Investigating γH2AX as a Biomarker of Radiosensitivity Using Flow Cytometry Methods

Background and Purpose. This project examined the in vitro？？γH2AX response in lymphocytes of prostate cancer patients who had a radiosensitive response after receiving radiotherapy. The goal of this project was to determine whether the γH2AX response, as measured by flow cytometry, could be used as a marker of individual patient radiosensitivity. Materials and Methods. Patients were selected from a randomized clinical trial evaluating the optimal timing of Dose Escalated Radiation and short-course Androgen Deprivation Therapy. Of 438 patients, 3% developed Grade 3 late radiation proctitis and were considered to be radiosensitive. Blood was drawn from 10 of these patients along with 20 matched samples from patients with Grade 0 proctitis. Dose response curves up to 10？Gy along with time response curves after 2？Gy (0–24？h) were generated for each sample. The γH2AX response in lymphocytes and lymphocyte subsets was analyzed by flow cytometry. Results. There were no significant differences between the radiosensitive and control samples for either the dose course or the time course. Conclusions. Although γH2AX response has previously been demonstrated to be an indicator of individual patient radiosensitivity, flow cytometry lacks the sensitivity necessary to distinguish any differences between samples from control and radiosensitive patients. 1. Introduction The severity of late normal tissue toxicity is a limiting factor during the administration of radiotherapy [1]. It has become clear that interpatient variability in the incidence of late normal tissue toxicity could be partially due to individual patient sensitivity to radiation [2] and the prediction of a patient’s radiosensitivity would facilitate improved patient treatment [3]. The induction and repair of chromosomal damage in irradiated lymphocytes are thought to be promising predictors of individual patient sensitivity to ionizing radiation [4]. Since DNA double-strand breaks (DSBs) are considered to be the critical lesion for DNA damage, the lack of repair, or misrepair, can have severe repercussions. As such, it is important to be able to quantitate the induction and disappearance of DSBs. Rogakou et al. in 1999 [5] showed that H2AX (an electrophoretic isoform of the histone H2A) is phosphorylated (γH2AX) at the sites of DSBs, and Sedelnikova et al. in 2003 [4] demonstrated that γH2AX foci corresponded one to one with DSBs. These results mean that the number of DSBs induced can be measured by counting the number of γH2AX foci [4]. Furthermore, by staining γH2AX with a fluorochrome-labelled