Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.

OBJECTIVE: Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. METHODS: Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. RESULTS: The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. CONCLUSIONS: This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.

Frequent IGF2/H19 domain epigenetic alterations and elevated IGF2 expression in epithelial ovarian cancer.

Overexpression of the imprinted insulin-like growth factor-II (IGF2) is a prominent characteristic of gynecologic malignancies. The purpose of this study was to determine whether IGF2 loss of imprinting (LOI), aberrant H19 expression, and/or epigenetic deregulation of the IGF2/H19 imprinted domain contributes to elevated IGF2 expression in serous epithelial ovarian tumors. IGF2 LOI was observed in 5 of 23 informative serous epithelial ovarian cancers, but this did not correlate with elevated expression of IGF2 H19 RNA expression levels were also found not to correlate with IGF2 transcript levels. However, we identified positive correlations between elevated IGF2 expression and hypermethylation of CCCTC transcription factor binding sites 1 and 6 at the H19 proximal imprint center (P = 0.05 and 0.02, respectively). Hypermethylation of CCCTC transcription factor sites 1 and 6 was observed more frequently in cancer DNA compared with lymphocyte DNA obtained from women without malignancy (P < 0.0001 for both sites 1 and 6). Ovarian cancers were also more likely to exhibit maternal allele-specific hypomethylation upstream of the imprinted IGF2 promoters when compared with normal lymphocyte DNA (P = 0.004). This is the same region shown previously to be hypomethylated in colon cancers with IGF2 LOI, but this was not associated with LOI in ovarian cancers. Elevated IGF2 expression is a frequent event in serous ovarian cancer and this occurs in the absence of IGF2 LOI. These data indicate that the epigenetic changes observed in these cancers at the imprint center may contribute to IGF2 overexpression in a novel mechanistic manner.

Imprinted expression of the canine IGF2R, in the absence of an anti-sense transcript or promoter methylation.

Imprinted genes are epigenetically modified in a parent of origin-dependent manner, and as a consequence, are differentially expressed. Although the evolution of genomic imprinting is a subject of intense debate, imprinted genes have been studied primarily in mice and humans and in a small number of marsupial mammals. Comparative studies involving rodents and primates are of limited value, as they belong to the same superordinal group of eutherian mammals (Euarchontoglires). On the other hand, comparisons involving marsupials may not be informative, due to phylogenetic distance. Canis familiaris belongs to Laurasiatheria, a sister-group of Euarchontoglires, and should prove useful in comparative studies of imprinted genes. Using RT-PCR we demonstrate monoallelic expression of the canine IGF2R in several tissues, including uterus and umbilical cord. In the case of umbilical cord, we identify the expressed allele as maternally derived. The canine IGF2R is thus an imprinted gene. Using bisulfite sequencing, we show that the canine IGF2R resembles the imprinted mouse Igf2r in having a CpG island in intron 2 that is hemi-methylated. However, it differs from the mouse gene in that maintenance of the monoallelic expression of canine IGF2R does not require expression of an anti-sense transcript from the paternally derived allele, or methylation of the repressed IGF2R promoter. In these two important features, the imprinted canine gene resembles the imprinted opossum IGF2R. Our data suggest that these features were properties of the ancestral imprinted IGF2R and that the anti-sense transcript (Air) and promoter methylation observed in mouse are derived features of the mouse Igf2r locus.