Mechanisms of osteopontin-stabilized amorphous calcium phosphate calcification in benign and pre-malignant breast disease

Mammographic calcifications are sentinel markers of ductal carcinoma in-situ (DCIS) with or without associated invasive breast cancer (IBC) but also occur in benign breast disease (BBD). Based on the size, shape and distribution of groups of calcifications, radiologists assess the need to perform a biopsy for pathologic diagnosis. The chemistry, mineralogy and cellular and molecular microenvironmental conditions of breast calcifications have been described in relationship to the pathology of breast lesions. However, detailed models to explain the development and expansion of calcifications are lacking, limiting our ability to define relationships of calcifications with prevalent and incident breast lesions. Here we apply a transdisciplinary geology, biology and medicine (GeoBioMed) approach targeting the internal structure, composition and occurrence of amorphous calcium phosphate (ACP) breast calcifications. BBD and DCIS biopsies from an extensively characterized Mayo Clinic cohort were evaluated with high-resolution microscopy and spectroscopy. Results show that calcifications are primarily composed of ACP, ACP at the threshold of transforming toward hydroxyapatite, and minor amounts of cholesterol and waxy substances. ACP nodule formation entails precipitation and coalescence of 100 nm-scale ACP spherules, 100 nm-thick alternating mineral- and organic matter-rich layering, fabric preserving (mimetic) diagenetic replacement of necrotic cells, and osteopontin stabilization within collagen containment. Based on these observations, we propose a model to explain the morphology and formation of ACP calcifications and classification linked to associated pathology. This work aims to guide development of methods to inhibit ACP calcifications within low-risk lesions that prompt unnecessary biopsies.