Introduction/Background Aberrant methylation of CpG islands acquired in tumor cells in promoter regions has been found in many cancers, including breast cancer. DNA hypermethylation of RAR-b and RASSF1A genes, have been associated with breast cancer in many studies which however focus on the analysis of breast tissue samples. In this study, we determined the frequency of aberrant promoter methylation of the RAR-b & RASSF1A genes through molecular analysis of cytological material obtained non-operatively with fine needle aspiration (FNAC).
Methodology Our study retrospectively analyzed 103 patients of our Breast Pathology Clinic. FNAC material was collected in Cytolyt (Hologic). DNA was extracted from the cytological material with Quick-gDNA MiniPrep kit (Zymo) and was subjected to complete bisulfite conversion with EZ DNA Methylation-Gold Kit (Zymo). A methylation specific PCR was carried out with primers specific for either the methylated or unmethylated status for each gene. The final PCR-products were analyzed in 2% agarose gels with electrophoresis.
Results Aberrant methylation was observed in both RAR-b and RASSF1A promoters. In fact, 88% and 84% of the C5 cytologically-classified samples (n=50) were methylated for RASSF1A and RAR-b respectively. Methylated RASSF1A and RAR-b promoters were also found in 81.2% and 62.5% in the C4 cytologically-classified samples (n=16). In the ‘grey zone’ of the C3 cytologically-classified samples (n=11) and RASSF1A and RAR-b methylation frequencies were 63.6% and 45.4% respectively. In comparison, over 70% of the C1 and C2 cytologically-classified samples were unmethylated with low methylation frequencies ranging from 0% to 33.3%.
Conclusion It seems that the higher the cytological diagnosis of the FNA material, the higher the methylation frequency of the RASS1AF and RAR-b promoters. DNA methylation assays during a non-operative FNA procedure could be a valuable pre-operative tool for clinicians in combination with cytology for more precise breast cancer detection and staging.
Disclosure Nothing to disclose.
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