RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients.

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.

RASSF1A and CDH1 hypermethylation as potential epimarkers in breast cancer.

Breast cancer is the most common cancer in women worldwide, representing 28.2% of all female malignancies. In addition to genetic changes, epigenetic events, as aberrant DNA methylation and histone modification, are responsible for cancer development. Many tumour suppressor genes are inactivated by DNA hypermethylation, which could be utilized for identification of new epigenetic biomarkers. To investigate the relation between DNA methylation level and breast cancer progression, we analysed DNA methylation in RASSF1A and CDH1 promoters using quantitative multiplex methylation-specific PCR in paraffin-embedded tumour tissues and blood samples from 92 breast cancer patients and 50 controls, respectively. The associations between RASSF1A and CDH1 methylation levels and clinico-pathological parameters were tested by Kruskal-Wallis and van der Waerden ANOVA tests. Out of 92 breast cancer patients, 76 (82.6%) manifested various levels of RASSF1A (range from 1.20 to 92.63%) and 20 (21.7%) of CDH1 (range from 1.20 to 79.62%) methylation. However, no methylation was found in 50 controls. Increasing trends in RASSF1A methylation were observed in tumour size, lymph node status and TNM stage, but only CDH1 methylation levels showed statistically significant differences between the patient subgroups in lymph node status and IHC subtype. Overall, stable relatively high RASSF1A methylation could be utilised as universal tumour marker and the less frequent but highly methylated CDH1 promoter can serve for identification of potentially metastasising tumours.

Epigenetic tools in potential anticancer therapy.

Human cancer represents a heterogeneous group of diseases that are driven by progressive genetic and epigenetic abnormalities. The latter alterations involve hypermethylation and hypomethylation of DNA, and changed patterns of histone modification, with resultant remodeling of the chromatin structure that cause deregulation of the transcription activity of many genes. Unlike the remarkable progress in understanding the processes by which DNA methyltransferases can regulate gene expression and histone deacetylases can induce alteration of chromatin structure, the roles of epigenetic events in tumors remain insufficiently explained. In contrast to genetic changes, the epigenetic alterations in cancer cells can be reversed by the inhibition of DNA methylation and histone deacetylation. Therefore, many inhibition agents for re-expression, predominantly of tumor-suppressor genes, have been identified and tested in laboratory models and numerous clinical trials. Despite in-vitro evidence that a single drug can lead to reactivation of methylated genes, inhibitors of DNA methyltransferases and histone deacetylases have been investigated in combination, or together with cytotoxic chemotherapy, radiotherapy, immunotherapy, or hormonal therapy to improve the therapeutic effect. Ongoing trials are recognizing that the identification of a target group of patients who are more likely to respond to the epigenetic therapy, defining of an optimal dose and schedule of treatment, and the development of more specific inhibitors with minimal unwanted side effects are necessary. Thus, new combinations of anticancer agents, including epigenetic modulators, may lead to a more effective control of cancer.

Redundant DNA methylation in colorectal cancers of Lynch-syndrome patients.

Lynch syndrome is an inherited disease resulting predominantly in colorectal cancer (CRC). The crucial cause is DNA mismatch repair (MMR) malfunction that is associated mostly with MLH1 or MSH2 germline mutations. A significant hallmark of repair defects is a high level of instability in microsatellites (MSI-H). In many sporadic unstable CRCs, the MLH1 gene is inactivated by promoter hypermethylation in addition to extensive promoter methylation in many tumor-suppressor genes known as CpG island methylation phenotype (CIMP). To investigate the possible role of epigenetic alterations in causing MMR deficiency and thereby Lynch syndrome, we evaluated the MLH1 specific and global hypermethylation in hereditary CRCs. Of 22 Lynch-syndrome-related CRCs, 18 (81.8%) demonstrated various levels of DNA methylation; of these, 14 (63.6%) and 4 (18.2%) were methylated in distal and both distal and proximal regions of the MLH1 promoter, respectively. However, only 7/18 (38.9%) of results were confirmed by bisulfite sequencing. Similar methylation patterns in tumors and frequently in matched normal DNA were found in twelve and four patients with MLH1 and MSH2 alterations documented by the absence of protein or presence of germline mutation, respectively. Moreover, the same results were observed in five stable CRCs. None of 22 Lynch-syndrome-related tumors presented CIMP in contrast to 3/10 (30%) stable carcinomas. The rather randomly distributed weak methylation patterns in hereditary CRCs indicate that epigenetic events are redundant in Lynch-syndrome etiology, in contrast to the widespread DNA methylation in sporadic unstable CRCs. These methylation-profile differences can lead to more effective molecular diagnosis of Lynch syndrome.