[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 Genome-wide copy number profiles were characterized in 41 primary bladder tumors using array-based comparative genomic hybridization (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic regions, some with high-level amplifications or homozygous deletions. High-level amplifications were detected for 192 genomic clones, most frequently at 6p22.3 (E2F3), 8p12 (FGFR1), 8q22.2 (CMYC), 11q13 (CCND1, EMS1, INT2), and 19q13.1 (CCNE). Homozygous deletions were detected in 51 genomic clones, with four showing deletions in more than one case: two clones mapping to 9p21.3 (CDKN2A/p16, in nine cases), one at 8p23.1 (three cases), and one at 11p13 (two cases). Significant correlations were observed between copy number gain of clones containing CCNE1 and gain of ERBB2, and between gain of CCND1 and deletion of TP53. In addition, there was a significant complementary association between gain of CCND1 and gain of E2F3. Although there was no significant relationship between copy number changes and tumor stage or grade, the linked behavior among genomic loci suggests that array CGH will be increasingly important in understanding pathways critical to bladder tumor biology.
INTRODUCTION Bladder cancer is a major cause of morbidity and mortality in the United States and Europe. Approximately 75% of cases are superficial (pTa, pT1, pTIS), 20% muscle-infiltrating (pT2–T4), and 5% metastatic at the time of diagnosis. The ability to identify carcinomas more likely to recur and/or progress would allow more aggressive treatment of these cases, which might lead to reduced morbidity and mortality from bladder cancer. A better understanding of the underlying molecular mechanisms leading to tumor formation and progression could result in the ability to identify more aggressive tumors, leading to improved survival and identification of potential therapeutic targets. The development and progression of bladder cancer is a multistep process, the result of a series of genetic alterations occurring over the lifetime of a tumor. The acquisition of chromosomal abnormalities by tumor cells is a central event in carcinogenesis and one that frequently decides the future malignant potential of a cancer. The search for specific alterations associated with the development and progression of solid tumors involves an intensive analysis of known genes and a search for genes the roles of which were previously unappreciated. Multiple studies have identified the prevalence and clinical significance of a limited number of genetic markers in bladder cancer. The recently developed array CGH3 technique allows high throughput Received 5/29/02; accepted 4/2/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grant R01CA47537. 2 To whom requests for reprints should be addressed, at University of California-San 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 necrosis factor; UCSC, University of California-Santa Cruz; RB, retinoblastoma.
analysis of copy number changes at high resolution throughout the genome (1, 2). This quantitative measurement of DNA copy number across the genome may facilitate oncogene identification (3) and can also be used for tumor classification (4). In this study, we have used array-based CGH for high resolution mapping of copy number changes in different stages of bladder carcinogenesis in 41 primary human tumors. Although a substantial body of work has suggested that low-stage tumors differ from muscle invasive tumors in their genetic alterations (5–10), our array CGH data did not show a significant association of genomic copy number alterations with tumor stage or grade. However, the high resolution of the array CGH technology allowed a precise identification of amplicons and regions containing homozygous deletions throughout the bladder cancer genome. Analysis of the patterns of alterations among pairs of known oncogenes and tumor suppressors revealed significant correlations between loci and concordant or complementary categorical behavior. These relationships agree with what is known about the relevant pathway biology and suggest that the ability to define 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. MATERIALS AND METHODS Tissue A series of 9 Ta, 7 T1, and 25 T2-T4 freshly frozen bladder tumors were obtained from the tissue banks of the UCSF Cancer Center and the Memorial Sloan-Kettering Cancer Center. An initial H&E-stained frozen section was examined to allow trimming of the block for exclusion of normal or necrotic tissue. A tumor sample was considered suitable for study if the proportion of tumor cells was higher than 70%. Ten 10 m sections were cut for DNA extraction, followed by a final 5-m section for validation of tumor tissue remaining in the block. Genomic DNA was obtained according to standard procedures using proteinase K digestion and phenol-chloroform extraction. Normal DNA was isolated from lymphocytes of healthy persons and was used as reference for two-color hybridizations. Array-based CGH Two arrays were used in this study.4 The first (Array1) consisted of 1777 clones covering the human genome at roughly a 1.5-Mb resolution [HumArray 1.11 (2)]. The second array (Array2) consisted of 380 clones specifically selected to contain important tumor suppressor and oncogene loci. The clones on Array1 were prepared by ligation-mediated PCR as recently described by Snijders et al. (2). DNA clones were robotically spotted in triplicate onto chromium-coated glass slides (PTI or Nanofilm), followed by UV crosslinking. For Array2, degenerate oligonucleotide-primed PCR products from 380 large-insert clones were robotically spotted in quadruplicate onto threedimensional link-activated slides [Surmodics, Inc., Eden Prairie, MN; according to Hodgson et al. (11)]. These slides underwent several pretreatment steps to block nonspecific binding, and the DNA was denatured before use. 4 For a listing of clones used, see http://cc.ucsf.edu/people/waldman/Veltman. CGHarray.htm.
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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 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 were set at log2ratio of 0.2 and ⫺0.2, respectively.
Each tumor sample was hybridized to both arrays, as described previously (1, 4), with modifications. One g of tumor DNA was labeled by random priming with fluorolink cy3-dUTP, and normal reference DNA was labeled in the same fashion with cy5-dUTP (Amersham Pharmacia, Piscataway, NJ). Unincorporated fluorescent nucleotides were removed using Sephadex G-50 spin columns. One-half of the labeled tumor sample was hybridized to Array1, and the remainder was hybridized to Array2. Test and reference DNAs were mixed with 100 g of Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD), were precipitated and were resuspended in 30 –50 l of a hybridization solution containing 50% formamide, 10% dextran sulfate, 2⫻ SCC, 4% SDS, and 100 g tRNA. The hybridization solution was heated to 72°C for 10 min to denature the DNA and then was incubated for 1 h at 37°C to allow blocking of the repetitive sequences. Hybridization was performed for 48 h in a moist chamber on a slowly rocking table, followed by a 15-min posthybridization wash in 50% formamide/2⫻ SSC at 45°C, and for 10 min in phosphate buffer at room temperature. Slides were mounted in 90% glycerol in phosphate buffer containing 4⬘,6-diamidino-2-phenylindole (DAPI; 0.3 g/ml). Sixteen-bit fluorescence intensity images were obtained using a charged coupled device camera (Sensys, Photometrics, equipped with a Kodak KAF 1400 chip) coupled to a 1⫻ magnification optical system. The acquired microarray images were analyzed using Genepix Pro 3.0 (Axon Instruments, Inc., Foster City, CA). DNA spots were automatically segmented, local background was subtracted, and the total intensity and the intensity ratio of the two dyes for each spot were calculated. Spots composed of less than nine pixels, showing bad correlations of the two fluorescent dyes (Genepix 3.0, R2 ⬍ 0.5), or showing autofluorescent particles over the target were discarded. Data Analysis Preprocessing. Log2 intensity ratios obtained for each array for each case were individually centered by subtracting the median of log2 intensity ratios for that case over all clones that met the quality control parameters described below. Data on the two arrays was then merged into one data set using the genomic mapping information from all of the clones. There were 19 clones in common on the two arrays. A matched-pair t test on each of the 19 revealed no clones with significantly different ratios at the 5% level. A series of eight normal versus normal hybridizations was used to define the set of clones having consistently good hybridization quality (data not shown). For each analysis, clones were excluded for which none or only one spot remained after the Genepix analysis. For all analyses, the 5% of clones with the 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 procedure resulted in the exclusion of 174 clones. In addition, all X-chromo-
some clones were excluded from data analysis (sex-mismatched reference samples were used for quality control). The final set, on which all of the analyses were performed, contained 1747 clones.5 Statistical Analysis. We considered three types of questions: (a) whether there were associations between copy number alterations and tumor stage or grade; (b) whether gene pairs exhibited significant correlations; and (c) whether gene pairs exhibited complementary or concordant behavior based on a categorical analysis. The association analyses consisted of statistical correlation with permutation-based assessment of significance, visualization by hierarchical clustering, and automatic pattern classification with cross-validation to assess predictive power, all as in Wilhelm et al. (4) and Olshen and Jain (12). For the gene pair correlations, we selected 24 clones containing known bladder cancer oncogenes or tumor suppressors and 22 clones that were most frequently aberrant. In this analysis, the values of clones spanning the same gene were averaged. We computed the pair-wise correlations of copy number for these clones. Permutation analysis under the null hypothesis of no association between clones was performed to establish the appropriate significance threshold for the correlation coefficient (4, 13). This form of permutation testing corrects for the multiple comparisons present in array analysis. We use a conservative method that computes a null distribution of the maximum magnitude correlation statistic across all genomic loci. We select our significance threshold to be at the 95th percentile of the permutation distribution [details can be found in Olshen and Jain (12)]. Because the foregoing analysis captures only linear relationships between values on a continuous scale, we also performed a categorical analysis to define associations among loci. We selected 10 gene pairs based on knowledge of signaling pathways (but independent of the array CGH data). These genes were also known a priori to be frequently gained or lost in bladder cancer. For each pair, we constructed 2 by 2 contingency tables with categories being “change” and “no change.” Change is defined as the most frequent aberration in a given gene (gain or loss using a conservative threshold of ⫾0.25 log2 value). Change may represent gain in the first gene of a pair and loss in the second. In other words, we addressed whether change in one gene makes the change in the other less (complementarity) or more (concordance) likely. The hypothesis was tested using an empirical null population of pairs constructed from the clones that were frequently changed in the data set. Here, “frequent” was defined to be 25%. The standard statistic for testing the difference in binomial proportions was used in the analysis. Null hypothesis of independence is not suitable here because there is bias toward concordance among frequently changed clones. 5 The data CGHarray.htm.
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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 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 All tumors (n ⫽ 41)
Ta (n ⫽ 9)
T1 (n ⫽ 7)
T2–4 (n ⫽ 25)
19a 30,948 1.1%
192 280,993 9.9%
129 191,945 6.8%
243 350,893 12.4%
200 293,479 10.3%
32 55,568 2.0%
226 338,778 11.9%
212 316,636 11.1%
354 504,271 17.8%
196 300,412 10.6%
0 0 0.0%
2 2,667 0.1%
2 4,228 0.1%
8 7,451 0.3%
0 765 0.0%
0 0 0.0%
7 7,188 0.3%
4 2,946 0.1%
4 4,337 0.2%
8 9,513 0.3%
Normals (n ⫽ 8) Copy number gain Average number of clones Average genome size (kb)b Average % of the genome Copy number loss Average number of clones Average genome size (kb) Average % of the genome Homozygous deletions Average number of clones Average genome size (kb) Average % of the genome High level amplifications Average number of clones Average genome size (kb) Average % of the genome Total genome covered (kb) Number of clones Average distance between clones (kb) Maximum distance between clones (kb) a b
2,839,983 1,747 1,630 16,729
Alterations were defined by log2ratio thresholds of 0.2 for copy number gain, ⫺0.2 for loss, 1 for high-level amplification and ⫺1 for homozygous deletion. 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 Quality Control. We tested the quality of the CGH arrays and intrinsic variability of the method by performing eight sexmismatched normal versus normal hybridizations. These control hybridizations were performed simultaneously with the tumor analyses, using the same batch of arrays, with identical labeling and hybridization conditions. The average replicate SD of these clones was 4%, and the average of the SE per clone, representing the average clone variability in the 8 normals, was 6% in this set of controls. Fig. 1 shows a representative profile and a histogram of the copy numbers across all clones in the 8 control experiments. On the basis of these controls, we used thresholds of 0.2 and ⫺0.2 (log2ratio) for calculating the frequencies of genomic copy number gains and losses, respectively, in the bladder tumor cases. As a further test of quality within the tumor data, we assessed the
concordance among clones in which more than one clone contained the same gene. In a hierarchical clustering constructed without averaging clones spanning the same gene (figure not shown), all of the gene pairs that spanned the same gene clustered together. The average correlation for the 27 pairs of overlapping clones was 0.87, validating the reproducibility of the array analysis within the tumor set. Genomic Profiles. Fig. 2 shows representative examples of the high-resolution analysis of the 41 transitional cell carcinomas of the bladder (9 Ta, 7 T1, and 25 T2– 4). Copy number gains and losses can easily be detected for small chromosome regions, chromosome arms, and whole chromosomes. Small genomic regions showing high-level amplifications (defined as log2ratio ⬎1), as well as regions indicating homozygous deletions (defined as log2ratio ⬍-1), can also be identified. 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 bottom, clones with at least one homozygous deletion.
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Table 2 Most frequently gained clones Clone name
Chromosome band
Base position (kb)a
RP11-167m06 RP11-177m16 RP11-163n11 RP11-229g06 RP11-88E14 RP11-22p23 RP11-54h13 GS1-27F18 GS1-207P11 RP11-27I15 RP11-10G10 RP11-142F22 RP11-208E21 VINT2_204G VLSI_MCL_271C CIT20B2328 CIT20B2935 VBAC_CAS_312B RMC20P071 RMC20P073
1q24.2 1q25.1 2p22.1 3q24 6p22.3 6p22.3 6p21.32 7q21.11 7q21.11 8q21.3 8q22.2 8q22.2 8q22.2 11q13.3 11q23 20q12 20q13.12 20q13.2 20q13.3 20q13.3
Chr1:156,894 Chr1:164,589 Chr2:38,637 Chr3:162,713 Chr6:17,673 Chr6:19,000 Chr6:35,350 Chr7:78,000 Chr7:82,458 Chr8:95,100 Chr8:99,811 Chr8:99,983 Chr8:100,226 Chr11:72,800 Chr11:129,748 Chr20:51,513 Chr20:55,271 Chr20:56,873 Chr20:65,253 Chr20:67,166
Genes contained in clones MPZL1, SAC GPR52 SMARCA3, CP SCA1, GMPR HLA-DRB5/DQA1/DQA2 GNAI1 GRM3 DORFIN, POLR2K, SPAG1
INT2 LSI YWHAB PRKCBP1
% of cases with copy number gainb 46% 48% 50% 50% 44% 50% 50% 57% 44% 46% 49% 52% 59% 54% 45% 49% 44% 49% 73% 48%
a
Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. 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. b
tumor genomes. To quantitate that fraction, each clone was assigned a genomic distance equal to the sum of one-half of the distance between its own center and that of its two neighboring clones. The average genomic distance between clones resulting from this calculation was ⬃1.6 Mb (Table 1). The entire tumor set involved an average of 281 Mb or 9.9% of the genome, and copy number loss affected 339 Mb or 11.9% of the genome. Only 8 of the 41 tumors showed any copy number alterations in less than 10% of their genome (4 Ta, 0 T1, and 4 T2– 4 cases). Although the number of tumors in each group is small, there were differences in the extent of the genome affected among the tumor stages. The group of T1 tumors contained on average the highest fraction of genomic alterations, both copy number gains and copy number losses (P ⬍ 0.01 for total changes). On average, superficial bladder tumors showed greater copy number losses (11.1% of the genome) than gains (6.8%). High-level amplifications were seen most frequently in the invasive tumors (0.3%). Fig. 3 shows a frequency plot of gains and losses for all of the clones. Although a large part of the genome is affected, the distribu-
tion of copy number alterations is not uniform throughout the genome. Tables 2–5 list the most frequently gained or lost clones in this series and those with high-level amplifications or homozygous deletions. High-level amplifications were identified in 22 of the 41 patients (2 Ta, 3 T1, and 17 T2– 4) and were present in 191 clones, of which 52 showed amplification in more than one case. Oncogenes contained in clones with high-level amplifications included E2F3 (6p22.3), EGFR (7p11.2), FGFR1 (8p12), CMYC (8q24.12-q24.22), CCND1/EMS/ INT2 (11q13), MDM2 (12q14.3-q15), ERBB2 (17q12), JUNB (19p13.2), CCNE (19q13.11), and CYP24 (20q13.2). Homozygous deletions were less common, with 74 clones identified in a total of 16 cases (5 Ta, 5 T1, and 6 T2– 4). Only four clones showed homozygous deletion in more than one case. Two were located within 1 Mb of each other at the CDKN2A/p16 locus on 9p21.3, with nine cases having homozygous deletion for one of these clones. The others were located at 8p23.1 and 11p13. On average, each tumor contained seven clones with high-level amplification and two clones with homozygous deletion.
Table 3 Clones showing high-level amplifications Clone
Chromosome band
Base position (kb)a
RP11-193j05 RP11-43b04 RP11-159c08 RP11-3d15 RP11-273j01 RP11-210F15 RP11-102K07 RP11-10G10 V204H V204F RMC11B021 V11B2685 V204A CIT11B2555 V204D VINT2_204G V11B2684 VBAC_EMS1 RP11-120p20 VPAK1_295A VCCNE_278A V19B2708
1q12 6p22.3 6p22.3 6p22.3 6p22.3 8p12 8q22.2 8q22.2 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.3 11q13.5 19q13.1 19q13.1
Chr1:156,000 Chr6:21,837 Chr6:22,387 Chr6:23,564 Chr6:23,974 Ch8:47,866 Chr8:99,764 Chr8:99,811 Chr11:72,300 Chr11:72,400 Chr11:72,414 Chr11:72,414 Chr11:72,600 Chr11:72,638 Chr11:72,700 Chr11:72,800 Chr11:73,143 Chr11:73,159 Chr11:73,279 Chr11:81,775 Chr19:30,935 Chr19:30,945
a
Gene
E2F3
POLR2K, SPAG1 DORFIN, POLR2K, SPAG1
INT2 INT2 CCND1 INT2 EMS1 EMS1 PAK1 CCNE1 CCNE1
Cases with highlevel amplificationsb 3 4 3 3 3 3 3 4 4 3 4 4 5 4 5 5 3 3 3 4 3 3
Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. 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. b
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Table 4 Most frequently lost clones
Clone
Chromosome band
Base position (kb)a
RP11-117P11 RP11-240A17 RP11-287P18 RP11-93D21 RP11-110I16 RP11-95G21 RP11-106O17 RP11-81P13 RP11-10I09 RP11-102F20 RP11-85P21 RP11-245K09 RP11-199K11 RP11-80b10 RP11-11A11 CIT11B2858 RP11-103p20 RP11-87e07 RP11-19L03
8p23.3 8p23.3 8p23.1 8p21.3 8p21.3 9q22.2 9q22.32 9q32 9q32 9q33.3 9q33.3 11p15.2 11p15.1 11p15.1 11p15.1 11p13 11p13 13q12.2 18q21.1
Chr8:1,000 Chr8:2,730 Chr8:12,500 Chr8:21,998 Chr8:23,170 Chr9:69,267 Chr9:74,000 Chr9:89,729 Chr9:92,823 Chr9:98,257 Chr9:99,760 Chr11:12,671 Chr11:18,000 Chr11:19,801 Chr11:20,607 Chr11:34,466 Chr11:38,993 Chr13:5,654 Chr18:48,110
Gene DLGAP2 HE2, DEFB3
AUH TXN ALAD, CHRAC17
TEAD1
WT1 HSPC166 SGCG, SACS
% of cases with copy number lossb 52% 59% 57% 62% 51% 51% 51% 58% 58% 51% 52% 54% 54% 73% 51% 51% 52% 51% 54%
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 loss. Using this threshold, we generated a frequency table. In this Table, the 20 most frequently lost clones are shown, ordered on chromosomal position.
Copy number gain at 6p22 was observed in 11 of the 41 cases analyzed. High-level amplifications were observed in three tumors at 6p22.3. In two of these cases, four clones spanning ⬃2.1 Mb were involved; in one case, only two of these clones were involved; and clone RP11-159C8 with the highest ratio (log2ratio ⫽ 3.7) contained E2F3. Almost 50% of all of the bladder cancers showed a loss of the 11p13 region. Two tumors showed intensity ratios indicating homozygous deletions in this region, one case showed 17 deleted clones spanning ⬃14 Mb at 11p13 with clone RP11-187A8 having the lowest ratio decrease. This clone was also the only one showing a log2ratio below ⫺1 in the second tumor, again suggesting homozygous deletion. Further support for the involvement of this specific clone came from three tumor cases that showed only a deletion of this clone at the 11p13 region. This clone contains the TNF-associated factor 6 (TRAF6) and the human recombination-activating gene RAG1. Array CGH also allowed the analysis of frequent break point regions mapped to 8p12. Seventeen cases showed a pattern of transition between distal loss and proximal gain in a small region composed of 17 clones spanning ⬃9.2 Mb. In six of these cases, the break point was flanked by clones RP11-258M15 and RP11-274F14, a region of 2.3 mb containing the candidate gene neuregulin 1 (NRG1). A second break point, located 8 Mb proximal to the first one, was present in another 8 cases, spanning a distance of only 1 Mb. The most promising candidate gene mapping to this break point region was 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 genes mapped to chromosome 9.6 Six of these genes were contained in clones present on the arrays used. There was a strong concurrence between the log2ratios for these clones obtained by array CGH and quantitative PCR for the associated genes, with an overall correlation of 0.78. Fourteen homozygous deletions of the CDKN2A/p16 gene were detected by quantitative PCR (mean log2ratio ⫽ ⫺2.16). All 14 6 Veltman, J., Bjerke, L., Moore, D., Carroll, P., Chew, K., Sudilovsky, D., and Waldman, F. M. Chromosome 9 gene copy number and expression alterations in bladder tumors, manuscript in preparation.
of these cases had deletions of the clone containing this gene in the array CGH analyses, with 11 cases having log2ratio at or below the ⫺1 threshold for homozygous deletion and 3 cases with ratios indicating single copy loss (log2ratios ⫽ ⫺0.63, ⫺0.68, ⫺0.74). In contrast, quantitative PCR analyses showed a normal copy number for CDKN2A/p16 in 20 cases (mean log2ratio ⫽ 0.01). In 18 of these cases, normal copy number was also detected by array CGH; 1 case showed copy number gain (log2ratio ⫽ 0.26); and 1 case showed a copy number loss (log2ratio ⫽ ⫺0.21; mean log2ratio for 20 cases ⫽ 0.02). The copy number correlation between array CGH and quantitative PCR for this specific region was 0.98. Statistical Associations. We explored three types of statistical relationships within the data set to determine whether: (a) there were associations between copy number alterations and tumor stage or grade; (b) gene pairs exhibited significant pair wise correlations; (c) gene pairs exhibited concordant or complementary categorical behavior (details in “Materials and Methods”). We found no significant relationship between genomic copy number alterations and tumor stage or grade using our methods. Given previous studies suggesting such a relationship (5–10) and given the large number of genomic loci being measured, our results likely indicate an insufficient sample size to reveal such associations (we have used statistical methods that correct for multiple testing). Our analysis of the correlation between gene pairs showed 22 significant correlations (P ⬍ 0.05) and 2 that almost met statistical significance (P ⬍ 0.1; see Fig. 4). These associations are present even in the absence of Ta tumors, suggesting that they are not driven by differences between tumor groups. Losses or gains of large genomic fragments account for significant correlations between 19 gene pairs on chromosomes 9, 11, 18, and 20. More interestingly, significant correlations were observed between copy number gain of ERBB2 (17q12) and gain of CCNE (19q13.11), between gain of AIB1 (20q12) and loss of PTEN (10q23), between loss of ABL1 (9q34.2) and loss of CDKN2A/p16 (9p21; possibly attributable to frequent loss of all of chromosome 9), and between loss of TP53 (17p13.3) and gain of CCND1/FGF3 (11q13; these last two significant at only P ⬍ 0.1). To determine whether paired genes from the same biological pathway showed complementary or concordant behavior (i.e., whether only one alteration of genes in a pathway is sufficient to modify the entire pathway or whether both of the alterations tend to occur together), a direct categorical complementarity analysis of pairs of genomic loci containing key components of important biological pathways was performed (TP53, MDM2, MYC, CDKN2A/p16, CCND1, CCNE1, BCL2, CDK4, E2F3). Copy number gain of CCND1 behaved complementarily with gain of E2F3 (P ⬍ 0.05) and gain of CCNE1 (P ⬍ 0.1). E2F3 had a concordant relationship with CCNE1 (P ⬍ 0.1). Finally, loss of TP53 showed complementarity with gain of MDM2 (P ⬍ 0.1). The results were robust to the threshold chosen for declaring gain/loss. The significant pairs are shown in Fig. 5.
Table 5 Clones showing homozygous deletions
Clone
Chromosome band
Base position (kb)a
RP11-287P18 RMC09P007 RP11-33O15 RP11-187A08
8p23.1 9p21.3 9p21.3 11p13
Chr8:12500 Chr9:23395 Chr9:24325 Chr11:39389
a
Gene P16/CDKI4 TRAF6/RAG1
Cases with homozygous deletionsb 3 9 3 2
Based on UCSC mapping position (http://genome.ucsc.edu/), version December 2000. b Alterations were defined by log2ratio thresholds of ⫺1 for homozygous deletion. Using this threshold, we generated a frequency table. Clones with deletions in at least two tumors are shown, ordered on chromosomal position.
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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 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 This study represents one of the first applications of genome-wide copy number analysis by array CGH. Snijders et al. (2) recently showed the potential for this technology to provide precise copy number measurements using an overlapping set of clones. The key biological value of high-resolution array CGH lies in its ability to detect small amplicons and deletions that potentially harbor specific oncogenes and tumor suppressor genes. High-level amplifications involving clones containing known oncogenes such as EGFR, CMYC, CCND1, and CCNE1 were easily detected, as well as small homozygous deletions containing the CDKN2A/p16 tumor suppressor gene at 9p21.3. Our analysis of clones containing high-level amplifications or homozygous deletions revealed two interesting candidate genes: transcription factor E2F3 on 6p22 and TNF-associated factor 6 (TRAF6) on 11p13. High-level amplifications as well as frequent copy number gain at 6p22 have previously been reported by others by low-resolution chromosomal CGH without oncogene identification (14). Array
CGH analysis strongly pointed to E2F3 as the target gene on 6p22. E2F3 is thought to be sequestered by unphosphorylated retinoblastoma protein and then released after RB phosphorylation by the CCND1/CDK4 complex in the G1 phase of the cell cycle (15). Once released, E2F3 is thought to act as a transcriptional regulator at the G1-S phase transition (16 –18). E2F-1 has been implicated in bladder carcinogenesis, but alterations of this gene appear to occur at the epigenetic level (19). A frequently deleted clone mapping to 11p13 contained both the TNF-associated factor 6 (TRAF6) and the human recombination activating gene RAG1. TRAF proteins are thought to be important regulators of cell death and responses to stress. The RAG proteins are known to initiate V(D)J recombination by facilitating doublestranded breaks. TRAF6 may be a more likely deletion target than is RAG1 in these tumors because it is presumed to function as a tumor suppressor. Frequent gains and losses could be defined at high resolution using array analysis, allowing a precise mapping of these genomic
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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 ⫺0.25, 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 were identified, containing candidate target genes neuregulin 1 (NRG1) and fibroblast growth factor receptor 1 (FGFR1). NRG1 interacts with the NEU/ERB family of receptor tyrosine kinases, known to be frequently overexpressed in bladder tumors. FGFR1 is a member of the fibroblast growth factor receptor family. Specific binding of fibroblast growth factors to these cell surface-expressed receptors activates tyrosine kinase activity. This activation allows coupling to downstream signal transduction pathways that regulate proliferation, migration, and differentiation of endothelial cells, thus enhancing angiogenesis (20). A recent study by Simon et al. (21) using fluorescence in situ hybridization (FISH)-based highthroughput tissue microarray analysis also showed frequent alterations of this gene in bladder cancers. Associations of alterations in pairs of known oncogenes and tumor suppressors could be characterized using array-based CGH. Genes functioning upstream or downstream of each other in the same biological pathway may exhibit complementary alteration, because an alteration of a single gene in this pathway may suffice for altering the effects of the entire pathway (e.g., enhanced proliferation, apoptosis). The retinoblastoma tumor suppressor pathway plays a critical role in the control of cellular proliferation by regulating the activity of the E2F family of transcription factors (22–23). Many of the genes involved in this pathway appear to be affected in bladder carcinogenesis, most notably cyclin D1 and cyclin E by amplification, and CDKN2A/p16 and RB1 by inactivation (24 –27). Clear associations were observed in a number of members of the RB proliferation pathway. There was a significant
complementary association between copy number gain of the locus containing cyclin D1 and gain of the transcription factor E2F3 locus. Also, there was a trend for complementarity between gain of the CCND1 and gain of CCNE1 loci. This is consistent with the idea that amplification of CCND1 and either CCNE1 or E2F3 may activate the RB pathway, but there is no advantage for more combined gene amplification. Geng et al. (28) showed that cyclin E can functionally replace cyclin D1 and suggested that cyclin E is the key downstream effector of cyclin D1. High-throughput tissue microarray analysis of a large series of bladder cancers (26) showed that cyclin E gene amplification was present in a subset of bladder carcinomas, especially during early invasion. These findings suggest that the RB pathway appears to be deregulated in a majority of the bladder cancers by a gene dosage increase of one of the activators cyclin D1, E2F3, or cyclin E. Significant correlation was observed in the gain of clones containing ERBB2 (17q12) and CCNE1 (19q13; P ⬍ 0.05). Activation of ERBB2 receptor tyrosine kinase pathway is thought to lie upstream from cyclin E activation; therefore, it is unclear why the gain of both genes would be selected for during tumor progression. It is possible, for example, that CCNE activation may play a larger role in the presence of activated receptor pathway. A different relationship may explain the correlation between cyclin D1 gain with p53 loss in individual tumors. Cells with both alterations may have a selective advantage because they play key roles in different cellular pathways controlling proliferation and programmed cell death, respectively. Interestingly the categorical analysis revealed a trend for complemen-
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tarity between p53 loss and MDM2 gain, consistent with the known interaction of these two proteins. Two distinct pathways are involved in bladder cancer development, with low-grade superficial lesions exhibiting clear clinical and prognostic differences from higher-grade CIS and invasive tumors. Genetic differences have been proposed to identify these tumor patterns, and candidate genes have been proposed to distinguish among them (29 –30). Array CGH has been used to differentiate among histological types of renal cancer (4). However, in this study, no statistically significant association was observed between the pattern of copy number alterations and tumor stage or grade. This may be explained by the presence of small genetic differences between superficial and invasive stages of bladder cancer, or the large heterogeneity within individual groups. Furthermore, the sample size for this study was relatively small, with limited power to discern such differences. In conclusion, array CGH detected the large chromosomal alterations previously identified by chromosomal CGH and other cytogenetic approaches. Higher-resolution analysis allowed specific identification of alterations in smaller copy number transition regions, which suggest the presence of specific candidate genes. Relationships were observed between alterations of loci containing genes known to be key players in bladder cancer biological pathways. This pilot study must be repeated with much larger numbers of well-characterized primary tumors and must be validated with other molecular, cytogenetic, and immunohistochemical approaches.
9.
10. 11.
12. 13.
14.
15. 16.
17.
18.
19.
ACKNOWLEDGMENTS
20.
We thank Karen Chew and the UCSF Cancer Center Tissue Core for assistance with the accrual and review of bladder tumor specimens at UCSF. We thank Joe Gray for his assistance with production of Array 2, and Dan Sudilovsky for help during the pathology review.
21.
22.
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