DNA double strand break quantification in skin biopsies

Background and purpose: Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gammaH2AX foci. The purpose of this study was to use this endogenous signalling system to quantify the in vivo response to radiation in normal tissue. Patients and methods: Skin biopsies were taken from prostate cancer patients undergoing radiotherapy with a curative intent. Biopsies were taken at locations corresponding to 5 different doses in the range below 1.1 Gy per fraction. Biopsies were taken from patients 30 min following the first fraction and then once again following the fraction given after the first weekend break in the treatment course. The DNA double strand breaks were visualised as gammaH2AX foci using immunohistochemistry. Images were acquired using a CCD-camera and a fluorescence microscope and the gammaH2AX foci were quantified using digital image analysis including the basic procedures of top-hat transformation, threshold setting and labelling. Results: Repeated assessments of the biopsies showed a high reproducibility in quantifying the number of foci per DNA area of the nucleated cells in epidermis. The reproducibility was equally good for the two biopsy occasions. A linear dose response was observed for the epidermis in the dose region 0-1 Gy. Conclusions: We have established a method to measure the relative amount of DNA double strand breaks by detecting gammaH2AX foci in patients exposed to radiotherapy. The method provides a tool to study induction and repair of DNA double strand breaks and has the potential to predict individual radiosensitivity. (C) 2004 Elsevier Ireland Ltd. All rights reserved.