Stromal expression of miR-21 identifies high-risk group in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. Expression of miR-21, an oncomiR, is frequently altered and may be distinctly expressed in the tumor stroma. Because tumor lesions are a complex mixture of cell types, we hypothesized that analysis of miR-21 expression at single-cell resolution could provide more accurate information to assess disease recurrence risk and BC-related death. We implemented a fully automated, tissue slide-based assay to detect miR-21 expression in 988 patients with BC. The miR-21(High) group exhibited shorter recurrence-free survival [hazard ratio (HR), 1.71; P < 0.001] and BC-specific survival (HR, 1.96; P < 0.001) in multivariate regression analyses. When tumor compartment and levels of miR-21 expression were considered, significant associations with poor clinical outcome were detected exclusively in tumor epithelia from estrogen receptor- and/or progesterone receptor-positive human epidermal growth factor receptor 2-negative cases [recurrence-free survival: HR, 3.67 (P = 0.006); BC-specific survival: HR, 5.13 (P = 0.002)] and in tumor stroma from TNBC cases [recurrence-free survival: HR, 2.59 (P = 0.013); BC-specific survival: HR, 3.37 (P = 0.003)]. These findings suggest that the context of altered miR-21 expression provides clinically relevant information. Importantly, miR-21 expression was predominantly up-regulated and potentially prognostic in the tumor stroma of TNBC.

Figure 1

Patterns, quantification, and scoring of miR-21 expression in breast cancer lesions. A and B: Representative images of multicolor detection of miR-21, snRNA U6, and CK19 in BC cell lines (A) and tissue cores (B). miR-21 heat map images were generated using histogram-based image segmentation analysis; the background signal was set on the basis of exposed slide areas (eg, tissue holes). Other categories were defined relative to the highest background intensity (black) as onefold (blue), twofold (cyan), threefold (orange), and fourfold or more (red) over background. Cases with a score of 1 to 2 and 3 to 4 were categorized low and high miR-21 expression, respectively. C: Summary of miR-21 expression levels, tumor compartment of expression, and molecular subtype on the basis of ER/PR/HER2 status in entire patient cohort. Original magnification, ×200 (A and B). Ca, tumor epithelia; Stroma, tumor stroma.

Figure 2

Kaplan-Meier plots of disease recurrence on the basis of miR-21 expression. Kaplan-Meier survival curves for breast cancer–specific recurrence were plotted by miR-21 category for all cases or major subtypes on the basis of ER/PR/HER2 status. Curves were also plotted by tumor compartment (miR-21Hi^Cancer, miR-21Hi^Stroma, and miR-21^Low).

Figure 3

Kaplan-Meier plots for disease outcome on the basis of miR-21 expression. Kaplan-Meier survival curves for breast cancer–specific deaths were plotted by miR-21 category for all cases or major subtypes on the basis of ER/PR/HER2 status. Curves were also plotted by tumor compartment (miR-21Hi^Cancer, miR-21Hi^Stroma, and miR-21^Low).