Gene Methylation and Cytological Atypia in Random Fine-Needle Aspirates for Assessment of Breast Cancer Risk

Abstract

Methods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density, and obtained random fine needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (p<0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number and BMI. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation

“…We enrolled 380 women with carefully documented menstrual and menopause status, and examined cumulative DNA methylation of eight genes in rFNA samples, and local breast steroid hormone concentration. We recently reported that cumulative DNA methylation of eight genes were not affected by menstrual or menopausal states, and showed significant positive associations with cytologic atypia (27). We now report on sex steroid hormone concentrations within breast tissue, its physiologic variation in a subset of 119 women, and explore relations of breast hormones with cytology, DNA methylation, and established clinical risk factors.…”

“…We have evaluated steroid hormones in rFNA and matching blood serum samples of 119 women whose rFNA samples were collected in PBS) with ascorbic acid from our recent published study (NU 08B2; ref. 27) where 380 pre-and postmenopausal women without a breast cancer history were recruited. Data on cellularity of the rFNA sample, cytomorphology, gene methylation (cumulative methylation index, CMI), along with clinical data including lifetime Gail risk and mammographic density of this study population have been described recently (27).…”

“…27) where 380 pre-and postmenopausal women without a breast cancer history were recruited. Data on cellularity of the rFNA sample, cytomorphology, gene methylation (cumulative methylation index, CMI), along with clinical data including lifetime Gail risk and mammographic density of this study population have been described recently (27). Menopausal status and menstrual phase was determined by serum concentrations of follicular stimulating hormone (FSH), progesterone, and estradiol as described previously (18).…”

“…The clinical characteristics of our study followed a non-Gaussian distribution (27), therefore data were reported as medians and interquartile ranges (IQR). Univariate analyses were performed using Mann-Whitney U test for two group comparisons and Kruskal-Wallis tests were performed for comparison more than two groups.…”

“…Therefore, the contribution of local breast hormone concentrations to these breastspecific risk factors is of interest and may provide mechanistic insights. Also of significant interest is the influence of local hormone concentration on the DNA methylation status of cancer-related genes, a potentially powerful risk indicator for breast cancer (23)(24)(25), which we have previously shown to be related to cytologic atypia (26,27).…”