Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients

Melanie Spitzwieser, Elisabeth Entfellner, Bettina Werner, Walter Pulverer, Georg Pfeiler, Stefan Hacker, Margit Cichna-Markl

Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma. We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing. Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = -0.485, p = 0.041) and tumor-distant tissues (r = -0.498, p = 0.036). From our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity.