Quantitative methylation-specific PCR for the detection of aberrant DNA methylation in liquid-based Pap tests.

The goal of cervical cancer screening programs that utilize the Papanicolaou (Pap) test is to identify and treat women with precancerous high-grade squamous intraepithelial lesions (HSIL) and invasive carcinoma. Based on the results of studies that use the 2001 Bethesda System for cervical cytology reporting, each Pap test interpretation is associated with a defined risk of cervical intraepithelial neoplasia grade 2 (CIN2) or more severe lesion (CIN2+) on histologic follow-up.1,2 Findings from the atypical squamous cells of undetermined significance (ASC-US)/low-grade squamous intraepithelial lesion (LSIL) Triage Study (ALTS), a randomized, multicenter clinical trial with 2-year follow-up, showed that the risk of underlying CIN2+ associated with abnormal cytologic findings on Pap tests interpreted as ASC-US, LSIL, atypical squamous cells cannot exclude HSIL (ASC-H), and HSIL are 17%, 25%, 50%, and 63%, respectively.2 Based on the etiologic role of oncogenic/high-risk human papillomavirus (HR-HPV) in cervical carcinogenesis, HR-HPV DNA testing has become a useful adjunct to the Pap test in some populations and clinical settings and has been incorporated into management algorithms for women with abnormal cytologic findings.3 In women of reproductive age and older women, HR-HPV DNA testing is useful in triaging those with ASC-US to colposcopy.3,4 The use of HR-HPV DNA testing in women with LSIL differs depending on patient age.3 In women of reproductive age and adolescent women with LSIL, the high prevalence of HR-HPV precludes its clinical utility in triaging them to colposcopy.3,5 However, because both the prevalence of HR-HPV and high-grade CIN (CIN2/3) decreases with age, HR-HPV DNA testing can be used in the initial management of postmenopausal women with LSIL to triage to colposcopy.3,6,7 In women with ASC-H, immediate referral to colposcopy is recommended given the increased prevalence of both HR-HPV and CIN2+.2,3 In light of the finding that many of the cervical lesions associated with mildly abnormal cytologic findings will spontaneously regress,8 the identification of other objective biomarkers that could help predict which women have underlying CIN2+ or are at increased risk of progression to CIN2+ could help to eliminate unnecessary colposcopic procedures and have a significant impact on the management of women with abnormal Pap tests. Aberrant DNA methylation of tumor suppressor genes is a frequent event in most human tumors and may occur early in neoplastic progression.9 Previous studies have identified several candidate tumor suppressor genes that are frequently methylated in invasive cervical carcinoma, such as CDH1, CDH13, CDKN2A, DAPK1, HIC1, IGSF4, RARB, and TWIST1.10–14 More recently, a genome-wide screening study that utilized global demethylation and expression microarray analysis of cervical cancer cell lines identified additional novel genes, including SPARC, TFPI2, SFRP1, MT1G, RRAD, and NMES1, that were methylated in a relatively high percentage of exfoliated cervical samples with invasive carcinoma but not in normal samples.15 The methylation profiles of several genes have also been examined in precursor squamous intraepithelial lesions in Pap test samples, with most genes demonstrating more frequent methylation in HSIL compared with LSIL.14,16,17 These findings suggest that detection of methylated tumor suppressor genes could potentially serve as a diagnostic and/or predictive molecular biomarker for CIN2+. The aims of the study were to 1) analyze the methylation profile of 4 genes using real-time quantitative methylation-specific polymerase chain reaction (PCR) (QMSP) to determine if each gene, and the genes in combination, could distinguish HSIL from LSIL and negative liquid-based Pap tests; 2) determine if the relative level of methylation was associated with histologic outcome, particularly in the distinction between CIN2 and CIN3; and 3) determine if there was an association between methylation profiles and HPV status and/or type.