AbstractBackground: Many established breast cancer risk factors are related to the timing and duration of exposure to reproductive hormones, which are known to drive breast epithelial cell proliferation. The epigenetic molecular clock hypothesis suggests that CpG island methylation records the cell division history of benign epithelium. In proliferative epithelium, such as breast, this may provide an individualized cell-based measure of cancer risk.Methods: Methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-β2 was measured by quantitative multiplex methylation-specific PCR in 290 benign and malignant breast epithelial cell samples obtained by palpation-directed fine-needle aspiration biopsy from 164 women. Univariate, multivariate, and unsupervised cluster analysis was used to establish the relationship between TSG methylation and a personal history of breast cancer, predicted breast cancer risk, and specific breast cancer risk factors.Results: RASSF1A methylation was highly correlated with breast cancer risk [odds ratio (OR), 5.28; 95% confidence interval (95% CI), 1.95-14.32; P = 0.001], atypical cytology (OR, 4.11; 95% CI, 1.30-12.98; P = 0.016), and benign breast disease requiring biopsy (OR, 6.12; 95% CI, 1.41-26.51; P = 0.016). RASSF1A methylation increased linearly between ages 32 and 55. Increasing parity was associated with decreased APC methylation.Conclusions: TSG methylation increases in benign breast epithelium with increasing age. Because it is independently related to a personal history of benign or malignant breast disease and to predicted breast cancer risk, it may have value for breast cancer risk stratification and as a surrogate endpoint marker in prevention trials. (Cancer Epidemiol Biomarkers Prev 2008;17(5):1051–9)