CORE Metadata, citation and similar papers at core.ac.uk Provided by Columbia University Academic Commons GENES, CHROMOSOMES & CANCER 47:755–765 (2008) Identification of Copy Number Gain and Overexpressed Genes on Chromosome Arm 20q by an Integrative Genomic Approach in Cervical Cancer: Potential Role in Progression Luigi Scotto,1† Gopeshwar Narayan,1†,‡ Subhadra V. Nandula,1† Hugo Arias-Pulido,2,3 Shivakumar Subramaniyam,1 Achim Schneider,4 Andreas M. Kaufmann,4 Jason D. Wright,5 Bhavana Pothuri,5 Mahesh Mansukhani,1 and Vundavalli V. Murty1,6* 1Departmentof Pathology,Columbia University Medical Center,NewYork,NY 2 Departmentof Tumor Molecular Biology,Instituto Nacional de Cancerolog|Ła,BogotaŁ,Colombia 3Division of Hematology/Oncology,The University of New Mexico Cancer Center,Albuquerque,NM 4 Departmentof Gynecology,ChariteŁUniversittsmedizin Berlin,Hindenburgdamm 30,Berlin,Germany 5Departmentof Gynecologic Oncology,Columbia University Medical Center,NewYork,NY 6Institute for Cancer Genetics,Columbia University Medical Center,NewYork,NY Recurrent karyotypic abnormalities are a characteristic feature of cervical cancer (CC) cells, which may result in deregulated expression of important genes that contribute to tumor initiation and progression. To examine the role of gain of the long arm of chromosome 20 (20q), one of the common chromosomal gains in CC, we evaluated CC at various stages of progression using single nucleotide polymorphism (SNP) array, gene expression profiling, and fluorescence in situ hybridization (FISH) anal- yses. This analysis revealed copy number increase (CNI) of 20q in >50% of invasive CC and identified two focal amplicons at 20q11.2 and 20q13.13 in a subset of tumors. We further demonstrate that the acquisition of 20q gain occurs at an early stage in CC development and the high-grade squamous intraepithelial lesions (HSIL) that exhibit 20q CNI are associated (P 5 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N 5 14), as targets of amplicon 1 (N 5 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N 5 6; one gene also commonly expressed with 20q gain). These include a number of functionally important genes in cell cycle regula- tion (E2F1, TPX2, KIF3B, PIGT, and B4GALT5), nuclear function (CSEL1), viral replication (PSMA7 and LAMA5), methylation and chromatin remodeling (ASXL1, AHCY, and C20orf20), and transcription regulation (TCEA2). Our findings implicate a role for these genes in CC tumorigenesis, represent an important step toward the development of clinically significant biomarkers, and form a framework for testing as molecular therapeutic targets. VC 2008 Wiley-Liss, Inc. INTRODUCTION which may ultimately facilitate in identification of About 500,000 new cases of cervical cancer (CC) critical genes in CC development. are diagnosed worldwide every year and the major- Chromosomal gain or amplification is a common ity of affected women with advanced stages of can- cellular mechanism of gene activation in tumori- cer die (Waggoner, 2003). This failure of response to treatment of advanced CC is due to the lack of This article contains Supplementary Material available at http:// understanding of its biology at molecular level and www.interscience.wiley.com/jpages/1045-2257/suppmat Supported by: NIH; Grant number: CA095647. targeted treatment regimens. Despite the docu- † mented etiologic role of HPV infection, the molec- These authors equally contributed to this work. {Present address: Department of Molecular and Human Genetics, ular basis of the genetic changes in progression in Banaras Hindu University, Varanasi, India. the multistep process of cervical tumorigenesis is *Correspondence to: Vundavalli V. Murty, Irving Cancer Research poorly understood (Gius et al., 2007). CC cells ex- Center, Room 605, Columbia University Medical Center, 1130 St. Nicholas Avenue, New York, New York 10032. hibit highly complex karyotypic alterations (Harris E-mail: [email protected] et al., 2003). Molecular characterization of these Received 22 January 2008; Accepted 23 April 2008 complex chromosomal alterations is therefore im- DOI 10.1002/gcc.20577 Published online 27 May 2008 in portant in understanding the genetic basis of CC, Wiley InterScience (www.interscience.wiley.com). VC 2008 Wiley-Liss, Inc. 756 SCOTTO ET AL. genesis. Specific genes amplify in distinct tumor and FISH. FISH slides were immediately fixed in types and their identification could be of prognostic 3:1 methanol and acetic acid, and stored at 48C significance as the copy number increase (CNI) of until hybridization. A total of 71 pap smears with a variety of genes correlate with tumor progression the diagnosis rendered by a cytopathologist as nor- and treatment resistance (Schwab, 1999). A large mal/squamous metaplasia/ASCUS (N 5 32), LSIL number of chromosomal regions are gained or (N 5 14), or HSIL (N 5 25) obtained from CUMC amplified in CC suggesting that gene amplification were used for FISH analysis. The diagnosis of all and CNI of specific genes is a common genetic HSILs was also confirmed by a biopsy. Of the 153 alteration in this tumor (Mitra et al., 1994; Narayan primary tumors, 117 were obtained as frozen tis- et al., 2003b, 2007; Rao et al., 2004). Using compar- sues and 36 specimens as formalin-fixed paraffin- ative genomic hybridization (CGH) approaches, embedded tissues. All primary invasive cancer we and others previously showed that chromosome specimens were obtained from patients evaluated 20 was one of the recurrently gained chromosomes at CUMC, Instituto Nacional de Cancerologia and a high level CNI at 20q11.2 and 20q13.1 (Santa Fe de Bogota, Colombia) (Pulido et al., regions has also been reported in invasive CC 2000), and the Department of Gynecology of Cam- (Harris et al., 2003; Narayan et al., 2003b, 2007; pus Benjamin Franklin, Charite´-Universita¨tsmedi- Rao et al., 2004; Wilting et al., 2006). These data zin Berlin (Germany) with appropriate informed suggest that the 20q genetic alterations contribute consent and approval of protocols by institutional to the CC pathogenesis. The identification of tar- review boards. All primary tumors were diagnosed get genes of 20q gain is likely to have a significant as squamous cell carcinoma (SCC) except five that impact on the understanding of the pathogenesis were diagnosed as adenocarcinoma (AC). Clinical and clinical management of CC. information such as age, stage and size of the tu- To unravel the role of chromosome 20 gain in mor, follow-up data after initial diagnosis and treat- CC tumorigenesis, we characterized the chromo- ment was collected from the review of institutional some 20 alterations using high throughput genomic medical records. Tissues were frozen at 2808Cim- and expression approaches. mediately after resection and were embedded with tissue freeze medium (OTC) before microdissection. MATERIALS AND METHODS All primary tumor specimens were determined to contain at least 60% tumor by examining hematoxy- Tumor Specimens and Cervical Cancer Cell Lines lin and eosin (H&E) stained adjacent sections. High A total of 253 specimens were used in the pres- molecular weight DNA and total RNA from tumor, ent study in various investigations. These include normal tissues, and cell lines were isolated by stand- 9 cell lines, 153 untreated primary tumors, 71 pap ard methods. The integrity of all RNA preparations smears, and 20 normal cervical tissues. The cell was tested by running formaldehyde gels and any lines (HT-3, ME-180, CaSki, MS751, C-4I, C-33A, samples that showed evidence of degradation were SW756, HeLa, and SiHa) were obtained from excluded from the study. American Type Culture Collection (ATCC, Mana- ssas, VA) and grown in tissue culture as per the Microarray Analysis supplier’s specifications. Twenty age-matched nor- The Affymetrix 250K NspI single nucleotide mal cervical tissues from hysterectomy specimens polymorphism (SNP) chip was used for copy num- obtained from Columbia University Medical Cen- ber analysis as per the manufacturer’s protocol. ter (CUMC), New York, were used as controls after Briefly, 250 ng of genomic DNA was digested with enrichment for epithelial cells by microdissection. NspI, generic linkers were added followed by PCR Cytologic specimens were collected using the amplification, end-labeling, and fragmentation fol- ThinPrep Test Kit (Cytc Corporation, Marlbor- lowing standard protocols. Hybridization, washing, ough, MA). After visualization of the cervical os acquisition of raw data using GeneChip Operating the ectocervix was sampled with a spatula and Software (GCOS), and generation of .CEL files endocervical cells obtained with a brush rotated was performed by the Affymetrix Core facility at 3608. Exfoliated cells were immediately placed in our institute. We used 79 CC cases (9 cell lines and PreservCyt Solution (Cytc Corporation, Marlbor- 70 primary tumors enriched for tumor cells by ough, MA) for routine processing by cytopatholo- microdissection) and 7 microdissected normal cer- gist. Pap smears were collected from normal and vical squamous epithelial samples as controls to precancerous lesions by simultaneous preparation serve as the reference for copy number analysis. of slides from the same spatula for both cytology SNP data of test samples and normal cervical epi- Genes, Chromosomes & Cancer DOI 10.1002/gcc 20q GENETIC ALTERATIONS IN CERVICAL CANCER 757 thelial specimens were loaded to dChip to calcu- to GEO (Accession number: GSE9750). To obtain late signal intensity values using the perfect a list of differentially expressed gene signatures, match/mismatch (PM/MM) difference model fol- we compared all normal with all tumor samples lowed by normalization of signals within chip and using the criteria of 1.75-fold change between the between chips using model-based expression (Li group means at 90% confidence interval and a sig- and Wong, 2001; Lin et al., 2004). DNA copy number nificance level of P < 0.05. All negative expression gains were obtained as determined by dChip using values for each probe set were truncated to 1 analysis of signal intensity values based on the Hid- before calculating fold changes and <10% of sam- den Markov Model.