Gene Expression Analysis of Chromosomal Regions with Gain or Loss of Genetic Material Detected by Comparative Genomic Hybridization Ba ´rbara Mele ´ ndez, 1 Ramo ´n Dı ´az-Uriarte, 2 Marta Cuadros, 1 A ´ ngel Martı ´nez-Ramı ´rez, 1 Jose ´ Ferna ´ ndez-Piqueras, 3 Ana Dopazo, 4 Juan-Cruz Cigudosa, 5 Carmen Rivas, 6 Joaquı ´n Dopazo, 2 Beatriz Martı ´nez-Delgado, 1 and Javier Benı ´tez 1 * 1 Department of Human Genetics, Spanish National Cancer Centre (CNIO), Madrid, Spain 2 Bioinformatics Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain 3 Human Molecular Laboratory, Universidad Auto ´ noma de Madrid, Madrid, Spain 4 Microarray Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain 5 Cytogenetics Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain 6 Department of Anatomical Pathology, Fundacio ´n Jime ´nez Dı ´az, Madrid, Spain Comparative genomic hybridization (CGH) has been widely used to detect copy number alterations in cancer and to identify regions containing candidate tumor-responsible genes; however, gene expression changes have been described only in highly amplified regions (amplicons). To study the overall impact of slight copy number changes on gene expression, we analyzed 16 T-cell lymphomas by using CGH and a custom-designed cDNA microarray containing 7,657 genes and expressed sequence tags related to tumorigenesis. We evaluated mean gene expression and variability within CGH-altered regions and explored the relationship between the effects of the gene and its position within these regions. Minimally overlapping CGH candidate areas (6q25, 13q21– q22, and 19q13.1) revealed a weak relationship between altered genomic content and gene expression. However, some candidate genes showed modified expression within these regions in the majority of tumors; these candidate genes were evaluated and confirmed in another independent series of 23 T-cell lymphomas by use of the same cDNA microarray and by FISH on a tissue microarray. When all the CGH regions detected for each tumor were considered, we found a significant increase or decrease in the mean expression of the genes contained in gained or lost regions, respectively. In addition, we found that the expression of a gene was dependent not only on its position within an altered region but also on its own mechanism of regulation: genes in the same altered region responded very differently to the gain or loss of genetic material. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http:// www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html. © 2004 Wiley-Liss, Inc. INTRODUCTION Human cancers are characterized by changes in the expression of oncogenes and tumor-suppressor genes, in many cases as a consequence of either the numerical or structural chromosomal abnormalities that occur during tumorigenesis. In hematologic disorders, many of these alterations are well known, although knowledge of the responsible cy- togenetic alterations in most solid tumors is still limited because of scarcity of good metaphase spreads, poor chromosomal morphology, and karyo- typic complexity. Comparative genomic hybridiza- tion (CGH) has facilitated the chromosomal char- acterization of solid tumors, because this technique allows gain or loss of genetic material to be identi- fied without needing to do a metaphase analysis (Kallioniemi et al., 1994). These regions are con- sidered candidates for containing tumor-relevant genes that are involved in the origin and/or devel- opment of the disease. In addition, the DNA re- quired for the CGH experiments can be obtained from fresh or paraffin-embedded tumor tissues, and consequently this technique has been used in a great number of studies of many types of tumors (reviewed in Knuutila et al., 1998, 1999, 2000; Struski et al., 2002). In some of these tumors, those Barbara Melendez is a postdoctoral fellow from the Exmo, Angel Martı ´nez-Ramı ´rez is a postgraduate fellow at the Instituto de Salud Carlos III, and Marta Cuadros is a fellow at the Colegio de Farma- ceuticos. Supported by: Comunidad Auto ´ noma de Madrid; Grant numbers: CAM 08.1/0013/00 and CAM 08.1/0020/00; Ayuntamiento de Ma- drid; Ramon y Cajal from the McyT (to R.D.-U.); Spanish MCyT (to R.D.-U.); Grant number: TIC2003-09331-C02-02. *Correspondence to: Dr. Javier Benı ´tez, Department of Human Genetics, Spanish National Cancer Centre (CNIO), c/ Melchor Ferna ´ndez Almagro 3, 28029 Madrid, Spain. E-mail: jbenitez@cnio.es Received 11 February 2004; Accepted 28 May 2004 DOI 10.1002/gcc.20105 Published online 20 September 2004 in Wiley InterScience (www.interscience.wiley.com). GENES, CHROMOSOMES & CANCER 41:353–365 (2004) © 2004 Wiley-Liss, Inc.