Alterations in DNA methylation are the most common molecular alterations in human malignancies. Detection of the aberrant DNA methylation associated with cancer-related genes is a promising approach to improve cancer prevention, diagnosis and treatment options. Bisulphite modification is a prerequisite for most popular techniques aiming at detecting changes in methylation, but has been limited by throughput capacity. In this study, we used a high-throughput methylation detection method to analyze DNA methylation at 8 imprinted DMRs in epithelial ovarian cancer. We found that the PCR-Luminex method precisely quantified the methylation status of specific DNA regions in somatic cells and was also relatively rapid, economical and easy to use.
In the epithelial ovarian cancers, the frequency of LOI was higher than that of LOH. In particular, LOI was most frequent at PEG1, IGF2 and H19 DMRs. The frequency, extent of changes in DNA methylation and loci affected varied considerably among the samples. Generally, we found that DNA methylation at imprinted DMRs was increased in both cell lines and primary material. Importantly, we showed that gain of DNA methylation in the imprinted DMRs was apparent in tumors with LOI, especially at PEG1 and H19. We also found DNA methylation changes in the absence of LOI. In other words, there were changes in DNA methylation at DMRs that were not associated with biallelic gene expression.
When we examined the clinical characteristics of the tumors, we found no significant differences in the frequency of LOI and aberrant DNA methylation between the localized early-stage and advanced-stage tumor groups. This suggested that the changes we identified occurred as a relatively early event of HOC. In general, the PEG1
DMRs appeared to be particularly prone to errors. This is similar to the previous findings in human sperm from subfertile men [21
]. In ZDBF2
DMRs, aberrant DNA methylation occurred in HOC. As with H19
, these DMRs are paternally methylated DMRs in somatic cells. In childhood cancers such as retinoblastoma, Wilms' tumor and osteosarcoma, changes primarily occur on the paternal allele first, followed by a second hit on the maternal allele [5
]. Similarly, methylation of paternally imprinted DMR in normal somatic cells might be a first hit and cause ovarian carcinogenesis. These observations suggest a role for altered genomic imprinting in the malignant transformation process.
A previous report had demonstrated the association between the abnormal genomic imprinting of H19
]. The aberrant hypermethylation in the CTCF binding site of the H19
gene was seen in the cases of HOC and correlated with IGF2
LOI. Our results for H19
were similar to those reported findings. The most frequent methylation error in HOC was seen in the PEG1
DMR. In our previous report, we showed that demethylation of PEG1
was present in growing oocytes from superovulated infertile women [23
]. This PEG1
DMR may be especially vulnerable to errors. LOI of PEG1 has subsequently also been implicated in the aetiology of lung adenocarcinomas, breast and colon cancer.
HOC is the leading cause of death from gynecologic malignancies because the majority of cases are not detected until the disease is well advanced. Our understanding of cancer as a clonal genetic disease has led to the identification of genetic alterations in many cancer types. However, ovarian cancer remains less well characterized. Only a few TSG genes acting in a recessive manner have been identified as somatically mutated or methylated in ovarian cancer, including TP53
(21% in the endometrioid subtype) [25
], and CDKN2A
(79% in the mucinous subtype) [27
]. Biomarkers provide useful tools in screening for cancer and are now emerging as highly informative for monitoring disease status [28
]. They can improve early detection and also the quality of life of patients with ovarian cancer. DNA methylation offers an additional tool that can be used in combination with other markers [29
]. In addition, it has been established that DNA methylation biomarkers are present in patient serum and other body fluids [30
]. To date, several methylated genes have been found to be highly prognostic for specific cancers, including those of the prostate [31
], breast [32
] and lung [33
]. Although some methylated markers such as RASSF1A and GSTP1 have potential as prognostic indicators individually [34
], 'methylation signature' panels could be much more informative [36
] and accurate for monitoring cancer progression. Methylation patterns have previously been suggested to be tumor and stage specific [37
]. Our work demonstrates that there is aberrant DNA methylation at several imprinted DMRs in HOC with changes at PEG1
being the most frequent and earliest alterations detected.