We examined normal and abnormal ovarian tissues for the presence of abnormal CpG island promoter methylation of 23 genes, as well as for p53 mutations and overexpression of Her-2/neu. Four genes (MINT31, HIC1, RASSF1, and CABIN1) were significantly more frequently methylated in OC tissue than in tissue from normal ovaries or ovaries with benign disease while a number of other genes, including APC, BRCA1, CDH1, and CDKN2B, were somewhat more likely to be methylated in OC tissue, although these differences did not reach statistical significance after adjustment for multiple comparisons. The frequency of hypermethylation of five of these genes in OC tissues (MINT31, 42%; RASSF1, 34%; CDH1, 18%; HIC1, 34%; APC, 22%) was similar to frequencies reported in previous studies. Methylation of MINT31 has been reported in 54% [15
], RASSF1 in 10%–50% [5
], and CDH1 in 26%–42% [20
], HIC1 in 16–35% [15
], and APC in 18%–22% [20
] of OC cases.
In contrast to most previous studies, this study included examination of DNA methylation of a large number of normal ovarian tissues from women without OC (N=68) as well as tissues from OCs (N=100), permitting a direct assessment of the risk of OC associated with gene specific DNA hypermethylation. This is important both for understanding the pathogenesis of OC as well as for assessment of the potential of aberrantly methylated genes as biomarkers for OC. Of the over 90 studies that have previously reported on aberrant methylation in OC, most have not included tissue from women without OC [10
]. In the largest study (n=215) of methylation in OC reported, no normal controls were included [40
]. Further, in some studies, “normal” tissue controls consisted of adjacent non-cancerous tissue obtained from women with OC [5
]. Only three studies have included normal tissues from women without OC [20
In our study, methylation of MINT31, HIC1, RASSF1A, APC, or BRCA1 each identified a large percentage (>20%) of OC cases and also had high specificity (>85%) for OC, while CDH1, TERC, CDH13, or CABIN1 were less sensitive, each identifying 14–18% of OC cases, while remaining specific (>90%). Rathi et al [20
] similarly reported hypermethylation of RASSF1A, HIC1, CDH1 (E-cadherin), APC, and CDH13 (H-cadherin) in 18–41% of 49 OC tissue and 15% of 39 nonmalignant tissues. Makarla et al [35
] detected hypermethylation of CDKN2A (p16), CDH1 (E-cadherin), CDH13 (H-cadherin), RASSF1A, and APC in 22–30% of 23 OC cases but in less than 15% of 23 benign cystadenomas and 16 normal tissues. Most recently, Tam et al. [45
] detected high rates of hypermethylation of HIC1 (52%), MINT31 (51%), APC (47%) in 89 OC tissues, while hypermethylation of these three genes was less frequent in 19 benign tumors (21%, 16%, and 26%, respectively) and 16 normal ovarian tissues (13%, 0%, and 25%, respectively).
This study is unique as it is the first to examine the relationship between genetic alterations and DNA methylation in OC. Interestingly, within tumor specimens, hypermethylation of three different genes (MINT31, RASSF1 and CDH13) was associated with overexpression of Her-2/neu. Recently, several studies of other cancers have noted associations between methylation of specific loci and genetic mutations (38
), with methylation of CDKN2A associated with K-ras mutations in non-small-cell lung cancer [12
], methylation of 5 genes (CDKN2A, MINT1, MINT2, MINT31 and MLH1) associated with mutations of BRAF in colon cancer [14
], and methylation of CDH13, PGR and HSD17B4 associated with overexpression of Her-2/neu in breast cancer [13
]. The mechanistic relationship between such mutations and epigenetic changes is unclear. Whether the existence of such associations is associated with specific OC histologic subtypes or a specific clinical course is unknown, although such associations have been proposed for NSCLC [12
], colon [14
] and breast cancers [13
In the present study we found that (after adjusting for stage of disease) methylation of CABIN1 was strongly associated with responsiveness to chemotherapy. A number of previous studies have examined associations between hypermethylation of specific genes or global methylation patterns and response to chemotherapy. Methylation of hMLH1 [46
], MCJ [38
], IGFBP3 [10
], p16 [47
] and BRCA1 [48
] have been associated with poor response to platinum-based chemotherapy and poor survival. Wei et al examined the overall pattern of methylation and found that, as compared to drug-sensitive cell lines, drug-resistant OC cell lines had increased number of methylated loci, and identified a group of OCs associated with poor survival [25
]. Clearly, whether methylation of any of these genes can predict therapy responsiveness and survival needs further investigation.
Our study has a number of limitations. We used a qualitative MSP assay to assess aberrant methylation, which does not allow for quantitative of methylation and may be associated with a lack of specificity [49
]. However, if conventional MSP lacks specificity, the potential non-specificity of the MSP assay would most likely have resulted in non-differential misclassification, and thus may have attenuated our study risk estimates. Therefore, due to misclassification, the relationship between aberrant methylation and ovarian cancer may be stronger than what we observed. Due to the qualitative nature of our assay, we could not assess the possibility that aberrant methylation of the genes studied is also at lower levels in normal as compared to OC tissues, as methylation quantity cannot be determined in the present study. This limitation is common to most previous studies as well. However, while quantitative MSP may be more sensitive under many conditions, conventional MSP may be more sensitive to detect methylation changes in samples with limited amount of DNA without losing specificity [50
]. Another potential weakness of this study is that the tissues analyzed included both ovarian surface epithelial cells as well as underlying ovarian stromal cells. However, while previously it was widely assumed that the pathogenesis of OCs was limited to changes in the ovarian surface epithelial cells, several studies have demonstrated that similar changes also occurred in adjacent stromal cells during the pathogenesis of epithelial OC. For example, SPARC has been reported to be up-regulated in stroma adjacent to epithelial OC [51
]. COX2 and iNOS expressing macrophages were not only present in OC associated stroma, but also in stroma associated with benign tumors [52
]. Further, similar genetic alterations were detected in both epithelial and stromal cells of OC [53
]. Thus far there has not been a study examining epigenetic changes in stromal cells in ovaries containing benign or malignant ovarian neoplasia. In the present study, we were not able to determine whether the aberrant methylation had occurred in epithelial or stromal cells. However, identifying the cell of origin of DNA hypermethylation will enhance our understanding the tumorigenesis of OC. Further, if changes in stromal cells are shown to be important in the early pathogenesis of OC, the use of histologically normal tissue adjacent to OC would make identification of such early epigenetic changes impossible.
Recent research shows that DNA methylation studies not only identify potential biomarkers for cancer diagnosis and prognosis, but also provide insight for tumorigenesis. Our current study confirms that this is also the case for ovarian cancer: DNA methylation is frequent in ovarian cancer, and certain methylation is associated with chemosensitivity. We also provide evidence for the complex interplay between genetic and epigenetic changes during tumorigenesis. Future studies are warranted to determine whether this interaction implies causal effect, or merely tumor specific association.