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

A genome-wide screening study for identification of hypermethylated genes in invasive cervical cancer (ICC) was carried out to augment our previously discovered panel of three genes found to be useful for detection of ICC and its precursor neoplasia. Putatively hypermethylated and silenced genes were reactivated in four ICC cell lines by treatment with 5-aza-2'-deoxycytidine and trichostatin A and identified on expression microarrays. Thirty-nine of the 235 genes up-regulated in multiple ICC cell lines were further examined to determine the methylation status of associated CpG islands. The diagnostic use of 23 genes that were aberrantly methylated in multiple ICC cell lines were then analyzed in DNA from exfoliated cells obtained from patients with or without ICC. We show, for the first time, that aberrant methylation of six genes (SPARC, TFPI2, RRAD, SFRP1, MT1G, and NMES1) is present in a high proportion of ICC clinical samples but not in normal samples. Of these genes, SPARC and TFPI2 showed the highest frequency of aberrant methylation in ICC specimens (86.4% for either) and together were hypermethylated in all but one ICC cases examined. We conclude that expression profiling of epigenetically reactivated genes followed by methylation analysis in clinical samples is a powerful tool for comprehensive identification of methylation markers. Several novel genes identified in our study may be clinically useful for detection or stratification of ICC and/or of its precursor lesions and provide a basis for better understanding of mechanisms involved in development of ICC.