Inefficient DNA-DSB repair via the homologous recombination pathway in prostate cancer patients with late radiation toxicity. Homo sapiens

Purpose: Severe late normal tissue damage limits radiotherapy treatment regimens. This study aims to validate γ-H2AX foci decay ratios and induced expression levels of DNA double strand break (DSB) repair genes, found in a retrospective study, as possible predictors for late radiation toxicity. Methods and Materials: Prospectively, decay ratios (initial/residual γ-H2AX foci numbers) and genome-wide expression profiles were examined in ex vivo irradiated lymphocytes of 198 prostate cancer patients. All patients were followed ≥2 years after radiotherapy, clinical characteristics were assembled and toxicity was recorded using the Common Terminology Criteria (CTCAE) v4.0. Results: No clinical factors were correlated with late radiation toxicity. Analysis of γ-H2AX foci uncovered a negative correlation between the foci decay ratio and toxicity grade. Significantly smaller decay ratios were found in grade≥3 compared to grade 0 patients (p=0.02), indicating less efficient DNA-DSB repair in radio-sensitive patients. Moreover, utilizing a foci decay ratio threshold determined in our previous retrospective study correctly classified 23 of the 28 grade≥3 patients (sensitivity, 82%) and 9 of the 14 grade 0 patients (specificity, 64%). Grade of toxicity also correlated with a reduced induction of the homologous recombination (HR) repair gene-set. The difference in average fold induction of the HR gene-set was most pronounced between grade 0 and grade≥3 patients (p=0.008). Conclusions: Reduced responsiveness of HR repair genes to irradiation and inefficient DSB repair correlate with an increased risk of late radiation toxicity. Using a decay ratio classifier, we could correctly classify 82% of the patients with grade≥3 toxicity. Additional studies are required to further optimize and validate the foci decay assay and to assess its predictive value for late radiation toxicity in patients prostate cancer Overall design: Between 2009 and 2013, we prospectively accrued 200 patients diagnosed with prostate cancer receiving curative external beam radiotherapy in combination with hormonal therapy at the Academic Medical Center (AMC) of the University of Amsterdam, with a follow-up of ≥2 years. After written informed consent, 40 ml whole blood was collected of all patients before start of treatment. Peripheral lymphocytes were isolated using Ficoll (Ficoll-Paque PLUS, GE Healthcare) gradient separation, and stored in liquid nitrogen. Development of late toxicities was monitored over more than 2 years after treatment using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Toxicity grade was determined mainly focusing on late gastrointestinal (GI) and genitourinary (GU) toxicities. Lymphocytes were cultured stimulated by phytohemagglutinin (concentration of 1μg/ml). After two weeks, half of the cells were irradiated at room temperature with 2Gy gamma rays from a 137Cs source, dose rate of approximately 0.5Gy/min and the other half of the cells was left untreated. Total cellular RNA was isolated and gene expression levels were measured using the Affymetrix HT HG-U133+ PM Array platform for 400 samples (2 per patient) and 12 technical replicates (all labelled as 'prospective' below). Samples were also included for 7 patients [4 with (overresponding, R) and 3 without (non-responding, NR) severe late radiation toxicity] from a previous retrospective study (International Journal of Radiation Oncology, Biology, Physics, 88(3):664-70; 2014) using total RNA isolated following an identical experimental protocol from whole-blood samples collected at least 2 years after radiation treatment. This was done either using previously isolated RNA stored at –80°C (14 samples, labelled as 'retrospective (old)' below) and freshly isolated RNA from newly collected whole-blood samples (14 samples, labelled as 'retrospective (new)' below). Samples were hybridized in two batches, the first batch consisting of 104 arrays on five array plates (2x16, 3x24) and the second batch of 336 arrays on five plates (2x24, 3x96). For all patients the irradiated (2Gy) and the control sample (0Gy) had by design been hybridrized in the same batch and, for the second and largest batch, on the same plate. Genes and genesets were determined that respond differently to irradiation between the four toxicity CTCAE 4.0 score-based patient groups. For 198 of the 200 patients from the prospective study the induction and decay of radiation-induced DNA–DSB breaks was monitored in ex vivo irradiated lymphocytes by detecting γ-H2AX foci 30min and 24h post radiation.