Promoter hypermethylation of p16, BRCA1 and RASSF1A genes in triple-negative breast cancer patients from Serbia

PURPOSE: In order to investigate if aberrant promoter methylation of p16, BRCA1 and RASSF1A genes contributes to biological behavior of triple-negative breast cancer (TNBC), marked as the most aggressive phenotype of breast cancer, we compared the hypermethylation pattern between TNBC and ER+PR+Her2- breast cancer.

METHODS: 131 patients with histologically confirmed breast cancers were included - 61 TNBC and 70 ER+PR+Her2- cases. The patients were followed up for 1-87 months (median 78). DNA from tumor tissues was isolated by the salting out procedure. The methylation status was assessed by nested methylation-specific PCR after bisulfite modification of DNA.

RESULTS: The frequency of p16 hypermethylated breast cancer cases was significantly higher in TNBC than in ER+PR+Her2- group (33; 54.1% vs. 20; 28.6%, p=0.00298). Co-methylated p16 and RASSF1A genes were more frequent in the TNBC than in ER+PR+Her2- group (20; 32.8% vs. 10; 14.3%, p=0.0225). The same result was observed when hypermethylated BRCA1 gene was added in the analysis: 12; 19.7% vs. 3; 4.3%, p=0.00791. Although there was significant difference in disease-free survival (DFS) and overall survival (OS) between TNBC and ER+PR+Her2- group, further analysis of co-methylation of p16 and RASSF1A (p16+RASSF1A+) showed that DFS was significantly shorter in the patients with both genes co-methylated in TNBC than in ER+PR+Her-2- group (8/20; 40% vs. 2/10; 20%, p=0.03272).

CONCLUSIONS: The obtained data indicate that hypermethylated p16 and RASSF1A cell-cycle inhibitor genes might be considered as biomarkers for bad prognosis in breast cancer. Hypermethylation of these genes may influence the clinical disease course, distinguishing a particular group of TNBC patients with even more aggressive phenotype.