The detection of methylated genes from cervical specimens is technically feasible and represents a source for detecting potential biomarkers of relevance to cervical carcinogenesis. In particular, there is the ultimate hope of finding methylation markers that, among HPV-infected women, would indicate the presence of CIN2+ and risk of cancer.
One striking conclusion of our survey of 51 studies is that methylation frequencies for the same gene vary widely between studies. This degree of heterogeneity combined with the low number of studies analyzing the same genes precluded us from performing a rigorous meta-analysis. In fact, we were unable to identify highly consistent results for most genes even when restricting analyses to studies of similar size or those that used common specimen sources or similar assays. These results suggest that the frequency of certain methylation markers may also vary for reasons related to differences in populations, specific features of assay protocols, chance or other unidentified factors. For example, CDH1 and FHIT, two genes with highly variable methylation frequencies in cancer between studies also show variable detection in normal tissue. In contrast, DAPK1 has been measured much more precisely over a wide range of heterogeneous studies, including those analyzing CDH1 and CDH13.
We described in detail the properties of 15 methylation markers that were analyzed in 5 or more studies. While we cannot discount the possibility of a promising biomarker among the remaining 53 candidates, there was insufficient information available to evaluate these candidates further.
The most important prerequisite for a potential biomarker is that it must be reliable in its measurement. Among the 15 genes analyzed in detail, 7 (CDH1, FHIT, TERT, CDH13, MGMT, TIMP3, HIC1) had a reported range of methylation frequencies in cervical cancers of greater than 60% between studies. Stratification for analysis method or specimen type used did not resolve the observed variations. However, since few studies used identical conditions to analyze the same markers, in most cases, numbers were insufficient to evaluate the influence of assay type and specimen on methylation frequencies. At the moment, assay reliability for these methylation markers therefore cannot be properly addressed since methods are poorly standardized. We acknowledge the possibility that the wide range of frequencies reported for some genes contrasting the more consistent measurement of few other genes in similar studies could be related to either unreliable assays for these specific genes or to biological variation.
Another prerequisite for a good biomarker is that it has high sensitivity and high specificity for disease detection, resulting in a high positive predictive value. Among the eight genes with limited variability, all but one (APC) had methylation frequencies below 5% in normal tissue. Of these, three had average frequencies of at least 30% in cancers (DAPK1, CADM1, and RARB). Based on these results, we conclude that there is currently no single methylation marker that has the appropriate performance to serve as a cervical cancer biomarker, especially since methylation frequencies in CIN2/CIN3/HSIL, the targets of most cervical cancer screening programs, were even lower for all genes.
Some groups have proposed the use of methylated gene panels to obtain an appropriate performance for cervical cancer screening. From the summary data in this review, the combination of DAPK1, CADM1, and RARB would appear the most promising. However, without a formal evaluation, it is unclear if methylation of these markers is mutually exclusive, entirely independent, or associated to some degree, which would affect the overall coverage of the marker panel and therefore has important impact on the sensitivity of marker combinations. We note that Feng et al. recently showed up to 74% sensitivity and 95% specificity for detecting cervical cancers using a panel of three candidate genes, DAPK1, RARB, and TWIST1 [19
]. Our review would support further evaluation of two of these genes (DAPK1 and RARB).
Finally, we consider the biological relevance to further inform our evaluation of candidate genes. Notably, a clear role of methylation in carcinogenesis has been demonstrated only for 6 genes (DAPK1, RASSF1, CDKN2A, RARB, MLH1, and GSTP1, see supplemental table 2
). Two of the 15 genes, TERT and GSTP1, encode for proteins with oncogenic functions, so their methylation is not likely to drive carcinogenesis. CDKN2A (encoding p16), is methylated in several tumors early in carcinogenesis. In cervical cancer, however, p16 is found strongly overexpressed due to HP-oncogene mediated release of E2F from RB. Recently, it has been demonstrated that p16 methylation does not affect protein expression in cervical cancer [20
]. Thus, most of the 15 genes analyzed in this study have no strong a-priori for potentially being important in cervical carcinogenesis. Furthermore, none of the 7 genes with the highest variation of methylation frequencies is among the genes with a clearly defined role of methylation in the carcinogenesis of other tumors. This evaluation suggests that the current genes may not be optimal candidates for cervical carcinogenesis. To date, only few agnostic profiling studies for aberrant methylation in cervical carcinogenesis have been performed [21
]. We therefore believe that, if methylation markers for cervical cancer screening are to be further pursued, future large well-conducted investigations should also incorporate a discovery effort specifically for methylated genes in cervical cancer. During the last years, several new platforms (e.g. microarray format, bead array format, 454 sequencing format) have been developed that allow for accurate high-throughput genome-wide DNA methylation profiling [24
]. Markers or marker panels identified in these approaches could be translated to smaller scaled assays such as Methylight to be used in cervical cancer screening.
In summary, to identify promising methylation candidates for cervical carcinogenesis, further tissue-based profiling studies using reliable and validated assays are needed. In addition, analyses of exfoliated tissue (e.g. stored in liquid based cytology media) will be critical as this would represent the most likely specimen used for cervical cancer screening and triage. Both efforts will require large well-powered epidemiologic studies designed to properly identify and then validate candidate methylation markers and panels of markers for utility in the early detection of cervical cancer.