Frequent promoter hypermethylation of BRCA2, CDH13, MSH6, PAX5, PAX6 and WT1 in ductal carcinoma in situ and invasive breast cancer.

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism to inactivate tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. In search of epigenetic events related to progression, we used MS-MLPA (ME-0002-B1, MRC-Holland, Amsterdam, The Netherlands) to compare the methylation status of 25 breast cancer-related genes between laser-microdissected ductal carcinoma in situ (DCIS) and adjacent invasive ductal cancer (IDC) lesions in 33 breast cancer patients. Using absolute methylation percentages or, alternatively, a 15% cut-off for methylation, promoter methylation in DCIS and IDC was not significantly different for any of the genes studied. Aberrant methylation in at least 50% of both the DCIS and adjacent IDC lesions was observed for PAX6, BRCA2, PAX5, WT1, CDH13 and MSH6. Methylation of MSH6, however, was also frequent in normal breast tissue. In contrast, CDKN2A, CHFR, PYCARD and one of the two analysed RB1 CpG loci were rarely (<5%) methylated in both lesions. CDKN2A and GSTP1 showed significantly (p < 0.002) higher mean methylation levels in increasing grades (I, II, III) of DCIS (1% versus 4% versus 7% for CDKN2A and 6% versus 26% versus 28% for GSTP1). The mean number of methylated genes per sample increased with increasing grades of DCIS (p = 0.014) and IDC (p = 0.109). In contrast to the observations in DCIS, none of the analysed genes showed significantly higher methylation levels with increasing grades of IDC. In conclusion, there were no differences in promoter methylation between DCIS and IDC in the 25 analysed genes, suggesting that DCIS, at the epigenetic level, is as advanced as IDC. Promoter hypermethylation of PAX6, BRCA2, PAX5, WT1, CDH13 and MSH6 seems to be a frequent early event in breast cancer and methylation levels of GSTP1 (and CDKN2A, although still low) seem to increase with increasing DCIS grade.