Background: There is growing evidence that methylation plays an important role in breast cancer (BC) development and that characterization of tumorspecific methylation profiles may allow the identification of specific biomarkers to discriminate BC subtypes. Significant differences in tumor-associated gene methylation patterns have been related to ER, PR and HER2 status. The contribution of DNA methylation and the precise targets of epigenetic alterations in male breast cancer (MBC) development have not yet well investigated. Materials and Methods: Using candidate-gene approach, we performed promoter methylation analysis of a panel of BC-related genes (hTERT, ESR1, RASSF1, AR, BCL2, MYC, WNT1, BRCA1, and CHEK2), in tumors and matched normal (blood) samples from 69 MBC patients. The analysis was performed by Pyrosequencing, a highly sensitive and reproducible method, which provides absolute quantitative information on bases at each CpG site analyzed. Methylation levels were determined by calculating the average of methylation for each gene, respectively in tumor and normal samples. ANOVA and Kruskal–Wallis tests were used to identify significant differences in methylation levels: (1) between normal and tumor samples; (2) among tumors stratified according to relevant clinical-pathologic features, including BRCA1/2 mutation. Results: Promoter methylation of all the 9 genes analyzed was found in the 69 MBC cases. A gene specific variability in methylation levels was observed, with MYC showing the lowest and CHEK2 the highest methylation level. Overall, methylation levels of each gene considered were higher in tumors than in the corresponding normal samples, and, in particular, significant differences emerged for hTERT (p < 0.0001), ESR1 (p < 0.0001), RASSF1 (p < 0.0001), MYC (p < 0.0001) and WNT1 (p = 0.047). Furthermore, significant association emerged between BRCA1/2 mutations and higher RASSF1 methylation level (p = 0.008), BRCA1/2 wild-type and higher AR methylation level (p = 0.016), HER2+ status and higher RASSF1 methylation level (p = 0.01), G3 (high tumor grade) and higher RASSF1 methylation level (p = 0.007) and between PR− status and higher WNT1 methylation level (p = 0.014). Conclusion: Overall, our results indicate that alterations in gene methylation profiles are common in MBC and that tumor-associated gene methylation patterns may identify specific MBC subgroups. Study supported by AIRC (IG 12780) to L.O. No conflict of interest.
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