Prostate microcalcification crystallography as a marker of pathology

Abstract

Prostate cancer remains the most common male cancer; however, treatment regimens remain unclear in some cases due to a lack of agreement in current testing methods. Therefore, there is an increasing need to identify novel biomarkers to better counsel patients about their treatment options. Microcalcifications offer one such avenue of exploration. Microfocus spectroscopy at the i18 beamline at Diamond Light Source was utilised to measure X-ray diffraction and fluorescence maps of calcifications in 10 µm thick formalin fixed paraffin embedded prostate sections. Calcifications predominantly consisted of hydroxyapatite (HAP) and whitlockite (WH). Kendall's Tau statistics showed weak correlations of 'a' and 'c' lattice parameters in HAP with GG (r_τ = - 0.323, p = 3.43 × 10^-4 and r_τ = 0.227, p = 0.011 respectively), and a negative correlation of relative zinc levels in soft tissue (r_τ = - 0.240, p = 0.022) with GG. Negative correlations of the HAP 'a' axis (r_τ = - 0.284, p = 2.17 × 10^-3) and WH 'c' axis (r_τ = - 0.543, p = 2.83 × 10^-4) with pathological stage were also demonstrated. Prostate calcification chemistry has been revealed for the first time to correlate with clinical markers, highlighting the potential of calcifications as biomarkers of prostate cancer.

Keywords: Biomarkers; Calcification; Prostate cancer.

Fig. 1

Diffractograms of examples of mineral phases in prostate calcifications. (a) Standard patterns for calcium phosphate mineral phases, hydroxyapatite and whitlockite, plus example diffractograms of three calcifications containing only hydroxyapatite (Calc 1), a mixture of hydroxyapatite and whitlockite (Calc 2) and only whitlockite (Calc 3). (b) Standard patterns for calcium carbonate mineral phases, aragonite and calcite, and example diffractograms of two calcifications containing calcite and aragonite (Calc 4) and only calcite (Calc 5). A diffractogram of paraffin is also included as this accounts for some of the diffraction peaks observed in the data due to the use of FFPE tissue. (c) A standard pattern of dorfmanite plus an example calcification containing this mineral phase (Calc 6).

Fig. 2

Comparison of calcification crystallography in different prostatic zones. (a) Average diffractograms for each prostatic zone, and standard patterns of hydroxyapatite and paraffin. (b) Box plot of HAP ‘c’ axis in calcifications in the different prostatic zones. Each point represents the average measurement for an individual calcification. PZ: Peripheral zone, CZ/TZ: Central/Transition zones, FMZ: Fibromuscular zone, HAP: hydroxyapatite. **p < 0.01.

Fig. 3

Crystallographic parameters of HAP and WH and zinc distribution averaged by calcification then by grade group (ISUP Grade Groups (GG) 1–5). (a and b) ‘a’ and ‘c’ axis values for HAP, (c and d) ‘a’ and ‘c’ axis values for WH. ‘a’ and ‘c’ axes represent physical dimensions of crystal units and can indicate ion substitutions. (e and f) CL measured along 002 and 030 for HAP, (g and h) CL measured along 0210 and 220 for WH. (i and j) Relative zinc levels in prostate calcifications and surrounding soft tissue, measured against total metal ion content (calcium, zinc and iron), presented as a ratio (0–1). Each point represents the average measurement for an individual calcification. HAP: hydroxyapatite, WH: whitlockite, CL: coherence length (a measure of crystallinity in a given direction in a crystal). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Crystallographic parameters of HAP and WH and zinc distribution averaged by calcification then by pathological stage (size or area of cancer, from inside the prostate gland (T2) to local spread (T4)). (a and b) ‘a’ and ‘c’ axis values for HAP, (c and d) ‘a’ and ‘c’ axis values for WH. ‘a’ and ‘c’ axes represent physical dimensions of crystal units, and can indicate ion substitutions. (e and f) CL measured along 002 and 030 for HAP, (g and h) CL measured along 0210 and 220 for WH. (i and j) Relative zinc levels in prostate calcifications and surrounding soft tissue, measured against total metal ion content (calcium, zinc and iron), presented as a ratio (0–1). Each point represents the average measurement for an individual calcification. HAP: hydroxyapatite, WH: whitlockite, CL: coherence length (a measure of crystallinity in a given direction in a crystal). *p < 0.05, **p < 0.01.