Array-Based Comparative Genomic Hybridization for Genome-Wide Screening of DNA Copy Number in Bladder Tumors1

[CANCER RESEARCH 63, 2872–2880, June 1, 2003] Array-based Comparative Genomic Hybridization for Genome-Wide Screening of DNA Copy Number in Bladder Tumors1

Joris A. Veltman, Jane Fridlyand, Sunanda Pejavar, Adam B. Olshen, James E. Korkola, Sandy DeVries, Peter Carroll, Wen-Lin Kuo, Daniel Pinkel, Donna Albertson, Carlos Cordon-Cardo, Ajay N. Jain, and Frederic M. Waldman2 Cancer Center [J. F., S. P., A. B. O., J. E. K., S. D., W-L. K.]; and Departments of Laboratory Medicine [D. P., D. A., A. N. J., F. M. W.] and Urology [P. C.], University of California-San Francisco, San Francisco, California 94143-0808; Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, the Netherlands [J. A. V.]; and Department of Epidemiology and Biostatistics [A. B. O.] and Department of Pathology [C. C-C.], Memorial Sloan-Kettering Cancer Center, New York, New York

ABSTRACT analysis of copy number changes at high resolution throughout the genome (1, 2). This quantitative measurement of DNA copy number Genome-wide copy number profiles were characterized in 41 primary across the genome may facilitate oncogene identification (3) and can bladder tumors using array-based comparative genomic hybridization also be used for tumor classification (4). (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic In this study, we have used array-based CGH for high resolution regions, some with high-level amplifications or homozygous deletions. mapping of copy number changes in different stages of bladder High-level amplifications were detected for 192 genomic clones, most carcinogenesis in 41 primary human tumors. Although a substantial frequently at 6p22.3 (E2F3), 8p12 (FGFR1), 8q22.2 (CMYC), 11q13 body of work has suggested that low-stage tumors differ from muscle (CCND1, EMS1, INT2), and 19q13.1 (CCNE). Homozygous deletions were invasive tumors in their genetic alterations (5–10), our array CGH detected in 51 genomic clones, with four showing deletions in more than data did not show a significant association of genomic copy number one case: two clones mapping to 9p21.3 (CDKN2A/p16, in nine cases), one alterations with tumor stage or grade. However, the high resolution of at 8p23.1 (three cases), and one at 11p13 (two cases). Significant correla- the array CGH technology allowed a precise identification of ampli- tions were observed between copy number gain of clones containing cons and regions containing homozygous deletions throughout the CCNE1 and gain of ERBB2, and between gain of CCND1 and deletion of bladder cancer genome. Analysis of the patterns of alterations among TP53. In addition, there was a significant complementary association between gain of CCND1 and gain of E2F3. Although there was no signif- pairs of known oncogenes and tumor suppressors revealed significant icant relationship between copy number changes and tumor stage or correlations between loci and concordant or complementary categor- grade, the linked behavior among genomic loci suggests that array CGH ical behavior. These relationships agree with what is known about the will be increasingly important in understanding pathways critical to relevant pathway biology and suggest that the ability to define bladder tumor biology. genomic alterations at high resolution, genome-wide, in larger sets of primary tumors may be an effective means for further elucidating the structure of pathways important in the progression of bladder cancer. INTRODUCTION

Bladder cancer is a major cause of morbidity and mortality in the MATERIALS AND METHODS United States and Europe. Approximately 75% of cases are superficial Tissue (pTa,pT1,pTIS), 20% muscle-infiltrating (pT2–T4), and 5% metastatic at the time of diagnosis. The ability to identify carcinomas more likely A series of 9 Ta,7T1, and 25 T2-T4 freshly frozen bladder tumors were to recur and/or progress would allow more aggressive treatment of obtained from the tissue banks of the UCSF Cancer Center and the Memorial these cases, which might lead to reduced morbidity and mortality from Sloan-Kettering Cancer Center. An initial H&E-stained frozen section was bladder cancer. A better understanding of the underlying molecular examined to allow trimming of the block for exclusion of normal or necrotic mechanisms leading to tumor formation and progression could result tissue. A tumor sample was considered suitable for study if the proportion of in the ability to identify more aggressive tumors, leading to improved tumor cells was higher than 70%. Ten 10 ␮m sections were cut for DNA survival and identification of potential therapeutic targets. extraction, followed by a final 5-␮m section for validation of tumor tissue The development and progression of bladder cancer is a multistep remaining in the block. Genomic DNA was obtained according to standard process, the result of a series of genetic alterations occurring over the procedures using proteinase K digestion and phenol-chloroform extraction. lifetime of a tumor. The acquisition of chromosomal abnormalities by Normal DNA was isolated from lymphocytes of healthy persons and was used as reference for two-color hybridizations. tumor cells is a central event in carcinogenesis and one that frequently decides the future malignant potential of a cancer. The search for Array-based CGH specific alterations associated with the development and progression of solid tumors involves an intensive analysis of known genes and a Two arrays were used in this study.4 The first (Array1) consisted of 1777 search for genes the roles of which were previously unappreciated. clones covering the human genome at roughly a 1.5-Mb resolution [HumArray Multiple studies have identified the prevalence and clinical signifi- 1.11 (2)]. The second array (Array2) consisted of 380 clones specifically selected to contain important tumor suppressor and oncogene loci. The clones cance of a limited number of genetic markers in bladder cancer. The 3 on Array1 were prepared by ligation-mediated PCR as recently described by recently developed array CGH technique allows high throughput Snijders et al. (2). DNA clones were robotically spotted in triplicate onto chromium-coated glass slides (PTI or Nanofilm), followed by UV cross- Received 5/29/02; accepted 4/2/03. linking. For Array2, degenerate oligonucleotide-primed PCR products from The costs of publication of this article were defrayed in part by the payment of page 380 large-insert clones were robotically spotted in quadruplicate onto three- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. dimensional link-activated slides [Surmodics, Inc., Eden Prairie, MN; accord- 1 Supported by National Cancer Institute Grant R01CA47537. ing to Hodgson et al. (11)]. These slides underwent several pretreatment steps 2 To whom requests for reprints should be addressed, at University of California-San to block nonspecific binding, and the DNA was denatured before use. Francisco, 2340 Sutter Street, Room S436, San Francisco, CA 94143-0808. Phone: (415) 476-3821; Fax: (415) 476-8218; E-mail: [email protected]. 3 The abbreviations used are: CGH, comparative genomic hybridization; TNF, tumor 4 For a listing of clones used, see http://cc.ucsf.edu/people/waldman/Veltman. necrosis factor; UCSC, University of California-Santa Cruz; RB, retinoblastoma. CGHarray.htm. 2872

Fig. 1. Normal versus normal control hybridizations. A, a representative genomic profile obtained from one of eight normal versus normal control hybridizations. Clones are ordered from chromosome 1 to 22 and within each chromosome on the basis of the UCSC mapping position (http:// genome.ucsc.edu/, version December 2000). Each dark square, the mean test over reference value of each clone after normalization and log2 transformation. Thresholds for copy number gain and loss Ϫ are shown at log2ratio of 0.2 and 0.2, respectively. Less than 10 of the 1745 clones included in the final data set crossed these thresholds for this control experiment. B, histogram of all of the ratios obtained in 8 normal versus normal control hybridizations. Thresholds for copy number gain and loss Ϫ were set at log2ratio of 0.2 and 0.2, respectively.

Each tumor sample was hybridized to both arrays, as described previously some clones were excluded from data analysis (sex-mismatched reference (1, 4), with modifications. One ␮g of tumor DNA was labeled by random samples were used for quality control). The final set, on which all of the priming with fluorolink cy3-dUTP, and normal reference DNA was labeled in analyses were performed, contained 1747 clones.5 the same fashion with cy5-dUTP (Amersham Pharmacia, Piscataway, NJ). Statistical Analysis. We considered three types of questions: (a) whether Unincorporated fluorescent nucleotides were removed using Sephadex G-50 there were associations between copy number alterations and tumor stage or spin columns. One-half of the labeled tumor sample was hybridized to Array1, grade; (b) whether gene pairs exhibited significant correlations; and (c) and the remainder was hybridized to Array2. Test and reference DNAs were whether gene pairs exhibited complementary or concordant behavior based on mixed with 100 ␮g of Cot-1 DNA (Life Technologies, Inc., Gaithersburg, a categorical analysis. The association analyses consisted of statistical corre- MD), were precipitated and were resuspended in 30–50 ␮l of a hybridization lation with permutation-based assessment of significance, visualization by solution containing 50% formamide, 10% dextran sulfate, 2ϫ SCC, 4% SDS, hierarchical clustering, and automatic pattern classification with cross-valida- and 100 ␮g tRNA. The hybridization solution was heated to 72°C for 10 min tion to assess predictive power, all as in Wilhelm et al. (4) and Olshen and Jain to denature the DNA and then was incubated for1hat37°C to allow blocking (12). For the gene pair correlations, we selected 24 clones containing known of the repetitive sequences. Hybridization was performed for 48 h in a moist bladder cancer oncogenes or tumor suppressors and 22 clones that were most chamber on a slowly rocking table, followed by a 15-min posthybridization frequently aberrant. In this analysis, the values of clones spanning the same wash in 50% formamide/2ϫ SSC at 45°C, and for 10 min in phosphate buffer gene were averaged. We computed the pair-wise correlations of copy number at room temperature. Slides were mounted in 90% glycerol in phosphate buffer for these clones. Permutation analysis under the null hypothesis of no associ- containing 4Ј,6-diamidino-2-phenylindole (DAPI; 0.3 ␮g/ml). ation between clones was performed to establish the appropriate significance Sixteen-bit fluorescence intensity images were obtained using a charged threshold for the correlation coefficient (4, 13). This form of permutation coupled device camera (Sensys, Photometrics, equipped with a Kodak KAF testing corrects for the multiple comparisons present in array analysis. We use 1400 chip) coupled to a 1ϫ magnification optical system. The acquired a conservative method that computes a null distribution of the maximum microarray images were analyzed using Genepix Pro 3.0 (Axon Instruments, magnitude correlation statistic across all genomic loci. We select our signifi- Inc., Foster City, CA). DNA spots were automatically segmented, local back- cance threshold to be at the 95th percentile of the permutation distribution ground was subtracted, and the total intensity and the intensity ratio of the two [details can be found in Olshen and Jain (12)]. Because the foregoing analysis dyes for each spot were calculated. Spots composed of less than nine pixels, captures only linear relationships between values on a continuous scale, we 2 Ͻ showing bad correlations of the two fluorescent dyes (Genepix 3.0, R 0.5), also performed a categorical analysis to define associations among loci. We or showing autofluorescent particles over the target were discarded. selected 10 gene pairs based on knowledge of signaling pathways (but inde- pendent of the array CGH data). These genes were also known a priori to be Data Analysis frequently gained or lost in bladder cancer. For each pair, we constructed 2 by Preprocessing. Log2 intensity ratios obtained for each array for each case 2 contingency tables with categories being “change” and “no change.” Change were individually centered by subtracting the median of log2 intensity ratios is defined as the most frequent aberration in a given gene (gain or loss using Ϯ for that case over all clones that met the quality control parameters described a conservative threshold of 0.25 log2 value). Change may represent gain in below. Data on the two arrays was then merged into one data set using the the first gene of a pair and loss in the second. In other words, we addressed genomic mapping information from all of the clones. There were 19 clones in whether change in one gene makes the change in the other less (complemen- common on the two arrays. A matched-pair t test on each of the 19 revealed tarity) or more (concordance) likely. The hypothesis was tested using an no clones with significantly different ratios at the 5% level. empirical null population of pairs constructed from the clones that were A series of eight normal versus normal hybridizations was used to define the frequently changed in the data set. Here, “frequent” was defined to be 25%. set of clones having consistently good hybridization quality (data not shown). The standard statistic for testing the difference in binomial proportions was For each analysis, clones were excluded for which none or only one spot used in the analysis. Null hypothesis of independence is not suitable here remained after the Genepix analysis. For all analyses, the 5% of clones with the because there is bias toward concordance among frequently changed clones. most extreme average test over reference ratio deviations from 1.0, and the 1% of clones with the largest SD in this set of normal controls was excluded. This 5 The data set is available at http://cc.ucsf.edu/people/waldman/Veltman. procedure resulted in the exclusion of 174 clones. In addition, all X-chromo- CGHarray.htm. 2873

Fig. 2. Genomic profiles from primary bladder cancers. Whole genome profiles are shown in A (stage Ta, G2), B (stage T1, G3), and C (stage T3, G3). Clones are ordered from chromosome 1 to 22 and within each chromosome on the basis of the UCSC mapping position (http://genome.ucsc.edu/, version December 2000). Each dark square, the mean test over reference value of each clone after normalization and log2 transformation. Thresholds for copy number gain and loss are shown at Ϫ log2ratio of 0.2 and 0.2, respectively. Individual chromosome profiles are shown in more detail below the entire genome profile. Vertical dashed lines, the centromeric location. In A, loss of one copy of the entire chromosome 2 is seen, except for a single pter clone. Five clones mapping to distal 2q appear to be homozygously deleted. A homozygous deletion of the 9p21 region is also seen in this case. For both cases B and C, deletion of distal 8p in combination with an amplicon just proximal to this region is present. An additional amplified region on 8q is present in case C. A complex chromosome 10 pattern is shown for case B, and a gain of the entire11q arm with multiple amplicons is shown for case C.

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Table 1 Copy number alterations and tumor stage

Normals All tumors Ta T1 T2–4 (n ϭ 8) (n ϭ 41) (n ϭ 9) (n ϭ 7) (n ϭ 25) Copy number gain Average number of clones 19a 192 129 243 200 Average genome size (kb)b 30,948 280,993 191,945 350,893 293,479 Average % of the genome 1.1% 9.9% 6.8% 12.4% 10.3% Copy number loss Average number of clones 32 226 212 354 196 Average genome size (kb) 55,568 338,778 316,636 504,271 300,412 Average % of the genome 2.0% 11.9% 11.1% 17.8% 10.6% Homozygous deletions Average number of clones 0 2280 Average genome size (kb) 0 2,667 4,228 7,451 765 Average % of the genome 0.0% 0.1% 0.1% 0.3% 0.0% High level amplifications Average number of clones 0 7448 Average genome size (kb) 0 7,188 2,946 4,337 9,513 Average % of the genome 0.0% 0.3% 0.1% 0.2% 0.3% Total genome covered (kb) 2,839,983 Number of clones 1,747 Average distance between clones (kb) 1,630 Maximum distance between clones (kb) 16,729 a Alterations were defined by log2ratio thresholds of 0.2 for copy number gain, Ϫ0.2 for loss, 1 for high-level amplification and Ϫ1 for homozygous deletion. b Size of a genomic alteration was defined as the sum of the affected clones, each representing one-half of the distance between its own center and that of its two neighboring clones.

RESULTS concordance among clones in which more than one clone contained the same gene. In a hierarchical clustering constructed without aver- Quality Control. We tested the quality of the CGH arrays and aging clones spanning the same gene (figure not shown), all of the intrinsic variability of the method by performing eight sex- gene pairs that spanned the same gene clustered together. The average mismatched normal versus normal hybridizations. These control hy- correlation for the 27 pairs of overlapping clones was 0.87, validating bridizations were performed simultaneously with the tumor analyses, the reproducibility of the array analysis within the tumor set. using the same batch of arrays, with identical labeling and hybridization conditions. The average replicate SD of these clones was 4%, and Genomic Profiles. Fig. 2 shows representative examples of the the average of the SE per clone, representing the average clone high-resolution analysis of the 41 transitional cell carcinomas of variability in the 8 normals, was 6% in this set of controls. Fig. 1 the bladder (9 Ta,7T1, and 25 T2– 4). Copy number gains and shows a representative profile and a histogram of the copy numbers losses can easily be detected for small chromosome regions, chro- across all clones in the 8 control experiments. On the basis of these mosome arms, and whole chromosomes. Small genomic regions Ͼ controls, we used thresholds of 0.2 and Ϫ0.2 (log2ratio) for calculat- showing high-level amplifications (defined as log2ratio 1), as ing the frequencies of genomic copy number gains and losses, respec- well as regions indicating homozygous deletions (defined as Ͻ tively, in the bladder tumor cases. log2ratio -1), can also be identified. As a further test of quality within the tumor data, we assessed the Copy number alterations are involved in a large fraction of most

Fig. 3. Genome-wide frequency of copy number alterations. The frequency of copy number gains (above 0, gray) and losses (below 0, black) throughout the genome for the 41 bladder cancers. Clones are ordered from chromosome 1 to 22 and within each chromosome on the basis of the UCSC mapping position (http://genome.ucsc.edu/, version December 2000). Diamonds at top, clones with at least one high-level amplification; diamonds at bot- tom, clones with at least one homozygous deletion.

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Table 2 Most frequently gained clones % of cases with Clone name Chromosome band Base position (kb)a Genes contained in clones copy number gainb RP11-167m06 1q24.2 Chr1:156,894 MPZL1, SAC 46% RP11-177m16 1q25.1 Chr1:164,589 GPR52 48% RP11-163n11 2p22.1 Chr2:38,637 50% RP11-229g06 3q24 Chr3:162,713 SMARCA3, CP 50% RP11-88E14 6p22.3 Chr6:17,673 SCA1, GMPR 44% RP11-22p23 6p22.3 Chr6:19,000 50% RP11-54h13 6p21.32 Chr6:35,350 HLA-DRB5/DQA1/DQA2 50% GS1-27F18 7q21.11 Chr7:78,000 GNAI1 57% GS1-207P11 7q21.11 Chr7:82,458 GRM3 44% RP11-27I15 8q21.3 Chr8:95,100 46% RP11-10G10 8q22.2 Chr8:99,811 DORFIN, POLR2K, SPAG1 49% RP11-142F22 8q22.2 Chr8:99,983 52% RP11-208E21 8q22.2 Chr8:100,226 59% VINT2_204G 11q13.3 Chr11:72,800 INT2 54% VLSI_MCL_271C 11q23 Chr11:129,748 LSI 45% CIT20B2328 20q12 Chr20:51,513 YWHAB 49% CIT20B2935 20q13.12 Chr20:55,271 PRKCBP1 44% VBAC_CAS_312B 20q13.2 Chr20:56,873 49% RMC20P071 20q13.3 Chr20:65,253 73% RMC20P073 20q13.3 Chr20:67,166 48% a Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. b Alterations were defined by log2ratio thresholds of 0.2 for copy number gain. Using this threshold, we generated a frequency Table. In this Table, the 20 most frequently gained clones are shown, ordered on chromosomal position. tumor genomes. To quantitate that fraction, each clone was assigned tion of copy number alterations is not uniform throughout the genome. a genomic distance equal to the sum of one-half of the distance Tables 2–5 list the most frequently gained or lost clones in this series between its own center and that of its two neighboring clones. The and those with high-level amplifications or homozygous deletions. average genomic distance between clones resulting from this calcu- High-level amplifications were identified in 22 of the 41 patients (2 ϳ lation was 1.6 Mb (Table 1). The entire tumor set involved an Ta,3T1, and 17 T2–4) and were present in 191 clones, of which 52 average of 281 Mb or 9.9% of the genome, and copy number loss showed amplification in more than one case. Oncogenes contained in affected 339 Mb or 11.9% of the genome. Only 8 of the 41 tumors clones with high-level amplifications included E2F3 (6p22.3), EGFR showed any copy number alterations in less than 10% of their genome (7p11.2), FGFR1 (8p12), CMYC (8q24.12-q24.22), CCND1/EMS/

(4 Ta,0T1,and4T2–4 cases). Although the number of tumors in each INT2 (11q13), MDM2 (12q14.3-q15), ERBB2 (17q12), JUNB group is small, there were differences in the extent of the genome (19p13.2), CCNE (19q13.11), and CYP24 (20q13.2). Homozygous affected among the tumor stages. The group of T1 tumors contained deletions were less common, with 74 clones identified in a total of 16 on average the highest fraction of genomic alterations, both copy cases (5 Ta,5T1,and6T2–4). Only four clones showed homozygous number gains and copy number losses (P Ͻ 0.01 for total changes). deletion in more than one case. Two were located within 1 Mb of each On average, superficial bladder tumors showed greater copy number other at the CDKN2A/p16 locus on 9p21.3, with nine cases having losses (11.1% of the genome) than gains (6.8%). High-level amplifi- homozygous deletion for one of these clones. The others were located cations were seen most frequently in the invasive tumors (0.3%). at 8p23.1 and 11p13. On average, each tumor contained seven clones Fig. 3 shows a frequency plot of gains and losses for all of the with high-level amplification and two clones with homozygous clones. Although a large part of the genome is affected, the distribu- deletion.

Table 3 Clones showing high-level amplifications Cases with high- Clone Chromosome band Base position (kb)a Gene level amplificationsb RP11-193j05 1q12 Chr1:156,000 3 RP11-43b04 6p22.3 Chr6:21,837 4 RP11-159c08 6p22.3 Chr6:22,387 E2F3 3 RP11-3d15 6p22.3 Chr6:23,564 3 RP11-273j01 6p22.3 Chr6:23,974 3 RP11-210F15 8p12 Ch8:47,866 3 RP11-102K07 8q22.2 Chr8:99,764 POLR2K, SPAG1 3 RP11-10G10 8q22.2 Chr8:99,811 DORFIN, POLR2K, SPAG1 4 V204H 11q13.3 Chr11:72,300 4 V204F 11q13.3 Chr11:72,400 3 RMC11B021 11q13.3 Chr11:72,414 INT2 4 V11B2685 11q13.3 Chr11:72,414 INT2 4 V204A 11q13.3 Chr11:72,600 5 CIT11B2555 11q13.3 Chr11:72,638 CCND1 4 V204D 11q13.3 Chr11:72,700 5 VINT2_204G 11q13.3 Chr11:72,800 INT2 5 V11B2684 11q13.3 Chr11:73,143 EMS1 3 VBAC_EMS1 11q13.3 Chr11:73,159 EMS1 3 RP11-120p20 11q13.3 Chr11:73,279 3 VPAK1_295A 11q13.5 Chr11:81,775 PAK1 4 VCCNE_278A 19q13.1 Chr19:30,935 CCNE1 3 V19B2708 19q13.1 Chr19:30,945 CCNE1 3 a Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. b Alterations were defined by log2ratio thresholds of 1 for high-level amplification. Using this threshold, we generated a frequency table. Clones with amplifications in at least three tumors are shown, ordered on chromosomal position. 2876

Table 4 Most frequently lost clones of these cases had deletions of the clone containing this gene in the

% of cases array CGH analyses, with 11 cases having log2ratio at or below the Chromosome Base position with copy Ϫ1 threshold for homozygous deletion and 3 cases with ratios indi- Clone band (kb)a Gene number lossb ϭϪ Ϫ Ϫ cating single copy loss (log2ratios 0.63, 0.68, 0.74). In RP11-117P11 8p23.3 Chr8:1,000 52% contrast, quantitative PCR analyses showed a normal copy number for RP11-240A17 8p23.3 Chr8:2,730 DLGAP2 59% ϭ RP11-287P18 8p23.1 Chr8:12,500 HE2, DEFB3 57% CDKN2A/p16 in 20 cases (mean log2ratio 0.01). In 18 of these RP11-93D21 8p21.3 Chr8:21,998 62% cases, normal copy number was also detected by array CGH; 1 case RP11-110I16 8p21.3 Chr8:23,170 51% ϭ RP11-95G21 9q22.2 Chr9:69,267 AUH 51% showed copy number gain (log2ratio 0.26); and 1 case showed a ϭϪ RP11-106O17 9q22.32 Chr9:74,000 51% copy number loss (log2ratio 0.21; mean log2ratio for 20 RP11-81P13 9q32 Chr9:89,729 TXN 58% cases ϭ 0.02). The copy number correlation between array CGH and RP11-10I09 9q32 Chr9:92,823 ALAD, CHRAC17 58% RP11-102F20 9q33.3 Chr9:98,257 51% quantitative PCR for this specific region was 0.98. RP11-85P21 9q33.3 Chr9:99,760 52% Statistical Associations. We explored three types of statistical RP11-245K09 11p15.2 Chr11:12,671 TEAD1 54% relationships within the data set to determine whether: (a) there were RP11-199K11 11p15.1 Chr11:18,000 54% RP11-80b10 11p15.1 Chr11:19,801 73% associations between copy number alterations and tumor stage or RP11-11A11 11p15.1 Chr11:20,607 51% grade; (b) gene pairs exhibited significant pair wise correlations; CIT11B2858 11p13 Chr11:34,466 WT1 51% RP11-103p20 11p13 Chr11:38,993 HSPC166 52% (c) gene pairs exhibited concordant or complementary categorical RP11-87e07 13q12.2 Chr13:5,654 SGCG, SACS 51% behavior (details in “Materials and Methods”). We found no signifi- RP11-19L03 18q21.1 Chr18:48,110 54% cant relationship between genomic copy number alterations and tumor a Based on UCSC mapping position (http://genome.ucsc.edu/), version December stage or grade using our methods. Given previous studies suggesting 2000. b Ϫ such a relationship (5–10) and given the large number of genomic loci Alterations were defined by log2ratio thresholds of 0.2 for copy number loss. Using this threshold, we generated a frequency table. In this Table, the 20 most frequently lost being measured, our results likely indicate an insufficient sample size clones are shown, ordered on chromosomal position. to reveal such associations (we have used statistical methods that correct for multiple testing). Our analysis of the correlation between gene pairs showed 22 Copy number gain at 6p22 was observed in 11 of the 41 cases significant correlations (P Ͻ 0.05) and 2 that almost met statistical analyzed. High-level amplifications were observed in three tumors at significance (P Ͻ 0.1; see Fig. 4). These associations are present even 6p22.3. In two of these cases, four clones spanning ϳ2.1 Mb were in the absence of T tumors, suggesting that they are not driven by involved; in one case, only two of these clones were involved; and a differences between tumor groups. Losses or gains of large genomic clone RP11-159C8 with the highest ratio (log2ratio ϭ 3.7) contained fragments account for significant correlations between 19 gene pairs E2F3. on chromosomes 9, 11, 18, and 20. More interestingly, significant Almost 50% of all of the bladder cancers showed a loss of the correlations were observed between copy number gain of ERBB2 11p13 region. Two tumors showed intensity ratios indicating homozy- (17q12) and gain of CCNE (19q13.11), between gain of AIB1 (20q12) gous deletions in this region, one case showed 17 deleted clones and loss of PTEN (10q23), between loss of ABL1 (9q34.2) and loss spanning ϳ14 Mb at 11p13 with clone RP11-187A8 having the of CDKN2A/p16 (9p21; possibly attributable to frequent loss of all of lowest ratio decrease. This clone was also the only one showing a chromosome 9), and between loss of TP53 (17p13.3) and gain log2ratio below Ϫ1 in the second tumor, again suggesting homozy- of CCND1/FGF3 (11q13; these last two significant at only P Ͻ 0.1). gous deletion. Further support for the involvement of this specific To determine whether paired genes from the same biological path- clone came from three tumor cases that showed only a deletion of this way showed complementary or concordant behavior (i.e., whether clone at the 11p13 region. This clone contains the TNF-associated only one alteration of genes in a pathway is sufficient to modify the factor 6 (TRAF6) and the human recombination-activating gene entire pathway or whether both of the alterations tend to occur RAG1. together), a direct categorical complementarity analysis of pairs of Array CGH also allowed the analysis of frequent break point genomic loci containing key components of important biological regions mapped to 8p12. Seventeen cases showed a pattern of tran- pathways was performed (TP53, MDM2, MYC, CDKN2A/p16, sition between distal loss and proximal gain in a small region com- CCND1, CCNE1, BCL2, CDK4, E2F3). Copy number gain of posed of 17 clones spanning ϳ9.2 Mb. In six of these cases, the break CCND1 behaved complementarily with gain of E2F3 (P Ͻ 0.05) and point was flanked by clones RP11-258M15 and RP11-274F14, a gain of CCNE1 (P Ͻ 0.1). E2F3 had a concordant relationship with region of 2.3 mb containing the candidate gene neuregulin 1 (NRG1). CCNE1 (P Ͻ 0.1). Finally, loss of TP53 showed complementarity A second break point, located 8 Mb proximal to the first one, was with gain of MDM2 (P Ͻ 0.1). The results were robust to the present in another 8 cases, spanning a distance of only 1 Mb. The most threshold chosen for declaring gain/loss. The significant pairs are promising candidate gene mapping to this break point region was shown in Fig. 5. FGFR1, a member of the fibroblast growth factor receptor family. Validation of Chromosome 9 Clones by Quantitative PCR. Quantitative real-time PCR analyses were performed on the same set of bladder cancers for the detection of copy number changes for seven Table 5 Clones showing homozygous deletions genes mapped to chromosome 9.6 Six of these genes were contained Cases with Chromosome Base position homozygous in clones present on the arrays used. There was a strong concurrence Clone band (kb)a Gene deletionsb between the log ratios for these clones obtained by array CGH and 2 RP11-287P18 8p23.1 Chr8:12500 3 quantitative PCR for the associated genes, with an overall correlation RMC09P007 9p21.3 Chr9:23395 P16/CDKI4 9 of 0.78. Fourteen homozygous deletions of the CDKN2A/p16 gene RP11-33O15 9p21.3 Chr9:24325 3 ϭϪ RP11-187A08 11p13 Chr11:39389 TRAF6/RAG1 2 were detected by quantitative PCR (mean log2ratio 2.16). All 14 a Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. 6 b Ϫ Veltman, J., Bjerke, L., Moore, D., Carroll, P., Chew, K., Sudilovsky, D., and Alterations were defined by log2ratio thresholds of 1 for homozygous deletion. Waldman, F. M. Chromosome 9 gene copy number and expression alterations in bladder Using this threshold, we generated a frequency table. Clones with deletions in at least two tumors, manuscript in preparation. tumors are shown, ordered on chromosomal position. 2877

Fig. 4. Gene correlation matrix. Twenty-four clones containing known bladder cancer oncogenes or tumor suppressor genes and 22 nonoverlapping clones that were most frequently aberrant were selected. The values of clones spanning the same gene were averaged. Permutation analysis was performed to establish the appropriate significance threshold for the correlation coefficient, and significant correlations are highlighted by yellow squares. The color scale reaches full saturation in green for significant positive correlations (copy number gain in one clone combined with copy number gain in the other clone or copy number loss in one clone combined with copy number loss in the other clone) and full saturation in red for significant negative correlations (copy number gain in one clone combined with copy number loss in the other clone). The gene names are printed in green when the log2ratio Ͼ Ͼ indicated a gain in 20% of cases, and the name is printed in red if the log2ratio indicated a loss in 20% of cases.

DISCUSSION CGH analysis strongly pointed to E2F3 as the target gene on 6p22. E2F3 is thought to be sequestered by unphosphorylated retinoblas- This study represents one of the first applications of genome-wide toma protein and then released after RB phosphorylation by the copy number analysis by array CGH. Snijders et al. (2) recently CCND1/CDK4 complex in the G phase of the cell cycle (15). Once showed the potential for this technology to provide precise copy 1 released, E2F3 is thought to act as a transcriptional regulator at the number measurements using an overlapping set of clones. The key G -S phase transition (16–18). E2F-1 has been implicated in bladder biological value of high-resolution array CGH lies in its ability to 1 detect small amplicons and deletions that potentially harbor specific carcinogenesis, but alterations of this gene appear to occur at the oncogenes and tumor suppressor genes. High-level amplifications epigenetic level (19). involving clones containing known oncogenes such as EGFR, CMYC, A frequently deleted clone mapping to 11p13 contained both the CCND1, and CCNE1 were easily detected, as well as small homozy- TNF-associated factor 6 (TRAF6) and the human recombination gous deletions containing the CDKN2A/p16 tumor suppressor gene at activating gene RAG1. TRAF proteins are thought to be important 9p21.3. regulators of cell death and responses to stress. The RAG proteins Our analysis of clones containing high-level amplifications or ho- are known to initiate V(D)J recombination by facilitating double- mozygous deletions revealed two interesting candidate genes: tran- stranded breaks. TRAF6 may be a more likely deletion target than scription factor E2F3 on 6p22 and TNF-associated factor 6 (TRAF6) is RAG1 in these tumors because it is presumed to function as a on 11p13. High-level amplifications as well as frequent copy number tumor suppressor. gain at 6p22 have previously been reported by others by low-resolu- Frequent gains and losses could be defined at high resolution tion chromosomal CGH without oncogene identification (14). Array using array analysis, allowing a precise mapping of these genomic 2878

Fig. 5. Gene complementarity/concordance analysis. Examples of the complementarity analysis of four gene pairs are shown; CCND1 versus E2F3, CCND1 versus CCNE1, E2F3 versus CCNE1, and p53 versus MDM2. Each dark square, the log2ratio value of the clone containing one of these genes for one case on the X-axis and the value for the other gene on the Y-axis. Thresholds for copy number gain and loss are shown at log2ratio of 0.25 and Ϫ0.25, respectively. E2F3/CCNE1 display concordant behavior, with the other three pairs exhibiting complementarity. Significance was assessed using an empirical null population of pairs constructed from the clones that were frequently changed in the data set (see “Materials and Methods”).

regions. For example, two frequent break points on chromosome 8 complementary association between copy number gain of the locus were identified, containing candidate target genes neuregulin 1 containing cyclin D1 and gain of the transcription factor E2F3 (NRG1) and fibroblast growth factor receptor 1 (FGFR1). NRG1 locus. Also, there was a trend for complementarity between gain of interacts with the NEU/ERB family of receptor tyrosine kinases, the CCND1 and gain of CCNE1 loci. This is consistent with the known to be frequently overexpressed in bladder tumors. FGFR1 is idea that amplification of CCND1 and either CCNE1 or E2F3 may a member of the fibroblast growth factor receptor family. Specific activate the RB pathway, but there is no advantage for more binding of fibroblast growth factors to these cell surface-expressed combined gene amplification. Geng et al. (28) showed that cyclin receptors activates tyrosine kinase activity. This activation allows E can functionally replace cyclin D1 and suggested that cyclin E is coupling to downstream signal transduction pathways that regulate the key downstream effector of cyclin D1. High-throughput tissue proliferation, migration, and differentiation of endothelial cells, microarray analysis of a large series of bladder cancers (26) thus enhancing angiogenesis (20). A recent study by Simon et al. showed that cyclin E gene amplification was present in a subset of (21) using fluorescence in situ hybridization (FISH)-based high- bladder carcinomas, especially during early invasion. These find- throughput tissue microarray analysis also showed frequent alter- ings suggest that the RB pathway appears to be deregulated in a ations of this gene in bladder cancers. majority of the bladder cancers by a gene dosage increase of one Associations of alterations in pairs of known oncogenes and of the activators cyclin D1, E2F3, or cyclin E. tumor suppressors could be characterized using array-based CGH. Significant correlation was observed in the gain of clones contain- Genes functioning upstream or downstream of each other in the Ͻ same biological pathway may exhibit complementary alteration, ing ERBB2 (17q12) and CCNE1 (19q13; P 0.05). Activation of because an alteration of a single gene in this pathway may suffice ERBB2 receptor tyrosine kinase pathway is thought to lie upstream for altering the effects of the entire pathway (e.g., enhanced from cyclin E activation; therefore, it is unclear why the gain of both proliferation, apoptosis). The retinoblastoma tumor suppressor genes would be selected for during tumor progression. It is possible, pathway plays a critical role in the control of cellular proliferation for example, that CCNE activation may play a larger role in the by regulating the activity of the E2F family of transcription factors presence of activated receptor pathway. A different relationship may (22–23). Many of the genes involved in this pathway appear to be explain the correlation between cyclin D1 gain with p53 loss in affected in bladder carcinogenesis, most notably cyclin D1 and individual tumors. Cells with both alterations may have a selective cyclin E by amplification, and CDKN2A/p16 and RB1 by inacti- advantage because they play key roles in different cellular pathways vation (24–27). Clear associations were observed in a number of controlling proliferation and programmed cell death, respectively. members of the RB proliferation pathway. There was a significant Interestingly the categorical analysis revealed a trend for complemen- 2879

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Joris A. Veltman, Jane Fridlyand, Sunanda Pejavar, et al.

Cancer Res 2003;63:2872-2880.

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