Array Comparative Genomic Hybridization Analysis of Colorectal Cancer Cell Lines and Primary Carcinomas

[CANCER RESEARCH 64, 4817–4825, July 15, 2004] Array Comparative Genomic Hybridization Analysis of Colorectal Cancer Cell Lines and Primary Carcinomas

Eleanor J. Douglas,1 Heike Fiegler,1 Andrew Rowan,2 Sarah Halford,2 David C. Bicknell,3 Walter Bodmer,3 Ian P. M. Tomlinson,2,4 and Nigel P. Carter1 1The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge; 2Molecular and Population Genetics Laboratory, Cancer Research UK, London; 3Cancer and Immunogenetics Laboratory, Cancer Research UK, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford; and 4Colorectal Cancer Unit, Cancer Research UK, St. Mark’s Hospital, Watford Road, Harrow, United Kingdom

ABSTRACT and SMAD4 mutations are more common in CINϩ CRCs (accompanied by loss of 17p13.1 and 18q21.1, respectively), whereas inacti- Array comparative genomic hybridization, with a genome-wide reso- vation of the BAX and TGFBIIR genes, by frameshift mutations in lution of ϳ1 Mb, has been used to investigate copy number changes in 48 tandem repeats, occurs in MSIϩ lesions (10). colorectal cancer (CRC) cell lines and 37 primary CRCs. The samples were divided for analysis according to the type of genomic instability that Genomic copy number changes are found frequently in cancers and they exhibit, microsatellite instability (MSI) or chromosomal instability are believed to contribute to their development and progression (CIN). Consistent copy number changes were identified, including gain of through inactivation of tumor suppressor genes, amplification of on- chromosomes 20, 13, and 8q and smaller regions of amplification such as cogenes, or more subtle gene dosage changes. Comparative genomic chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent find- hybridization (CGH; Ref. 11) was developed to allow genome-wide ing along with deletion of discrete regions such as chromosome 4q34-q35. screening for such copy number changes, and CGH investigations into The overall pattern of copy number change was strikingly similar between CRC have revealed consistent gains and losses (12–16). In particular, cell lines and primary cancers with a few obvious exceptions such as loss gain of chromosome 20q is a widespread finding in primary CRCs of chromosome 6 and gain of chromosomes 15 and 12p in the former. A (67%) as is loss of 18q (49%; Ref. 16). Other consistent regions of -greater number of aberrations were detected in CIN؉ than MSI؉ sam copy number gain are 7p, 8q, 13q, and 12p along with deletions of 8p ples as well as differences in the type and extent of change reported. For example, loss of chromosome 8p was a common event in CIN؉ cell lines and 4p. Overall, a smaller number of aberrations have been detected and cancers but was often found to be gained in MSI؉ cancers. In in diploid compared with aneuploid cancers (16). CGH has also been addition, the target of amplification on chromosome 8q appeared to differ, used to investigate any differences in chromosomal gains and losses -with 8q24.21 amplified frequently in CIN؉ samples but 8q24.3 amplifi- between MSIϩ and CINϩ cancers (17, 18). It was found that chro -cation a common finding in MSI؉ samples. A number of genes of interest mosomal imbalances were more frequent in CINϩ than MSIϩ can are located within the frequently aberrated regions, which are likely to be cers, and cell lines and differences in the particular chromosomes of importance in the development and progression of CRC. involved were also detected. Gains of 4q (15%) and 8q (8%) and losses of 9q (21%), 1p (18%), and 11q (18%) were the most common INTRODUCTION findings in MSIϩ cancers. However, gain of 8q (50%), 13q (35%), and 20q (25%) and loss of 18q (55%), 15q (35%), and 17p (30%) Colorectal cancer (CRC) is the second most common malignancy in were detected most frequently in CINϩ cancers (17). In addition, the Western world and is responsible for ϳ20,000 deaths in the United different types of aberration were detected depending on the particular Kingdom per annum. The lifetime risk in the general population is mismatch repair defect identified (18). ϳ5% (1). The majority of CRC cases are sporadic, but the autosomal, Conventional CGH has a limited resolution and can only detect dominantly inherited syndromes familial adenomatous polyposis and losses of ϳ10 Mb or greater (19, 20). High-level amplifications have hereditary nonpolyposis CRC account for up to 5% of cases. Thus far, a maximum resolution of 3 Mb (21). The resolution of CGH has been a handful of genes have been identified in which somatic mutations improved by replacing the metaphase chromosomes as the hybridiza- contribute to the pathogenesis of CRC, including APC, SMAD4, p53, tion target with mapped and sequenced clones (bacterial artificial KRAS, and ␤-catenin (2, 3). The earliest aberration detected in CRC chromosomes, P1-derived artificial chromosome, and cosmids) ar- is mutation of APC (found as germ line mutations in familial ade- rayed onto glass slides (22, 23). The resolution of this matrix (22) or nomatous polyposis patients), which generally appears to be necessary array (23) CGH is limited only by the insert size and density of the for cancer initiation (4). Mutant KRAS is found in ϳ50% of CRCs, mapped sequences used. The capability of the technique to provide mutant p53 in ϳ60%, and mutant SMAD4 in 10–20% (5, 6). CRCs high-resolution mapping of variation in copy number has been dem- can be categorized according to the type of genomic instability that onstrated in the analysis of breast tumors, where the fine structure of they are thought to exhibit, namely chromosomal instability (CIN), amplicons was resolved and potential candidate genes identified (24). characterized by an aneuploid/polyploid karyotype (6, 7) or microsat- In this study, we describe the use of array CGH with a resolution of ellite instability (MSI), characterized by defective mismatch repair ϳ1 Mb (25) to investigate copy number changes occurring in CRC by and a near-diploid karyotype (8, 9). Other CRCs are near-diploid and the analysis of 48 cell lines and 37 primary cancers. MSIϪ. Genetic pathways defined at the chromosomal level are mir- rored by genetic changes at the level of the gene. For example, p53 MATERIALS AND METHODS Received 2/2/04; revised 4/22/04; accepted 5/14/04. Grant support: This work was supported by the Wellcome Trust and Cancer Research Cell Lines and Primary Cancers. DNA was extracted from 48 CRC cell United Kingdom. lines derived from local collaborators or public sources. In almost all cases the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with MSI status and ploidy were already known, and several authors have reported 18 U.S.C. Section 1734 solely to indicate this fact. molecular changes, such as mutation of APC, KRAS, and p53, in some of the Note: Supplementary data for this article online at http://www.sanger.ac.uk/Teams/ cell lines (10, 26, 27). Fresh frozen samples of primary sporadic CRCs, derived Team70/supplemental-data/. from patients at St. Mark’s Hospital, were obtained. All of the cancers Requests for reprints: Nigel P. Carter, The Wellcome Trust Sanger Institute, Well- Ͼ come Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom. Phone: contained 70% neoplastic tissue on histological review. MSI analysis (28) 44-0-1223-494860; Fax: 44-0-1223-494919; E-mail: [email protected]. was undertaken, and ploidy was assessed using flow cytometry (data not 4817

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. ARRAY CGH ANALYSIS OF COLORECTAL CANCER shown). DNA was extracted from 7 MSIϩ and 30 CINϩ cancers using Data Analysis. The determination of significant copy number changes standard methods; no MSIϪCINϪ (or MSIϩCINϩ) cancers were included in detected in array CGH for cancer and cancer cell lines, where much of the the study because these are very rarely represented in the set of known CRC genome has been altered, is not straightforward. Measurement variation will cell lines. Cell line and cancer details can be downloaded from Supplementary vary from hybridization to hybridization, so it is important to set statistically Table 1 (Excel spreadsheet). Cell line DNAs were hybridized against a normal defined thresholds for copy number change by analysis of the variation for female lymphoblastoid cell line C0009-SAH (HRC160; European Collection each hybridization. We have approached this by identifying regions of modal of Cell Cultures no. 93010702) obtained from the European Collection of Cell copy number (normalized linear ratio of 1.0) in each hybridization, using an Cultures (Salisbury, United Kingdom). Primary cancers were hybridized arbitrary threshold determined from independent normal versus normal hy- against a sex-matched control DNA pool created from 20 normal blood DNAs bridizations and then using the variation of the defined modal region to set obtained from Human Random Collection of the European Collection of Cell thresholds specific to that hybridization. Specifically, the largest contiguous Cultures. Clones that had been shown previously to report copy number chromosomal region reporting a modal copy number was identified. A region changes in hybridizations of individual normal DNAs against the pool of was considered to be modal if 95% of the clones fell within 99% confidence normal DNAs (data not shown) were considered to be polymorphic in the intervals calculated from the linear ratios of autosomal clones in a normal male normal population and were excluded from analysis. versus normal female hybridization (data not shown). The size of the region DNA Labeling. Test and control DNAs were differentially labeled using a used varied from 100 Mb to 246 Mb. The SD of measurements in these modal Bioprime labeling kit (Invitrogen, Carlsbad, CA), as described previously (25), regions ranged from 0.03 to 0.14. The linear ratios reported by the clones with minor modification. Briefly, a 130.5-␮l reaction was set up containing within the defined modal region were used to calculate the coefficient of 450 ng of DNA and 60 ␮lof2.5ϫ random primer solution. The DNA was variation and 99% confidence intervals for each hybridization. Clones that fell denatured for 10 min at 100°C, and 1.5 ␮l of 1 mM Cy5-dCTP or Cy3-dCTP outside the 99% confidence intervals were identified as reporting significant (NEN Life Science Products, Boston, MA) and 3 ␮l of Klenow fragment were copy number changes. We have shown previously that our arrays demonstrate added on ice to a final reaction volume of 150 ␮l. The reaction was incubated close to theoretical copy number changes in response to autosomal single copy overnight at 37°C and stopped by adding 15 ␮l of stop buffer. Unincorporated gains and losses (25). For the cell lines, gains were considered to be single nucleotides were removed using G50 spin columns (Pharmacia Biotech, Pis- copy or greater if the reported ratio was greater or equal to the theoretical value cataway, NJ) according to the manufacturer’s instructions. for a single copy gain (1.5) minus the 99% confidence interval at this value for Microarray Hybridization and Image Analysis. Hybridization to the each hybridization and double copy number or greater if the reported ratio was array was carried out as described previously (25). Briefly, test and control- greater or equal to the theoretical value for a double copy amplification (2.0) labeled DNAs were combined, precipitated together with 135 ␮l of human minus the 99% confidence interval at this value for each hybridization. Cot1 DNA (Roche, Mannheim, Germany), and resuspended in 60 ␮lof Deletions were considered to be single copy or greater if the reported ratio was hybridization buffer [50% formamide, 10% dextran sulfate, 0.1% Tween 20, greater or equal to the theoretical value for a single copy deletion (0.5) plus the 2ϫ SSC, 10 mM Tris-HCl (ph7.4)], and 6 ␮l of yeast tRNA (100 ␮g/␮l; 99% confidence interval at this value for each hybridization. In no hybridiza- Invitrogen, Carlsberg, CA). This hybridization solution was incubated for 1 h tion did these defined thresholds overlap the 99% confidence intervals for at 37°C before application to the array. A prehybridization solution of 80 ␮lof modal clones. The theoretical values for single and double copy number herring sperm DNA (10 mg/ml; Sigma-Aldrich Company Ltd., Dorset, United changes were calculated relative to a diploid karyotype; therefore, classifica- Kingdom) and 135 ␮l of human Cot1 DNA (Roche), precipitated together and tion of copy number changes, which occurred in samples with an increased resuspended in 160 ␮l of hybridization buffer, was applied to the array and ploidy, would be underestimates of the true level of copy number gain or loss. incubated at 37°C for 1 h. The arrays were scanned on an Axon 4000B scanner For the primary cancers, the observed changes were not classified as single or (Axon Instruments, Burlingame, CA), and images were quantified using the double copy number changes because the cancer samples are inevitably con- software program “Spot” (29). The fluorescence intensity ratio for each clone taminated with DNA from surrounding normal tissue. Contaminating normal (after local background subtraction) was calculated and normalized by dividing tissue results in suppression of the reported ratios in regions of amplification each ratio by the median ratio of all of the autosomal clones and plotted against or deletion. The exact amount of contamination for each sample could not be the mapped genomic position (NCBI31; freeze date, November 2002). readily determined; hence, precise thresholds could not be set. Copy number

Fig. 1. A, genome-wide frequency of copy number changes for 48 colorectal cancer cell lines. The total frequency of all significant gains reported by each clone are shown in blue, and all losses are shown in pink. Gains and deletions of single copy or greater within the total are shown in black, and amplifications of double copy or greater are shown in yellow. Clones are ordered according to their mapped positions along the chromosomes. B, genome-wide frequency of copy number changes for 37 primary colorectal cancers. The frequency of significant gains reported by each clone are shown in blue, significant losses are shown in pink.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. ARRAY CGH ANALYSIS OF COLORECTAL CANCER change of the sex chromosomes was not analyzed because the cell lines were chromosome 20 gained most frequently was 20q13.3 (RP4-563E14 at all hybridized against female control DNA. To identify trends in copy number 62.3Mb) in 65% of cell lines and 81% of cancers. Gain of chromo- gain, the “mean percentage of gain by clones” (MPG) for each particular some 13 was also a common finding in both cell lines (MPG ϭ 43%) chromosome or region of interest was used. The MPG was determined by and cancers (MPG ϭ 46%), along with gain of chromosome 7 calculating the percentage of samples with a significant copy number gain for (MPG ϭ 22% of cell lines; 46% of cancers) and the long arm of each clone and calculating the mean percentage gain for all of the clones in the ϭ chromosome or region of interest. An equivalent calculation, “mean percent- chromosome 8 (MPG 31% of cell lines; 27% of cancers). The most age of loss by clones” (MPL) was applied for copy number losses. The MPL common region of copy number loss was the long arm of chromosome was determined by calculating the percentage of samples with a significant 18 (MPL ϭ 52% of cell lines; 32% of cancers). In particular, three copy number loss for each clone and calculating the mean percentage loss for clones detected the most frequent copy number loss: two at 18q21.1 all of the clones in the chromosome or region of interest. The genomic (RP11-313C14 at 43.9Mb in 60% of cell lines and cancers and locations of all of the clones named specifically in the text were confirmed by RP11-25O3 at 49.7Mb in 60% of cell lines and 49% of cancers) and bacterial artificial chromosome end sequencing and fluorescence in situ hy- one at 18q12.2 (RP11-19F9 at 35Mb in 56% of cell lines and 59% of bridization. Raw, normalized log ratios for all of the cell lines and cancers can 2 cancers). In addition, chromosome 8p was also a consistent region of be downloaded from Supplementary Table 1 (Excel spreadsheet). ϭ Metaphase Fluorescence in Situ Hybridization. Degenerate oligonucleo- copy number loss (MPL 33% of cell lines; 29% of cancers). tide primed amplified clone DNA (amplified using primers degenerate oligo- The overall pattern of copy number change was very similar in the nucleotide primed 1, 2, and 3) created for array construction (25) was labeled cell lines and cancers (Fig. 1). However, loss of chromosome 6 was a with biotin-16-dUTP (Roche) or digoxigenin-11-dUTP (Roche) by nick trans- significantly more common event (P Ͻ 0.005, ␹2 test) in the cell lines lation. Hybridizations were carried out using conventional methods to chro- (MPL ϭ 21%) than the cancers (MPL ϭ 1%). In addition, gain of mosomes prepared from a normal male lymphoblastoid cell line (HRC575; chromosome 15 occurred more frequently in the cell lines European Collection of Cell Cultures no. 94060845). Biotin-labeled probes (MPG ϭ 21%) than the cancers (MPG ϭ 1%; P Ͻ 0.005, ␹2 test). were detected using Avidin TexasRed (Molecular Probes Inc., Eugene, OR), Furthermore, gain of chromosome 12, in particular, the short arm, was and digoxigenin-labeled probes were detected with a combination of mouse a common finding in the cell lines (MPG ϭ 32%) but not the cancers antidigoxigenin (Vector Laboratories Ltd., Peterborough, United Kingdom) ϭ Ͻ ␹2 and goat antimouse-FITC (Sigma-Aldrich Company Ltd.) antibodies. (MPG 3%; P 0.005, test). Although chromosome 12p was not generally gained in the cancers, one particular clone, RP11-59H1 at 12.9 Mb, was gained in 22% of cancers and 31% of cell lines. The RESULTS most frequently gained clone in the cell lines (38%) was RP11-4N23 The most common finding in both cell lines and cancers was gain at 13.6 Mb. The total number of clones reporting significant gains and of chromosome 20. The MPG was 49% for cell lines and 50% for losses was calculated, and the median number per sample was com- cancers (for definition, see “Materials and Methods”). The region of pared between the cell lines and the cancers. The cell lines showed a

Table 1 Clones reporting amplifications (double copy number or greater) and deletions (single copy or greater) in Ն2 cell lines Amplifications Deletions

Chromosome Clone Position in Mb No. of cell lines Chromosome Clone Position in Mb No. of cell lines 13 RP11-44J9 26.2 9 18 RP11-313C14 44.0 5 8 RP11-28I2 127.3 7 18 RP11-25O3 50.0 5 8 RP1-80K22 128.4 7 4 RP11-226A18 182.4 4 13 RP11-10M8 37.2 7 18 7 clones 28–56 4 13 RP11-235O20 96.3 7 18 3 clones 73–77 4 8 RP11-269I24 131.3 6 4 RP11-495H13 18.0 3 13 5 clones 34–109 6 8 19 clones 3–35 3 8 10 clones 123–140 5 16 RP11-167B4 6.3 3 13 9 clones 22–107 5 18 17 clones 39–76 3 6 RP11-524H19 54.7 4 20 RP5-855L24 14.6 3 7 RP11-99O17 25.6 4 1 RP11-241C9 213.9 2 8 9 clones 117–130 4 3 RP11-425D6 81.4 2 13 24 clones 25–105 4 3 RP11-81P15 86.9 2 20 4 clones 52–64 4 4 RP11-84H6 179.8 2 7 RP11-550A18 29.3 3 4 RP11-451F20 184.8 2 8 10 clones 58–135 3 8 17 clones 0–40 2 10 RP11-382A18 18.4 3 9 RP11-109M15 16.2 2 13 23 clones 20–114 3 9 RP11-15P13 20.3 2 17 RP5-986F12 35.8 3 13 RP11-95G6 27.9 2 20 9 clones 41–63 3 13 RP11-10M8 37.2 2 6 RP1-283K11 2.3 2 13 RP11-247M1 45.8 2 6 6 clones 38–43 2 13 RP11-524F1 58.7 2 6 4 clones 51–54 2 16 RP11-556H2 78.9 2 6 3 clones 65–68 2 17 RP11-104O19 4.1 2 6 5 clones 135–138 2 17 RP5-1050D4 4.8 2 7 7 clones 1–82 2 17 RP11-243K12 6.1 2 8 32 clones 58–144 2 18 7 clones 24–43 2 11 RP11-93M11 73.0 2 18 4 clones 60–72 2 11 RP11-569A20 106.7 2 12 RP11-283I3 0.3 2 12 3 clones 3–42 12 4 clones 10–16 2 12 4 clones 24–27 2 13 25 clones 47–112 2 16 RP11-283C7 46.8 2 16 3 clones 81–82 2 17 4 clones 33–37 2 20 9 clones 20–61 2 4819

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. ARRAY CGH ANALYSIS OF COLORECTAL CANCER median number of aberrant clones of 975 (range ϭ 598-2556), and the (MPL ϭ 38%) as shown in Fig. 2, C and D. The most frequently cancers showed a median number of aberrant clones of 536 deleted clone defining the peak of the copy number change in the cell (range ϭ 48–1152). This difference is statistically significant lines (8%) was RP11-226A18 at 182.4 Mb. Examples of 4q34-q35 (P Ͻ 0.005, Mann-Whitney U test). loss are shown in Fig. 2, E and F. The frequency with which each clone on the array reported a The genome-wide pattern of copy number changes in the MSIϩ high-level amplification (double copy number or greater) in the cell and CINϩ cell lines and cancers was compared (Supplementary Fig. lines was calculated and is displayed in Fig. 1 as yellow bars. Clones 1). CINϩ samples reported significantly more aberrations than MSIϩ reporting amplifications in Ն2 cell lines are listed in Table 1. The samples (cell lines P Ͻ 0.005; cancers P Ͻ 0.05, Mann-Whitney U most common region of amplification of chromosome 20 was again at test). Gain of chromosome 20 was the most common overall finding, 62.3 Mb (reported by clone RP4-563E14; amplified in 4 cell lines). but when the analysis was split by the microsatellite status of the The most common region of amplification of chromosome 13 was samples, a marked difference emerged. Gain of chromosome 20 13q12.13-q12.2 (RP11-44J9 at 26.2 Mb; amplified in 9 cell lines), and occurred more often in CINϩ cell lines (MPG ϭ 51%) and CINϩ the most frequently amplified region of chromosome 8 was 8q24.21 cancers (MPG ϭ 59%) than in MSIϩ cell lines (MPG ϭ 6%) and (126-128 Mb; reported in 7 cell lines). The most frequently amplified MSIϩ cancers (MPG ϭ 12%; P Ͻ 0.005 for both cell lines and region of chromosome 17 was 35.8 Mb (RP5-986F12), and the most cancers, ␹2 test). However, clone RP11-563E14 was gained in 14% of frequent amplification of 7p was 29.3 Mb (RP11-550A18). The fre- MSIϩ cell lines and 43% of MSIϩ cancers and gained (single copy quency with which each clone reported a deletion (single copy or or greater) in 7% of MSIϩ cell lines. Loss of 18q was also a more greater) in the cell lines is displayed as black bars in Fig. 1. Clones frequent event in CINϩ cell lines (MPL ϭ 71%) and CINϩ cancers reporting deletions in Ն2 cell lines are listed in Table 1. Consistent (MPL ϭ 42%) than MSIϩ cell lines (MPL ϭ 12%) and MSIϩ regions of deletion included chromosome 4q34.3 (RP11-226A18; cancers (MPL ϭ 1%). However, loss of chromosome 18q21.1-21.2 deleted in 4 cell lines) and, again, chromosome 18q21.1. (44–47 Mb, including clones RP11-313C14 and RP11-46D1) was Small regions of consistent copy number change were also ob- detected in MSIϩ cell lines (MPL ϭ 21%) and MSIϩ cancers served (Tables 2 and 3). For example, chromosome 17q11.2-q12 (MPL ϭ 29%). Loss of the short arm of chromosome 17 occurred (33–38 Mb) was a consistent region of gain in both cell lines more frequently in CINϩ cell lines (MPL ϭ 38%) and cancers (MPG ϭ 21%) and cancers (MPG ϭ 22%). RP5-986F12 at 35.8 Mb (MPL ϭ 29%) than MSIϩ cell lines (MPL ϭ 3%) and cancers was the most frequently amplified clone (double copy number or (MPL ϭ 1%). In addition, loss of chromosome 8p was a frequent greater) occurring in 6% of cell lines. Gain of this clone occurred as event in CINϩ cell lines (MPL ϭ 47%) and cancers (MPL ϭ 37%) part of both broad and narrow regions of copy number change (Figs. but occurred much less frequently in MSIϩ cell lines (MPL ϭ 3.4%). 2, A and B). In addition, chromosome 4q34-q35 was a common region Loss of 8p was never found in MSIϩ cancers but was frequently of copy number loss in cell lines (MPL ϭ 35%) and cancers gained (MPG ϭ 28%; Fig. 3). The overall peak of copy number gain

Table 2 Small regions of copy number gain Region Cell lines Cancers Peak clone (highest % gain)

Gainc Amplificationd Start End Gainb (single copy) (double copy) Gainb Position Chr (Mb)a (Mb)a (%) (%) (%) (%) Name (Mb) Band Candidate genes 1 2.9 2.9 2 2 14 RP4-785P20 2.9 1p36.32-p36.33 SKI, MEL1, PRKCZ, DVL1, CENTB5 1 113 118 15 6 2 5 RP4-787H6 115.9 1p13.1 VANGL1 2 102.3 111.8 31 10 2 3 RP11-519H15 107.2 2q12.3 2 126 130 17 6 4 11 RP11-207G14 130 2q21.1 MEKK3, RAB6C 3 125 125 13 2 – 30 RP11-25C10 125 3p25.2 RAF-1, FBLN1 3 172.3 175.3 40 10 – 32 RP11-163H6 173 3q26.31 PLD1, ECT2 5 147 152.6 17 4 – 11 CTB-137O22 150 5q33.1 G3BP 5 72.9 81.3 12.5 4 2 – RP11-30D15 79.2 5q14.1 RASGRF2 6 37.8 68.1 19 12.5 8 24 RP11-524H19 54.7 6p12.1 6 134.8 137.9 19 10 4 16 RP1-32B1 135.4 6q23.3 MYB 7 0.7 0.7 52 10 2 57 RP11-449P15 0.7 7p22.3 CENTA1, MAFK 8 117 128.4 50 31 15 40 RP1-80K22 128.4 8q24.21 C-MYC 8 133.8 133.8 48 27 10 30 RP6-98A24 133.8 8q24.22 WISP2 8 144.3 145.3 48 25 4 51 RP11-472K18 144.2 8q24.3 RIG-E, GLI4, RHPN1 9 83.2 85.1 23 2 – 11 RP11-423O13 83.3 9q21.33 9 95.3 98 29 2 – 19 RP11-80H12 97.2 9q31.1 10 76 80.8 23 4 – 11 RP11-90J7 80 10q22.3 DLG5 11 67.9 74.3 32 8 4 16 RP11-804L21 (cancers) 69.9 11q13.3 CCND1, FGF3, FGF4, FGF19, ORAOV1, MYEOV, EMS1 RP11-93M11 (cell lines) 73 11q13.4 CENTD2, P2RY2, P2RY6, RAB6A 12 12.9 15.6 38 23 4 22 RP11-59H1/RP11-4N23 12.9/13.6 12p13.1-p13.2 MGP 12 67.6 73.6 19 4 4 5 RP11-101K2 71.2 12q15 TM4SF3, PTPRR, PTPRB, GRP49, RAB21 12 116 120.5 23 6 2 8 RP11-8A1 116 12q24.21 16 0.2 2.2 40 8 – 49 RP11-243K18 0.3 16p13.3 MPF 16 64.2 82.4 21 13 4 24 RP11-303E16 80.9 16q23.2 MAF 17 80.7 81.2 48 13 – 43 RP11-567O16 81.2 17q25.3 RAB40B 17 33.2 37.8 21 10 6 22 RP5-986F12 35.8 17q12 AATF, TBC1D3 17 42.1 49.3 17 4 2 24 RP11-361K8 47 17q21.32 GIP 22 39.3 39.4 6 3 – 27 RP3-355C18 39.4 22q13.2 MKL1 a Start and end positions (Mb) are the midpoints of the most extreme clones in the region. b Clones with ratios that fell outside of 99% confidence intervals of modal values were identified as reporting significant copy number changes. c Gains were considered to be single copy or greater if the reported ratio was greater or equal to the theoretical value for a single copy gain (log2 ϭ 0.58) minus the 99% confidence interval for each hybridization. d Amplifications were considered to be double copy or greater if the reported ratio was greater or equal to the theoretical value for a double copy amplification (log2 ϭ 1) minus the 99% confidence interval for each hybridization. 4820

Table 3 Small regions of copy number loss Region Cell lines Cancers Peak clone (highest % loss)

Start End Lossb Deletionc Lossb Position Chr (Mb)a (Mb)a (%) (%) (%) Name (Mb) Band Candidate genes 1 19.7 27.2 30 2 13 RP11-509F14 23.6 1p36.11 E2F2, ID3 4 45.8 47.9 30 2 11 RP11-100N21 47.5 4p12 4 57.7 59.9 33 2 19 RP11-355L4 57.7 4q12 IGFBP7 4 107 112.6 33 2 19 RP11-75N20 110 4q25 4 179.7 186.3 35 8 38 RP11-226A18 182.4 4q34.3 CASP3 6 112.4 114.5 27 2 8 RP1-142L7 112.4 6q21 TUBE1 9 9.7 27.3 23 4 11 RP11-109M15/RP11-15P13 16.2/20.3 9p21-p22 CDKN2A/B, MLLT3, MTAP, NSGX 10 88.2 89.9 23 2 11 RP11-765C10 89.9 10q23.31 PTEN 10 126.7 132.6 17 2 22 RP11-16P8 128.3 10q26.2 BCCIP, DDX32 11 62.7 64.1 21 – 30 RP11-424O11 64.1 11q13.1 BAD, HRASLS2, HRASL3, LGAS12, RARRES3, FLRT1 16 5.6 10.6 19 6 14 RP11-167B4 6.3 16p13.3 17 4.1 6.1 31 4 51 RP11-243K12 6.1 17p13.2 DHX33, NAL1 17 9.5 12 38 2 32 RP11-471L13 12 17p12 ELAC2 20 12.6 15.6 35 6 3 RP5-855L24 14.6 20p12.1 FLRT3 a Start and end positions (Mb) are the midpoints of the most extreme clones in the region. b Clones with ratios that fell outside of 99% confidence intervals of modal values were identified as reporting significant copy number changes. c Losses were considered to be single copy or greater if the reported ratio was greater or equal to the theoretical value for a single copy deletion (log2 ϭϪ1) plus the 99% confidence interval for each hybridization. on chromosome 8q was band 8q24.21 from 126–128Mb. However, in CINϩ cell lines and 1 MSIϩ cell line and clones reporting deletions MSIϩ cancers a different region 8q24.3 (144–145Mb) was the most (single copy or greater) in Ն2 cell lines (CINϩ and MSIϩ) are listed frequently gained (RP11-472K18 at 144.3Mb in 36% of MSIϩ cell in Table 4. There were no clones that were amplified in Ͼ1 MSIϩ cell lines and 57% of MSIϩ cancers and RP11-349C2 at 145.3 Mb in 36% line. Only one clone reported a deletion in Ͼ1 MSIϩ cell line. This of MSIϩ cell lines and 71% of MSIϩ cancers; see Fig. 3). clone, RP11-241C9, is mapped to chromosome 1q41 at 214Mb. The frequency with which the clones reported amplifications (dou- Copy number losses reported by clones representing three genes, ble copy number or greater) and deletions (single copy or greater) in APC, p53, and SMAD4, were compared with previously published the CINϩ and MSIϩ cell lines is displayed in Supplementary Fig. 1 loss of heterozygosity (LOH) data in the cell lines (10, 27, 30). In the as yellow and black bars. Clones reporting amplifications in Ն2 case of APC, it was found that LOH occurs most often in cell lines

Fig. 2. A and B, two examples of copy number gain of chromosome 17q11.2-q12 in cell line Colo 678 (A) and a primary cancer (B). RP5-986F12 (35.8 Mb) is indicated with an arrow. C and D, chromosome 4 (177-188 Mb). Frequency of copy number losses (gray), gains (open), and deletions (black) in the cell lines (C) and primary cancers (D). RP11-226A18 (182.4 Mb) is indicated with an arrow. E and F, two examples of copy number loss of chromosome 4q34-q35 in cell line SW1417 (E) and a primary cancer (F).

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Fig. 3. Frequency of copy number gains and losses reported by the chromosome 8 clones in MSIϩ cell lines (A), CINϩ cell lines (B), MSIϩ cancers (C), and CINϩ cancers (D). Expanded region of chromosome 8 (97–146Mb) showing frequency of copy number gains (blue), gains of single copy or greater (black), amplifications of double copy number or greater (yellow)inMSIϩ cell lines (E), and frequency of copy number gains (blue)in MSIϩ cancers (F). The 8q24.21 is underlined in red, and 8q24.3 is underlined in green.

with a normal copy number at this locus (17 of 22 samples), probably involved in calcium-mediated signal transduction and cellular prolif- by mitotic recombination, as suggested previously (30). Conversely, eration (35); and (d) EEF1A2, a transcription elongation factor found LOH at SMAD4 is generally associated with copy number loss (25 of to be amplified and overexpressed in ovarian cancer (36). Gain of 26 samples), and in cases where LOH was not detected, copy number chromosome 13 was also a common finding, and the most frequently is normal (11 of 12 cases). LOH at p53 is not associated with copy amplified region was 13q12.13-q12.2 from 26–28 Mb. Genes of number change, suggesting a mechanism of mitotic recombination or interest that map to this region include FLT3, a tyrosine kinase gene conversion. receptor in which activating mutations have been found in acute myeloid leukemia (37) and FLT1, a vascular endothelial growth factor DISCUSSION receptor found to be expressed in gastric and breast carcinoma cells (38, 39). The most frequent region of copy number loss was the long A genomic microarray with a resolution of ϳ1 Mb was used to arm of chromosome 18, particularly 18q21.1, including the known investigate chromosomal imbalances in a set of 48 CRC cell lines and tumor suppressor genes SMAD2 (40) and SMAD4 (41), which func- 37 primary CRCs. The collection included both MSIϩ and CINϩ tion in the transforming growth factor signaling pathway to mediate samples. Analysis of the array hybridizations revealed consistent growth inhibition. Allelic loss at 18q21.1 has been detected in up to regions of copy number change. Many of the findings were in agree- 60% of CRCs (42), and mutation of SMAD4 has been identified in ment with those observed previously in conventional CGH investiga- 50–60% of CRCs with loss of 18q21 (10). tions (12–16), for example, gain of chromosomes 20, 13, and 8q and loss of chromosomes 18q and 8p. However, the increased resolution In addition to large-scale rearrangements, smaller regions of copy of array CGH allowed the identification of the most frequently altered number change were also detected. For example, amplification of regions within these large scale gains and losses and also the detection 17q11.2-q12 (33–38 Mb) was a common finding, and the peak of this of previously unreported changes, for example, amplification of amplification was reported by clone RP5-986F12 at 35.8 Mb. Genes 17q11.2-q12 and deletion of chromosome 1q41. The most common of interest that are located in this region include AATF, which func- region of gain was chromosome 20, especially 20q13.3 (RP4-563E14 tions as an antagonist to apoptosis (43), and TBC1D3, a Rab GTPase at 62.3Mb). Candidate genes that map to this region include the amplified in 15% of prostate cancers (44). Consistent regions of following: (a) LIVIN, an inhibitor of apoptosis that has been associ- deletion (single copy or greater) were also identified, for example, ated with the progression of bladder cancer (31) and detected at high RP11-226A18 at 4q34–35 (182.4 Mb). The proapoptotic gene levels in a CRC cell line (32); (b) PTK6, a protein kinase, which when caspase-3, which is down-regulated in gastric cancer (45), maps to overexpressed in mammary cells leads to sensitization to epidermal this region. In addition, RP11-241C9 at 1q41 (213.9 Mb) was the only growth factor and partial transformation (33) and also shows in- clone reporting a deletion in Ͼ1 MSIϩ cell line. Transforming growth creased expression in primary colon tumors (34); (c) HD54, which is factor ␤2 is located in this deleted region. 4822

Table 4 Clones reporting amplifications (double copy or greater) in Ն2 CINϩa cell lines and 1 MSIϩ cell line and clones reporting deletions (single copy or greater) in Ն2 cell lines (CINϩ and MSIϩ) Amplifications Deletions

CINϩ cell lines MSIϩ cell lines CINϩ cell lines MSIϩ cell lines

Position No. of Position No. of Position No. of Position No. of Chr Clone in Mb cell lines Chr Clone in Mb cell lines Chr Clone in Mb cell lines Chr Clone in Mb cell lines 13 RP11-44J9 26.2 9 3 4 clones 60–65 1 18 RP11-313C14 44.0 5 1 RP11-241C9 213.9 2 13 RP11-570F6 26.0 7 3 5 clones 76–87 1 18 RP11-25O3 50.0 5 13 RP11-10M8 37.2 7 6 18 clones 2–24 1 4 RP11-226A18 182.4 4 13 RP11-235O20 96.3 7 7 RP11-502P9 11.6 1 18 7 clones 28–56 4 8 RP11-28I2 127.3 6 7 6 clones 26–36 1 18 3 clones 73–77 4 8 RP1-80K22 128.4 6 7 2 clones 82–84 1 4 RP11-495H13 18.0 3 13 RP11-266E6 34.1 6 7 RP11-126C19 117.8 1 8 19 clones 3–35 3 13 RP11-125A7 41.2 6 7 RP11-298A10 142.6 1 18 17 clones 39–76 3 13 RP11-384G23 52.9 6 8 RP11-174I12 124.2 1 3 RP11-425D6 81.4 2 13 RP11-184L18 70.4 6 8 RP11-269I24 131.3 1 3 RP11-81P15 86.9 2 13 RP11-40E6 109.3 6 8 RP11-472K18 144.3 1 4 RP11-84H6 179.8 2 8 9 clones 123–140 5 9 RP11-295G24 129.1 1 4 RP11-451F20 184.8 2 13 9 clones 22–106 5 12 RP11-101K2 71.2 1 8 17 clones 0–40 2 8 11 clones 117–130 4 15 RP11-86K22 55.8 1 9 RP11-109M15 16.2 2 13 24 clones 25–105 4 15 RP11-41P8 92.2 1 9 RP11-15P13 20.3 2 20 RP5-1162C3 53.5 4 16 RP11-167B4 6.3 2 6 RP11-524H19 54.7 3 16 RP11-556H2 78.9 2 7 RP11-99O17 25.6 3 17 RP11-104O19 4.1 2 8 9 clones 58–134 3 17 RP5-1050D4 4.8 2 13 22 clones 20–114 3 17 RP11-243K12 6.1 2 17 RP5-986F12 35.8 3 18 7 clones 24–43 2 20 6 clones 41–64 3 18 4 clones 60–72 2 6 RP1-283K11 2.3 2 20 RP5-855L24 14.6 2 6 13 clones 38–68 2 7 RP11-510K8 1.5 2 7 RP11-550A18 29.3 2 8 32 clones 57–141 2 10 RP11-382A18 18.4 2 11 RP11-93M11 73.0 2 11 RP11-569A20 106.7 2 12 13 clones 0–27 2 13 26 clones 47–112 2 16 RP11-283C7 46.8 2 16 4 clones 80–81 2 17 4 clones 33–37 2 20 11 clones 20–63 2 a CIN, chromosomal instability; MSI, microsatellite instability.

Within the limitations of our analysis, imposed by both the un- cancers are more likely to be causative in the initiation and progres- known ploidy of the cancer samples and the variable degree of sion of the cancer rather than a consequence of a generally unstable contamination by normal cells, the overall pattern of copy number karyotype and likely to contain target genes of the rearrangement. For change in the cell lines and cancers was markedly similar; however, example, gain of the whole of chromosome 20 was a common finding some differences were detected. The number of aberrations (all of the in CINϩ cancers but a much less frequent event in the MSIϩ gains and losses) was significantly greater in the cell lines than the samples; yet, gain of a particular clone, RP11-563E14 at 62.3 Mb, was cancers, and loss of chromosome 6 and gain of chromosome 15 were frequently detected in the MSIϩ cancers and cell lines, again sug- far more common events in the cell lines. It has been observed gesting that this region could be an important target of the gain of previously that loss of chromosome 6, especially 6q, occurs more chromosome 20. Dividing the analysis by microsatellite stability frequently in cell lines than primary tumors (26). The additional status has also been informative in focusing the attention on a partic- changes seen in the cell lines could have arisen if they were advan- ular region of chromosome 18q. Although loss of the entire long arm tageous to cells growing in cell culture conditions and may not always of chromosome 18 is not a common event in MSIϩ samples, loss of be relevant to cancer formation. Gain of the short arm of chromosome 18q21.1–21.2 (44–47 Mb) is a frequent occurrence, suggesting that 12 was another more frequent occurrence in the cell lines than the this area is likely to contain candidate genes that may be the target of cancers. However, one clone, RP11-59H1 at 12.9 Mb, was gained in the aberration. The pattern of copy number changes reported by the the cancers, perhaps suggesting that this region of 12p, in particular, chromosome 8 clones differs distinctly between the MSIϩ and CINϩ could be the target of the general increase in copy number seen in the samples. Loss of 8p is a common event in CINϩ cell lines and cell lines. Genes of interest that map to this region include MGP, a cancers; however, this is not the case in the MSIϩ samples. Not only matrix GLA protein that has been shown previously to be overex- is loss of 8p not frequently detected, but in the MSIϩ cancers, 8p is pressed in breast cancer (46). actually a common region of copy number gain, suggesting that loss The analysis was split by the type of genomic instability that the of 8p is an important event, specifically in the development of CINϩ samples exhibited, chromosomal (CINϩ) or microsatellite (MSIϩ) CRCs. Furthermore, gain of the long arm of chromosome 8 was a instability. Overall, MSIϩ samples had significantly fewer aberra- common finding in all of the samples. However, although gain of tions. This finding follows expectation; MSIϩ cancers tend to main- 8q24.21 was the most prominent finding in the CINϩ samples, a tain a diploid karyotype, whereas aneuploidy is a common feature of different region, 8q24.3, stands out in the MSIϩ samples, and RP11- CINϩ cancers (8, 9) and agrees with findings reported previously (17, 472K18 and RP11-349C2 are the most frequently gained clones. 18). The copy number changes that were detected in the MSIϩ Candidate genes that map to this region include RIG-E, a receptor that 4823

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Eleanor J. Douglas, Heike Fiegler, Andrew Rowan, et al.

Cancer Res 2004;64:4817-4825.

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