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 CellLines 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 ResearchUK, St. Mark’s Hospital, Watford Road, Harrow, United Kingdom

ABSTRACT

Array comparative genomic hybridization, with a genome-wide reso-lution of �1 Mb, has been used to investigate copy number changes in 48colorectal cancer (CRC) cell lines and 37 primary CRCs. The sampleswere divided for analysis according to the type of genomic instability thatthey exhibit, microsatellite instability (MSI) or chromosomal instability(CIN). Consistent copy number changes were identified, including gain ofchromosomes 20, 13, and 8q and smaller regions of amplification such aschromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent find-ing along with deletion of discrete regions such as chromosome 4q34-q35.The overall pattern of copy number change was strikingly similar betweencell lines and primary cancers with a few obvious exceptions such as lossof chromosome 6 and gain of chromosomes 15 and 12p in the former. Agreater number of aberrations were detected in CIN� than MSI� sam-ples as well as differences in the type and extent of change reported. Forexample, loss of chromosome 8p was a common event in CIN� cell linesand cancers but was often found to be gained in MSI� cancers. Inaddition, the target of amplification on chromosome 8q appeared to differ,with 8q24.21 amplified frequently in CIN� samples but 8q24.3 amplifi-cation a common finding in MSI� samples. A number of genes of interestare located within the frequently aberrated regions, which are likely to beof importance in the development and progression of CRC.

INTRODUCTION

Colorectal cancer (CRC) is the second most common malignancy inthe Western world and is responsible for �20,000 deaths in the UnitedKingdom per annum. The lifetime risk in the general population is�5% (1). The majority of CRC cases are sporadic, but the autosomal,dominantly inherited syndromes familial adenomatous polyposis andhereditary nonpolyposis CRC account for up to 5% of cases. Thus far,a handful of genes have been identified in which somatic mutationscontribute to the pathogenesis of CRC, including APC, SMAD4, p53,KRAS, and �-catenin (2, 3). The earliest aberration detected in CRCis mutation of APC (found as germ line mutations in familial ade-nomatous polyposis patients), which generally appears to be necessaryfor cancer initiation (4). Mutant KRAS is found in �50% of CRCs,mutant p53 in �60%, and mutant SMAD4 in 10–20% (5, 6). CRCscan be categorized according to the type of genomic instability thatthey are thought to exhibit, namely chromosomal instability (CIN),characterized by an aneuploid/polyploid karyotype (6, 7) or microsat-ellite instability (MSI), characterized by defective mismatch repairand a near-diploid karyotype (8, 9). Other CRCs are near-diploid andMSI�. Genetic pathways defined at the chromosomal level are mir-rored by genetic changes at the level of the gene. For example, p53

and SMAD4 mutations are more common in CIN� CRCs (accompa-nied by loss of 17p13.1 and 18q21.1, respectively), whereas inacti-vation of the BAX and TGFBIIR genes, by frameshift mutations intandem repeats, occurs in MSI� lesions (10).

Genomic copy number changes are found frequently in cancers andare believed to contribute to their development and progressionthrough inactivation of tumor suppressor genes, amplification of on-cogenes, or more subtle gene dosage changes. Comparative genomichybridization (CGH; Ref. 11) was developed to allow genome-widescreening for such copy number changes, and CGH investigations intoCRC have revealed consistent gains and losses (12–16). In particular,gain of chromosome 20q is a widespread finding in primary CRCs(67%) as is loss of 18q (49%; Ref. 16). Other consistent regions ofcopy number gain are 7p, 8q, 13q, and 12p along with deletions of 8pand 4p. Overall, a smaller number of aberrations have been detectedin diploid compared with aneuploid cancers (16). CGH has also beenused to investigate any differences in chromosomal gains and lossesbetween MSI� and CIN� cancers (17, 18). It was found that chro-mosomal imbalances were more frequent in CIN� than MSI� can-cers, and cell lines and differences in the particular chromosomesinvolved were also detected. Gains of 4q (15%) and 8q (8%) andlosses of 9q (21%), 1p (18%), and 11q (18%) were the most commonfindings in MSI� cancers. However, gain of 8q (50%), 13q (35%),and 20q (25%) and loss of 18q (55%), 15q (35%), and 17p (30%)were detected most frequently in CIN� cancers (17). In addition,different types of aberration were detected depending on the particularmismatch repair defect identified (18).

Conventional CGH has a limited resolution and can only detectlosses of �10 Mb or greater (19, 20). High-level amplifications havea maximum resolution of 3 Mb (21). The resolution of CGH has beenimproved by replacing the metaphase chromosomes as the hybridiza-tion target with mapped and sequenced clones (bacterial artificialchromosomes, P1-derived artificial chromosome, and cosmids) ar-rayed onto glass slides (22, 23). The resolution of this matrix (22) orarray (23) CGH is limited only by the insert size and density of themapped sequences used. The capability of the technique to providehigh-resolution mapping of variation in copy number has been dem-onstrated in the analysis of breast tumors, where the fine structure ofamplicons was resolved and potential candidate genes identified (24).In this study, we describe the use of array CGH with a resolution of�1 Mb (25) to investigate copy number changes occurring in CRC bythe analysis of 48 cell lines and 37 primary cancers.

MATERIALS AND METHODS

Cell Lines and Primary Cancers. DNA was extracted from 48 CRC celllines derived from local collaborators or public sources. In almost all cases theMSI status and ploidy were already known, and several authors have reportedmolecular changes, such as mutation of APC, KRAS, and p53, in some of thecell lines (10, 26, 27). Fresh frozen samples of primary sporadic CRCs, derivedfrom patients at St. Mark’s Hospital, were obtained. All of the cancerscontained �70% neoplastic tissue on histological review. MSI analysis (28)was undertaken, and ploidy was assessed using flow cytometry (data not

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

United Kingdom.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 with18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article online at http://www.sanger.ac.uk/Teams/Team70/supplemental-data/.

Requests for reprints: Nigel P. Carter, The Wellcome Trust Sanger Institute, Well-come Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom. Phone:44-0-1223-494860; Fax: 44-0-1223-494919; E-mail: [email protected].

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shown). DNA was extracted from 7 MSI� and 30 CIN� cancers usingstandard methods; no MSI�CIN� (or MSI�CIN�) cancers were included inthe study because these are very rarely represented in the set of known CRCcell lines. Cell line and cancer details can be downloaded from SupplementaryTable 1 (Excel spreadsheet). Cell line DNAs were hybridized against a normalfemale lymphoblastoid cell line C0009-SAH (HRC160; European Collectionof Cell Cultures no. 93010702) obtained from the European Collection of CellCultures (Salisbury, United Kingdom). Primary cancers were hybridizedagainst a sex-matched control DNA pool created from 20 normal blood DNAsobtained from Human Random Collection of the European Collection of CellCultures. Clones that had been shown previously to report copy numberchanges in hybridizations of individual normal DNAs against the pool ofnormal DNAs (data not shown) were considered to be polymorphic in thenormal population and were excluded from analysis.

DNA Labeling. Test and control DNAs were differentially labeled using aBioprime labeling kit (Invitrogen, Carlsbad, CA), as described previously (25),with minor modification. Briefly, a 130.5-�l reaction was set up containing450 ng of DNA and 60 �l of 2.5� random primer solution. The DNA wasdenatured for 10 min at 100°C, and 1.5 �l of 1 mM Cy5-dCTP or Cy3-dCTP(NEN Life Science Products, Boston, MA) and 3 �l of Klenow fragment wereadded on ice to a final reaction volume of 150 �l. The reaction was incubatedovernight at 37°C and stopped by adding 15 �l of stop buffer. Unincorporatednucleotides were removed using G50 spin columns (Pharmacia Biotech, Pis-cataway, NJ) according to the manufacturer’s instructions.

Microarray Hybridization and Image Analysis. Hybridization to thearray was carried out as described previously (25). Briefly, test and control-labeled DNAs were combined, precipitated together with 135 �l of humanCot1 DNA (Roche, Mannheim, Germany), and resuspended in 60 �l ofhybridization buffer [50% formamide, 10% dextran sulfate, 0.1% Tween 20,2� SSC, 10 mM Tris-HCl (ph7.4)], and 6 �l of yeast tRNA (100 �g/�l;Invitrogen, Carlsberg, CA). This hybridization solution was incubated for 1 hat 37°C before application to the array. A prehybridization solution of 80 �l ofherring sperm DNA (10 mg/ml; Sigma-Aldrich Company Ltd., Dorset, UnitedKingdom) and 135 �l of human Cot1 DNA (Roche), precipitated together andresuspended in 160 �l of hybridization buffer, was applied to the array andincubated at 37°C for 1 h. The arrays were scanned on an Axon 4000B scanner(Axon Instruments, Burlingame, CA), and images were quantified using thesoftware program “Spot” (29). The fluorescence intensity ratio for each clone(after local background subtraction) was calculated and normalized by dividingeach ratio by the median ratio of all of the autosomal clones and plotted againstthe mapped genomic position (NCBI31; freeze date, November 2002).

Data Analysis. The determination of significant copy number changesdetected in array CGH for cancer and cancer cell lines, where much of thegenome has been altered, is not straightforward. Measurement variation willvary from hybridization to hybridization, so it is important to set statisticallydefined thresholds for copy number change by analysis of the variation foreach hybridization. We have approached this by identifying regions of modalcopy number (normalized linear ratio of 1.0) in each hybridization, using anarbitrary threshold determined from independent normal versus normal hy-bridizations and then using the variation of the defined modal region to setthresholds specific to that hybridization. Specifically, the largest contiguouschromosomal region reporting a modal copy number was identified. A regionwas considered to be modal if 95% of the clones fell within 99% confidenceintervals calculated from the linear ratios of autosomal clones in a normal maleversus normal female hybridization (data not shown). The size of the regionused varied from 100 Mb to 246 Mb. The SD of measurements in these modalregions ranged from 0.03 to 0.14. The linear ratios reported by the cloneswithin the defined modal region were used to calculate the coefficient ofvariation and 99% confidence intervals for each hybridization. Clones that felloutside the 99% confidence intervals were identified as reporting significantcopy number changes. We have shown previously that our arrays demonstrateclose to theoretical copy number changes in response to autosomal single copygains and losses (25). For the cell lines, gains were considered to be singlecopy or greater if the reported ratio was greater or equal to the theoretical valuefor a single copy gain (1.5) minus the 99% confidence interval at this value foreach hybridization and double copy number or greater if the reported ratio wasgreater or equal to the theoretical value for a double copy amplification (2.0)minus the 99% confidence interval at this value for each hybridization.Deletions were considered to be single copy or greater if the reported ratio wasgreater or equal to the theoretical value for a single copy deletion (0.5) plus the99% confidence interval at this value for each hybridization. In no hybridiza-tion did these defined thresholds overlap the 99% confidence intervals formodal clones. The theoretical values for single and double copy numberchanges were calculated relative to a diploid karyotype; therefore, classifica-tion of copy number changes, which occurred in samples with an increasedploidy, would be underestimates of the true level of copy number gain or loss.For the primary cancers, the observed changes were not classified as single ordouble copy number changes because the cancer samples are inevitably con-taminated with DNA from surrounding normal tissue. Contaminating normaltissue results in suppression of the reported ratios in regions of amplificationor deletion. The exact amount of contamination for each sample could not bereadily determined; hence, precise thresholds could not be set. Copy number

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

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ARRAY CGH ANALYSIS OF COLORECTAL CANCER



change of the sex chromosomes was not analyzed because the cell lines wereall hybridized against female control DNA. To identify trends in copy numbergain, the “mean percentage of gain by clones” (MPG) for each particularchromosome or region of interest was used. The MPG was determined bycalculating the percentage of samples with a significant copy number gain foreach clone and calculating the mean percentage gain for all of the clones in thechromosome or region of interest. An equivalent calculation, “mean percent-age of loss by clones” (MPL) was applied for copy number losses. The MPLwas determined by calculating the percentage of samples with a significantcopy number loss for each clone and calculating the mean percentage loss forall of the clones in the chromosome or region of interest. The genomiclocations of all of the clones named specifically in the text were confirmed bybacterial artificial chromosome end sequencing and fluorescence in situ hy-bridization. Raw, normalized log2 ratios for all of the cell lines and cancers canbe downloaded from Supplementary Table 1 (Excel spreadsheet).

Metaphase Fluorescence in Situ Hybridization. Degenerate oligonucleo-tide primed amplified clone DNA (amplified using primers degenerate oligo-nucleotide primed 1, 2, and 3) created for array construction (25) was labeledwith biotin-16-dUTP (Roche) or digoxigenin-11-dUTP (Roche) by nick trans-lation. Hybridizations were carried out using conventional methods to chro-mosomes prepared from a normal male lymphoblastoid cell line (HRC575;European Collection of Cell Cultures no. 94060845). Biotin-labeled probeswere detected using Avidin TexasRed (Molecular Probes Inc., Eugene, OR),and digoxigenin-labeled probes were detected with a combination of mouseantidigoxigenin (Vector Laboratories Ltd., Peterborough, United Kingdom)and goat antimouse-FITC (Sigma-Aldrich Company Ltd.) antibodies.

RESULTS

The most common finding in both cell lines and cancers was gainof chromosome 20. The MPG was 49% for cell lines and 50% forcancers (for definition, see “Materials and Methods”). The region of

chromosome 20 gained most frequently was 20q13.3 (RP4-563E14 at62.3Mb) in 65% of cell lines and 81% of cancers. Gain of chromo-some 13 was also a common finding in both cell lines (MPG � 43%)and cancers (MPG � 46%), along with gain of chromosome 7(MPG � 22% of cell lines; 46% of cancers) and the long arm ofchromosome 8 (MPG � 31% of cell lines; 27% of cancers). The mostcommon region of copy number loss was the long arm of chromosome18 (MPL � 52% of cell lines; 32% of cancers). In particular, threeclones detected the most frequent copy number loss: two at 18q21.1(RP11-313C14 at 43.9Mb in 60% of cell lines and cancers andRP11-25O3 at 49.7Mb in 60% of cell lines and 49% of cancers) andone at 18q12.2 (RP11-19F9 at 35Mb in 56% of cell lines and 59% ofcancers). In addition, chromosome 8p was also a consistent region ofcopy number loss (MPL � 33% of cell lines; 29% of cancers).

The overall pattern of copy number change was very similar in thecell lines and cancers (Fig. 1). However, loss of chromosome 6 was asignificantly more common event (P � 0.005, �2 test) in the cell lines(MPL � 21%) than the cancers (MPL � 1%). In addition, gain ofchromosome 15 occurred more frequently in the cell lines(MPG � 21%) than the cancers (MPG � 1%; P � 0.005, �2 test).Furthermore, gain of chromosome 12, in particular, the short arm, wasa common finding in the cell lines (MPG � 32%) but not the cancers(MPG � 3%; P � 0.005, �2 test). Although chromosome 12p was notgenerally gained in the cancers, one particular clone, RP11-59H1 at12.9 Mb, was gained in 22% of cancers and 31% of cell lines. Themost frequently gained clone in the cell lines (38%) was RP11-4N23at 13.6 Mb. The total number of clones reporting significant gains andlosses was calculated, and the median number per sample was com-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 �2 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 58 RP11-28I2 127.3 7 18 RP11-25O3 50.0 58 RP1-80K22 128.4 7 4 RP11-226A18 182.4 4

13 RP11-10M8 37.2 7 18 7 clones 28–56 413 RP11-235O20 96.3 7 18 3 clones 73–77 48 RP11-269I24 131.3 6 4 RP11-495H13 18.0 3

13 5 clones 34–109 6 8 19 clones 3–35 38 10 clones 123–140 5 16 RP11-167B4 6.3 3

13 9 clones 22–107 5 18 17 clones 39–76 36 RP11-524H19 54.7 4 20 RP5-855L24 14.6 37 RP11-99O17 25.6 4 1 RP11-241C9 213.9 28 9 clones 117–130 4 3 RP11-425D6 81.4 2

13 24 clones 25–105 4 3 RP11-81P15 86.9 220 4 clones 52–64 4 4 RP11-84H6 179.8 27 RP11-550A18 29.3 3 4 RP11-451F20 184.8 28 10 clones 58–135 3 8 17 clones 0–40 2

10 RP11-382A18 18.4 3 9 RP11-109M15 16.2 213 23 clones 20–114 3 9 RP11-15P13 20.3 217 RP5-986F12 35.8 3 13 RP11-95G6 27.9 220 9 clones 41–63 3 13 RP11-10M8 37.2 26 RP1-283K11 2.3 2 13 RP11-247M1 45.8 26 6 clones 38–43 2 13 RP11-524F1 58.7 26 4 clones 51–54 2 16 RP11-556H2 78.9 26 3 clones 65–68 2 17 RP11-104O19 4.1 26 5 clones 135–138 2 17 RP5-1050D4 4.8 27 7 clones 1–82 2 17 RP11-243K12 6.1 28 32 clones 58–144 2 18 7 clones 24–43 2

11 RP11-93M11 73.0 2 18 4 clones 60–72 211 RP11-569A20 106.7 212 RP11-283I3 0.3 212 3 clones 3–4 212 4 clones 10–16 212 4 clones 24–27 213 25 clones 47–112 216 RP11-283C7 46.8 216 3 clones 81–82 217 4 clones 33–37 220 9 clones 20–61 2

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median number of aberrant clones of 975 (range � 598-2556), and thecancers showed a median number of aberrant clones of 536(range � 48–1152). This difference is statistically significant(P � 0.005, Mann-Whitney U test).

The frequency with which each clone on the array reported ahigh-level amplification (double copy number or greater) in the celllines was calculated and is displayed in Fig. 1 as yellow bars. Clonesreporting amplifications in �2 cell lines are listed in Table 1. Themost common region of amplification of chromosome 20 was again at62.3 Mb (reported by clone RP4-563E14; amplified in 4 cell lines).The most common region of amplification of chromosome 13 was13q12.13-q12.2 (RP11-44J9 at 26.2 Mb; amplified in 9 cell lines), andthe most frequently amplified region of chromosome 8 was 8q24.21(126-128 Mb; reported in 7 cell lines). The most frequently amplifiedregion of chromosome 17 was 35.8 Mb (RP5-986F12), and the mostfrequent amplification of 7p was 29.3 Mb (RP11-550A18). The fre-quency with which each clone reported a deletion (single copy orgreater) in the cell lines is displayed as black bars in Fig. 1. Clonesreporting deletions in �2 cell lines are listed in Table 1. Consistentregions of deletion included chromosome 4q34.3 (RP11-226A18;deleted in 4 cell lines) and, again, chromosome 18q21.1.

Small regions of consistent copy number change were also ob-served (Tables 2 and 3). For example, chromosome 17q11.2-q12(33–38 Mb) was a consistent region of gain in both cell lines(MPG � 21%) and cancers (MPG � 22%). RP5-986F12 at 35.8 Mbwas the most frequently amplified clone (double copy number orgreater) occurring in 6% of cell lines. Gain of this clone occurred aspart of both broad and narrow regions of copy number change (Figs.2, A and B). In addition, chromosome 4q34-q35 was a common regionof copy number loss in cell lines (MPL � 35%) and cancers

(MPL � 38%) as shown in Fig. 2, C and D. The most frequentlydeleted clone defining the peak of the copy number change in the celllines (8%) was RP11-226A18 at 182.4 Mb. Examples of 4q34-q35loss are shown in Fig. 2, E and F.

The genome-wide pattern of copy number changes in the MSI�and CIN� cell lines and cancers was compared (Supplementary Fig.1). CIN� samples reported significantly more aberrations than MSI�samples (cell lines P � 0.005; cancers P � 0.05, Mann-Whitney Utest). Gain of chromosome 20 was the most common overall finding,but when the analysis was split by the microsatellite status of thesamples, a marked difference emerged. Gain of chromosome 20occurred more often in CIN� cell lines (MPG � 51%) and CIN�cancers (MPG � 59%) than in MSI� cell lines (MPG � 6%) andMSI� cancers (MPG � 12%; P � 0.005 for both cell lines andcancers, �2 test). However, clone RP11-563E14 was gained in 14% ofMSI� cell lines and 43% of MSI� cancers and gained (single copyor greater) in 7% of MSI� cell lines. Loss of 18q was also a morefrequent event in CIN� cell lines (MPL � 71%) and CIN� cancers(MPL � 42%) than MSI� cell lines (MPL � 12%) and MSI�cancers (MPL � 1%). However, loss of chromosome 18q21.1-21.2(44–47 Mb, including clones RP11-313C14 and RP11-46D1) wasdetected in MSI� cell lines (MPL � 21%) and MSI� cancers(MPL � 29%). Loss of the short arm of chromosome 17 occurredmore frequently in CIN� cell lines (MPL � 38%) and cancers(MPL � 29%) than MSI� cell lines (MPL � 3%) and cancers(MPL � 1%). In addition, loss of chromosome 8p was a frequentevent in CIN� cell lines (MPL � 47%) and cancers (MPL � 37%)but occurred much less frequently in MSI� cell lines (MPL � 3.4%).Loss of 8p was never found in MSI� cancers but was frequentlygained (MPG � 28%; Fig. 3). The overall peak of copy number gain

Table 2 Small regions of copy number gain

Chr

Region Cell lines Cancers Peak clone (highest % gain)

Candidate genesStart

(Mb)aEnd

(Mb)aGainb

(%)

Gainc

(single copy)(%)

Amplificationd

(double copy)(%)

Gainb

(%) NamePosition

(Mb) Band

1 2.9 2.9 2 2 14 RP4-785P20 2.9 1p36.32-p36.33 SKI, MEL1, PRKCZ, DVL1, CENTB51 113 118 15 6 2 5 RP4-787H6 115.9 1p13.1 VANGL12 102.3 111.8 31 10 2 3 RP11-519H15 107.2 2q12.32 126 130 17 6 4 11 RP11-207G14 130 2q21.1 MEKK3, RAB6C3 125 125 13 2 – 30 RP11-25C10 125 3p25.2 RAF-1, FBLN13 172.3 175.3 40 10 – 32 RP11-163H6 173 3q26.31 PLD1, ECT25 147 152.6 17 4 – 11 CTB-137O22 150 5q33.1 G3BP5 72.9 81.3 12.5 4 2 – RP11-30D15 79.2 5q14.1 RASGRF26 37.8 68.1 19 12.5 8 24 RP11-524H19 54.7 6p12.16 134.8 137.9 19 10 4 16 RP1-32B1 135.4 6q23.3 MYB7 0.7 0.7 52 10 2 57 RP11-449P15 0.7 7p22.3 CENTA1, MAFK8 117 128.4 50 31 15 40 RP1-80K22 128.4 8q24.21 C-MYC8 133.8 133.8 48 27 10 30 RP6-98A24 133.8 8q24.22 WISP28 144.3 145.3 48 25 4 51 RP11-472K18 144.2 8q24.3 RIG-E, GLI4, RHPN19 83.2 85.1 23 2 – 11 RP11-423O13 83.3 9q21.339 95.3 98 29 2 – 19 RP11-80H12 97.2 9q31.1

10 76 80.8 23 4 – 11 RP11-90J7 80 10q22.3 DLG511 67.9 74.3 32 8 4 16 RP11-804L21 (cancers) 69.9 11q13.3 CCND1, FGF3, FGF4, FGF19, ORAOV1,

MYEOV, EMS1RP11-93M11 (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 MGP12 67.6 73.6 19 4 4 5 RP11-101K2 71.2 12q15 TM4SF3, PTPRR, PTPRB, GRP49, RAB2112 116 120.5 23 6 2 8 RP11-8A1 116 12q24.2116 0.2 2.2 40 8 – 49 RP11-243K18 0.3 16p13.3 MPF16 64.2 82.4 21 13 4 24 RP11-303E16 80.9 16q23.2 MAF17 80.7 81.2 48 13 – 43 RP11-567O16 81.2 17q25.3 RAB40B17 33.2 37.8 21 10 6 22 RP5-986F12 35.8 17q12 AATF, TBC1D317 42.1 49.3 17 4 2 24 RP11-361K8 47 17q21.32 GIP22 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 � 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 � 1) minus

the 99% confidence interval for each hybridization.

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on chromosome 8q was band 8q24.21 from 126–128Mb. However, inMSI� cancers a different region 8q24.3 (144–145Mb) was the mostfrequently gained (RP11-472K18 at 144.3Mb in 36% of MSI� celllines and 57% of MSI� cancers and RP11-349C2 at 145.3 Mb in 36%of MSI� cell lines and 71% of MSI� cancers; see Fig. 3).

The frequency with which the clones reported amplifications (dou-ble copy number or greater) and deletions (single copy or greater) inthe CIN� and MSI� cell lines is displayed in Supplementary Fig. 1as yellow and black bars. Clones reporting amplifications in �2

CIN� cell lines and 1 MSI� cell line and clones reporting deletions(single copy or greater) in �2 cell lines (CIN� and MSI�) are listedin Table 4. There were no clones that were amplified in �1 MSI� cellline. Only one clone reported a deletion in �1 MSI� cell line. Thisclone, RP11-241C9, is mapped to chromosome 1q41 at 214Mb.

Copy number losses reported by clones representing three genes,APC, p53, and SMAD4, were compared with previously publishedloss of heterozygosity (LOH) data in the cell lines (10, 27, 30). In thecase of APC, it was found that LOH occurs most often in cell lines

Table 3 Small regions of copy number loss

Chr

Region Cell lines Cancers Peak clone (highest % loss)

Candidate genesStart

(Mb)aEnd

(Mb)aLossb

(%)Deletionc

(%)Lossb

(%) NamePosition

(Mb) Band

1 19.7 27.2 30 2 13 RP11-509F14 23.6 1p36.11 E2F2, ID34 45.8 47.9 30 2 11 RP11-100N21 47.5 4p124 57.7 59.9 33 2 19 RP11-355L4 57.7 4q12 IGFBP74 107 112.6 33 2 19 RP11-75N20 110 4q254 179.7 186.3 35 8 38 RP11-226A18 182.4 4q34.3 CASP36 112.4 114.5 27 2 8 RP1-142L7 112.4 6q21 TUBE19 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 PTEN10 126.7 132.6 17 2 22 RP11-16P8 128.3 10q26.2 BCCIP, DDX3211 62.7 64.1 21 – 30 RP11-424O11 64.1 11q13.1 BAD, HRASLS2, HRASL3, LGAS12, RARRES3, FLRT116 5.6 10.6 19 6 14 RP11-167B4 6.3 16p13.317 4.1 6.1 31 4 51 RP11-243K12 6.1 17p13.2 DHX33, NAL117 9.5 12 38 2 32 RP11-471L13 12 17p12 ELAC220 12.6 15.6 35 6 3 RP5-855L24 14.6 20p12.1 FLRT3a 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 � �1) plus the 99% confidence

interval for each hybridization.

Fig. 2. A and B, two examples of copy numbergain of chromosome 17q11.2-q12 in cell line Colo678 (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 copynumber losses (gray), gains (open), and deletions(black) in the cell lines (C) and primary cancers(D). RP11-226A18 (182.4 Mb) is indicated with anarrow. E and F, two examples of copy number lossof chromosome 4q34-q35 in cell line SW1417 (E)and a primary cancer (F).

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with a normal copy number at this locus (17 of 22 samples), probablyby mitotic recombination, as suggested previously (30). Conversely,LOH at SMAD4 is generally associated with copy number loss (25 of26 samples), and in cases where LOH was not detected, copy numberis normal (11 of 12 cases). LOH at p53 is not associated with copynumber change, suggesting a mechanism of mitotic recombination orgene conversion.

DISCUSSION

A genomic microarray with a resolution of �1 Mb was used toinvestigate chromosomal imbalances in a set of 48 CRC cell lines and37 primary CRCs. The collection included both MSI� and CIN�samples. Analysis of the array hybridizations revealed consistentregions of copy number change. Many of the findings were in agree-ment with those observed previously in conventional CGH investiga-tions (12–16), for example, gain of chromosomes 20, 13, and 8q andloss of chromosomes 18q and 8p. However, the increased resolutionof array CGH allowed the identification of the most frequently alteredregions within these large scale gains and losses and also the detectionof previously unreported changes, for example, amplification of17q11.2-q12 and deletion of chromosome 1q41. The most commonregion of gain was chromosome 20, especially 20q13.3 (RP4-563E14at 62.3Mb). Candidate genes that map to this region include thefollowing: (a) LIVIN, an inhibitor of apoptosis that has been associ-ated with the progression of bladder cancer (31) and detected at highlevels in a CRC cell line (32); (b) PTK6, a protein kinase, which whenoverexpressed in mammary cells leads to sensitization to epidermalgrowth factor and partial transformation (33) and also shows in-creased expression in primary colon tumors (34); (c) HD54, which is

involved in calcium-mediated signal transduction and cellular prolif-eration (35); and (d) EEF1A2, a transcription elongation factor foundto be amplified and overexpressed in ovarian cancer (36). Gain ofchromosome 13 was also a common finding, and the most frequentlyamplified region was 13q12.13-q12.2 from 26–28 Mb. Genes ofinterest that map to this region include FLT3, a tyrosine kinasereceptor in which activating mutations have been found in acutemyeloid leukemia (37) and FLT1, a vascular endothelial growth factorreceptor found to be expressed in gastric and breast carcinoma cells(38, 39). The most frequent region of copy number loss was the longarm of chromosome 18, particularly 18q21.1, including the knowntumor suppressor genes SMAD2 (40) and SMAD4 (41), which func-tion in the transforming growth factor signaling pathway to mediategrowth inhibition. Allelic loss at 18q21.1 has been detected in up to60% of CRCs (42), and mutation of SMAD4 has been identified in50–60% of CRCs with loss of 18q21 (10).

In addition to large-scale rearrangements, smaller regions of copynumber change were also detected. For example, amplification of17q11.2-q12 (33–38 Mb) was a common finding, and the peak of thisamplification was reported by clone RP5-986F12 at 35.8 Mb. Genesof interest that are located in this region include AATF, which func-tions as an antagonist to apoptosis (43), and TBC1D3, a Rab GTPaseamplified in 15% of prostate cancers (44). Consistent regions ofdeletion (single copy or greater) were also identified, for example,RP11-226A18 at 4q34–35 (182.4 Mb). The proapoptotic genecaspase-3, which is down-regulated in gastric cancer (45), maps tothis region. In addition, RP11-241C9 at 1q41 (213.9 Mb) was the onlyclone reporting a deletion in �1 MSI� cell line. Transforming growthfactor �2 is located in this deleted region.

Fig. 3. Frequency of copy number gains andlosses reported by the chromosome 8 clones inMSI� cell lines (A), CIN� cell lines (B), MSI�cancers (C), and CIN� cancers (D). Expandedregion of chromosome 8 (97–146Mb) showing fre-quency of copy number gains (blue), gains of singlecopy or greater (black), amplifications of doublecopy number or greater (yellow) in MSI� cell lines(E), and frequency of copy number gains (blue) inMSI� cancers (F). The 8q24.21 is underlined inred, and 8q24.3 is underlined in green.

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Within the limitations of our analysis, imposed by both the un-known ploidy of the cancer samples and the variable degree ofcontamination by normal cells, the overall pattern of copy numberchange in the cell lines and cancers was markedly similar; however,some differences were detected. The number of aberrations (all of thegains and losses) was significantly greater in the cell lines than thecancers, and loss of chromosome 6 and gain of chromosome 15 werefar more common events in the cell lines. It has been observedpreviously that loss of chromosome 6, especially 6q, occurs morefrequently in cell lines than primary tumors (26). The additionalchanges seen in the cell lines could have arisen if they were advan-tageous to cells growing in cell culture conditions and may not alwaysbe relevant to cancer formation. Gain of the short arm of chromosome12 was another more frequent occurrence in the cell lines than thecancers. However, one clone, RP11-59H1 at 12.9 Mb, was gained inthe cancers, perhaps suggesting that this region of 12p, in particular,could be the target of the general increase in copy number seen in thecell lines. Genes of interest that map to this region include MGP, amatrix GLA protein that has been shown previously to be overex-pressed in breast cancer (46).

The analysis was split by the type of genomic instability that thesamples exhibited, chromosomal (CIN�) or microsatellite (MSI�)instability. Overall, MSI� samples had significantly fewer aberra-tions. This finding follows expectation; MSI� cancers tend to main-tain a diploid karyotype, whereas aneuploidy is a common feature ofCIN� cancers (8, 9) and agrees with findings reported previously (17,18). The copy number changes that were detected in the MSI�

cancers are more likely to be causative in the initiation and progres-sion of the cancer rather than a consequence of a generally unstablekaryotype and likely to contain target genes of the rearrangement. Forexample, gain of the whole of chromosome 20 was a common findingin CIN� cancers but a much less frequent event in the MSI�samples; yet, gain of a particular clone, RP11-563E14 at 62.3 Mb, wasfrequently detected in the MSI� cancers and cell lines, again sug-gesting that this region could be an important target of the gain ofchromosome 20. Dividing the analysis by microsatellite stabilitystatus has also been informative in focusing the attention on a partic-ular region of chromosome 18q. Although loss of the entire long armof chromosome 18 is not a common event in MSI� samples, loss of18q21.1–21.2 (44–47 Mb) is a frequent occurrence, suggesting thatthis area is likely to contain candidate genes that may be the target ofthe aberration. The pattern of copy number changes reported by thechromosome 8 clones differs distinctly between the MSI� and CIN�samples. Loss of 8p is a common event in CIN� cell lines andcancers; however, this is not the case in the MSI� samples. Not onlyis loss of 8p not frequently detected, but in the MSI� cancers, 8p isactually a common region of copy number gain, suggesting that lossof 8p is an important event, specifically in the development of CIN�CRCs. Furthermore, gain of the long arm of chromosome 8 was acommon finding in all of the samples. However, although gain of8q24.21 was the most prominent finding in the CIN� samples, adifferent region, 8q24.3, stands out in the MSI� samples, and RP11-472K18 and RP11-349C2 are the most frequently gained clones.Candidate genes that map to this region include RIG-E, a receptor that

Table 4 Clones reporting amplifications (double copy or greater) in �2 CIN�a cell lines and 1 MSI� cell line and clones reporting deletions (single copy or greater)in �2 cell lines (CIN� and MSI�)

Amplifications Deletions

CIN� cell lines MSI� cell lines CIN� cell lines MSI� cell lines

Chr ClonePositionin Mb

No. ofcell lines Chr Clone

Positionin Mb


Positionin Mb


Positionin Mb

No. ofcell lines

13 RP11-44J9 26.2 9 3 4 clones 60–65 1 18 RP11-313C14 44.0 5 1 RP11-241C9 213.9 213 RP11-570F6 26.0 7 3 5 clones 76–87 1 18 RP11-25O3 50.0 513 RP11-10M8 37.2 7 6 18 clones 2–24 1 4 RP11-226A18 182.4 413 RP11-235O20 96.3 7 7 RP11-502P9 11.6 1 18 7 clones 28–56 48 RP11-28I2 127.3 6 7 6 clones 26–36 1 18 3 clones 73–77 48 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 3–35 313 RP11-125A7 41.2 6 7 RP11-298A10 142.6 1 18 17 clones 39–76 313 RP11-384G23 52.9 6 8 RP11-174I12 124.2 1 3 RP11-425D6 81.4 213 RP11-184L18 70.4 6 8 RP11-269I24 131.3 1 3 RP11-81P15 86.9 213 RP11-40E6 109.3 6 8 RP11-472K18 144.3 1 4 RP11-84H6 179.8 28 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 28 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 20.3 220 RP5-1162C3 53.5 4 16 RP11-167B4 6.3 26 RP11-524H19 54.7 3 16 RP11-556H2 78.9 27 RP11-99O17 25.6 3 17 RP11-104O19 4.1 28 9 clones 58–134 3 17 RP5-1050D4 4.8 2

13 22 clones 20–114 3 17 RP11-243K12 6.1 217 RP5-986F12 35.8 3 18 7 clones 24–43 220 6 clones 41–64 3 18 4 clones 60–72 26 RP1-283K11 2.3 2 20 RP5-855L24 14.6 26 13 clones 38–68 27 RP11-510K8 1.5 27 RP11-550A18 29.3 28 32 clones 57–141 2

10 RP11-382A18 18.4 211 RP11-93M11 73.0 211 RP11-569A20 106.7 212 13 clones 0–27 213 26 clones 47–112 216 RP11-283C7 46.8 216 4 clones 80–81 217 4 clones 33–37 220 11 clones 20–63 2

a CIN, chromosomal instability; MSI, microsatellite instability.

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shares homology to epidermal growth factor receptors and is highlyexpressed in leukemic cells (47), and GLI4, a member of the Kruppelfamily of transcription factors found to be overexpressed in braintumors (48).

A number of genes located within the reported regions of copynumber change are associated with transforming growth factor signaltransduction, suggesting that the abrogation of this pathway may beimportant in the development of CRC. For example, the only clone toreport a deletion in �1 MSI� cell line was RP11-241C19 at 1q41;transforming growth factor �2, which has been shown to inducegrowth inhibition and apoptosis (49, 50), maps to this region. Inaddition, caspase-3, which maps to the 4q34-q45 frequent region ofdeletion, has been implicated in transforming growth factor �-medi-ated apoptosis (51), and SMAD2 and SMAD4, key mediators oftransforming growth factor � signaling (52), are located within themost common region of loss of chromosome 18. Furthermore, BAD,a member of the BCL-2 family of proapoptotic genes, maps to11q13.1, a region of frequent copy number loss. This protein isinhibited by the 14-3-3� protein but promotes cell death on its release,which occurs via the cleavage of 14-3-3� by caspase-3 (53). Thetumor suppressor CDKN2A (P16-INK4A), an activator of caspase-3-mediated apoptosis (54), is located within the region of deletion onchromosome 9p21-p22. Additional genes that act in this pathway arelocated within common regions of gain. For example, FLT1, a recep-tor for the vascular endothelial growth factor that promotes angiogen-esis, cancer invasion, and metastasis in breast cancer, is located withinthe most frequently amplified region of chromosome 13. Stimulationof this receptor in epithelial cells results in decreased caspase-3activity and prevention of apoptosis (55). In addition, RAF-1, whichfunctions downstream of the Ras family of GTP-ases in the promotionof cell cycle progression (56), is located within the 3p25.2 region ofcopy number gain. It has been shown that RAF-1 is activated byvascular endothelial growth factor (57) and that RAF-1 signaling canlead to the inhibition of apoptosis via the phosphorylation of BAD(58).

Overall, the analysis of CRC cell lines and cancers by array CGHhas revealed consistent regions of copy number change ranging fromgain of whole chromosomes to the loss of a single clone. The genome-wide pattern of copy number change was strikingly similar betweencell lines and cancers, although a few obvious differences, loss ofchromosome 6 and gain of chromosomes 15 and 12p, were reported.CIN� samples had a significantly greater number of aberrations thanMSI� samples, particularly gain of chromosome 20 and loss ofchromosomes 18q and 8p. In addition, the target of chromosome 8qgain appeared to differ depending on microsatellite status with8q24.21 frequently gained in CIN� samples and 8q24.3 gain associ-ated with MSI� samples. A number of genes of interest are locatedwithin frequently aberrated regions, which, on additional investiga-tion, may prove to be of importance in the pathogenesis of CRC.

ACKNOWLEDGMENTS

We thank the Wellcome Trust Sanger Institute Mapping Core Group, CarolScott, and Elizabeth Huckle for clone selection and verification, the WellcomeTrust Sanger Institute Micorarray Facility for printing the arrays, Judy Fantesfor fluorescence in situ hybridization analysis of clones RP11-59H1 andRP11-4N23, Sarah Edkins for the cell line Colo320 HSR, and Ying Liu forsome karyotype data.

REFERENCES

1. HMSO. Mortality Statistics. Cause. England and Wales, London: HMSO; 1992.2. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:

159–70.

3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67.

4. Powell SM, Zilz N, Beazer-Barclay Y, et al. APC mutations occur early duringcolorectal tumorigenesis. Nature (Lond) 1992;359:235–7.

5. Forrester K, Almoguera C, Han K, Grizzle WE, Perucho M. Detection of highincidence of K-ras oncogenes during human colon tumorigenesis. Nature (Lond)1987;327:298–303.

6. Fodde R, Smits R, Clevers H. APC, signal transduction and genetic instability incolorectal cancer. Nature Rev Cancer 2001;1:55–67.

7. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers.Nature (Lond) 1998;396:643–9.

8. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet1991;25:229–53.

9. Ilyas M, Straub J, Tomlinson IP, Bodmer WF. Genetic pathways in colorectal andother cancers. Eur J Cancer 1999;35:335–51.

10. Woodford-Richens KL, Rowan AJ, Gorman P, et al. SMAD4 mutations in colorectalcancer probably occur before chromosomal instability, but after divergence of themicrosatellite instability pathway. Proc Natl Acad Sci USA 2001;98:9719–23.

11. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridizationfor molecular cytogenetic analysis of solid tumors. Science (Wash DC) 1992;258:818–21.

12. Ried T, Knutzen R, Steinbeck R, et al. Comparative genomic hybridization reveals aspecific pattern of chromosomal gains and losses during the genesis of colorectaltumors. Genes Chromosomes Cancer 1996;15:234–45.

13. Korn WM, Yasutake T, Kuo WL, et al. Chromosome arm 20q gains and othergenomic alterations in colorectal cancer metastatic to liver, as analyzed by compar-ative genomic hybridization and fluorescence in situ hybridization. Genes Chromo-somes Cancer 1999;25:82–90.

14. Al-Mulla F, Keith WN, Pickford IR, Going JJ, Birnie GD. Comparative genomichybridization analysis of primary colorectal carcinomas and their synchronous me-tastases. Genes Chromosomes Cancer 1999;24:306–14.

15. Aragane H, Sakakura C, Nakanishi M, et al. Chromosomal aberrations in colorectalcancers and liver metastases analyzed by comparative genomic hybridization. Int JCancer 2001;94:623–9.

16. De Angelis PM, Clausen OP, Schjolberg A, Stokke T. Chromosomal gains and lossesin primary colorectal carcinomas detected by CGH and their associations with tumourDNA ploidy, genotypes and phenotypes. Br J Cancer 1999;80:526–35.

17. Li LS, Kim NG, Kim SH, et al. Chromosomal imbalances in the colorectal carcino-mas with microsatellite instability. Am J Pathol 2003;163:1429–36.

18. Snijders AM, Fridlyand J, Mans DA, et al. Shaping of tumor and drug-resistantgenomes by instability and selection. Oncogene 2003;22:4370–9.

19. Bentz M, Plesch A, Stilgenbauer S, Dohner H, Lichter P. Minimal sizes of deletionsdetected by comparative genomic hybridization. Genes Chromosomes Cancer 1998;21:172–5.

20. Kallioniemi OP, Kallioniemi A, Piper J, et al. Optimizing comparative genomichybridization for analysis of DNA sequence copy number changes in solid tumors.Genes Chromosomes Cancer 1994;10:231–43.

21. Kirchhoff M, Gerdes T, Maahr J, et al. Deletions below 10 megabasepairs aredetected in comparative genomic hybridization by standard reference intervals. GenesChromosomes Cancer 1999;25:410–3.

22. Solinas-Toldo S, Lampel S, Stilgenbauer S, et al. Matrix-based comparative genomichybridization: biochips to screen for genomic imbalances. Genes ChromosomesCancer 1997;20:399–407.

23. Pinkel D, Segraves R, Sudar D, et al. High resolution analysis of DNA copy numbervariation using comparative genomic hybridization to microarrays. Nat Genet 1998;20:207–11.

24. Albertson DG, Ylstra B, Segraves R, et al. Quantitative mapping of ampliconstructure by array CGH identifies CYP24 as a candidate oncogene. Nat Genet2000;25:144–6.

25. Fiegler H, Carr P, Douglas EJ, et al. DNA microarrays for comparative genomichybridization based on DOP-PCR amplification of BAC and PAC clones. GenesChromosomes Cancer 2003;36:361–74.

26. Abdel-Rahman WM, Katsura K, Rens W, et al. Spectral karyotyping suggestsadditional subsets of colorectal cancers characterized by pattern of chromosomerearrangement. Proc Natl Acad Sci USA 2001;98:2538–43.

27. Rowan AJ, Lamlum H, Ilyas M, et al. APC mutations in sporadic colorectal tumors:A mutational “hotspot” and interdependence of the “two hits.” Proc Natl Acad SciUSA 2000;97:3352–7.

28. Halford S, Sasieni P, Rowan A, et al. Low-level microsatellite instability occurs inmost colorectal cancers and is a nonrandomly distributed quantitative trait. CancerRes 2002;62:53–7.

29. Jain AN, Tokuyasu TA, Snijders AM, et al. Fully automatic quantification ofmicroarray image data. Genome Res 2002;12:325–32.

30. Sieber OM, Heinimann K, Gorman P, et al. Analysis of chromosomal instability inhuman colorectal adenomas with two mutational hits at APC. Proc Natl Acad SciUSA 2002;99:16910–5.

31. Gazzaniga P, Gradilone A, Giuliani L, et al. Expression and prognostic significanceof LIVIN, SURVIVIN and other apoptosis-related genes in the progression ofsuperficial bladder cancer. Ann Oncol 2003;14:85–90.

32. Ashhab Y, Alian A, Polliack A, Panet A, Ben Yehuda D. Two splicing variants of anew inhibitor of apoptosis gene with different biological properties and tissue distri-bution pattern. FEBS Lett 2001;495:56–60.

4824




33. Kamalati T, Jolin HE, Mitchell PJ, et al. Brk, a breast tumor-derived nonreceptorprotein-tyrosine kinase, sensitizes mammary epithelial cells to epidermal growthfactor. J Biol Chem 1996;271:30956–63.

34. Llor X, Serfas MS, Bie W, et al. BRK/Sik expression in the gastrointestinal tract andin colon tumors. Clin Cancer Res 1999;5:1767–77.

35. Nourse CR, Mattei MG, Gunning P, Byrne JA. Cloning of a third member of the D52gene family indicates alternative coding sequence usage in D52-like transcripts.Biochim Biophys Acta 1998;1443:155–68.

36. Anand N, Murthy S, Amann G, et al. Protein elongation factor EEF1A2 is a putativeoncogene in ovarian cancer. Nat Genet 2002;31:301–5.

37. Reilly JT. FLT3 and its role in the pathogenesis of acute myeloid leukaemia. LeukLymphoma 2003;44:1–7.

38. Zhang H, Wu J, Meng L, Shou CC. Expression of vascular endothelial growth factorand its receptors KDR and Flt-1 in gastric cancer cells. World J Gastroenterol2002;8:994–8.

39. Wu H, Li Y, Zhu G, et al. Expression of vascular endothelial growth factor and itsreceptor (Flt-1) in breast carcinoma [in Chinese]. Zhonghua Yi Xue Za Zhi 2002;82:708–11.

40. Eppert K, Scherer SW, Ozcelik H, et al. MADR2 maps to 18q21 and encodes aTGFbeta-regulated MAD-related protein that is functionally mutated in colorectalcarcinoma. Cell 1996;86:543–52.

41. Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene athuman chromosome 18q21.1. Science (Wash DC) 1996;271:350–3.

42. Thiagalingam S, Lengauer C, Leach FS, et al. Evaluation of candidate tumoursuppressor genes on chromosome 18 in colorectal cancers. Nat Genet 1996;13:343–6.

43. Page G, Lodige I, Kogel D, Scheidtmann KH. AATF, a novel transcription factor thatinteracts with Dlk/ZIP kinase and interferes with apoptosis. FEBS Lett 1999;462:187–91.

44. Pei L, Peng Y, Yang Y, et al. PRC17, a novel oncogene encoding a Rab GTPase-activating protein, is amplified in prostate cancer. Cancer Res 2002;62:5420–4.

45. Kania J, Konturek SJ, Marlicz K, Hahn EG, Konturek PC. Expression of survivin andcaspase-3 in gastric cancer. Dig Dis Sci 2003;48:266–71.

46. Chen L, O’Bryan JP, Smith HS, Liu E. Overexpression of matrix Gla protein mRNAin malignant human breast cells: isolation by differential cDNA hybridization. On-cogene 1990;5:1391–5.

47. Mao M, Yu M, Tong JH, et al. RIG-E, a human homolog of the murine Ly-6 family,is induced by retinoic acid during the differentiation of acute promyelocytic leukemiacell. Proc Natl Acad Sci USA 1996;93:5910–4.

48. Ruppert JM, Kinzler KW, Wong AJ, et al. The GLI-Kruppel family of human genes.Mol Cell Biol 1988;8:3104–13.

49. Fynan TM, Reiss M. Resistance to inhibition of cell growth by transforming growthfactor-beta and its role in oncogenesis. Crit Rev Oncog 1993;4:493–540.

50. Markowitz SD, Roberts AB. Tumor suppressor activity of the TGF-beta pathway inhuman cancers. Cytokine Growth Factor Rev 1996;7:93–102.

51. Saltzman A, Munro R, Searfoss G, et al. Transforming growth factor-beta-mediatedapoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activationand Bcl-XL downregulation. Exp Cell Res 1998;242:244–54.

52. Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression andcancer progression. Nat Genet 2001;29:117–29.

53. Won J, Kim DY, La M, et al. Cleavage of 14-3-3 protein by caspase-3 facilitates badinteraction with Bcl-x(L) during apoptosis. J Biol Chem 2003;278:19347–51.

54. Katsuda K, Kataoka M, Uno F, et al. Activation of caspase-3 and cleavage of Rb areassociated with p16-mediated apoptosis in human non-small cell lung cancer cells.Oncogene 2002;21:2108–13.

55. Yilmaz A, Kliche S, Mayr-Beyrle U, Fellbrich G, Waltenberger J. p38 MAPKinhibition is critically involved in VEGFR-2-mediated endothelial cell survival.Biochem Biophys Res Commun 2003;306:730–6.

56. Chang F, Steelman LS, McCubrey JA. Raf-induced cell cycle progression in humanTF-1 hematopoietic cells. Cell Cycle 2002;1:220–6.

57. Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh DA. Role of Raf in vascularprotection from distinct apoptotic stimuli. Science (Wash DC) 2003;301:94–6.

58. Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase Raf-1 to mitochondria.Cell 1996;87:629–38.

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2004;64:4817-4825. Cancer Res Eleanor J. Douglas, Heike Fiegler, Andrew Rowan, et al. Colorectal Cancer Cell Lines and Primary CarcinomasArray Comparative Genomic Hybridization Analysis of

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