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Abstract: Background/Introduction: Breast microcalcifications are deposits of calcium oxalate, found mostly in benign tissue, or calcium phosphate in the form of hydroxyapatite, found in benign and malignant tissue. Differences in the crystallographic properties and chemical make-up of hydroxyapatite breast microcalcifications have previously been noted in differing breast pathologies. Purpose: Ductal carcinoma in-situ (DCIS) is a precancerous breast lesion, which has the potential to form invasive breast cancer. Currently there are no definitive markers to determine DCIS invasiveness, therefore this work aims to elucidate differences in the calcification chemistry between invasive and non-invasive cases of DCIS, ultimately developing a novel biomarker for DCIS progression. Methods: 75 formalin fixed paraffin embedded archive breast tissue samples were used subject to NHS REC approval (ref. 18/LO/0945). X-ray diffraction was carried out at 12keV on beamline i18 at Diamond Light Source, UK to determine crystallographic properties of 279 breast calcifications. SEM-EDS experiments were carried out on a Hitachi SU3500 at 11kV under low vacuum. Results: Significant differences (P < 0.05) were observed in the proportion of magnesium whitlockite found as a secondary phase in breast calcifications from invasive (3.51 %) and non-invasive (2.82 %) DCIS samples. Additionally, crystallographic features of hydroxyapatite, the bulk of calcifications in both cases, were found to differ between the two groups. The d-spacing between crystallographic planes, was significantly (P < 0.001) larger in invasive compared to non-invasive DCIS cases. Finally, the calcium to phosphate ratio measured using EDS was significantly lower (P < 0.001) in invasive samples (1.60) compared to non-invasive samples (1.67), which more closely reflected stoichiometric hydroxyapatite. Conclusion(s): Differences in calcification chemistry and crystallographic structure between invasive and non-invasive DCIS cases have been demonstrated in this study. Therefore, calcification chemistry is a key candidate for novel DCIS progression biomarkers and could ultimately lower treatment expenditure and improve patient quality of life by reducing overtreatment.

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Abstract: Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign – B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.

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Abstract: With the increasing demand for new materials, analytical techniques which are able to rapidly characterize a large number of samples are becoming indispensable. Thin film technology has the potential to improve the amount of information contained on as-deposited samples by creating compositionally graded libraries. Conventionally, raster scan methods are used to interrogate such libraries but, in this paper, a different approach is presented to provide a method of high-throughput data collection and analysis using an X-ray diffraction (XRD) probe. An extended X-ray beam was used to illuminate the libraries, and a large area detector was used to collect the data. A new algorithm "Bandit" has been employed to analyze the collected data and extract the crystallographic information. The results of the technique have been compared with the raster scans showing that the algorithm provides reliable data at a significantly increased data acquisition speed.