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Discovery of Novel Methylation Biomarkers in Cervical Carcinoma by Global Demethylation and Microarray Analysis

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Discovery of Novel Methylation Biomarkers in Cervical Carcinoma by Global Demethylation and Microarray Analysis 114 Discovery of Novel Methylation Biomarkers in Cervical Carcinoma by Global Demethylation and Microarray Analysis Pavel Sova,1 Qinghua Feng,1 Gary Geiss,2 Troy Wood,1 Robert Strauss,2 Vania Rudolf,1 Andre Lieber,2 and Nancy Kiviat1 1Department of Pathology, School of Medicine at South Lake Union and 2Division of Medical Genetics, University of Washington, Seattle, Washington Abstract A genome-wide screening study for identification of hyper- RRAD, SFRP1, MT1G, and NMES1) is present in a high methylated genes in invasive cervical cancer (ICC) was proportion of ICC clinical samples but not in normal carried out to augment our previously discovered panel of samples. Of these genes, SPARC and TFPI2 showed the three genes found to be useful for detection of ICC and highest frequency of aberrant methylation in ICC speci- its precursor neoplasia. Putatively hypermethylated and mens (86.4% for either) and together were hypermethylated silenced genes were reactivated in four ICC cell lines by in all but one ICC cases examined. We conclude that treatment with 5-aza-2V-deoxycytidine and trichostatin A expression profiling of epigenetically reactivated genes and identified on expression microarrays. Thirty-nine of followed by methylation analysis in clinical samples is a the 235 genes up-regulated in multiple ICC cell lines were powerful tool for comprehensive identification of methyl- further examined to determine the methylation status of ation markers. Several novel genes identified in our study associated CpG islands. The diagnostic use of 23 genes that may be clinically useful for detection or stratification of were aberrantly methylated in multiple ICC cell lines were ICC and/or of its precursor lesions and provide a basis for then analyzed in DNA from exfoliated cells obtained from better understanding of mechanisms involved in develop- patients with or without ICC. We show, for the first time, ment of ICC. (Cancer Epidemiol Biomarkers Prev that aberrant methylation of six genes (SPARC, TFPI2, 2006;15(1):114–23) Introduction The control of ICC is based upon detection and treatment of make this approach impractical in resource poor settings. the cervical intraepithelial neoplasia stage III (CIN-3) precur- Lastly, our studies showed that vaccines that prevent HPV sor lesions and carcinoma in situ (CIS). In the United States infection will eventually be central to cervical cancer control and other developed countries, the incidence of ICC has (3); however, because such vaccines will not offer protection to dramatically decreased as a result of the identification and most of the millions of women already infected with oncogenic ablation of the precursor CIN-3/CIS lesions achieved via HPVs, screening will be necessary for the foreseeable future. regular Papanicolaou screening with referral of all women Thus, development of more accurate molecular screening with abnormal Papanicolaou smears (atypical squamous cells methods that could be automated would be of significant of undetermined significance and greater) to repeat cytology interest. or colposcopy and biopsy. This approach is costly and Epigenetic silencing of a variety of genes by hypermethy- extremely burdensome to the health care system but deemed lation of promoter-associated CpG islands is now recognized necessary in light of the low reproducibility of cytology and as a frequent and early event in the pathogenesis of many the low sensitivity of a single cytologic smear (range, 30-60%) cancers, including ICC, making such changes of potential to detect CIN-3 (1). More recently, testing for oncogenic interest as biomarkers for identification of individuals with or human papillomavirus (HPV) types has been introduced to at risk for cancer (4). Currently, relatively little is known about help triage women with atypical squamous cells of undeter- specific patterns of CpG island hypermethylation in ICC. mined significance. Nevertheless, current recommendations Most previous studies examining DNA hypermethylation in (2) based on a screening approach, which include HPV-based ICC have generally focused on testing of genes known to be triage of atypical squamous cells of undetermined significance, aberrantly methylated in other cancers (the candidate gene will send over three million women each year for colposcopy approach; refs. 5-7). In the largest such study thus far, we and biopsy, although most are at little risk of ICC. Further- studied 21 candidate hypermethylated genes and identified a more, the cost and infrastructure requirements of primary panel of three (DAPK1, RARB, and TWIST1), which were screening based on cytology (with or without HPV testing) sensitive and specific for ICC and its immediate precursor CIS. We estimated that detection of hypermethylation of these three genes in exfoliated cell samples would identify histologically Received 5/4/05; revised 8/23/05; accepted 11/14/05. confirmed CIN-3 or worse with a sensitivity of 60% and Grant support: NIH grant CA097275 (N.B. Kiviat). a specificity of 95% in unscreened women over age 35, The costs of publication of this article were defrayed in part by the payment of page charges. presenting to community based clinics for reasons other than This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cervical neoplasia (8). This study also showed that exfoliated Note: Supplementary data for this article are available at Cancer Epidemiology Biomakers and cell samples could be used to identify women who had Prevention Online (http://cebp.aacrjournals.org/). acquired hypermethylated changes in their cervical epithelial Requests for reprints: Pavel Sova, Department of Pathology, University of Washington, 815 cells that are highly associated with CIS/ICC (8). However, Mercer Street, Box 358050, Seattle, WA 98109-4325. Phone: 206-897-1584; Fax: 206-897-1334. E-mail: [email protected] whereas the performance of this panel of hypermethylated Copyright D 2006 American Association for Cancer Research. genes compared favorably with the performance of exfoliated doi:10.1158/1055-9965.EPI-05-0323 cervical cytology and detection of high-risk types of HPV Cancer Epidemiol Biomarkers Prev 2006;15(1). January 2006 Downloaded from cebp.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Epidemiology, Biomarkers & Prevention 115 DNA, hypermethylated genes with increased sensitivity and and 30 Ag were labeled by incorporating amino-allyl dUTP in specificity for ICC and precursor neoplasia will need to be cDNA using reverse transcriptase (Invitrogen) according to identified if detection of DNA hypermethylation in exfoliated the manufacturer’s instructions. For C4I, CaSki, and SiHa cells, cell samples is to have clinical utility for cervical cancer total RNA was amplified and labeled with amino-allyl dUTP screening. The present study was undertaken to find such using the Amino Allyl MessageAmpTM aRNA amplification kit genes using a genome-wide examination of the methylation (Ambion, Austin, TX). Genomic DNA from untreated cells pattern of cervical cancer cell lines. lines was isolated using the QiaAmp DNA Mini kit (Qiagen). A number of different approaches to genome-wide identi- cDNA Array Hybridization. Human cDNA microarrays fication of cancer-associated hypermethylated genes have been consisting of 17,568 sequence-verified clones from the Re- developed, including restriction landmark genome scanning, search Genetic IMAGE (Integrated Molecular Analysis of methylation-sensitive arbitrarily primed PCR, and methylated Genomes and their Expression) clone set, available through CpG island amplification (4). An alternative approach is to Invitrogen (http://clones.invitrogen.com) were printed by the treat cells with epigenetic modifying drugs to allow expression Fred Hutchinson Cancer Research Center’s DNA microarray of hypermethylated/silenced genes and to then compare the facility. Labeled cDNA or aRNA were coupled from both expression profile by microarray analysis of untreated and directions with Cy3 and Cy5 dyes according to the manufac- drug-treated cells to identify putatively hypermethylated turer’s instructions (Amersham Biosciences, Piscataway, NJ). genes. The methylation status of such genes can then be Replicate hybridizations to microarrays were done with three confirmed in untreated cells using methylation-specific PCR independent RNA isolates for C4I, HeLa, and SiHa and two analyses. Global demethylation and expression microarray independent RNA isolates for CaSki; each isolate was analysis has been successfully used to identify a number of hybridized twice with a dye swap. Microarray slides were novel genes hypermethylated in association with colorectal scanned at wavelengths of 532 and 635 nm using a dual-laser and pancreatic cancer (9, 10). In the present study, we treated V GenePix4000A microarray scanner (Axon Instruments, Union four ICC cell lines (C4I, CaSki, HeLa, and SiHa) with 5-aza-2 - City, CA), and fluorescence data were extracted using the deoxycytidine (DAC), a DNA methyltransferase inhibitor, and GenePix Pro 3.0 program. trichostain A, a histone deacetylase inhibitor and compared the pretreatment and post-treatment expression profiles to identify Array Data Analysis and Statistical Methods. Raw hybrid- putatively hypermethylated genes. Genes with low initial ization signals were normalized using the locfit (LOWESS) expression that were strongly up-regulated after treatment in method in MIDAS
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