[CANCER RESEARCH 62, 1134 –1138, February 15, 2002] Silence of Chromosomal Amplifications in Colon Cancer 1 Petra Platzer, 2 Madhvi B. Upender, 2 Keith Wilson, 2 Joseph Willis, James Lutterbaugh, Arman Nosrati, James K. V. Willson, David Mack, Thomas Ried, and Sanford Markowitz 3 Howard Hughes Medical Institute, Cleveland, Ohio 44106 [P. P., J. L., S. M.]; Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH Bethesda, Maryland 20892 [M. B. U., T. R.]; Eos Biotechnology, South San Francisco, California 94080 [K. W., D. M.]; Department of Pathology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio 44106 [J. W.]; and Cancer Center and Department of Medicine at Case Western Reserve University, Ireland Cancer Center, Department of Medicine, and Research Institute at University Hospitals of Cleveland, Cleveland, Ohio 44106 [A. N., J. K. V. W., S. M.] ABSTRACT Oncogene activation by gene amplification is a major pathogenetic mechanism in human cancer. Using comparative genomic hybridization, we determined that metastatic human colon cancers commonly acquire numerous extra copies of chromosome arms 7p, 8q, 13q, and 20q. We then examined the consequence of these amplifications on gene expression using DNA microarrays. Of 55,000 transcripts profiled, 2,146 were deter- mined to map to one of the four common colon cancer amplicons and to also be expressed in normal or malignant colon tissues. Of these, only 81 transcripts (3.8%) demonstrated a 2-fold increase over normal expression among cancers bearing the corresponding chromosomal amplification. Chromosomal amplifications are common in colon cancer metastasis, but increased expression of genes within these amplicons is rare. INTRODUCTION Chromosomal aberrations may act as a fundamental pathophysio- logical event in human carcinogenesis (1). Common examples include inactivation of tumor suppressor genes by chromosomal deletion, and creation of oncogenic fusion genes by chromosomal translocation (1). Additionally, as exemplified by the HER2 gene (ERBB2), chromo- some amplification can activate a target gene to become an oncogene by inducing its expression to levels substantially greater than normal (2). However, cancer-associated chromosome amplifications may span entire chromosome arms, and it has been unresolved whether this class of chromosome aberration can act by altering expression of thousands of amplified genes or rather acts by deregulating only a select few of such amplified genes. In this study, we have combined comparative genomic hybridization and DNA microarray expression profiling to examine the expression of over 2000 genes that were identified as residing on chromosome arms that were amplified in metastatic colon cancers. We have found for nearly all these genes that chromosome amplification does not result in up-regulation of gene expression, or alternatively, that amplified genes that also dem- onstrate increased expression levels are quite rare. MATERIALS AND METHODS DNA Microarray Analysis. We designed two custom expression moni- toring DNA microarrays using Affymetrix GeneChip technology (3) that contained essentially all expressed human genes in the public domain at the time of design. Briefly, we selected the sequences for inclusion on the arrays using genes predicted from the available human genome sequences and se- quences derived from the expressed mRNA and EST databases in GenBank (4). Consensus sequences representing human expressed sequences were gen- erated using the Clustering and Alignment Tool software (DoubleTwist, Oak- land, CA) using the mRNAs (nt) and EST (dbest) databases in GenBank. Prediction of the expressed genome from the human genome sequence was done using Ab initio exon prediction (5). The arrays were hybridized with labeled cRNA derived from 10 g of total RNA using standard protocols (6). The intensity data from the arrays were analyzed using a statistically based analysis methodology that allows for estimating expression levels and providing confidence intervals for these estimates. This method uses a gamma distribution model of the intensity data for normalization to control for the systematic variation attributable to nonbiological factors, such as array-to-array variability, and attributable to variation in sample quality. For each probeset, a single measure or average intensity was calculated using Tukey’s trimean of the intensity of the constit- uent probes (7). Comparative Genomic Hybridization. Total genomic DNA from normal and tumor tissue was labeled with digoxigenin and biotin, respectively, using nick translation. Two g each of digoxigenin-labeled normal DNA and biotin- labeled tumor DNA were ethanol precipitated together in the presence of 10 g of salmon sperm DNA and 60 g of Cot-I fraction of human DNA (Life Technologies, Inc., Gaithersburg, MD). Hybridization conditions were as described previously (8). Briefly, probes were dried and resuspended in 10 l of hybridization solution (50% formamide, 2  SSC, and 10% dextran sulfate). DNA was denatured for 5 min at 80°C; repetitive sequences were allowed to preanneal for 1.5 h at 37°C and hybridized to normal human metaphase preparations. Normal metaphase slides were prepared from peripheral blood lymphocytes. Slides were treated with RNase (100 g/ml) for 45 min, fixed, and dehydrated. DNA was denatured at 80°C for 1.5 min in 70% deionized formamide, 2  SSC. The probe mixture was applied to the slide, covered with an 18-mm 2 coverslip, sealed with rubber cement, and hybridized for 48 h at 37°C in a humidified chamber. Probe signals were detected using an amplifi- cation procedure and counterstained with DAPI, as described previously (8). Slides were mounted in antifade solution. Microscopy and Image Analysis. Images were acquired with a cooled charge coupled device camera (Photometrics, Tuscon, Arizona) mounted on a Leica DMRBE epifluorescence microscope using filters specific for DAPI, fluorescein, and rhodamine (Chroma Technologies, Brattleboro, VT). CGH ratio profiles were calculated using Leica Q-CGH software (Leica Imaging Systems, Cambridge, United Kingdom) as described (9). RESULTS CGH of Metastatic Colon Cancer. To determine the relationship between chromosomal amplification and gene expression profiles, we characterized both processes in 23 independent metastatic colon can- cers. This included 15 samples of metastatic tumor tissue resected from colon cancer liver metastases and an additional eight cell lines that were derived from biopsies of such colon cancer hepatic metas- tases. All metastatic tissue samples were dissected free of tissue contaminants and were confirmed by histology examination to be comprised of at least 70% malignant epithelial cells. We focused this study on colon cancer metastases, because they have had maximal opportunity in vivo to select for chromosome amplifications that could confer an aggressive cancer phenotype. Received 11/19/01; accepted 1/2/02. 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 This work was supported by NIH Grant CA88130 and by a grant from the National Colon Cancer Research Alliance. S. D. M. is an associate investigator of the Howard Hughes Medical Institute. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Howard Hughes Medical Institute, Room 200, 11001 Cedar Road, Cleveland, OH 44106. Phone: (216) 844-8237; Fax: (216) 844-8230; E-mail: sxm10@po.cwru.edu. 4 The abbreviation used are EST, expressed sequence tag; DAPI, 4,6-diamidino-2- phenylindole; CGH, comparative genomic hybridization; EGFR, epidermal growth factor receptor. 1134 Research. on February 21, 2016. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from