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The objective of the present study was to evaluate the epigenetic changes occurring in early stages of breast cancer. The present study investigated the methylation profile of the ATM, p14 ARF  and p16 INK4a  promoters in total blood and plasma cell-free DNA (cfDNA) from women with impalpable breast lesions compared with in total blood of a control cohort of women without breast lesions. The samples were evaluated using the methylation-specific PCR method. The Fisher's exact test was used to evaluate statistical significance between the methylation and clinical variables. A total of 111 women were evaluated, including 56 women with impalpable breast cancer (39/56 also had paired plasma cfDNA) and 55 women in the control cohort (55 blood DNA). For blood DNA from women with malignant impalpable breast lesions, p16 INK4a  exhibited the greatest percentage of methylation (48%), followed by ATM (37.5%) and p14 ARF  (27%) promoters, regardless of age variation. For plasma cfDNA, the methylation rates for ATM, p14 ARF  and p16 INK4a  were 26, 26 and 10%, respectively. The methylation rates for the blood DNA of controls were the lowest for ATM (9%), p14 ARF  (7%) and p16 INK4a  (7%). The women with impalpable breast lesions (benign and malignant lesions) exhibited the highest methylation rate, regardless of age, compared with the paired plasma cfDNA and controls. This epigenetic change was statistically significant for the promoters of ATM (P=0.009) and p16 INK4a  (P=0.001) (impalpable breast lesions vs. control). The present study demonstrated that epigenetic changes occurring in the ATM and CDKN2A genes detectable in liquid biopsy were associated with the development of impalpable breast lesions.

Methylation profiling in promoter sequences of ATM and CDKN2A (p14ARF/p16INK4a) genes in blood and cfDNA from women with impalpable breast lesions