Loss of Heterozygosity Analysis and DNA Copy Number Measurement on 8P in Bladder Cancer Reveals Two Mechanisms of Allelic Loss

Research Article

Loss of Heterozygosity Analysis and DNA Copy Number Measurement on 8p in Bladder Cancer Reveals Two Mechanisms of Allelic Loss

Jacqui Adams, Sarah V. Williams, Joanne S. Aveyard, and Margaret A. Knowles

Cancer Research UK Clinical Centre, St. James’ University Hospital, Leeds, United Kingdom

Abstract invasive tumors have 8p LOH; ref. 1). 8p LOH is also found in Many epithelial tumors show deletion of the short arm of several other tumor types, including prostate and colorectal chromosome 8 that is related to aggressive disease or adverse carcinomas, where it is also associated with a more aggressive prognosis. In undissected samples of urothelial cell carcinoma clinical phenotype. of the bladder, at least two regions of loss of heterozygosity The pattern of 8p LOH is complex with at least two regions of deletion (2–6), consistent with the presence of two or more (LOH) were identified previously within a small region of 8p11-p12. LOH analysis on a panel of pure tumor DNA samples relevant tumor suppressor genes. A distal region of LOH at 8p21- confirmed this and identified tumors with allelic imbalance, p23 has been identified and mapped in several cancer types, some with clear breakpoints in 8p12. This suggests either that including prostate (7, 8), oral and laryngeal (9, 10), colorectal these samples contained genetically distinct subclones or that (11–13), non–small cell lung (13), liver (13), breast (14), and ovarian breakpoints in 8p12 may confer a selective advantage without (15). A second more proximal region on 8p11-p12 shows LOH in LOH. To assess the mechanism of LOH and to map breakpoints many tumor types, including bladder (2), colorectal (16), prostate precisely, a panel of bladder cancer cell lines was examined. (3, 4, 6, 17–19), renal (20), breast (21, 22), and oropharyngeal (5). Microsatellite analysis of 8p markers identified regions of LOH in this region is associated with tumor stage and grade in contiguous homozygosity that coincided with regions of LOH bladder cancer and nonpapillary renal cell carcinoma (2, 20); with invasive behavior in prostate cancer (3, 18); and with lymphatic, in tumors. Fluorescence in situ hybridization analysis was carried out on seven cell lines predicted to have 8p LOH using vascular, and perineural microinvasion in colorectal cancer (23). a chromosome 8 paint, a chromosome 8 centromeric probe, Two independent regions of interstitial loss of 8p11.2-p12 have and a series of single-copy genomic probes. This revealed been reported in colorectal cancers, suggesting the presence of at overall underrepresentation of 8p and overrepresentation of least two suppressor genes (16). Homozygous deletions have been 8q. Several breakpoints and one interstitial deletion were found in prostate cancer at D8S87 (8p12; ref. 24) and between identified in 8p12. Two cell lines with extensive interstitial D8S87 and D8S133 on 8p12-21 (25). Unbalanced chromosome regions of homozygosity showed no reduction in DNA copy translocations with breakpoints around 8p12, resulting in loss of number by fluorescence in situ hybridization analysis, distal 8p, are reported in breast, colon, and squamous cell lung indicating that, in addition to large deletions and rearrange- cancers (26). Breakpoints clustered in a small region around ments of 8p, small regions of interstitial LOH on 8p12 may be D8S505 in 8p12 have been mapped in three breast cancer cell lines, generated by mitotic recombination. This implicates both and in two cases, these were accompanied by additional and major DNA double-strand break repair mechanisms in the complex rearrangements (27). The 8p12 region is also a site of amplification in breast cancer generation of 8p alterations. (Cancer Res 2005; 65(1): 66-75) (28, 29) with a potential target being FGFR1, which maps to 8p12. Coamplification of FGFR1 (8p12) with CCND1 (11q13) has been f Introduction identified in 5% to 8% of primary breast cancers (28), and in 3% of primary breast tumors, there is a recurrent rearrangement Alterations affecting the short arm of chromosome 8 are involving the proximal portions of 8p and 11q resulting in the frequent in human cancers. Most are deletions and these have formation of a hybrid amplified structure composed of 8p and 11q commonly been identified and mapped by loss of heterozygosity sequences (30). (LOH) analysis, which uses highly polymorphic genetic markers Functional assays have confirmed the importance of chromo- to detect loss of one parental allele in a tumor sample compared some arm 8p in tumorigenesis. Introduction of a whole human with constitutional DNA from the patient. LOH is frequently chromosome 8 suppressed invasiveness of a highly metastatic rat found in the region of tumor suppressor genes that require prostate cancer (31), with the 8p12-p21 region being necessary for biallelic inactivation for effect. This is commonly achieved via suppression of metastasis (32). 8p12-pter was also found to be LOH and a small mutation in the gene on the retained allele. In critical for suppression of invasiveness in colon carcinoma cells bladder cancer, 23% of tumors show 8p LOH, and there is a (33). Another study found 8p22-p23 to suppress soft agar significant association with muscle invasion (56% of muscle clonogenicity in colorectal carcinoma cells (34). Our previous LOH studies on 193 cases of urothelial cell carcinoma (UCC) of the bladder identified two regions of deletion, Note: J. Adams and S.V. Williams contributed equally to this work. a large telomeric region (8p21.1-pter) and a more proximal region Requests for reprints: Margaret A. Knowles, Cancer Research UK Clinical Centre, at 8p11.2-12 (2). With the recent availability of the correct order for St. James’ University Hospital, Beckett Street, Leeds LS9 7TF, United Kingdom. Phone: 44-113-2064913; Fax: 44-113-2429886; E-mail: [email protected]. the microsatellite markers used in that study, the more proximal I2005 American Association for Cancer Research. region of deletion is now defined as two separate regions.

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To determine the nature of the genetic events that occur on from the Genome Database (http://www.gdb.org). For analysis of cell lines, 32 proximal 8p and to refine the localization of the regions of deletion, primers were end labeled using T4 polynucleotide kinase and [g P]dATP we have examined a panel of tumors from which DNA could be and PCR reactions were carried out, as described previously (35), using j extracted from pure microdissected tumor cells and a panel of annealing temperatures 5 C below the calculated theoretical melting temperature of the primers. The number of alleles seen at each locus was bladder cancer cell lines, several of which are predicted to have 8p recorded. LOH. We confirmed that at least two regions of deletion exist on For analysis of tumors, the forward primer was fluorescently labeled proximal 8p in tumor samples. We used fluorescence in situ with either FAM, TET, or HEX dyes. Sequences from tumor DNA and hybridization (FISH) analysis in cell lines to determine overall corresponding normal DNA were amplified, the products were denatured overrepresentation of 8p and 8q and to identify cytogenetic and electrophoresed in 4.25% polyacrylamide gels containing 7 mol/L urea, breakpoints in 8p in cell lines with partial LOH. We also used FISH and products were analyzed by Genescan software (Applied Biosystems, to obtain evidence that LOH on 8p is achieved in some cases Warrington, United Kingdom). Each informative result was repeated at least without DNA copy number change. twice. Allele ratios were visually compared with normal DNA. LOH was scored when the T:N ratio for one allele was V0.2. Fluorescence In situ Hybridization. Cells were harvested and slides Materials and Methods were prepared using standard cytogenetic techniques. Chromosome Tumor Samples. Bladder tissues were obtained by surgical resection 8 centromere was detected using a fluorescein-labeled a-satellite probe from St. James’ University Hospital (Leeds, United Kingdom). All patients (Roche, Lewes, United Kingdom) or digoxigenin-labeled D8Z2 (Qbiogene, gave informed consent. Tissues were snap frozen in liquid nitrogen, Ilkirch, France). Five cosmid probes mapped to a 7.9-Mb region on 8p12 embedded in cryoembedding compound (Leica, Milton Keynes, United were selected by screening the LA08NC01 flow sorted chromosome 8 cosmid Kingdom), and stored at À80jC. Fresh frozen blood was used as a source of library (36): 128C10 containing D8S339, 138B12 containing D8S259, 57F7 normal DNA and kept at À20jC until DNA was extracted. containing D8S283, 73E9 containing D8S535, and 129E11 containing Cell Lines. Fourteen cell lines were used in this study. Thirteen were D8S135. Two PAC clones from this region, 96p7 containing D8S505 and derived from transitional cell carcinomas: 253J, J82, VMCUB-II, JOVN, RT4, 124d8 containing D8S87, were provided by Dr. Paul Edwards (University of UMUC3, T24, HT1197, HT1376, RT112, SW1710, 5637, and SD. There was Cambridge, Cambridge, United Kingdom; ref. 27). BAC clones 155L11, one EBV immortalized lymphoblastoid cell line, J82-EBV (kindly provided by 263C3, 14I17, 395I14, 561E1, 529P14, 16I12, and 241P12 were obtained from Dr. C. O’Toole). All cell lines were grown under standard conditions. the Sanger Centre and form part of the human genome sequence tiling path Microdissection of Samples and DNA Extraction. Sections (5 Am) of (http://www.sanger.ac.uk/HGP/). The relative positions of 8p12 probes and frozen or paraffin-embedded samples were cut and stained with H&E. microsatellite loci are shown in Fig. 1. Equivalent distances from 8pter for Sections containing >80% tumor cells were used directly for DNA extraction. the other probes are 14I17 (26.17-26.34 Mb), 395I14 (25.12-25.31 Mb), 561E1 For other samples, cells were selectively captured by laser capture (24.2-24.4 Mb), 529P14 (21.6-21.8 Mb), 16I12 (18.4-18.6 Mb), and 241P12 (9.8- microdissection (Arcturus, Mountain View, CA) to minimize normal DNA 10 Mb). Chromosome 8 paint (Qbiogene) and MYC (Qbiogene) were used to contamination, and the DNA was extracted using the QIAmp DNA midi kit assess the chromosome 8 and 8q24 content of each cell line. (Qiagen, Crawley, United Kingdom). DNA from blood was extracted using Probes were labeled with biotin-14-dATP (Life Technologies, Rockville, the Nucleon DNA extraction kit (Nucleon Biosciences, Lanarkshire, United MD) or digoxigenin-11-dUTP (Roche), denatured, and applied to denatured Kingdom). Both tumor and corresponding normal blood DNA were stored slides. Commercial probes were hybridized according to the manufacturer’s at À80jC. instructions. Hybridization was allowed to proceed at 37jC for 15 to 40 PCR Analysis for Microsatellite Markers on 8p. Genomic DNA was hours and detected following incubation with FITC-conjugated avidin DCS extracted from cell lines using proteinase K digestion and phenol- (Vector Laboratories, Inc., Burlingame, CA) and rhodamine-conjugated anti- chloroform extraction. Eighteen chromosome 8–specific microsatellite digoxigenin (Roche). Slides were mounted in Citifluor (Citifluor Ltd., markers were used. The primer sequences for these markers were obtained London, United Kingdom) containing 4V,6-diamidino-2-phenylindole

Figure 1. Map of 8p12-p11 showing the positions of microsatellite markers, cosmid, and PAC and BAC clones used for FISH analysis and the position of breakpoints identified in cell lines RT112, SD, J82, and VMCUB II. Distances from 8pter are in Mb (National Center for Biotechnology Information Build 34 of human genome). www.aacrjournals.org 67 Cancer Res 2005; 65: (1). January 1, 2005

(Cambio, Cambridge, United Kingdom). Images were captured using SmartCapture2 (Digital Scientific, Cambridge, United Kingdom). At least 10 metaphase spreads were analyzed for each hybridization. A probe was scored as retained when probe signal was seen with each 8 centromere in >80% of cells. Deletion was recorded when the number of probe signals was lower than the number of centromere signals and was confirmed by repetition.

Results Identification of Regions of Interstitial LOH in 8p12 in Bladder Tumor Samples. Fifty-five tumor samples were studied. Ten (18%) showed clear LOH within 8p12 with minimal signal from the lost allele (T:N ratio V0.2) as expected for samples of clonal tumors with at least 80% tumor cells. Five tumors showed allelic imbalance within 8p12, which given the purity of samples used implies aneuploidy rather than LOH. Two of these samples showed clear retention of equal copy number of both alleles for at least one marker within the region of allelic imbalance, indicating that a breakpoint was present within the region of interest. Forty-one of the tumors were also analyzed for LOH in 8p21. Three had LOH in 8p21 but not 8p12, and seven had allelic imbalance (T:N ratio 0.2-0.6) in 8p21 but equal representation of both alleles in 8p12, again suggesting breakpoints within 8p. This gives an overall frequency of 23.6% with clear LOH anywhere on 8p and an additional 22% with allelic imbalance. Of the 10 tumors with LOH in 8p12, 6 had loss of all of 8p12 extending distally and 4 had partial loss. These are shown in Fig. 2 together with the two tumors that showed allelic imbalance with breakpoints in 8p12 (tumors 5 and 6). When correct marker order is applied, our previous LOH analysis on undissected samples (2) defined two regions, shown as regions 1 and 2 in Fig. 2. Our current results with pure tumor samples are compatible with this. Only one tumor (tumor 4) had an interstitial region of LOH. This involved a single marker (D8S513) that lies within region 1. Apart from this single case, regions of LOH extended beyond 8p12 toward the centromere or telomere Figure 2. Pattern of 8p breakpoints in bladder tumor samples. Selected and thus could target distant genes. Nevertheless, these six tumors samples with partial LOH and allelic imbalance on 8p from a total of 55 tumors studied. all showed at least one breakpoint within a relatively small region of 8p. The estimated distance between D8S1791 and D8S283, at the margins of the region containing the breakpoints, is 4.5 Mb based suggesting these as possible sites of LOH. Differences in observed on National Center for Biotechnology Information Build 34 of the and expected allele frequencies are shown in Fig. 3. human genome. Six cell lines (SD, RT4, 5637, UMUC3, VMCUB-II, and the Identification of Bladder Tumor Cell Lines with Predicted lymphoblastoid line J82-EBV), which had two alleles at multiple 8p LOH. To determine likely 8p LOH status, we screened 13 UCC loci, showed an interstitial region in which several contiguous loci cell lines with 18 highly polymorphic microsatellite loci mapping to had only one allele. For SD, 5637, and J82-EBV, this included chromosome 8. For one cell line (J82), a paired immortalized D8S283, D8S513, D8S259, and D8S278 (probability of homozygosity lymphoblast cell line was available (J82-EBV) and classic LOH at all four loci 0.00797). VMCUB-II showed one allele at three of analysis was possible. For the other cell lines, the presence of these loci, D8S283, D8S513, and D8S259. The cell line RT4 showed contiguous regions with single alleles allowed prediction of regions a region of homozygosity including D8S282, NEFL, D8S137, of LOH. Results are shown in Table 1. Lower than expected D8S339, and D8S278 (probability of homozygosity 0.0001) and frequencies of heterozygosity were found at all loci. Two cell lines UMUC3 showed only one allele at three of these loci (NEFL, D8S137, (T24 and 253J) had one allele at all loci assessed, suggesting loss of and D8S339). During our previous LOH analysis of bladder tumors, an entire parental copy of chromosome 8. Three cell lines (HT1197, these markers have all been assessed in the normal DNA of JOVN, and HT1376) had only one allele at all 8p loci but two alleles individuals with bladder cancer. In these samples, the expected at one of the two 8q loci assessed (D8S84), suggesting loss of one allele frequencies (Table 1) were observed (data not shown). Based parental copy of 8p with retention of 8q. The remaining cell lines on the allele frequencies, seven bladder cell lines that were predicted had two alleles at one or more loci on 8p, demonstrating retention to have partial deletion of 8p were selected for FISH analysis. of both parental alleles for at least part of 8p. This is illustrated by They were RT112, SD, RT4, UMUC3, VMCUB-II, 5637, and J82. J82, which had LOH distal to D8S339 when compared with J82-EBV. Assessment of Chromosome 8 Content of Bladder Tumor One allele was seen in z11 of the 13 UCC cell lines analyzed at Cell Lines by FISH. The chromosome 8 content of the cell lines D8S135, D8S283, D8S513, D8S259, D8S339, D8S137, NEFL, and LPL was examined using chromosome 8 paint, a single-copy MYC

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Table 1. Allele frequencies on 8p in bladder tumour cell lines.

Expressed Observed Locus J82-EBV J82 RT112 SD RT4 UMUC3 VMCUB-II 5637 HT1197 T24 253J JOVN HT1376 SW1710 hetero- hetero- c zygosity* zygosity

b b D8S264 2112222 21 111 1 2 8346 b b D8S261 2111122 21 111 1 2 7731 LPL 2 1 1 1 2 1 1 1 1 1 1 1 1 2 83 15 b D8S282 2111122 2 1 1 1 1 1 2 72 31 b NEFL 211111211 111 1 2 8115 D8S137 11111111111112628 D8S339 21111111111111800 b b D8S278 1111122 11 111 1 2 6423 b D8S259 1111211 1111112 5915 D8S283 11111111111111800 b D8S513 11111211ND111127315 b D8S535 2211222 2111111 6238 b D8S505 2211222 2111112 8046 D8S87 2 2 1 2 1 1 1 2 1 1 1 1 1 2 68 31 D8S135 11111211111111538 b b b D8S255 2221122 2 1 1 1 1 1 2 72 46

b D8S205 22221 2 2 2 1 1 1 1 1 1 78 46 (8q) D8S84 2 2 2 1 1 1 1 2 2 1 1 2 2 2 57 54 (8q)

NOTE: 1 and 2 represent the number of alleles detected in each cell line (ND, not done). *The expected heterozygosity values for each marker were obtained from the Genome Database. cThe observed heterozygosity values were calculated as a percentage of tumour cell lines containing two alleles. bImbalanced. Shaded area shows loci with observed heterozygosity at V15%. Boxed areas show regions of contiguous interstitial homozygosity.

probe, and a series of single-copy probes on 8p, which were 8 material in markers are known from a multicolor FISH study, examined with 8 centromere by two-color FISH (Fig. 4). The cell which will be described elsewhere (37). For three cell lines, 5637, lines were heterogenous as shown by varying chromosome 8 copy UMUC3, and RT4, the multicolor FISH study was on clones derived number. The most common chromosome 8 content for each cell by single cell cloning from the uncloned cell lines used here, and line is illustrated in Fig. 5. The origins of non–chromosome some differences in chromosomes 8 were seen (37).

Figure 3. Frequency of heterozygosity for chromosome 8 microsatellite markers in bladder cancer cell lines. Columns, percentage of the expected frequency of heterozygosity (Genome Database).

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Figure 4. FISH results on chromosome 8 in bladder tumor cell lines. Positions of microsatellite markers are shown relative to FISH probes to allow comparison of LOH and FISH data. Numbers of FISH signals for each probe are shown. Gray areas, regions of contiguous homozygosity for microsatellite markers; pale gray areas, maximum extent of these regions.

J82-EBV was diploid with two apparently normal chromosomes 8. chromosome 8 markers in RT4. UMUC3 was hypertriploid with J82 was triploid, with two subclones differing in their chromosome two or three apparently normal chromosomes 8, two derivative 8 content. Some cells contained one normal chromosome 8, two chromosomes 8, a deleted chromosome 8, and a derivative 11, derivative chromosomes 8, and a derivative chromosome 3, which which included part of 8q. This resulted in gain of 8q and of included part of 8p, whereas the remaining cells had two normal distal 8p23. Some of the derivatives were present in only a pro- chromosomes 8 and the same two derivative chromosomes 8 but portion of cells. UMUC3 had breaks in 8p proximal to 129E11 no derivative 3 with 8p (Fig. 4). This resulted in slight gain of (D8S135), at 8p22-23.1 between 16I12 and 241P12, and at 8p23 8 from 8qter to 8p12 and loss of two copies from 8p12 to 8p21.3 distal to 241P12. and one copy from 8p21.3 to 8pter. J82 had breaks on 8p at 8p12 VMCUB-II was triploid with some cells having three apparently between 128C10 (D8S339) and 263C6 and at 8p21.3 between 561E1 normal chromosomes 8 and a chromosome 8 with a deletion in 8p12 and 529P14. and some cells having four apparently normal chromosomes 8. This RT112 was diploid with one apparently normal chromosome resulted in a gain of all of chromosome 8, except for the small deleted 8 and two derivative chromosomes 8, resulting in gain of 8q, two region. VMCUB-II had breaks in 8p12 either side of the small copies of 8p11, and loss of 8p from 8p12 to 8pter. RT112 had deletion. 5637 was triploid with one apparently normal chromosome breaks on 8p at 8p10-11 proximal to 129E11 (D8S135) and at 8p12 8, two copies of a derivative chromosome 8, and a derivative between 129E11 (D8S135) and 124d8 (D8S87). SD was triploid with chromosome 4, which included distal 8p. This resulted in a loss of 1 or 2 normal chromosomes 8, 1 or 2 copies of a derivative chro- 8p21.3-22 to 8pter. 5637 had breaks in 8p at 8p21.3-22 between mosome 8, a deleted chromosome 8 and a derivative chromosome 529P14 and 16I12. 20 which included part of 8q. This resulted in gain of 8q, especially Thus, five of seven cell lines showed gain of 8q, whereas four distal 8q, and loss of 8p, particularly 8p12 to 8p23. SD had breaks showed loss of part of 8p and a further two had part of 8p on 8p at 8p10-11 proximal to 129E11 (D8S135), at 8p12 between underrepresented when compared with 8q and 8 centromere. 155L11 and 96p7 (D8S505) and at 8p22-23.1 between 16I12 and Four cell lines had breakpoints in 8p12 within a region spanning 241P12 (Fig. 5). f9.3 Mb: between 128C10 (D8S339) and 263C6 in J82, between RT4 was tetraploid with three apparently normal chromosomes 129E11 (D8S135) and 124d8 (D8S87) in RT112, between 155L11 8 and one apparently balanced translocation with a breakpoint in and 96p7 (D8S505) in SD, and either side of 155L11 in VMCUB-II 8p22-23.1 between 16I12 and 241P12. There was no loss or gain of (Fig. 1). One of the breakpoints in VMCUB-II may be the same as

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Figure 5. Illustration of chromosome 8 content of the cell lines studied by FISH. Pale gray, chromosome 8; dark gray, material from other chromosomes. Probes noted are those closest to the breakpoints but still retained. Asterisk, one or two copies of these chromosomes present in each cell. www.aacrjournals.org 71 Cancer Res 2005; 65: (1). January 1, 2005

Figure 6. FISH analysis of 5637 and SD. A, 5637 + 8 paint (red) showing normal 8(arrow) and two copies of der(8) (arrowheads). B, 5637 + 14I17 (green) and 8 centromere (red); 14I17 is present on normal 8 (arrow) and both der(8) (arrowheads). C, 5637 + 241P12 (green) and 8 centromere (red); 241P12 is present on normal 8 (arrow) and der(4) (broad arrow) but not on either der(8) (arrowheads). D, SD + 8 paint (red) and 20 paint (green) showing normal 8 (arrow), der(8;21) (broad arrow), del(8) (arrowhead), der(20) with 8q (broad arrowhead), and chromosome 20 and derivatives with 20. E, SD + 155L11 (green) and 8 centromere (red); 155L11 is present on normal 8 (arrow) and del(8) (arrowhead) but not der(8;21) (broad arrow). F, SD + 16I12 (green) and 8 centromere (red); 16I12 is present on two normal chromosomes 8 (arrows) but not del(8) (arrowhead) or der(8;21) (broad arrow).

that in SD. Three cell lines had breakpoints in 8p10-11, and five showed retention of all four copies. In VMCUB-II, single alleles were cell lines had breakpoints in distal 8p of which three were in the found at D8S259, D8S283, and D8S513, and no loss of two FISH same 9Mb interval between 16I12 and 241P12. probes containing D8S259 and D8S283, respectively, was detected. Comparison of Cytogenetic Data with Allele Frequencies. The deletion of 155L11 in 80% of cells in VMCUB-II is in a distinct Figure 4 shows the relationship of copy number detected by FISH location, separated by the heterozygous D8S535 and D8S505 loci. and regions of predicted LOH in the cell lines. In RT112 and SD, Similarly, UMUC3 had three contiguous homozygous markers cytogenetically detected losses fitted well with predicted 8p LOH. (D8S339, D8S137, and NEFL) but showed no deletion of FISH However, in four of the other five cell lines, there were regions probes. If, as predicted, the regions of homozygosity in these cell of predicted LOH that were not consistent with cytogenetic obser- lines represent small interstitial regions of LOH as observed in tumor vations. In 5637, homozygosity of seven loci from D8S513 to NEFL sample 6, the observed retention of copy number by FISH analysis conferred a high probability of LOH, but this was not accompanied indicates that LOH was generated via a copy number neutral by copy number loss. Five FISH probes within this region showed mechanism such as mitotic recombination (gene conversion). retention on all three copies of chromosome 8 (Fig. 6). In RT4, LOH was found in J82 by comparison with J82-EBV for five markers with single alleles denoted probable LOH within the markers including and telomeric to D8S339. This is compatible region D8S278 to D8S282, but five FISH probes within this region with the cytogenetic finding of reduction in copy number of the

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FISH probe 263C6. However, the FISH probe containing D8S339 Interestingly, the immortalized lymphoblasts from this patient were did not show a reduction in copy number, although LOH was homozygous at four contiguous markers (D8S513, D8S283, D8S259, recorded for D8S339, possibly indicating that the breakpoint lies and D8S278). Normal tissue from the patient was not available, so within the cosmid probe. Interestingly, J82-EBV was homozygous it is unclear whether this represents allelic loss that occurred for four markers (D8S513-D8S278) so that the true extent of LOH during immortalization of the lymphocytes or the true constitu- could not be determined. tional genotype of this patient. We have found no evidence for an excess of this haplotype in other patient or normal samples studied. FISH analysis of seven cell lines with predicted partial LOH of 8p Discussion confirmed the presence of frequent underrepresentation of 8p in This study has revealed considerable complexity in the keeping with several previous cytogenetic reports of monosomy 8, genomic events that occur on proximal 8p in bladder cancer. isochromosomes of 8q, and deletions of 8p in bladder cancer, all of We have confirmed by LOH analysis on pure tumor DNA which suggest loss of material from 8p and the presence of one or samples that LOH is found in this region in UCC. Although it more tumor suppressor genes in the region. The 8p breakpoints was not the objective of this study to carry out extensive identified by FISH are compatible with the regions of LOH found in deletion mapping by LOH analysis, we confirmed the existence primary bladder tumors and in other cancers including colorectal of at least two regions of LOH in 8p12-p11 that are compatible cancer and prostate cancer (3, 6, 16). This suggests that the same with our previous findings (2) and identified one tumor with a tumor suppressor gene(s) may be involved in the pathogenesis of very small region of loss. A recent study showed two similar these distinct cancers. regions of LOH in 8p12 in microdissected breast tumor samples, Breakpoints in 8p12 were mapped in four cell lines (J82, RT112, although the use of different markers precludes direct compar- SD, and VMCUB-II). This breakpoint region is similar to that ison (38). We found one case with LOH at a single locus only identified in three breast cancer cell lines (27), suggesting that this (D8S513). The two critical regions of LOH defined in our a consistent breakpoint region. The interstitial deletion in VMCUB- previous study (2) lie between D8S535 and D8S278 and between II could indicate the location of a gene affected by breaks in this D8S135 and D8S535, and this small region of loss lies within the region, although the significance of a loss of one of four copies in first of these. The markers that flank this region (D8S283 and 80% of cells is not clear and this is not coincident with the single D8S535) are only 62.8 kb apart (Ensembl v20.34c.1), and there are no small interstitial region of LOH in tumor 4. This region also other known polymorphic markers that could be used to confirm contains two Genescan predictions but no known genes. In J82, that there is true regional LOH. The region contains two Genescan RT112, and SD, an affected gene could lie at considerable distance predictions but no known genes. It is possible that a single distal to this breakpoint region, as all have a long region of unidentified gene spans the entire region or that an unrecognized 5V homozygosity extending either to 8pter (J82 and RT112) or to exon or promoter region of a nearby gene is affected by this event. D8S264 (SD). Further studies of a larger tumor panel will be needed before At least four cell lines, 5637, UMUC3, RT4, and VMCUB-II, had conclusions can be drawn on the significance of this finding. regions of predicted LOH with multiple homozygous microsatellite An unexpected finding was that, in DNA samples with >80% markers but did not show regional underrepresentation of 8p. This tumor cell content, some tumors showed allelic imbalance with could represent gene conversion, resulting in retention of copy clear breakpoints within 8p. If these samples contain only one number for a mutated allele, and this might be achieved via mitotic tumor clone, it is difficult to reconcile this finding with the biallelic recombination. Homozygosity for seven contiguous markers, which f inactivation of a tumor suppressor gene and equally difficult to span 8.8 Mb in 5637, almost certainly indicates LOH and all five explain how such events might confer a selective advantage on the FISH markers in the region were retained. We have previously tumor cell. An alternative explanation might be that these samples, found evidence for this mechanism on 15q, where small, apparently albeit morphologically homogeneous, contain more than one interstitial regions of LOH are found in bladder cancer (39). evolving subclone. The two tumors with allelic imbalance depicted Analysis of tumor samples must now be carried out to assess the in Fig. 2 were both grade 3 tumors and might be expected to show generality of this mechanism of LOH on 8p, but this preliminary a high level of genomic instability. Future studies can address this information indicates a much greater complexity in the nature of 8p issue by examining multiple small microdissected samples from breakage mechanisms than previously expected. Reported studies such tumors. to date indicate frequent breakage of 8p, commonly associated A major aim was to define breakpoints in 8p more precisely. with translocation and concomitant loss of large regions of 8p. Microsatellite analysis of a panel of bladder cancer cell lines Such observations are compatible with repair of DNA double- indicated frequent reduction to homozygosity in the region of strand breaks by the nonhomologous end-joining pathway of interest. Observed frequencies of heterozygosity for all markers were repair. However, the LOH detected here in the absence of copy significantly lower than expected. No cell lines showed heterozy- number changes would most probably be achieved via the gosity for D8S283, which lies within LOH region 1 found in tumors. alternative mechanism of double-strand break repair, homologous We predict from allele counts that most cell lines have allelic loss recombination. affecting the same region(s) of LOH identified in tumors. Two other The region of homozygosity at 8p21 identified in RT4 (including regions, one close to D8S339 and the other at D8S135, are also the markers D8S339, D8S137, and NEFL) and UMUC3, which is predicted to represent critical regions of allelic loss in these UCC cell overlapped by larger regions of homozygosity in other lines, has lines. Thus, as in tumor samples studied previously (2), there were been identified as a minimal region of LOH in several other cancer three distinct regions of predicted allelic loss: one in the region of types including oral and laryngeal (10), hepatocellular (40), and NEFL, one focused on D8S283, and one more proximal between breast (21). The region containing D8S259, D8S283, and D8S513 has D8S87 and D8S255. In J82, the only cell line for which paired normal also been implicated in colorectal (16), prostate (3, 6, 24), and DNA was available, LOH extended from D8S339 to 8pter. breast (37, 41) cancers. www.aacrjournals.org 73 Cancer Res 2005; 65: (1). January 1, 2005

Complex rearrangements and patterns of 8p LOH have been There is an aphidicolin-induced common fragile site on 8p11- described in other cancer types, but despite great efforts to q11 (50, 51), the location of which has not been mapped identify the target gene or genes, to date no good candidates precisely. If this is on 8p11, it is possible that some of the breaks have been identified. Many cancers have LOH involving 8p21- we have identified have occurred at this site. However, it is clear pter and most genes examined to date have mapped to this that many breaks in bladder tumors and cell lines occur in 8p12 part of the chromosome. These include TNFRSF10B (DR5), or 8p21 where no fragile sites are known. Whatever the LZTS1, DBC2, and DLC1. Of these, a promising candidate for an mechanism by which these breaks are generated, chromosomal 8p21-p22 tumor suppressor gene is DLC1, which although not mu- breakage alone is unlikely to account for the finding of 8p loss tated shows significant loss of expression in relevant tumors (42, 43). as a clonal event in specific subgroups of cancers. Selection for In 8p12-p11, less attention has focused on possible candidate loss or breakage in the region clearly indicates that a biological genes. In bladder cancer, a mutation screen did not identify advantage is conferred. It will be important to determine mutations in POLB and PPP2CB (44). Mutations of sFRP1,an whether fragile sites are involved in these events, as a propensity antagonist of Frizzled receptors, which lies centromeric to all to breakage at particular sites due to structural features provides probes and markers studied here, have been reported in colorectal no clear information on the likely location of the gene or genes cancer (45) but not bladder cancer (46). In breast cancer, whose loss provides a selective advantage. Indeed, involvement deletions, translocations, and complex rearrangements sometimes of an unidentified fragile site could lead to inappropriate including amplification proximal to the breakpoints in 8p have concentration of efforts on candidate genes in that region. been described (27, 47). In a recent study of 43 breast and In conclusion, our study has confirmed that LOH of 8p12-p11 is pancreatic tumor cell lines, in which all but two lines showed common in bladder cancer and we have identified several regions chromosome 8 imbalance by CGH and half had 8p loss, seven involved. The breakpoints identified here in UCC cell lines lie close breakpoints were found within the NRG1 gene (48). These to breakpoints identified previously in breast cell lines (27), but as breakpoints were scattered over a 1.1-Mb region that lies telomeric they do not target a single gene, it will be important to examine to the breakpoints described here (Fig. 1). NRG1 encodes a series breakpoints in a larger series of cell lines and tumors and to of ligands for the ERBB family of receptors, including the determine whether these affect specific genomic motifs. Most heregulins and NEU differentiation factors, generated by alterna- importantly, we have shown that two mechanisms may be involved tive splicing of 17 exons. In the study of Adelaide et al. (49), mRNA in the generation of genomic alterations on proximal 8p, possibly expression of a range of isoforms was examined in cell lines with involving aberrations in two distinct pathways of DNA double- chromosomal breaks within the gene. In none was expression strand repair. eliminated, nor was there any consistent pattern of isoform expression in cell lines with breakpoints, so that the relationship of this gene to 8p alterations is not clear. However, the known role Acknowledgments for overexpression of receptors for NRG1 encoded ligands in breast and bladder cancer and the existence of recurrent Received 7/11/2004; revised 8/31/2004; accepted 10/27/2004. Grant support: Cancer Research UK. breakpoints in the gene make this an attractive candidate for The costs of publication of this article were defrayed in part by the payment of page future assessment. Indeed, four of the potential interstitial regions charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of LOH identified here in cell lines include NRG1, which lies at the We thank Dr. Paul Edwards for provision of clones for use as FISH probes and for telomeric end of the region examined by FISH. helpful discussions and Carolyn Hurst for help with DNA preparations.

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