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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2003 American Association for Cancer Research. ARRAY CGH IN BLADDER CANCER 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 transfor- mation. 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).

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