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Genetic Polymorphisms in the DNA Double-Strand Break Repair Genes XRCC3, XRCC2, and NBS1 Are Not Associated with Acute Side Effects of Radiotherapy in Breast Cancer Patients

Divisions of ¹ Toxicology and Cancer Risk Factors and ² Clinical Epidemiology, German Cancer Research Center, Heidelberg, Germany; ³ Department of Epidemiology, Division of Cancer Prevention and Population Science, Roswell Park Cancer Institute, Buffalo, New York; ⁴ Department of Gynecological Radiology, Heidelberg University Hospital, Heidelberg, Germany; ⁵ Clinic for Radiotherapy and Radio-Oncology, St. Vincentius-Kliniken Karlsruhe; ⁶ Clinic for Radiotherapy, Karlsruhe Hospital GmbH, Karlsruhe, Germany; and ⁷ Department of Radiation Oncology, Universitätsklinikum Mannheim, Mannheim, Germany

Requests for reprints: Odilia Popanda, Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany. Phone: 49-6221-423315; Fax: 49-6221-423359. E-mail: o.popanda{at}dkfz-heidelberg.de

	Introduction

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Clinical sensitivity to ionizing radiation varies considerably among patients, and radiation-induced adverse effects developing in normal tissue can be therapy limiting in >10% of patients (1). Ionizing radiation induces both DNA single-strand breaks and double-strand breaks (DSB), with the DSBs generally considered the lethal event (2). Several syndromes associated with increased radiosensitivity are caused by deficiencies in genes of DSB repair, leading to the hypothesis that the individual repair capacity for these lesions should be an important determinant of individual radiosensitivity (3, 4). Here, three polymorphisms in genes involved in homologous recombination (XRCC3 Thr²⁴¹Met, XRCC2 Arg¹⁸⁸His, and NBS1 Glu¹⁸⁵Gln) with potential functional effects (5-8) were evaluated for a possible association with the risk of developing acute skin reactions following radiotherapy in a prospective epidemiologic study.

	Materials and Methods

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Study subjects included female breast cancer patients receiving primary radiotherapy after breast-conserving surgery as reported (ref. 9; reference no. 37/98 of the ethical committee of the University of Heidelberg). All the patients were given a typical breast radiation treatment with an average biologically effective radiation dose of 54.0 ± 4.8 Gy. Clinical radiation reaction developing in the skin within the radiation field of the breast was documented at regular time intervals during treatment, and the severity of acute side effects was assessed using a classification system based on the Common Toxicity Criteria of the U.S. NIH (10). Seventy-seven of the 446 participants presented with increased acute toxicity (at least one moist desquamation or interruption of radiotherapy due to toxicity) by the end of treatment (9, 11).

DNA extraction, analysis of the XRCC3 Thr²⁴¹Met polymorphism (rs861539), and quality control was as described (12). The XRCC2 Arg¹⁸⁸His (rs3218536) and the NBS1 Glu¹⁸⁵Gln (rs1805794) polymorphisms were measured as other variants in (12) by melting curve analysis of sequence-specific hybridization probes (LightCycler, Roche Diagnostics, Mannheim, Germany). For XRCC2 Arg¹⁸⁸His, PCR primers were 5'-TTGATATGCTCCGGCTAGTTA-3' and 5'-CTGCCATGCCTTACAGAGATAA-3', and probes were 5'-CTTGTAAATGACTATCACCTGGTTCTT-FITC-3' and 5'-LCRed640-TGCAACGACACAAACTATAATGCAGAAAGCp-3'; for NBS1 Glu¹⁸⁵Gln, primers were 5'-TTTTATGGATGTAAACAGCCTCT-3' and 5'-AAACCTTCCATTAATAATACCGAA-3', and probes were 5'-LCRed640-AGAAGCAGCCTCCACAAATTGAAAGGTp-3' and 5'-TGAATTCCTGAAAGCAGTTCAGTCC-FITC-3' (Tib Molbiol, Berlin, Germany). Genotype information was incomplete for two samples because of an inadequate amount of DNA.

Occurrence of acute skin toxicity was analyzed using Cox proportional hazards model in relation to biologically effective radiation dose instead of time in days during radiotherapy, thereby adjusting for differences in radiation dose when acute skin toxicity occurred and for the total dose received (13). As possible confounders, differences by treating hospitals and body mass index were included in all models (9).

	Results

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Frequencies of rare alleles were 0.41, 0.07, and 0.32 for XRCC3 ²⁴¹Met, XRCC2 ¹⁸⁸His, and NBS1 ¹⁸⁵Gln, respectively, consistent with published reports (0.39, XRCC3; 0.06, XRCC2; and 0.32, NBS1) in European populations (8, 12, 14). All genotype distributions were in Hardy-Weinberg equilibrium. Overall, we did not observe a significant association between the genetic polymorphisms investigated and the risk of acute skin toxicity after radiotherapy (Table 1 ). Analysis yielded differences in the associations between XRCC3 and NBS1 polymorphisms and acute skin toxicity for patients with normal weight and overweight (Table 1), which were, however, not statistically significant. When combined effects of alleles were examined, there was neither a significant trend (P_trend = 0.08) for increased protection associated with increasing number of potentially protective alleles (XRCC3 ²⁴¹Thr, XRCC2 ¹⁸⁸His, and NBS1 ¹⁸⁵Gln) in normal weight patients nor significant risk alterations (hazard ratio for carriers of three and more alleles, 0.26; 95% confidence interval, 0.04-1.66; data not shown).

	Discussion

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To our knowledge, this is the first study to investigate the association between XRCC2 Arg¹⁸⁸His and NBS1 Glu¹⁸⁵Gln polymorphisms and acute side effects of radiotherapy. For XRCC3 Thr²⁴¹Met, the Thr/Thr genotype was reported to be correlated with increased risk of late effects in irradiated breast cancer patients, such as s.c. fibrosis and telangiectasia (15). This finding was not confirmed in another study of late-adverse radiotherapy effects in gynecologic tumors (16). Our results weakly implicated the XRCC3 ²⁴¹Met allele as risk allele and may differ from the other studies because we focused on acute side effects rather than late effects of radiotherapy, which are not necessarily related (17).

All three polymorphisms investigated were predicted to have an maximal effect on cellular, and possibly clinical, function using a algorithm based on allele frequency, potential functional effect, and results from previous epidemiologic studies (18). The XRCC3 ²⁴¹Met allele has in fact been associated with an increased number of micronuclei in peripheral lymphocytes of humans exposed to ionizing radiation (5, 6). No differences in homology-directed repair of DSB have, however, been found between the wild-type and the variant XRCC3 protein (19), and DSB repair in vitro and acute normal tissue reaction after radiotherapy of breast cancer patients were not correlated (20). Thus, the polymorphisms investigated, although apparently functional, may not give total information about variability in gene function. In the future, a more detailed haplotype analysis of the genes and a comprehensive analysis, including variants in genes of both DSB repair pathways (homologous recombination and nonhomologous end joining), and consideration of late effects of radiotherapy will be necessary.

	Acknowledgments

 
We thank the staff of the participating clinics for their contribution to the data collection; Belinda Kaspereit, Kati Smit, and Peter Waas in DKFZ for excellent technical assistance; and all the patients who participated in the study.

	Footnotes

 
Grant support: German Office for Radiation Protection project no. St. Sch. 4116 and 4233 and USAMRMC FY01 Breast Cancer Research Program grant DAMD17-02-1-0500.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/26/06; accepted 3/ 2/06.

	References

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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? Radiother Oncol 2002;64:131–40.[Medline]
2. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature 2001;411:366–74.[CrossRef][Medline]
3. Digweed M, Sperling K. Nijmegen breakage syndrome: clinical manifestation of defective response to DNA double-strand breaks. DNA Repair (Amst) 2004;3:1207–17.
4. Gatti RA. The inherited basis of human radiosensitivity. Acta Oncol 2001;40:702–11.[CrossRef][Medline]
5. Aka P, Mateuca R, Buchet JP, Thierens H, Kirsch-Volders M. Are genetic polymorphisms in OGG1, XRCC1 and XRCC3 genes predictive for the DNA strand break repair phenotype and genotoxicity in workers exposed to low dose ionising radiations? Mutat Res 2004;556:169–81.[Medline]
6. Angelini S, Kumar R, Carbone F, et al. Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes. Mutat Res 2005;570:105–17.[Medline]
7. Medina PP, Ahrendt SA, Pollan M, Fernandez P, Sidransky D, Sanchez-Cespedes M. Screening of homologous recombination gene polymorphisms in lung cancer patients reveals an association of the NBS1–185Gln variant and p53 gene mutations. Cancer Epidemiol Biomarkers Prev 2003;12:699–704.[Abstract/Free Full Text]
8. Rafii S, O'Regan P, Xinarianos G, et al. A potential role for the XRCC2 R188H polymorphic site in DNA-damage repair and breast cancer. Hum Mol Genet 2002;11:1433–8.[Abstract/Free Full Text]
9. Twardella D, Popanda O, Helmbold I, et al. Personal characteristics, therapy modalities and individual DNA repair capacity as predictive factors of acute skin toxicity in an unselected cohort of breast cancer patients receiving radiotherapy. Radiother Oncol 2003;69:145–53.[CrossRef][Medline]
10. Cancer Therapy Evaluation Program. Common Toxicity Criteria. NIH 1998; Available from: http://ctep.cancer.gov/reporting/ctc.html.
11. Popanda O, Ebbeler R, Twardella D, et al. Radiation-induced DNA damage and repair in lymphocytes from breast cancer patients and their correlation with acute skin reactions to radiotherapy. Int J Radiat Oncol Biol Phys 2003;55:1216–25.[CrossRef][Medline]
12. Popanda O, Schattenberg T, Phong CT, et al. Specific combinations of DNA repair gene variants and increased risk for non-small cell lung cancer. Carcinogenesis 2004;25:2433–41.[Abstract/Free Full Text]
13. Chang-Claude J, Popanda O, Tan XL, et al. Association between polymorphisms in the DNA repair genes, XRCC1, APE1, and XPD and acute side effects of radiotherapy in breast cancer patients. Clin Cancer Res 2005;11:4802–9.[Abstract/Free Full Text]
14. Kuschel B, Auranen A, McBride S, et al. Variants in DNA double-strand break repair genes and breast cancer susceptibility. Hum Mol Genet 2002;11:1399–407.[Abstract/Free Full Text]
15. Andreassen CN, Alsner J, Overgaard M, Overgaard J. Prediction of normal tissue radiosensitivity from polymorphisms in candidate genes. Radiother Oncol 2003;69:127–35.[CrossRef][Medline]
16. De Ruyck K, Van Eijkeren M, Claes K, et al. Radiation-induced damage to normal tissues after radiotherapy in patients treated for gynecologic tumors: association with single nucleotide polymorphisms in XRCC1, XRCC3, and OGG1 genes and in vitro chromosomal radiosensitivity in lymphocytes. Int J Radiat Oncol Biol Phys 2005;62:1140–9.[CrossRef][Medline]
17. Bentzen SM, Overgaard M. Relationship between early and late normal-tissue injury after postmastectomy radiotherapy. Radiother Oncol 1991;20:159–65.[Medline]
18. Millikan RC, Player JS, Decotret AR, Tse CK, Keku T. Polymorphisms in DNA repair genes, medical exposure to ionizing radiation, and breast cancer risk. Cancer Epidemiol Biomarkers Prev 2005;14:2326–34.[Abstract/Free Full Text]
19. Araujo FD, Pierce AJ, Stark JM, Jasin M. Variant XRCC3 implicated in cancer is functional in homology-directed repair of double-strand breaks. Oncogene 2002;21:4176–80.[CrossRef][Medline]
20. El Awady RA, Mahmoud M, Saleh EM, et al. No correlation between radiosensitivity or double-strand break repair capacity of normal fibroblasts and acute normal tissue reaction after radiotherapy of breast cancer patients. Int J Radiat Biol 2005;81:501–8.[Medline]

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