Mammary microcalcifications in breast cancer: simple bystanders or active phenomenon mediated by epithelial to mesenchymal transition?

Abstract

Mammary microcalcifications have a crucial role in breast cancer detection, but the processes that induce their formation are unknown. Moreover, recent studies have described the occurrence of the epithelial–mesenchymal transition (EMT) in breast cancer, but its role is not defined. In this study, we hypothesized that epithelial cells acquire mesenchymal characteristics and become capable of producing breast microcalcifications. Moreover, we wanted to further investigate the evolution of the breast lesions with microcalcifications hypothesizing and investigating a pathological mechanism able to describe this phenomenon. Methods Breast sample biopsies with microcalcifications underwent energy dispersive X-ray microanalysis to better define the elemental composition of the microcalcifications. Images by mammographic exams were used to extrapolate pixel-wise values of density-related microcalcification features. Breast sample biopsies without microcalcifications were used as controls. The ultrastructural phenotype of breast cells near to calcium deposits was also investigated to verify EMT in relation to breast microcalcifications. The mesenchymal phenotype and tissue mineralization were studied by immunostaining for vimentin, BMP-2, β2-microglobulin, β-catenin and osteopontin (OPN). Results The complex formation of calcium hydroxyapatite was strictly associated with malignant lesions whereas calcium-oxalate is mainly reported in benign lesions. Notably, for the first time, we observed the presence of magnesium-substituted hydroxyapatite, which was frequently noted in breast cancer but never found in benign lesions. In the 69% of the cases, Mg-Hap wasradiologically detectable as the “casting type” microcalcification. Moreover, pixel measured in correspondence to microcalcifications revealed that Mg-HAp microcalcifications were significantly more radiodense than those made of HA and CO. Morphological studies demonstrated that epithelial cells with mesenchymal characteristics were significantly increased in infiltrating carcinomas with microcalcifications and in cells with ultrastructural features typical of osteoblasts close to microcalcifications. These data were strengthened by the rate of cells expressing molecules typically involved during physiological mineralization (i.e. BMP-2, OPN) that discriminated infiltrating carcinomas with microcalcifications from those without microcalcifications. Conclusions We found significant differences in the elemental composition of calcifications between benign and malignant lesions. Observations of cell phenotype led us to hypothesize that under specific stimuli, mammary cells, which despite retaining a minimal epithelial phenotype (confirmed by cytokeratin expression), may acquire some mesenchymal characteristics transforming themselves into cells with an osteoblast-like phenotype, and are able to contribute to the production of breast microcalcifications