gamma H2AX assay in ex vivo irradiated tumour specimens: A novel Method to determine tumour radiation sensitivity in patient-derived material

Purpose: To establish a clinically applicable protocol for quantification of residual gamma H2AX foci in ex vivo irradiated tumour samples and to apply this method in a proof-of-concept feasibility study to patient-derived tumour specimens. Material and methods: Evaluation of gamma H2AX foci formation and disappearance in excised FaDu tumour specimens after (a) different incubation times in culture medium, 4 Gy irradiation and fixation after 24 h (cell recovery), (b) 10 h medium incubation, 4 Gy irradiation and fixation after various time points (double strand break repair kinetics), and (c) 10 h medium incubation, irradiation with graded single radiation doses and fixation after 24 h (dose-response). The optimised protocol was applied to patient-derived samples of seminoma, prostate cancer and glioblastoma multiforme. Results: Post excision or biopsy, tumour tissues showed stable radiation-induced gamma H2AX foci values in oxic cells after >6 h of recovery in medium. Kinetics of foci disappearance indicated a plateau of residual foci after >12 h following ex vivo irradiation. Fitting the dose-response of residual gamma H2AX foci yielded slopes comparable with in situ irradiation of FaDu tumours. Significant differences in the slopes of ex vivo irradiated patient-derived tumour samples were found. Conclusion: A novel clinically applicable method to quantify residual gamma H2AX foci in ex vivo irradiated tumour samples was established. The first clinical results suggest that this method allows to distinguish between radiosensitive and radioresistant tumour types. These findings support further translational evaluation of this assay to individualise radiation therapy.