Analysis On The Aberrant Methylation Of Female Breast Cancer-related Genes

Objective: Breast cancer(BC) is one of the most common cancers in women in the world. The clinical relevance of aberrant methylation of key genes is being increasingly recognized in BC. The inactivation of functional genes by epigenetic alterations is a critical event in the breast tumorigenesis, which may potentially be used as biomarkers for the diagnosis and prognosis of the disease. Our study aimed(1) to evaluate the promoter methylation status of seven candidate genes in breast cancer and to explore their potential use as a biomarker for the diagnosis of BC;(2) to systematically review the promoter methylation of BRCA1 gene and its relationship to the clinical outcomes of breast cancer patients.Methods: This study included two parts. First, we analyzed the relation between the methylation of seven genes(BRCA1, GSTP1, P16INK4 A, MGMT, PTEN, RARβ2 and CCND2) and the breast cancer using a case-control study design and molecular epidemiological methods. We recruited 70 BC patients as cases and 20 patients with benign breast disease(BBD) as controls. The methylation-specific PCR(MSP) was carried out to measure the methylation status of selected genes in the promoter region. The protein expression of the selected gene was evaluated by the immunohistochemistry. Discriminant validity of these genes was examined with sensitivity, specificity and the area under the receiver operator characteristic(ROC) curve(AUC). Then, we performed a meta-analysis following the PRISMA guideline to clarify the role of BRCA1 methylation in the prognosis of patients with BC. Relevant articles were identified by searching Pub Med, Web of Science and Embase database until December 2014. The pooled hazard ratio(HR) and 95% confidence interval(CI) were applied to estimate the prognostic effect of BRCA1 methylation. Random or fixed effect model was chosen based on the heterogeneity test.Results:(1) Findings from the molecular epidemiological studies revealed that 94.3% BC samples were hypermethylated in at least one gene. The frequency of hypermethylation of BRCA1, GSTP1, P16INK4 A, MGMT, PTEN, RARβ2 and CCND2 in BC tissues was 24.3%, 31.4%, 40.0%, 27.1%, 48.6%, 55.7 and 67.1%, respectively, which was higher than that in controls(0%, 0%, 20.0%, 25.0%, 40.0%, 40.0% and 40.0%, respectively). Immunohistochemical analysis demonstrated that BRCA1 and GSTP1 methylation were significantly associated with the down-regulation of the gene expression. When we combined BRCA1 and GSTP1 as the biomarker for diagnosing BC, the AUC reached to 0.721(95% CI: 0.616-0.827). The sensitivity was 44.3% and specificity was 100.0% when we set the cut-point was 1(hypermethylation in at least on gene). If we used all seven candidate genes, the AUC was 0.741(95% CI: 0.631-0.850). If we set the cut-point as 3(hypermethylation in at least three genes), the sensitivity and specificity was 58.6% and 80.0%, respectively.(2) A total of 3374 patients from 11 studies were included in the meta-analysis. BRCA1 methylation was found to be significantly correlated with a poor overall survival(OS) of breast cancer patients, with the combined HR(95% CI) of 2.11(1.42-3.13). After adjusting for potential confounders using the Cox regression model, the pooled HR(95% CI) of BRCA1 methylation on patients’ overall survival was 1.83(1.08-3.09). If we used the disease free survival(DFS) as the outcome index, the combined HR(95% CI) was 2.30(1.35-3.93) for univariate analysis and 3.72(95%CI: 1.65-8.38) for multivariate analysis, respectively. Subgroup analysis on specimen types revealed that the pooled HR(95% CI) for OS was 1.75(1.25-2.46) when using the formalin-fixed paraffin-embedded(FFPE) specimen, 1.38(0.16-11.84) when using the fresh frozen tissues, and 5.97(2.35-15.13) when using the serum. As for the DFS, the pooled HR(95% CI) was 1.85(0.99-3.49) when using the FFPE specimen, and 2.78(1.47-5.28) when using the fresh frozen tissues, respectively. BRCA1 promoter methylation remained a favorable factor in regard to DFS in triple-negative breast cancer(TNBC)(pooled HR: 0.38; 95% CI: 0.24-0.62). But in non-TNBC cases, the HR(95% CI) was 1.58(1.20-2.07).Conclusion: Promoter hypermethylation of BRCA1, GSTP1, P16INK4 A, MGMT, PTEN, RARβ2 and CCND2 was frequently observed in BC patients and related with a variety of clinical pathological features. Hypermethylation of BRCA1 and GSTP1 was more common in cancerous tissues and might be used as promising biomarkers for the diagnosis of BC. The meta-analysis further provided evidence that BRCA1 methylation is associated with a poor survival of BC. Our study underscores the potential utility of DNA methylation in specific genes as a promising biomarker for the diagnosis and prognosis of breast cancer.