Figure 4. Evaluation of HRR gene mutation, COSMIC signature 3, and integrated assessment of HRR deficiency classification with responses to DNA-damaging therapeutics.

(A) Distribution of COSMIC single base substitution mutational signatures (CSigs) across PC patient-derived xenograft (PDX) lines. The classes of COSMIC mutation signatures are color-coded, and tumors are ordered in decreasing frequency of CSig3. (B and C) Confocal immunostaining and quantitation of γH2AX foci in short-term cultures of the indicated PDX line 3 hours and 24 hours after exposure to 6 Gy IR or sham treatment (Ct). Foci counts for each time point are the mean ± SD of 3 replicate experiments. Intergroup comparison in change of foci count per cell was performed by a Welch’s t test. (P value ** < 0.01; *** < 0.001). Scale bar: 10 µm. (D and E) Immunofluorescence microscopy quantitation of RAD51 foci in LNCaP_Tet_shBRCA2 with or without DOX and with or without IR; (D) representative images of cells with and without designated treatments; (E) percentage of 50 cells counted with more than 5 RAD51 foci/cell. Each measurement represents the mean ± SD of 3 independent measurements. Significance was determined by Fisher’s exact test. Scale bar: 10 μm. (F) Quantitation of RAD51 immunofluorescence foci count in short-term cultures of LuCaP PDX lines exposed to IR or sham treatment. The percentage of 50 cells counted with more than 5 RAD51 foci/cell is plotted with each measurement representing the mean ± SD of 3 independent measurements. Significance was determined by Fisher’s exact test. (G) Cell viability assessments of LuCaP PDX lines grown in vitro and measured 72 hours after treatment with vehicle or 50 μM or 100 μM carboplatin. Each measurement represents the mean ± SD of 3 (LuCaP81 and LuCaP174.1) or 6 (remainder) replicate experiments. Comparison of cell viability was performed using paired t tests with Bonferroni’s corrections (P value * ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001).