[Multiplexed methylation analysis--a new technology to analyse the methylation pattern of laser microdissected cells of normal breast tissue, DCIS and invasive ductal carcinoma of the breast].

Abstract

Aims: DNA methylation has been shown to play an important role in breast cancer pathogenesis, but up until now it is not clear how the tissue components contribute to the overall methylation of the sample, because microdissection does not provide sufficient material for most standard methylation assays.

Methods: We developed a technology to analyse several methylation markers in a limited number of cells dissected from tissue sections. To evaluate the technology, we analysed, among others, the methylation markers PITX2, RASSF1A and TFF1 in 79 samples of three PITX2 methylation positive invasive ductal carcinomas. The microdissected samples were from the invasive part, the intraductal part, the stroma, tumor infiltrating lymphocytes, chest wall muscle, adipose tissue and healthy ducts.

Results: The multiplexed methylation analysis allows for the quantitative analysis of methylation patterns in microdissected samples with as few as 100 genome copies. In all analysed patients PITX2 and RASSF1A were highly methylated in invasive and intraductal carcinoma cells compared to other tissue components. TFF1 behaved inversely. PITX2 showed some methylation in normal adjacent breast tissue. The methylation of the individual markers varied little within one tissue type and between blocks.

Conclusions: This technology is a powerful tool to analyse the methylation of multiple markers in different microdissected tissue components. Methylation patterns may differ significantly between different markers and tissue components. This technology may help to analyse different transitions states of breast and other cancers.