Flow cytometric assessment of DNA double-strand break and repair kinetics in prediction of intrinsic radiosensitivity

Document Type : Original Article


1 Medical Physics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

2 Reza Radiotherapy Oncology Center, Mashhad, Iran

3 Immunology Research Center, Bu-Ali Research Institute, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran



Objective(s): To enhance the efficiency of radiotherapy (RT), implementation of individual-based treatment is essential. In this way, determining individual intrinsic radiosensitivity (IRS) can be useful to achieve minimal adverse effects of RT. The present study aimed to identify IRS of breast cancer (BC) patients through determination of radiation-induced DNA double-strand breaks (DSBs), repair kinetics, and acute normal tissue complications induced by RT.
Materials and Methods: DSBs induction and its repair kinetics in 50 BC patients’ lymphocytes were analyzed by flow cytometric analysis of H2AX Ser-139 phosphorylation at 30 min, 3 and 24 hr after in vitro irradiation. In vivo skin dosimetry was done by GAFChromic films and acute skin toxicity was scored by radiation oncologists according to the criteria of Radiation Therapy and Oncology Group (RTOG) in all patients with similar prescribed treatment.
Results: The average surface dose for patients ranged from 0.92 to 1.9 Gy and correlation analysis showed no significant relationship with weekly acute skin reactions. Formation of γH2AX after 30 min, slope of dose-response curve and repair kinetics of DSBs after 3 and 24 hr (intrinsic radiosensitivity) were significantly correlated with the RTOG scores following irradiation (clinical radiosensitivity) (r=0.48 and P-value<0.0001, r=0.72 and P-value<0.0001, r=0.48 and P-value<0.001, and finally r=0.53 and P-value<0.001, respectively; (using Pearson’s correlation test). 
Conclusion: Flow cytometric analysis of DNA DSBs by γH2AX measurement has the potential to be developed into a clinical predictor for identifying the overreactor patients prior to RT. Our result suggests that the slope-related quantity based on the linear pattern of the dose-response curve has the merit to predict overreactor patients with a sensitivity of 89% and a specificity of 94%.


1. Andreassen CN, Alsner J, Overgaard J. Does variability in normal tissue reactions after radiotherapy have a genetic basis–where and how to look for it? Radiat Oncol J 2002;64:131-140.
2. Brown KR, Rzucidlo E. Acute and chronic radiation injury. J Vasc Surg 2011;53:15S-21S.
3. Deneuve S, Mirjolet C, Bastogne T, Duclos M, Retif P, Zrounba P, et al. Proof of concept of a binary blood assay for predicting radiosensitivity. Cancers 2021;13:2477.
4. Carlotto A, Hogsett VL, Maiorini EM, Razulis JG, Sonis ST. The economic burden of toxicities associated with cancer treatment: Review of the literature and analysis of nausea and vomiting, diarrhoea, oral mucositis and fatigue. Pharmacoeconomics 2013;31:753-766.
5. Pixberg C, Koch R, Eich HT, Martinsson U, Kristensen I, Matuschek C, et al. Acute toxicity grade 3 and 4 after irradiation in children and adolescents: Results from the IPPARCA collaboration. Int J Radiat Oncol Biol Phys 2016;94:792-799.
6. Li K, Yang L, Hu Q-y, Chen X-z, Chen M, Chen Y. Oral mucosa dose parameters predicting grade≥ 3 acute toxicity in locally advanced nasopharyngeal carcinoma patients treated with concurrent intensity-modulated radiation therapy and chemotherapy: An independent validation study comparing oral cavity versus mucosal surface contouring techniques. Transl Oncol 2017;10:752-759.
7. Banerjee R, Chakraborty S, Nygren I, Sinha R. Small bowel dose parameters predicting grade≥ 3 acute toxicity in rectal cancer patients treated with neoadjuvant chemoradiation: An independent validation study comparing peritoneal space versus small bowel loop contouring techniques. Int J Radiat Oncol Biol Phys 2013;85:1225-1231.
8. Gill S, Thomas J, Fox C, Kron T, Rolfo A, Leahy M, et al. Acute toxicity in prostate cancer patients treated with and without image-guided radiotherapy. Radiat Oncol 2011;6:1-7.
9. Herschtal A, Martin RF, Leong T, Lobachevsky P, Martin OA. A bayesian approach for prediction of patient radiosensitivity. Int J Radiat Oncol Biol Phys 2018;102:627-634.
10. Fitzgerald TJ, Wang T, Goel HL, Huang J, Stein G, Lian J, et al. Prostate carcinoma and radiation therapy: therapeutic treatment resistance and strategies for targeted therapeutic intervention. Expert Rev Anticancer Ther 2008;8:967-974.
11. Eschrich SA, Pramana J, Zhang H, Zhao H, Boulware D, Lee J-H, et al. A gene expression model of intrinsic tumor radiosensitivity: prediction of response and prognosis after chemoradiation. Int J Radiat Oncol Biol Phys 2009;75:489-496.
12. Shahbazian D, Bindra RS, Kluger HM, Glazer PM. Radiation sensitivity and sensitization in melanoma. Pigment Cell Melanoma Res 2013;26:928-930.
13. Ocolotobiche EE, Dauder RM, Güerci AM. Radiosensitivity of radiotherapy patients: The effect of individual DNA repair capacity. Mutat Res-Gen Tox En 2021;867:503371.
14. Yan D, Shen M, Du Z, Cao J, Tian Y, Zeng P, et al. Developing ZNF Gene Signatures Predicting Radiosensitivity of Patients with Breast Cancer. J Oncol 2021;2021: 9255494.
15. Mohammadi C, Khoei SG, Fayazi N, Mohammadi Y, Najafi R. miRNA as promising theragnostic biomarkers for predicting radioresistance in cancer: A systematic review and meta‐analysis. Crit Rev Oncol Hemat 2021;157:103183.
16. Maeda J, Froning CE, Brents CA, Rose BJ, Thamm DH, Kato TA. Intrinsic radiosensitivity and cellular characterization of 27 canine cancer cell lines. PloS One 2016;11:e0156689.
17. Bahreyni-Toossi M-T, Azimian H, Aghaee-Bakhtiari SH, Mahmoudi M, Sadat-Darbandi M, Zafari N. Radiation-induced DNA damage and altered expression of p21, cyclin D1 and Mre11 genes in human fibroblast cell lines with different radiosensitivity. Mutat Res 2021;823:111760.
18. Cox JD. Toxicity criteria of the radiation therapy oncology group (RTOG) and the European organization for research and treatment of cancer (EORTC). Int J Radiat Oncol Biol Phys 1995;31:1341-1346.
19. Bahreyni-Toossi M-T, Mohamadian N, Ghorbani M, Khorshidi F, Akbari F, Knaup C. Skin dosimetry in radiotherapy of breast cancer: a comparison between EBT and EBT3 radiochromic films. J Biomed Phys Eng 2016;6:51.
20. Kern R, Correa SC, Scandolara TB, Carla da Silva J, Pires BR, Panis C. Current advances in the diagnosis and personalized treatment of breast cancer: lessons from tumor biology. J Pers Med 2020;17:399-420.
21. Eschrich SA, Pramana J, Zhang H, Zhao H, Boulware D, Lee J-H, et al. A gene expression model of intrinsic tumor radiosensitivity: prediction of response and prognosis after chemoradiation. Int J Radiat Oncol Biol Phys 2009;75:489-496.
22. Huang R-X, Zhou P-K. DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target Ther 2020;5:1-27.
23. Goodarzi AA, Jeggo PA. Irradiation induced foci (IRIF) as a biomarker for radiosensitivity. Mutat Res 2012;736:39-47.
24. Martin NT, Nahas SA, Tunuguntla R, Fike F, Gatti RA. Assessing ‘radiosensitivity’with kinetic profiles of γ-H2AX, 53BP1 and BRCA1 foci. Radiother Oncol 2011;101:35-38.
25. Djuzenova CS, Elsner I, Katzer A, Worschech E, Distel LV, Flentje M, et al. Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci. Radiat Oncol 2013;8:1-12.
26. Kawashima S, Kawaguchi N, Taniguchi K, Tashiro K, Komura K, Tanaka T, et al. γ‑H2AX as a potential indicator of radiosensitivity in colorectal cancer cells. Oncol Lett 2020;20:2331-2337.
27. Chua MLK, Horn S, Somaiah N, Davies S, Gothard L, A’Hern R, et al. DNA double-strand break repair and induction of apoptosis in ex vivo irradiated blood lymphocytes in relation to late normal tissue reactions following breast radiotherapy. Radiat Environ Biophys. 2014;53:355-364.
28. Mumbrekar KD, Fernandes DJ, Goutham HV, Sharan K, Vadhiraja BM, Satyamoorthy K, et al. Influence of double-strand break repair on radiation therapy-induced acute skin reactions in breast cancer patients. Int J Radiat Oncol Biol Phys 2014;88:671-676.
29. Goutham HV, Mumbrekar KD, Vadhiraja BM, Fernandes DJ, Sharan K, Parashiva GK, et al. DNA double-strand break analysis by γ-H2AX foci: a useful method for determining the overreactors to radiation-induced acute reactions among head-and-neck cancer patients. Int J Radiat Oncol Biol Phys 2012;84:e607-e612.
30. Granzotto A, Benadjaoud MA, Vogin G, Devic C, Ferlazzo ML, Bodgi L, et al. Influence of nucleoshuttling of the ATM protein in the healthy tissues response to radiation therapy: toward a molecular classification of human radiosensitivity. Int J Radiat Oncol Biol Phys 2016;94:450-460.
31. Bahreyni-Toossi M-T, Azimian H, Sarrafzadeh O, Mohebbi S, Soleymanifard S. Automatic detection of micronuclei by cell microscopic image processing. Mutat Res 2017;806:9-18.
32. Pouliliou SE, Lialiaris TS, Dimitriou T, Giatromanolaki A, Papazoglou D, Pappa A, et al. Survival fraction at 2 Gy and γH2AX expression kinetics in peripheral blood lymphocytes from cancer patients: Relationship with acute radiation-induced toxicities. Int J Radiat Oncol Biol Phys 2015;92:667-674.
33. Mahmoud AS, Hassan AM, Ali AA, Hassan NM, Yousif AA, Elbashir FE, et al. Detection Of radiation-induced DNA damage in breast cancer patients by using gamma H2AX biomarker: A possible correlation with their body mass index. Genome Integr 2022;13:1.
34. Bulat T, Keta O, Korićanac L, Žakula J, Petrović I, Ristić-Fira A, et al. Radiation dose determines the method for quantification of DNA double strand breaks. An Acad Bras Cienc. 2016;88:127-136.
35. Raavi V, Perumal V, Paul SF. Potential application of γ-H2AX as a biodosimetry tool for radiation triage. Mutat Res Rev Mutat Res 2021;787:108350.
36. Bourton EC, Plowman PN, Smith D, Arlett CF, Parris CN. Prolonged expression of the γ‐H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment. Int J Cancer 2011;129:2928-2934.
37. Beaton LA, Marro L, Malone S, Samiee S, Grimes S, Malone K, et al. Investigating γ H2AX as a biomarker of radiosensitivity using flow cytometry methods. ISRN Radiol. 2013;2013:704659.