In this analysis, the combined results for PCR assays designed to detect methylation of HS3ST2 or CDH1 were positive in 100% of cervical cancer tissues and 83% of cytology specimens interpreted as CIN3. Methylation was not detected in any of 12 histologically normal tissue specimens for either marker. The two genes are of further interest as heparin sulfate proteoglycans have been demonstrated to mediate binding of the human papillomavirus (HPV) to cell surfaces [14
] and e-cadherin has been reported to play an important role in mounting an immune response to HPV infection by mediating the adhesion of dendritic and Langerhans cells [15
Our results provide support for evaluation of HS3ST2 and CDH1 and other candidate markers in a large epidemiologic study where there are cytology specimens associated with a range of cytologic interpretations and well-defined outcomes determined in prospective follow-up. The equally sensitive detection of methylation of HS3ST2 and CDH1 in squamous cell and adenocarcinoma is potentially important for further follow-up, particularly among adenocarcinoma in situ (AIS) because cytology is insensitive in identifying AIS and adenocarcinomas are suspected to account for a disproportionate percentage of rapidly developing cancers [16
Although several studies have evaluated methylation markers in cervical cancers [17
] data for cervical precursor lesions are more limited. Steenbergen et al [21
] reported methylation of TSLC1 gene at frequencies of 35% and 58% in precancerous lesions and cervical cancers, respectively. Li et al [22
] reported methylation of TSLC1 gene at a frequency of 65% in cervical cancers. Feng et al [23
] reported methylation of at least one of the three genes (DAPK1, RARβ and TWIST1) at frequencies of 57% and 74% in CIN3 lesions and cervical cancers, respectively.
Similar to these previous studies, we found that detection of specific methylation markers was generally lower in cytologic specimens showing CIN3 than in cancer tissues. Both lesion severity and specimen type could have contributed to this difference in our study.
Higher levels of methylation in cancer as opposed to CIN3 may reflect progressive accumulation of methylation events as putative precursors progress to carcinoma. This might be expected if methylation confers cells with a relative growth advantage. Although CIN3 represents the most proximate histologic precursor of invasive carcinoma, the peak age of detection for CIN3 lesions is 10–20 years younger than invasive cancer. Therefore, many CIN3 lesions seem to persist and expand for long periods prior to invasion, which itself is not recognized as an inevitable occurrence. These data suggest that assessment of methylation among women infected with carcinogenic HPV types stratified by severity of concurrent lesions, age, HPV type and other factors may be of interest in understanding progression.
We acknowledge that the disparity observed between CIN3 cytology and cancer tissues may reflect inherent differences between the specimen types. Cytology sampling always includes many ”normal” cells and also includes many superficial epithelial cells, whereas cancer tissues may consist of purer populations of tumor cells. Consequently, useful assays must have high analytical sensitivity to detect rare but important changes in samples, yet at the same time not detect random insignificant changes that lack biological consequences. The importance of these concerns is demonstrated by the wide range of semi-quantitative methylation values that were determined in the cytology specimens, even though all had been interpreted by experts as showing definite CIN3.
Finally, we acknowledge inconsistencies in the published literature regarding methylation of the same genes. For example, Kang et al [24
] and Yang et al [25
] reported DAPK1 methylation of 73% and 60%, respectively, compared to our report of 21%. Yang et al also found DAPK1 methylation more common in SCC than in AC. Such discrepancies underscore many of the technical issues related to assay development. Specifically, in the Kang et al and Yang et al articles, methylation was conducted with a standard/conventional protocol, not real time PCR. Our present manuscript uses real time PCR with TaqMan probe Technology where two additional primers are used (forward and reverse) and a sequence specific probe in the middle makes the assay more specific. Standard or conventional PCR may thus result in higher methylation frequencies due to the lack of specificity. Reasons for discrepancy by cell type are more difficult to explain although the combination of using different assays, having varying and usually small sizes, and geographically different patient populations [26
] are possibilities.
In summary, iterative cytologic screening has demonstrated effectiveness in reducing cervical cancer incidence and mortality, but burdens resulting from repeated testing have prompted efforts to develop highly sensitive tests that can provide safety with fewer lifetime screens. Identifying abnormal methylation events is a promising avenue of research for developing such tests, but further efforts to identify and validate a clinical useful marker panel are needed.