Microcalcification crystallography as a potential marker of DCIS recurrence

Abstract

Ductal carcinoma in-situ (DCIS) accounts for 20-25% of all new breast cancer diagnoses. DCIS has an uncertain risk of progression to invasive breast cancer and a lack of predictive biomarkers may result in relatively high levels (~ 75%) of overtreatment. To identify unique prognostic biomarkers of invasive progression, crystallographic and chemical features of DCIS microcalcifications have been explored. Samples from patients with at least 5-years of follow up and no known recurrence (174 calcifications in 67 patients) or ipsilateral invasive breast cancer recurrence (179 microcalcifications in 57 patients) were studied. Significant differences were noted between the two groups including whitlockite relative mass, hydroxyapatite and whitlockite crystal maturity and, elementally, sodium to calcium ion ratio. A preliminary predictive model for DCIS to invasive cancer progression was developed from these parameters with an AUC of 0.797. These results provide insights into the differing DCIS tissue microenvironments, and how these impact microcalcification formation.

Figure 1

SEM images of microcalcifications. (a) An SEM image of an ‘average’ sized microcalcification for the control and case groups. Scale bars = 100 µm. (b,c) Box plots for the longest axis (b) and microcalcification area (c) measured from SEM images. n = 181 and 175 for control and case respectively, ***p < 0.001.

Figure 2

Box plots for HAP parameters determined by XRD. (a) ‘a’ axis lattice parameter, (b) ‘c’ axis lattice parameter, (c) CL for 030, (d) CL for < 00ℓ > . n = 179 and 174 for control and case respectively, *p < 0.05, **p < 0.01.

Figure 3

Box plots for WH parameters determined by XRD. (a) Whitlockite wt %, (b) ‘a’ axis lattice parameter, (c) ‘c’ axis lattice parameter, (d) CL for 220, (e) CL for 1010. n = 179 and 174 for control and case respectively, *p < 0.05, **p < 0.01.

Figure 4

Box plots of EDX analysis of microcalcifications. (a) Ca:P ratio, (b) Na:Ca ratio, (c) Mg:Ca ratio. n = 179 and 174 for control and case respectively, ***p < 0.001.

Figure 5

ROC curve for validation results. 200 repeats are plotted in colour, with ROC curve produced from averaging the repeats in black and a marked optimal cut point calculated from Youden’s index (yellow dot). Dotted line represents an AUC of 0.50.

Figure 6

Measurements taken across microcalcifications. XRD data was collected along the longest axis and a perpendicular axis passing through the centre, at 11 µm intervals, marked as double headed black arrows. The longest axis was also measured from SEM images for size analysis. Microcalcification area was measured by outlining each microcalcification (dotted line). The same area was used for EDX analysis. Scale bar = 100 µm.