γH2AX Expression in Tumors Exposed to Cisplatin and Fractionated Irradiation

Abstract

Purpose: Is retention of γH2AX foci useful as a biomarker for predicting the response of xenograft tumors to cisplatin with X-ray? Is a similar approach feasible using biopsies from patients with cervical cancer? Experimental Design: Mice bearing SiHa, WiDr, or HCT116 xenograft tumors were exposed to cisplatin and/or three daily doses of 2 Gy. Tumors were excised 24 h after treatment and single cells were analyzed for clonogenic fraction and retention of γH2AX foci. Tumor biopsies were examined using 47 paraffin-embedded sections from untreated tumors and 24 sections from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. Results: Residual γH2AX measured 24 h after cisplatin injection accurately predicted surviving fraction in SiHa and WiDr xenografts. When a clinically equivalent protocol using cisplatin and fractionated irradiation was employed, the fraction of xenograft cells lacking γH2AX ranked survival accurately but underestimated tumor cell kill. Residual γH2AX foci were detected in clinical samples; on average, only 25% of tumor nuclei exhibited one or more γH2AX foci before treatment and 74% after the start of treatment. Conclusion: γH2AX can provide useful information on the response of human tumors to the combination of cisplatin and radiation, but prediction becomes less accurate as more time elapses between treatment and tumor biopsy. Use of residual γH2AX as a biomarker for response is feasible when cell survival exceeds ∼20%, but heterogeneity in endogenous and treatment-induced γH2AX must be considered.Prediction of tumor response to radiation remains an important goal in radiotherapy (1, 2). However, prediction is complicated by the use of combined modality therapy and the lack of a simple but effective predictor of response in situ. Ionizing radiation and many cancer therapeutics cause phosphorylation of histone H2AX at sites of individual DNA double-strand breaks, providing an exceptionally sensitive method for detection of this critical lesion (3, 4). Production of γH2AX foci can be detected microscopically or by using flow cytometry, and the extent of phosphorylation has been used as a biomarker of both exposure and DNA repair capacity (5-9). The fraction of cells that retain of γH2AX foci, measured 24 h after exposure to either radiation (6) or cisplatin (10), is correlated with the fraction of cells that lose clonogenicity in SiHa and WiDr tumor cells. These in vitro results and the availability of biopsies from patients treated with cisplatin and radiation provided the motivation to examine the response of xenograft tumors to the combination of cisplatin and radiation and then to measure γH2AX expression in clinical biopsies.The presence of residual γH2AX was examined using three human tumor xenograft models growing subcutaneously in immunodeficient mice given a single dose of cisplatin, 2 Gy × three daily fractions of X-ray, or the combination. To determine the importance of tumor cell location relative to functional blood vessels, tumor cells were sorte...

Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness

“…The fact that phosphorylation remains at the sites of DSBs until the end of repair processes before the foci are dephosphorylated [32][33][34][35][36] facilitates the study of foci disappearance along with the quantitative evaluation of residual DSBs [20,21,23,29,[37][38][39][40][41]. In several studies both these endpoints have been correlated with cellular radiation sensitivity in vitro and in vivo either in tumour or normal tissue samples [20,21,23,28,29,31,37,39,41,42]. Taking together, the cH2AX assay is simple, sensitive and straightforward method to quantify DSBs in cells and tissues and therefore promising for translation into clinical trials.…”

PARP Inhibitors

“…• Iniparib is the first PARP inhibitor to be tested in combination with chemotherapy in metastatic triple-negative breast cancer. The randomized phase II trial of gemcitabine and carboplatin given with or without iniparib in patients with metastatic triple-negative breast cancer showed that the addition of iniparib to chemotherapy resulted in an improved clinical benefit rate (56% vs 34%; P=0.01) and overall response rate (52% vs 32%; P=0.02) as well as an improvement in progression-free survival (5.9 vs 3.6 months; P= 0.01) and overall survival ( PAR, have been proposed as pharmacodynamic markers of DNA damage and PARP inhibition [13,29,47,48]. In the phase I trial of olaparib, examination of γH2AX foci in plucked eyebrow-hair follicle samples showed sustained induction of γH2AX foci, and the level of PAR in PBMCs was reduced by more than 90% after treatment with olaparib.…”

Translational research in phase I trials

“…Bañuelos and colleagues assessed the value of H2Ax phosphorylation as a predictive biomarker of response to DNAdamaging agents that cause double-strand breaks (DSBs) [47,48].…”