The applicability of cyclin D2 and RARbeta2 methylated markers for the development of a breast tumor screening assay was evaluated. Overall, 76 volunteers (mean age, 50.4 years), including clinically healthy women and women with breast lesions, were enrolled in a blind study of methylation of the cyclin D2 and RARbeta2 genes. Methylation-specific PCR (MSP) was conducted using total circulating DNA (cirDNA) from the blood, including the cell-free and cell-surface-bound DNA fractions. Only 1 of the 25 women of the clinically healthy group displayed methylated cyclin D2 gene (4%). However, 42% of the patients with primary diagnosis of breast fibroadenoma showed an aberrant methylation of at least one of the tested genes in the cirDNA. The number of patients with breast lesions (mastopathy) not diagnosed as fibroadenoma that displayed methylation was lower (33%). A long-term follow-up study based on a quantitative evaluation of cyclin D2 and RARbeta2 methylation in cirDNA will provide the data on applicability of these markers for breast tumor predictive diagnostics.
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