No abstract

Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left-right asymmetry development (PITX2) and to correlate results with known clinicopathological features of breast cancer. Formalin-fixed, paraffin-embedded (FFPE) tissues of breast carcinomas (n = 149) were used for DNA extraction. DNA was modified by bisulphite conversion. Detection of the methylation level of the genes mentioned above was performed by methylation-sensitive high-resolution melting assay (MS-HRM). Based on MS-HRM results for RASSF1A and PITX2, we subdivided the samples into four groups according to methylation level (≤50 % methylated, >50 % methylated, 100 % methylated and completely unmethylated alleles). All degrees of methylation status for both genes underwent analysis of dependence with known clinicopathological features, and we found significant associations. In 134 of 149 (89.9 %) primary breast carcinomas, the RASSF1A promoter was methylated. Total hypermethylation of PITX2 was observed in 60 of 135 (44.4 %) breast cancer cases. RASSF1A hypermethylation had significant association with increased age (p < 0.05), tumour grade (p < 0.0001) and stage (p < 0.0001) in the 100 % methylated group. There was significant association of PITX2 hypermethylation with tumour grade (p < 0.0001) and stage (p < 0.0001). Association between the methylation level of both investigated genes and tumour type was significant for ductal invasive carcinoma cases only (p < 0.0001). This study shows different levels of heterogeneous methylation acquired by MS-HRM assay of the promoter region of RASSF1A and PITX2 and its relationship with clinicopathological features of 149 breast cancer patients. We noticed that immunohistopathological subtypes of breast cancer contain distinct promoter methylation patterns. All these data suggest that hypermethylation of the CpG island promoters of RASSF1A and PITX2 might play an essential role in the very early stages of breast cancer pathogenesis.

Hypermethylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, the present study focused on the DNA methylation profile of invasive breast carcinoma. The aim of the study was to evaluate the prognostic significance of Ras association domain family 1 isoform A (RASSF1A) promoter methylation status in operable breast cancer, and to analyze the utility of this biomarker regarding its association with metastatic and nonmetastatic axillary nodal status. For this purpose, formalin-fixed, paraffin-embedded tissue specimens from 116 breast cancer patients with known axillary nodal status were subjected to assessment of RASSF1A promoter methylation status by methylation-specific polymerase chain reaction (MSP) and methylation-sensitive high-resolution melting assay, and the results were subsequently validated by bisulfite sequencing. A multinomial logistic regression model was used to model the dependence of distinct levels of methylation status of the RASSF1A promoter on the nodal status. Promoter region CpG hypermethylation was identified by MSP in 97 (83.6%) of 116 primary breast tumors, while hypermethylation of RASSF1A was confirmed by MS-HRM in 107 (92.2%) of 116 cases of breast cancer. Based on the results of the multinomial logistic regression model, there was no significant difference between the frequency of RASSF1A promoter methylation and axillary lymph node status of patients in general. However, upon adjustment of pN stage, an association was identified between pN0 lymph node-negative status (without axillary metastases) and percentage of RASSF1A methylation in two groups of heterogeneous methylated alleles with ≤50% methylated (P<0.05) and >50% methylated alleles (P<0.0001). If a patients' nodal status changes from pN- to pN+ then the risk of having >50% methylated alleles increases by 7%. The present study revealed a specific phenomenon, suggesting that the presence of heterogeneous methylated alleles in the RASSF1A gene is significantly associated with lymph node-negative status in breast cancer patients. Furthermore, greater significance with negative axillary nodal status was observed with a higher level of heterogeneous methylated alleles in the RASSF1A gene.