Identification of novel DNA methylation markers in cervical cancer Hung-Cheng Lai 1,2 * , Ya-Wen Lin 2,3 , Tim H.M. Huang 4 , Pearlly Yan 4 , Rui-Lan Huang 1 , Hui-Chen Wang 1 , Joseph Liu 4 , Michael W.Y. Chan 5 , Tang-Yuan Chu 6 , Chien-An Sun 7 , Cheng-Chang Chang 1 and Mu-Hsien Yu 1 1 Department of Obstetrics and Gynecology, Tri-Service General Hospital, Taipei, Taiwan, Republic of China 2 Laboratory of Epigenetics, National Defense Medical Center, Taipei, Taiwan, Republic of China 3 Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China 4 Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 5 Department of Life Science, National Chung Cheng University, Min-Hsiung Chia-Yi, Taiwan, Republic of China 6 Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital, Tzu Chi University, Hualien, Taiwan, Republic of China 7 School of Public Health, National Defense Medical Center, Taipei, Taiwan, Republic of China Testing for DNA methylation has potential in cancer screening. Most previous studies of DNA methylation in cervical cancer used a candidate gene approach. The aim our study was to identify novel genes that are methylated in cervical cancers and to test their potential in clinical applications. We did a differential meth- ylation hybridization using a CpG island (CGI) microarray con- taining 8640 CGI tags to uncover methylated genes in squamous cell carcinomas (SCC) of the uterine cervix. Pooled DNA from cancer tissues and normal cervical swabs were used for compari- son. Methylation-specific polymerase chain reaction, bisulfite sequencing and reverse transcription polymerase chain reaction were used to confirm the methylation status in cell lines, normal cervices (n 5 45), low-grade lesions (n 5 45), high-grade lesions (HSIL; n 5 58) and invasive squamous cell carcinomas (SCC; n 5 22 from swabs and n 5 109 from tissues). Human papillomavirus (HPV) was detected using reverse line blots. We reported 6 genes (SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1) more fre- quently methylated in SCC tissues (81.5, 94.4, 89.9, 80.4, 77.8 and 20.4%, respectively) than in their normal controls (2.2, 0, 6.7, 11.9, 11.1 and 0%, respectively; p < 0.0001). Parallel testing of HPV and PAX1 methylation in cervical swabs confers an improved sensitivity than HPV testing alone (80% vs. 66%) with- out compromising specificity (63% vs. 64%) for HSIL/SCC. Test- ing PAX1 methylation marker alone, the specificity for HSIL/SCC is 99%. The analysis of these novel DNA methylations may be a promising approach for the screening of cervical cancers. ' 2008 Wiley-Liss, Inc. Key words: cervical cancer; epigenetics; hpv; methylation; microarray In addition to genetic changes, epigenetic alterations such as DNA methylation and histone modifications can result in heritable gene silencing without changes to genetic sequences and are recognized as important causes of cancer. 1–3 DNA methylation mostly occurs at the 5 0 cytosine in the palindromic sequence, 5 0 - CpG-3 0 . CpG islands are CpG-rich areas of 1 kb that are usually located in the vicinity of genes, often near the promoters of widely expressed genes. 4,5 Methylation of CpG sites in the human ge- nome is catalyzed by a family of DNA methyltransferases (DNMTs). DNMT1 is a maintenance methyltransferase with a preference for hemimethylated DNA whereas DNMT3a and DNMT3b are de novo methyltransferases with approximately equal preferences for methylated and unmethylated DNA. 2,6 The addition of methyl groups by DNMTs recruits complexes with transcription repressors that modify histones and thus silence genes. Global DNA hypomethylation and site-specific hypermeth- ylation result in genomic instability and transcriptional gene inac- tivation, respectively, both of which are associated with cancer. 7,8 As epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly observed in human cancers, DNA methylation could serve as a marker for early diagnosis of cancer and as a means of assessing the prognosis of cancer patients. 3 Cervical cancer is one of the main causes of death of women worldwide. 9 Infection with oncogenic human papillomavirus (HPV) is the most significant risk factor in the etiology of cervical cancer and is present in nearly all the cases of cervical cancer. 10 Although HPV infection is necessary for the development of cervi- cal cancer, its presence alone is insufficient to cause cervical cancer. The molecular mechanism responsible for the inefficiency of HPV- initiated cervical carcinogenesis remains elusive. 11 In addition to genetic changes, epigenetic changes may play a role in the develop- ment of cervical cancer, 12–16 which indicates that DNA methylation may be useful as a marker for cervical cancer screening. 12,17 Most previous studies of DNA methylation in cervical cancer used candidate gene approaches involving classical tumor sup- pressor genes or genes that are known to be methylated in other cancers. There is a need for a genome-wide approach to identify hypermethylated genes in invasive cervical cancer. A technique involving inhibition of DNA methyltransferases and histone deacetylases coupled with the use of expression microarrays was recently developed to study cervical cancer cell lines. 12 Yet to- date, there are no data on genome-wide analysis of methylation in cervical cancer samples. Elucidation of methylation changes could identify new tumor suppressor genes and biomarkers, which could further our understanding of cervical carcinogenesis and would be useful for cervical cancer screening. We conducted a genome- wide differential analysis of the methylation status of cervical can- cer using a CpG island microarray to discover novel genes methyl- ation-silenced in cervical cancer. A panel of cervical scrapings and tissues from patients with pathologically proven cervical lesions was used to test the diagnostic utility of the methylation status of these genes in combination with HPV testing. Material and Methods Patients Patients with normal uterine cervixes (n 5 45) and patients with LSIL (n 5 45) HSIL (n 5 58), and invasive squamous cell carcinoma (SCC; n 5 109 with tissue DNA and n 5 22 with cervi- cal swab DNA) of the uterine cervix participated in our study. The This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. The application for patents using these DNA sequences in cancer screen- ing is in processing. Grant sponsor: Tri-Service General Hospital; Grant number: TSGH- C96-2-S014; Grant sponsor: National Science Council, Republic of China; Grant numbers: NSC95-2622-B-016-001, NSC95-2314-B-016-058- MY2; Grant sponsors: C.Y. Chai Foundation for Advancement of Educa- tion, Sciences and Medicine, Human Medical Research Foundation; Grant number: B962114-1. *Correspondence to: Department of Obstetrics and Gynecology, Tri- Service General Hospital, National Defense Medical Center, 5F, 325, Sec 2, Cheng-Gong Rd., Neihu district, Taipei city 114, Taiwan, Republic of China. Fax: 1886-2-8792-7199. E-mail: hclai@ndmctsgh.edu.tw Received 19 January 2008; Accepted after revision 23 January 2008 DOI 10.1002/ijc.23519 Published online 8 April 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 161–167 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer