Summary:
Breast cancer and ovarian cancer are the most frequent forms of cancer in women after the age of 40 years old and consitute the most lethal gynecological malignities. In a high percentage, almost 30%, both types are hereditary diseases and connected with specific mutations in some genes, but the rest of the percentage, almost 70%, it has the sporadic form, which means that there is not a germline mutation but is due to epigenetics changes and it is not associated with family members. One of the most important epigenetic changes is the methylation of DNA.
It has been proved, experimentally, that cancer cells, in contrast with the normal ones, are characterized by hypermethylation on the CpG islands of the promoter region of several genes leading to their transcriptional silencing. That means that they can not be translated to proteins and will not be functional. It has been found that the majority of these genes normally act like tumor suppressor genes.
The purpose of this study is to investigate the methylation on the CpG island of the promoter region of NR2F1 gene and was examined in breast and ovarian cancer. This specific gene, according to the international bibliography, relates to the process of relapse and metastasis, because it has been associated with cancer dormancy. In this study, methylation was examined in tumor samples from patients’ peripheral blood with primary and metastatic breast cancer and also in tissue samples from patients with primary ovarian cancer. In the breast cancer case, samples from patients’ peripheral blood were used for the extraction of ctDNA and CTCs. In particular, 25 samples of ctDNA and 97 samples of CTCs were examined in both primary and metastatic tumors.
In the ovarian cancer case, 61 FFPEs samples from primary tumors were examined. These FFPEs samples were used for the extraction of genomic DNA, followed by sodium bisulfite conversion. Then, real time methylation specific PCR (MSP) was performed for the detection of methylation. The guiding principle of this method consists of the selective amplification of the methylated sequence, which is achieved by using the appropriate pairs of primers.
In breast cancer, methylation of the promoter of NR2F1 was observed in 10% of the samples in primary tumors and the percentage of methylation in metastatic tumors
10
was 15%. While the percentage of promoter’s methylation in NR2F1 for ovarian cancer was 8.2%. According to our results, no statistically significant correlation was found between methylation of gene and the available clinicopathological characteristics of patients, but the total number of samples was limited. However, there was an important correlation with Progression Free Survival in the case of ovarian cancer.
In the last decade, a study for the detection of methylation of the promoter of NR2F1 gene, in breast cancer case, was conducted from our research group, but the results were not capable to extract some conclusions about the gene. In this study, methylation of NR2F1 gene promoter was detected for the first time in ovarian cancer. However, it is necessary to further investigate the methylation status of this gene. Τhe prognostic and predictive significance of the methylation of this gene can also be determined, with the association of the results to more clinicopathological characteristics.
Keywords:
CTCs, ctDNA, FFPEs, DNA methylation, real-time MSP, NR2F1, epigenetics, metastasis, dormancy, ovarian cancer, breast cancer
