Abstract
BackgroundBreast 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.MethodsWe 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.ResultsTumor-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).ConclusionsFrom 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.
Highlights
Breast carcinogenesis is a multistep process involving genetic and epigenetic changes
Methylation-sensitive high resolution melting (MS-HRM) Primer sequences for adenomatous polyposis coli (APC) [32], BRCA1 [33], Cyclin-dependent kinase inhibitor 2A (CDKN2A) [34] and phosphatase and tensin homolog (PTEN) [35] were taken from literature, those for CDKN2A, Estrogen receptor 1 (ESR1), HER2/neu and LINE-1 were designed in-house
Association between the DNA methylation status of individual genes with the age and clinicopathological parameters For none of the genes investigated in the present study, we found a correlation between the age of the healthy women and the DNA methylation status in their breast tissues
Summary
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, been found in tumors, and in tumor-surrounding tissue appearing histologically normal. Breast carcinogenesis is a multistep process involving genetic and epigenetic changes, in particular aberrations in the extent of DNA methylation [2,3,4]. Tumors of many cancer types including breast cancer are frequently characterized by the co-occurrence of global DNA hypomethylation and gene-specific hypermethylation [5]. Several studies have already evaluated the applicability of gene-specific hypermethylation as diagnostic [8, 9], prognostic [10,11,12] or predictive [13, 14] biomarker in breast cancer
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