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Page 2 of 7 Bonfiglio et al. J Cancer Metastasis Treat 2019;5:29 I http://dx.doi.org/10.20517/2394-4722.2018.88

Keywords: Microcalcifications, breast cancer, bone metastasis, breast osteoblast-like cells, bone morphogenetic
proteins 2, pentraxin-3

INTRODUCTION
[1,2]
Bone metastasis from breast cancer represent the main disability associated to breast cancer . Indeed, the
occurrence of bone metastatic lesions affected the patients’ quality of life by inducing pain, hypercalcemia,
[2]
bone fracture and spinal compression . Also, the progression of metastatic lesions is often the cause of the
[2]
patient’s death .
In this context, the early identification of breast cancer lesions with high propensity to form bone metastasis
could improve the patient’s survival allowing to clinicians to choose the more appropriate therapeutical
protocol.

In the last years, several studies investigated the cellular and molecular mechanisms involved in the breast
[3-6]
cancer osteotropism . In particular, our group described, for the first time, a new breast cancer cell type
[7]
showing an osteoblast-like phenotype, the breast osteoblast-like cells (BOLCs) . As the real osteoblasts, it
is demonstrated that these cells are capable to product calcium crystals made of hydroxyapatite (HA) of
[7]
magnesium substituted hydroxyapatite (MgHAp) in a process similar to the physiological mineralization .
Several molecules have been associated to the process of the formation of microcalcifications in breast
cancer. Among them, deserve particular mention the bone morphogenetic proteins 2 (BMP-2) and
[8]
pentraxin-3 (PTX3). BMP-2 is currently considered the most powerful osteoblast induction factor , whereas
it is recently demonstrated the ability of PTX3 to induce both osteoblast proliferation and activity [9,10] . In
[7]
particular, studies of Scimeca et al. highlighted the essential role of PTX3 in bone metabolisms founding
a correlation between the impairment of PTX3 expression and the inhibition of osteoblast activity. In these
reports authors suggested a direct role of PTX3 in the assembly of HA crystals. Surprisingly, the presence of
BOLCs into breast cancer lesions is also associated to the development of bone metastatic lesions at five years
from diagnosis. Therefore, the presence of microcalcifications made of HA or MgHAp in breast lesions could
be considered as a negative prognostic factor for bone metastatic diseases as well as the BOLCs.

Starting from these considerations, the aim of this study was to correlate the microcalcifications’
characteristics, such as morphology and elemental compositions, with the occurrence of bone metastatic
lesions at 5 years from diagnosis.

METHODS
In this retrospective study, we enrolled 70 patients from which we collected one breast biopsy each. Our
study protocol was approved by independent ethical committee. From each biopsy, paraffin serial sections
were obtained to perform histological classifications, immunohistochemical analyses and energy dispersive
X-ray (EDX) evaluation. Exclusion criteria were history of previously or concomitant other neoplastic
diseases, autoimmune diseases, viral chronic infections (HBV, HCV, and HIV), and any antitumoral
treatment received before biopsy.

Histology
After fixation in 10% buffered formalin for 24 h, breast tissues were paraffin embedded. Four μm thick
[11]
sections were stained with haematoxylin-eosin (H&E) .
EDX microanalysis
The EDX microanalysis is a technology that performs the elemental and chemical analysis of a sample in a
transmission electron microscope. When the electron beam in an electron microscope hits a thin sample,