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Bonfiglio et al. J Cancer Metastasis Treat 2019;5:29 I http://dx.doi.org/10.20517/2394-4722.2018.88 Page 3 of 7

some atoms of the sample will be excited or ionized. When they return into their ground state, they will
emit characteristic X-rays. The X-ray emission at different wavelengths may then be measured by a photon-
energy-sensitive detector [12,13] .

The EDX detector system performs a simultaneous display of all mid-energy (1-20 keV) X-rays collected
during any individual analysis period. Therefore, it is possible to detect those elements with N.A. > 10.
The minimal detectable elemental concentration, which requires some signal averaging, is approximately
0.1 mmol/kg of dry specimen (i.e., 10 ppm), whereas spatial resolution ranges from about 10 nm to a few
micrometers [12,13] .

All breast samples underwent ultrastructural microanalysis. Following to the identification of
microcalcifications, six-micrometer-thick paraffin sections were embedded in Epon resin as previously
[12]
described . Briefly, sections were de-paraffinized, hydrated, osmium tetroxide-fixed, dehydrated in
ethanol and propylene oxide and infiltrated in Epon. The embedding capsules were positioned over areas
containing microcalcifications identified by Toluidine Blue staining previously. Unstained ultra-thin
sections of approximately 100-nm-thick were mounted on copper grids for microanalysis. EDX spectra
of microcalcifications were acquired with a Hitachi 7100FA transmission electron microscope (Hitachi,
Schaumburg, IL, USA) and an EDX detector (Thermo Scientific, Waltham, MA, USA) at an acceleration
voltage of 75 KeV and magnification of 12,000. Spectra were semi-quantitatively analyzed by the Noram
System Six software (Thermo Scientific, Waltham, MA, USA) using the standardless Cliff-Lorimer k-factor
method [12,13] . The EDX microanalysis apparatus was calibrated using an X-ray microanalysis standard
(Micro-Analysis Consultants Ltd., Cambridgeshire, UK).

Immunohistochemistry
We employed immunohistochemical techniques to study the expression of molecules associated to
microcalcifications production, PTX3 and BMP-2. Briefly, antigen retrieval was performed on 3-μm-thick
paraffin sections using EDTA citrate pH 7.8 buffers for 30 min at 95 °C. Sections were then incubated for
1 h at room temperature with the following primary antibodies diluted 1:100: BMP-2 (clone N/A; Novus
Biologicals, USA) and PTX3 (clone MNB1; Abcam, UK). Reactions were revealed by HRP-DAB Detection
Kit (UCS Diagnostic, Italy). To assess the background of immuno-staining we included a negative control
for each reaction by incubating the sections with secondary antibodies (HRP) and detection system (DAB).
Reactions have been set-up by using specific control tissues as indicated in the data sheets.

Statistical analysis
2
Separate χ tests were used to assess the associations between morphological appearance and experimental
groups and between elemental composition and experimental groups. Difference between the
expression of biomarkers evaluated by immunohistochemistry were evaluated by Mann Whitney test.
Immunohistochemical values were reported as mean value ± standard error.

RESULTS
Histology
The study of H&E sections allowed us to classify breast biopsies in ductal infiltrating carcinomas according
[14]
to Nottingham Histological system . Specifically, we observed 15/70 G1 infiltrating carcinomas, 38/70
G2 infiltrating carcinomas and 17/70 G3 infiltrating carcinomas. Also, based on the presence of metastatic
lesions at 5 years from diagnosis, biopsies collected in the study were classified as follow: 30 infiltrating
carcinomas of patients with clinical evidence of bone metastasis (BM+) (59.65 ± 1.23 years) and 40
infiltrating carcinomas of patients without clinical evidence of bone metastasis (BM-) (57.91 ± 0.96 years).
Microcalcifications were detectable in 63.3% of BM+ and in 62.5% of BM-. From morphological point of
view, we observed 63.3% of psammomabodies and 36.7% of polymorphous calcifications in BM+ and 62.5%
of psammomabodies and 37.5% of polymorphous calcifications in BM- [Figure 1A-C].