The Proliferative Activity of Mammary Epithelial Cells in Normal Tissue Predicts Breast Cancer Risk in Premenopausal Women

Abstract

The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67(+) cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12-48.0]. Conversely, the frequency of ER(+) or p27(+) cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER(+): OR = 0.70, 95% CI, 0.33-1.50; p27(+): OR = 0.89, 95% CI, 0.45-1.75). Notably, high Ki67(+)/low p27(+) and high Ki67(+)/low ER(+) cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67(+)/low p27(+) and low Ki67(+)/low ER(+) cell frequencies, respectively, among premenopausal women (Ki67(hi)/p27(lo): OR = 5.08, 95% CI, 1.43-18.1; Ki67(hi)/ER(lo): OR = 4.68, 95% CI, 1.63-13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926-34. ©2016 AACR.

Figure 1. Imaging analysis of the normal breast tissue in the TMAs

A, Tissue and subsequently completed cell segmentation in normal breast tissue using the inForm software. Segmented normal epithelium (red), cell (green) and stained positive cells (pink). Only cells segmented in the normal epithelium were analyzed. B, Spearman correlation between the percentage of p27⁺ cells on p27/ER and p27/Ki67 co-stained slides (r=0.68; N=211). C, Representative double-immunofluorescence images of staining for p27/ER and p27/Ki67 of normal breast tissues from the indicated groups of women. Nuclei are visualized by DAPI (blue) stain. Scale bar: 25 μm.