Gamma-H2AX Foci Decay Ratio as a Stronger Predictive Factor of Late Radiation Toxicity Than Dose-Volume Parameters in a Prospective Cohort of Prostate Cancer Patients.

PURPOSE: Late radiation toxicity is a major dose-limiting factor in curative cancer radiation therapy. Previous studies identified several risk factors for late radiation toxicity, including both dose-volume factors and genetic predisposition. Herein, we investigated the contribution of genetic predisposition, particularly compared with dose-volume factors, to the risk of late radiation toxicity in patients treated with highly conformal radiation therapy. METHODS AND MATERIALS: We included 179 patients with prostate cancer who underwent treatment with curative external beam radiation therapy between 2009 and 2013. Toxicity was graded according to the Common Terminology Criteria for Adverse Events version 4.0. Transcriptional responsiveness of homologous recombination repair genes and γ-H2AX foci decay ratios (FDRs) were determined in ex vivo irradiated lymphocytes in a previous analysis. Dose-volume parameters were retrieved by delineating the organs at risk (OARs) on CT planning images. Associations between risk factors and grade ≥2 urinary and bowel late radiation toxicities were assessed using univariable and multivariable logistic regression analyses. The analyses were performed using the highest toxicity grade recorded during the follow-up per patient. RESULTS: The median follow-up period was 31 months. One hundred and one patients (56%) developed grade ≥2 late radiation toxicity. Cumulative rates for urinary and bowel grade ≥2 late toxicities were 46% and 17%, respectively. In the multivariable analysis, factors significantly associated with grade ≥2 late toxicity were transurethral resection of the prostate (P = .013), γ-H2AX FDR <3.41 (P = .008), and rectum V70 >11.52% (P = .017). CONCLUSIONS: Our results suggest that impaired DNA double-strand break repair in lymphocytes, as quantified by γ-H2AX FDR, is the most critical determining factor of late radiation toxicity. The limited influence of dose-volume parameters could be due to the use of increasingly conformal techniques, leading to improved dose-volume parameters of the organs at risk.

Enhancement of Radiation Effectiveness in Cervical Cancer Cells by Combining Ionizing Radiation with Hyperthermia and Molecular Targeting Agents.

Hyperthermia (HT) and molecular targeting agents can be used to enhance the effect of radiotherapy (RT). The purpose of this paper is to evaluate radiation sensitization by HT and different molecular targeting agents (Poly [ADP-ribose] polymerase 1 inhibitor, PARP1-i; DNA-dependent protein kinase catalytic subunit inhibitor, DNA-PKcs-i and Heat Shock Protein 90 inhibitor, HSP90-i) in cervical cancer cell lines. Survival curves of SiHa and HeLa cells, concerning the combined effects of radiation with hyperthermia and PARP1-i, DNA-PKcs-i or HSP90-i, were analyzed using the linear-quadratic model: S(D)/S(0) = exp - (αD + ßD²). The values of the linear-quadratic (LQ) parameters α and ß, determine the effectiveness at low and high doses, respectively. The effects of these sensitizing agents on the LQ parameters are compared to evaluate dose-dependent differences in radio enhancement. Combination of radiation with hyperthermia, PARP1-i and DNA-PKcs-i significantly increased the value of the linear parameter α. Both α and ß were significantly increased for HSP90-i combined with hyperthermia in HeLa cells, though not in SiHa cells. The Homologous Recombination pathway is inhibited by hyperthermia. When hyperthermia is combined with DNA-PKcs-i and PARP1-i, the Non-Homologous End Joining or Alternative Non-Homologous End Joining pathway is also inhibited, leading to a more potent radio enhancement. The observed increments of the α value imply that significant radio enhancement is obtained at clinically-used radiotherapy doses. Furthermore, the sensitizing effects of hyperthermia can be even further enhanced when combined with other molecular targeting agents.

Prostate Cancer Patients with Late Radiation Toxicity Exhibit Reduced Expression of Genes Involved in DNA Double-Strand Break Repair and Homologous Recombination.

Severe late damage to normal tissue is a major limitation of cancer radiotherapy in prostate cancer patients. In a recent retrospective study, late radiation toxicity was found to relate to a decreased decay of γ-H2AX foci and reduced induction of DNA double-strand break repair genes. Here, we report evidence of prognostic utility in prostate cancer for γ-H2AX foci decay ratios and gene expression profiles derived from ex vivo-irradiated patient lymphocytes. Patients were followed ≥2 years after radiotherapy. Clinical characteristics were assembled, and toxicity was recorded using the Common Terminology Criteria (CTCAE) v4.0. No clinical factor was correlated with late radiation toxicity. The γ-H2AX foci decay ratio correlated negatively with toxicity grade, with a significant difference between grade ≥3 and grade 0 patients (P = 0.02). A threshold foci decay ratio, determined in our retrospective study, correctly classified 23 of 28 patients with grade ≥3 toxicity (sensitivity 82%) and 9 of 14 patients with grade 0 toxicity (specificity 64%). Induction of homologous recombination (HR) repair genes was reduced with increasing toxicity grade. The difference in fold induction of the HR gene set was most pronounced between grade 0 and grade ≥3 toxicity (P = 0.008). Notably, reduced responsiveness of HR repair genes to irradiation and inefficient double-strand break repair correlated with severe late radiation toxicity. Using a decay ratio classifier, we correctly classified 82% of patients with grade ≥3 toxicity, suggesting a prognostic biomarker for cancer patients with a genetically enhanced risk for late radiation toxicity to normal tissues after radiotherapy. Cancer Res; 77(6); 1485-91. ©2017 AACR.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 µM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Predicting Radiosensitivity with Gamma-H2AX Foci Assay after Single High-Dose-Rate and Pulsed Dose-Rate Ionizing Irradiation.

Gamma-H2AX foci detection is the standard method to quantify DNA double-strand break (DSB) induction and repair. In this study, we investigated the induction and decay of γ-H2AX foci of different tumor cell lines and fibroblasts with known mutations in DNA damage repair genes, including ATM, LigIV, DNA-PKcs, Rad51 and Rad54. A radiation dose of 2.4 Gy was used for either an acute single high-dose-rate (sHDR) exposure or a pulsed dose-rate (pDR) exposure over 24 h. The number of γ-H2AX foci was determined at 30 min and 24 h after sHDR irradiation and directly after pDR irradiation. In a similar manner, γ-H2AX foci were also examined in lymphocytes of patients with differences in normal tissue toxicity after a total radiation dose of 1 Gy. In an initial count of the number of foci 30 min after sHDR irradiation, repair-proficient cell types could not be distinguished from repair-deficient cell types. However at 24 h postirradiation, while we observed a large decrease in foci numbers in NHEJ-proficient cells, the amount of γ-H2AX foci in cell types with mutated NHEJ repair remained at high levels. Except for IRS-1SF cells, HR-deficient cell types eventually did show a moderate decrease in foci number over time, albeit to a lesser extent than their corresponding parentals or repair-proficient control cells. In addition, analysis of γ-H2AX foci after sHDR exposure of patients with different sensitivity status clearly showed individual differences in radiation response. Radiosensitive patients could be distinguished from the more radioresistant patients with γ-H2AX foci decay ratios (initial number of foci divided by residual number of foci). Significantly higher decay ratios were observed in patients without toxicities, indicating more proficient repair compared to patients with radiation-induced side effects. After pDR irradiation, no consistent correlation could be found between foci number and radiosensitivity. In conclusion, γ-H2AX formation is a rapid and sensitive cellular response to DNA DSBs. Decay ratios after sHDR exposure elucidated large differences in γ-H2AX foci kinetics between the repair-proficient or -deficient cell types and patients. This assay may be useful for measuring cellular radiosensitivity and could serve as a clinically useful test for predicting radiosensitivity ex vivo before treatment.

Reduced activity of double-strand break repair genes in prostate cancer patients with late normal tissue radiation toxicity.

PURPOSE: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. METHODS AND MATERIALS: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. RESULTS: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. CONCLUSIONS: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells.

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+ßD(2)). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a (137)Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Decay of γ-H2AX foci correlates with potentially lethal damage repair in prostate cancer cells.

To determine the relationship between ionizing radiation-induced levels of γ-H2AX foci and cell survival in cultured prostate cancer cell lines, three prostate cancer cell lines: LNCaP (wt TP53), DU145 (mut TP53) and PC3 (TP53 null), were studied. For γ-H2AX foci induction, cells were irradiated with a single dose of 2 Gy and foci levels were studied at 30 min and 24 h after irradiation. Cell survival was determined by clonogenic assay, directly and 24 h after irradiation with doses ranging from 0 to 8 Gy. Irradiation was performed with a Siemens Stabilipan 250 KeV X-ray machine at a dose rate of approximately 3 Gy/min. Survival curves were analyzed using the linear-quadratic model S(D)/S(0)=exp-(αD+ßD2). LNCaP cells clearly demonstrated potentially lethal damage repair (PLDR) which was assessed as increased survival levels after delayed plating as compared to cells plated immediately after irradiation. DU145 cells demonstrated only a slight PLDR and PC3 cells did not show PLDR at all. Levels of γ-H2AX foci were significantly decreased in all cell lines at 24 h after irradiation, compared to levels after 30 min. The LNCaP cells which demonstrated a clear PLDR also showed the largest decay in the number of γ-H2AX foci. In addition, the PC cells which did not show PLDR had the lowest decay of γ-H2AX foci. A clear correlation was demonstrated between the degree of decay of γ-H2AX foci and PLDR.