for metaphase proximal sequences, unless these are merely conse- spreads, using digoxigenin-dUTP and biotin-UTP (both from quences of the breakage event, or initial translocation Roche Diagnostics) haptens and mouse anti-digoxin (Sigma) or deletion that makes the chromosome unstable. followed by alexafluor-594 anti-mouse (Molecular Probes,

Oncogene 8p in carcinomas JCM Pole et al 5705 Invitrogen) for the digoxin-labelled DNA and streptavidin- chromosome clones from outside the 8p12 region were from alexafluor-488 conjugate (Molecular Probes, Invitrogen), the 1 Mb cloneset of Fiegler et al. (2003). Labelling of genomic biotinylated anti-streptavidin (Vector laboratories) and DNA, hybridization and post-hybridization washes were streptavidin-alexafluor-488 conjugate in three layers for carried out as previously described (Garcia et al., 2005). biotin-labelled DNA. Estimates of breakpoint position from Arrays were scanned using an Axon Genepix 4100 and the fibre-FISH were made by comparing the length of a whole data acquired using Genepix4.1 software (Molecular Devices, BAC of known length to the length of a BAC fragment, Union City, CA, USA). Data analysis was carried out using averaged over 20 captured images. Microsoft Excel, followed by CGHAnalyzer (http://acgh. Distances between sequences were estimated as described by afcri.upenn.edu; Margolin et al., 2005) to compare array (van den Engh et al., 1992), by hybridizing BACs to interphase CGH profiles and generate Figures 2a and b. Results were nuclei. The average separation of FISH signals in interphase calculated relative to the median Cy3:Cy5 ratio for the whole nuclei is proportional to the square root of the genomic array excluding sex chromosomes, and expressed on a log2 distance between the hybridization targets, up to 1 Mb scale. The array CGH data will be available on our website separation, so it easily distinguishes sequences that are http://www.path.cam.ac.uk/Bpawefish. adjacent from sequences that are a few hundred kilobases or more apart (van den Engh et al., 1992). Distances between Acknowledgements BAC signals were measured in arbitrary units, using Cytovi- sion software (Applied Imaging), except one unit was subtracted from values to give a zero result for zero distance, We thank Huai-En Huang, Rachel Lyman, Nicholas J and averaged over at least 15 examples. Roberts, Joanne M Staines, Melody Tabiner, and Katherine Williams for contributing to the FISH data and cell culture. Supported by Cancer Research UK, Bio- Array CGH technology and Biological Sciences Research Council Array CGH was performed essentially by the methods of (BBSRC), Breast Cancer Campaign, Wellbeing, Medical Fiegler et al. (2003), on an in-house array (Huang et al., 2004; Research Council (studentship for SLC), Cambridge Com- Garcia et al., 2005), that comprised BACs approximately monwealth Trust (studentships for AEA, KAB) and Sackler 10 Mb apart across the whole genome; 1–1.5 Mb apart over Fund (KAB). chromosome 8; and at near-tiling path density over 8p12, with 58 BAC clones covering 9.6 Mb of 8p12 from 31.0 (RP11- 473A17) to 40.6 Mb (RP11-51K12). All but one gap between Note: successive BAC ends were 0.3Mb or less, the one larger gap Further analysis of 8p12 amplicons was recently pub- being 0.6 Mb near the proximal end. Bacterial artificial lished by Gelsi-Boyer et al., 2005. Mole Cancer Res.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene