Hypermethylation of cyclin D₂ and DAP kinase is associated with the lobular subtype of breast cancer

Background

Promoter hypermethylation is a common inactivation mechanism in the development and progression of neoplastic transformation. For mammary carcinoma numerous genes have been described to be silenced due to aberrant methylation, some of them already in intraductal carcinomas. Much less is known about the association of hypermethylation events with the different histological subtypes of breast cancer.

Methods

Genomic DNA was isolated from fresh-frozen and formalin-fixed paraffin-embedded biopsies and was treated with bisulfite for subsequent methylation analysis. Altogether 40 lobular breast cancer, 89 ductal breast cancer, and 20 normal breast tissue samples were analyzed. For this purpose, real-time PCR-based quantitative methylation assays were developed for the following genes: p16INK4a, cyclin D₂, RASSF1A, GSTp1, RIZ-1, HIN-1, APC, DAP kinase, Twist, and SOCS-1.

Results

A stringent threshold for scoring a sample as 'methylated' (mean of the methylation level in the control group plus twice the standard deviation) was established for every gene analyzed. Differences in DNA methylation between ductal and lobular breast cancer concerning frequency, intensity, age dependence and concurrence of hypermethylation were uncovered. The most frequently hypermethylated genes in the whole series of 129 breast cancer specimens were cyclin D₂ (75.2%), RASSF1A (71.3%), and HIN-1 (66.7%). The p16INK4a and the RIZ-1 genes were only rarely methylated. A quantitative analysis of the methylation levels using the Mann–Whitney test revealed a statistical significant association of the methylation of the genes DAP kinase and cyclin D₂ with the lobular subtype. By contrast, a mere qualitative scoring of methylation data did not reveal any significant differences.

Conclusions

The results presented in this study demonstrate that subtype-specific patterns of aberrant gene methylation exist in breast cancer, which will help to elucidate the underlying biological differences. These subtype-specific patterns could only be revealed by using stringent real-time PCR-based quantitative methylation assays.