Endogenous and radiation-induced expression of gamma H2AX in biopsies from patients treated for carcinoma of the uterine cervix

Background and purpose: The possibility of using gamma H2AX foci as a marker of DNA damage and as a potential predictor of turnout, response to treatment was examined using biopsies from 3 sets of patients with advanced carcinoma of the cervix. The relation between endogenous gamma H2AX expression and hypoxia was also examined. Materials and methods: Set I consisted of 26 biopsies that included pre-treatment and 24 h post-radiation treatment samples. Pre-treatment biopsies from 12 patients in Set 2 were used to develop image analysis Software while pre-treatment biopsies from 33 patients in Set 3 were examined for the relation between staining for the hypoxia marker pimonidazole and endogenous gamma H2AX expression. Formalin-fixed paraffin-embedded sections were analyzed after antigen retrieval and fluorescence antibody labeling for the hypoxia markers CAIX or pimonidazole in combination with gamma H2AX staining. Results: Before treatment, 24 +/- 19% of cells contained gamma H2AX foci, with most positive cells containing fewer than 5 foci per nucleus. Twenty-four hours after exposure to the first fraction of 1.8-2.5 Gy. 38 +/- 19% contained foci. CAIX positive cells were 1.4 times more likely to exhibit endogenous gamma H2AX foci, and pimonidazole-positive cells were 2.8 times more likely to contain gamma H2AX foci. For 18 patients for whom both pre-treatment and 24 h post-irradiation biopsies were available, local control was unrelated to the fraction of cells that retained gamma H2AX foci. However, 24 h after irradiation, tumours that had received 2.5 Gy showed a significantly higher fraction of cells with residual gamma H2AX foci than tumours given 1.8 Gy. Conclusions: Endogenous gamma H2AX foci are enriched in hypoxic tumour regions. Small differences in delivered dose can produce quantifiable differences in residual DNA damage that can overshadow inter-tumour differences in response. (C) 2009 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 94 (2010) 82-89