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Identification of Novel Methylation Markers in Cervical Cancer Using Restriction Landmark Genomic Scanning

¹ Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland; ² Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York; ³ Division of Human Cancer Genetics, The Ohio State University, Columbus, Ohio; ⁴ Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri; ⁵ Departments of Pathology and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan; ⁶ Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York; and ⁷ Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Requests for reprints: Sophia S. Wang, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Boulevard, Room 5104, Rockville, MD 20852-7234. Phone: 301-402-5374; E-mail: wangso{at}mail.nih.gov.

Key Words: DNA methylation • cancer • cervix • CpG island • RLGS • Methylight

Aberrant methylation of CpG islands in gene promoters often represents an early clonal event in carcinogenesis. Accordingly, defining methylation profiles may be useful for developing marker panels for early detection or predicting the risk of cancer precursors. To identify specific genes frequently methylated in cervical cancer, we conducted methylation profiling of 20 primary human cervical cancers using NotI-based restriction landmark genomic scanning (RLGS). Of 2,172 RLGS fragments analyzed (average, 1,753 CpG islands per patient), 186 RLGS fragments were lost in at least one tumor and 40 were lost in three or more. Methylation was identified in 19 (95%) of 20 tumor samples compared with normal DNA. Bisulfite sequencing was conducted to confirm RLGS results. Of the confirmed markers frequently methylated, we developed Methylight assays for two corresponding genes, nucleolar protein 4 (NOL4), and lipoma HMGIC fusion partner–like protein 4 (LHFPL4), which were methylated in 85% and 55% of cancers, respectively. Using these assays, we further confirmed frequent CpG island methylation in the original cancers and in another independent series of 15 cervical cancers. We also showed methylation at a reduced frequency in a set of carefully reviewed cytology specimens demonstrating cells exfoliated from cancer precursor lesions. In summary, we identified, for the first time, NOL4 and LHFPL4 as novel methylation targets specific for cervical cancer. Inclusion of NOL4 and LHFPL4 in evaluating methylation panels for early detection, risk prediction, and etiologic research on cervical cancer is warranted. [Cancer Res 2008;68(7):2489–97]