Breast hormone concentrations in random fine-needle aspirates of healthy women associate with cytological atypia and gene methylation

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cyto-morphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r ¼ 0.34, P_adj ¼ 0.001 and r ¼ 0.69, P_adj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (P_adj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P ¼ 0.026 and P_adj ¼ 0.208). Breast androgens were significantly correlated with breast density (Spearman r ¼ 0.27, P_adj ¼ 0.02 for testosterone) and CMI (Spearman r ¼ 0.3, P_adj ¼ 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study.