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Clinical Cancer Research Vol. 11, 4802-4809, July 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Association between Polymorphisms in the DNA Repair Genes, XRCC1, APE1, and XPD and Acute Side Effects of Radiotherapy in Breast Cancer Patients

Jenny Chang-Claude1, Odilia Popanda2, Xiang-Lin Tan1, Silke Kropp1, Irmgard Helmbold1, Dietrich von Fournier3, Wulf Haase4, Marie Luise Sautter-Bihl5, Frederik Wenz6, Peter Schmezer2 and Christine B. Ambrosone7

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

Requests for reprints: Jenny Chang-Claude, German Cancer Research Center, Division of Clinical Epidemiology, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. Phone: 49-6221-422373; Fax: 49-6221-422203; E-mail: j.chang-claude{at}dkfz-heidelberg.de.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Several DNA repair gene polymorphisms have been described, which affect DNA repair capacity and modulate cancer susceptibility. We evaluated the association of six polymorphisms in the DNA repair genes: XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), APE1 (Asp148Glu), and XPD (Lys751Gln and Asp312Asn), with the risk of acute skin reactions following radiotherapy.

Design: We conducted a prospective study of 446 female patients with breast cancer who received radiotherapy after breast-conserving surgery. Individual genetic polymorphisms were determined using melting point analysis of sequence-specific hybridization probes. The development of acute skin reactions (moist desquamation) associated with DNA repair gene polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose.

Results: Overall, the development of acute toxicity, which presented in 77 patients, was not associated with the genetic variants studied, although the hazard ratios (HR) were generally below 1. Risks were however differential by body mass index. Among normal-weight patients only, both carriers of the APE1 148Glu and the XRCC1 399Gln alleles had decreased risk of acute skin reactions after radiotherapy (HR, 0.49 and 0.51, respectively). The results for XRCC1 were confirmed by haplotype analysis. When considering joint effects, we observed that compared with homozygote carriers of the wild-type allele in both genes, the risk was most strongly reduced in carriers of both APE1 148Glu and XRCC1 399Gln alleles with normal weight [HR, 0.19; 95% confidence interval (95% CI), 0.06-0.56] but not in those with overweight (HR, 1.39; 95% CI, 0.56-3.45; Pinteraction = 0.009).

Conclusion: The XRCC1 399Gln or APE1 148Glu alleles may be protective against the development of acute side effects after radiotherapy in patients with normal weight.

Severity of damage to normal tissue after therapeutic radiotherapy is predominantly influenced by factors related to radiation exposure, which however are not sufficient to explain fully the patient-to-patient variability (1, 2). There is considerable evidence that both patient- and treatment-related factors as well as intrinsic factors of individual radiosensitivity could influence the variability of side effects observed (3–7).

Because radiation therapy exerts its cytotoxic effects through damage to cells, proteins, and DNA, the individual capacity to repair damaged DNA may modify the response of the normal tissue. The individual DNA repair capacity consists of several pathways: nucleotide and base excision repair (BER), homologous recombination, end joining, mismatch repair, and telomere metabolism. BER of single-strand breaks as well as nonhomologous end joining and homologous repair of double-strand breaks are considered the most important pathways involved in repair of radiation-induced DNA damage (8, 9).

DNA repair gene polymorphisms in the BER genes XRCC1 and APE1 were shown to affect cellular repair activity such as prolongating cell cycle delay in the G2 phase after irradiation (10) or decreasing mutagen sensitivity towards bleomycin, a radiation-mimicking agent that induces double-strand breaks in DNA (11). Studies associating repair gene polymorphisms and clinical radiosensitivity, however, are rare. Only the gene variants of XRCC1, XRCC3, and ATM have been shown to correlate with hypersensitivity to radiotherapy (12–14). It is likely that further DNA repair gene polymorphisms may be associated with ionizing radiation hypersensitivity.

The XRCC1 gene is mapped at human chromosome 19q13.2-13.3 (15) and shows three relatively common polymorphisms in codon 194 (Arg/Trp), 280 (Arg/His), and 399 (Arg/Gln) affecting the amino acid sequence (16). The protein acts as a scaffold to coordinate other BER proteins at the repair site (8). Cells defective in XRCC1 have increased sensitivity to ionizing radiation, UV, hydrogen peroxide, and mitomycin (17). APE1 is the rate-limiting enzyme in the BER pathway (18). It cleaves 5' of DNA abasic sugar residues generated from exogenous factors, such as ionizing radiation and environmental carcinogens, as well as endogenous agents from normal cellular metabolism (19). To date, studies addressing the possible combined effects of variants in these two genes involved in the BER pathway in relation to clinical radiation sensitivity have not been published.

XPD protein functions as an ATP-dependent 5'-3' helicase joint to the basal TFIIH complex (20). There are two genetic polymorphisms causing amino acid changes in codon 312 (Asp to Asn) and in codon 751 (Lys to Gln; ref. 16) and there is evidence that subjects homozygous for the variant genotypes of XPD have suboptimal DNA repair capacity for benzo(a)pyrene adducts and UV DNA damage (21, 22). Several studies suggest that XPD protein may participate in the repair of ionizing radiation–induced oxidative damage (23, 24). Elevated chromatid aberration frequency found in lymphocytes containing a mutated XP gene after exposure to ionizing radiation suggests a role for nucleotide excision repair proteins in the repair of ionizing radiation–induced damage (25). However, the association between XPD polymorphisms and clinical radiosensitivity has not yet been investigated.

We therefore evaluated the possible association of genetic variants in the three genes, XRCC1, APE1, and XPD, and the risk of acute normal skin reactions after therapeutic radiotherapy in a prospective epidemiologic study of breast cancer patients receiving radiotherapy of the breast without chemotherapy after breast-conserving surgery.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study subjects and data collection. We recruited female breast cancer patients receiving primary radiotherapy of the breast after breast-conserving surgery, who were Caucasians, regardless of age, at radiotherapy units of the Women's Clinic in Heidelberg, the St. Vincentius Clinic in Karlsruhe, the City Hospital of Karlsruhe, and the University Hospital of Mannheim from June 1998 to March 2001. Details of the choice of patients and data collection have been previously reported (26). Women were excluded if they were currently or ever treated with chemotherapy to avoid confounding of side effects data. The study was approved by the ethical committee of the University of Heidelberg and informed consent was obtained from all patients enrolled.

Clinical radiation reaction developing in the skin within the radiation field of the breast was documented four times during the study: (a) before the beginning of radiotherapy and at a cumulative dose of (b) 36 to 42 Gy, (b) 44 to 50 Gy, and (d) about 60 to 66 Gy (end of radiotherapy). The severity of acute side effects was assessed using a modified classification system based on the common toxicity criteria of the NIH (27). For more detailed categorization of skin reaction, grade 2a was defined as tender/bright erythema or moderate edema, grade 2b as severe erythema, and grade 2c as at least one moist desquamation or interruption of radiotherapy due to toxicity. Development of acute side effects of grade ≥2c was considered to indicate increased sensitivity for acute effects ("clinical radiosensitivity") in our study (28).

Clinical data on tumor characteristics and therapy regime were abstracted from patient records. Participants completed a self-administered questionnaire at the first visit before commencement of radiotherapy, which elicited information on demographic factors, medical history, family history of cancer, and lifestyle.

All the patients were given a common breast radiation treatment including CT-based planning, simulation, verification, and quality assurance and received conformal tangential irradiation with lateral and medial wedge fields. The standard radiotherapy regime included irradiation of the whole breast followed by an electron boost at three radiology departments, either 50 Gy given in 5 x 2.0 Gy fractions per week or 50.4 Gy in 5 x 1.8 Gy fractions per week. In the fourth radiology department, 56 Gy of whole breast irradiation were applied in 5 x 2.0 Gy fractions per week without an electron boost. The biologically effective radiotherapy dose (BED) was calculated to account for differences in fractionation and overall treatment time, using the formula given the number of fractions n, the fraction size of d, an {alpha}/ß ratio of 10 Gy for acute skin reactions (29), a time factor {gamma}/{alpha} of 0.7 Gy/d, the overall treatment time of T, and a starting time for compensatory proliferation T0 of 21 days (30).

A total of 478 patients participated with complete clinical and epidemiologic information, of whom 84 developed acute clinically relevant toxicity by the end of radiotherapy. Blood samples collected before starting radiotherapy were available for 446 patients (average age, 60.3 ± 9.0 years) who were included in this analysis. Of these patients, 77 presented with acute toxicity of grade >2c. The average biologically effective radiotherapy dose by censoring was 54.0 ± 4.8 Gy with range 35.5 to 64.5 Gy.

Laboratory methods: Blood sample processing. Blood samples (~15 mL) were collected using standard venipuncture techniques before starting radiotherapy. Genomic DNA was purified from lymphocytes extracted from whole blood using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. All DNA preparations were stored at 4°C until use.

Genotyping by fluorescence-based melting curve analysis. Detection of polymorphisms was done by rapid capillary PCR with melting curve analysis using fluorescence-labeled hybridization probes in a LightCycler (Roche Diagnostics, Mannheim, Germany). PCR primers and probes for melting point analyses are given in Table 1 (31). Probes were designed and prepared by Tib Molbiol (Berlin, Germany). The melting point analysis uses fluorescence resonance energy transfer for detecting a polymorphic site. Therefore, the 3'-end of the sensor probe was labeled with FITC and the 5'-end of the anchor probe, which was placed 1 nucleotide downstream of the sensor probe, was labeled with LC Red 640 and the 3'-end was phosphorylated. The sensor probe was designed for a perfect match either to the wild-type or the variant allele sequence. Thus, in the allele with the sequence deviating from the sensor, a 1-nucleotide mismatch between sensor and target DNA sequence was formed and caused destabilization of the hybrid yielding to a melting point (Tm) shift of 5°C to 10°C.


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  		Table 1. Primers and probes for genotyping single-nucleotide polymorphisms

 
Analysis was done in 10 µL volumes in glass capillaries (Roche Diagnostics) using Qiagen reagents: 1 x PCR buffer, 2.5 mmol/L total MgCl2, 0.2 mmol/L deoxynucleotide triphosphates, 0.1% bovine serum albumin in DMSO, 1 x Q-solution (not for APE1 Asp148Glu), 0.5 unit Taq DNA polymerase, 0.5 µmol/L of each primer, 0.15 µmol/L of each probe, and 10 ng of DNA. For determination of APE1 Asp148Glu, 0.2 µmol/L of anchor and sensor were used. Reaction conditions were as follows: initial denaturation at 95°C for 3 minutes then 40 cycles of denaturation at 95°C for 15 seconds; annealing at 60°C (XPD Lys751Gln and Asp312Asn and XRCC1 Arg280His), 58°C (XRCC1 Arg194Trp and Arg280His and APE1 Asp148Glu), or 55°C (XRCC1 Arg399Gln) for 10 seconds; and elongation at 72°C for 12 seconds. Melting curve analysis was done with an initial denaturing step at 95°C for 30 seconds and 20 seconds at 40°C (55°C for XPD Lys751Gln), slow heating to 75°C with a ramping rate of 0.3°C/s (0.4°C/s for XPD Lys751Gln and Asp312Asn), and continuous fluorescence detection. Melting curves were converted to melting peaks by plotting the negative derivatives of fluorescence against temperature (–dF/dT). A negative control containing all reagents but water instead of the DNA template was included to each amplification set. Melting curves were evaluated by two independent observers who were blinded to the analysis of the clinical data. In addition, 10% of randomly selected samples were repeated independently to verify genotyping results and 100% concordance was found. For each polymorphism, PCR fragments of homozygous common allele, homozygous variant allele, and heterozygous samples were sequenced to confirm genotypes. Because of an inadequate amount of DNA, a few samples did not generate complete information for all polymorphisms.

Statistical methods. Each polymorphism was tested for deviation from Hardy-Weinberg equilibrium by comparing the observed and expected genotype frequencies using the {chi}2 test with 1 degree of freedom. Linkage disequilibrium between different markers in XRCC1 and in XPD was estimated using the "Estimating Haplotypes" program (32, 33). Haplotypes for XRCC1 and XPD were reconstructed using the PHASE version 2 software (34).

The effect of the genetic variants on risk of developing clinical radiosensitivity was evaluated by Cox proportional hazards model using the procedure PHREG of SAS, Release 8.1 (35, 36). Here, the event of interest was the occurrence of clinical radiosensitivity defined as skin reactions scored grade ≥2c. Risk of skin toxicity increases with the BED (26); therefore, we modeled the occurrence of this event in relation to BED received instead of time in days during radiotherapy. In this way, we adjusted for differences in radiation dose when skin toxicity of grade ≥2c was recorded and for the total dose received. Differences by treating hospital were accounted for by including the variables, hospital, photon beam energy for whole breast irradiation, and boost irradiation in the model. Comorbidities (e.g., diabetes and smoking) were previously not found to be risk determinants of acute toxicity (26). We showed however that higher body mass index (BMI) was significantly associated with an increased risk for acute toxicity [hazard ratio (HR), 1.09 per 1 kg/m2; ref. 26]. BMI (as a continuous variable) was therefore included as a possible confounder in all models. Effect modification by BMI was investigated by separate analyses for the two groups subdivided at the median BMI of 24.9: normal weight (BMI ≤ 25.0) and overweight/obese (BMI > 25.0). Interactions between BMI group and the genotypes were measured by using multiplicative terms in the model, including the main effects and evaluated by the likelihood ratio tests.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Overall, the variant allele frequencies were 0.37 for XRCC1 399Gln, 0.06 for XRCC1 280His and 194Trp, and 0.48 for APE1 148Glu in our study population. The XPD 312Asn and XPD 751Gln alleles occurred at a frequency of 0.37 and 0.38, respectively. All the genotype distributions were in Hardy-Weinberg equilibrium. The allele frequencies observed were similar to those previously reported in Caucasian subjects (37) and a German population (31). Strong association (linkage disequilibrium) was found among the three XRCC1 polymorphisms and between the two XPD polymorphisms (data not shown).

Seventy-seven of the 446 participants presented with increased acute toxicity by the end of treatment. We found no significant association between the genetic variants of XRCC1, APE1, and XPD and the development of increased acute toxicity in multivariate analysis although the HRs were generally below one (Table 2).


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  		Table 2. Association of XRCC1, APE1, and XPD polymorphisms with risk of acute skin reaction after radiotherapy in breast cancer patients

 
Further analysis yielded differences for patients with normal weight and overweight (Table 3). Compared with noncarriers, carriers of the APE1 148Glu had a decreased risk of developing clinical radiosensitivity in normal-weight patients [HR, 0.49; 95% confidence interval (95% CI), 0.21-1.15] but not in overweight patients (HR, 1.05; 95% CI, 0.55-2.00; Pinteraction = 0.14). There was a similar differential risk for carriers of XRCC1 399Gln, with a HR of 0.51 (95% CI, 0.22-1.19) for normal-weight patients and 1.28 (95% CI, 0.72-2.27) for overweight patients (Pinteraction = 0.08). The XPD variants were associated with a nonsignificantly decreased risk of clinical radiosensitivity in both normal-weight and overweight patients.


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  		Table 3. Association of XRCC1 and APE1 polymorphisms with risk of acute skin reaction after radiotherapy in breast cancer patients stratified by BMI

 
To further elucidate the relevance of XRCC1 and XPD variants, haplotypes for the three XRCC1 polymorphisms (Arg194Trp, Arg280His, and Arg399Gln) and the two XPD polymorphisms (Asp312Asn and Lys751Gln) were reconstructed. A total of five XRCC1 haplotypes were found, but the haplotype CAA (Arg/His/Gln) occurred in only one patient (Table 4). The rare variants, XRCC1 194Trp and XRCC1 280His, were not found to occur on the same haplotype. The results showed that the haplotype effects reflect that of the 399Gln allele. Thus, compared with the common CGG (Arg/Arg/Arg) haplotype, the CGA (Arg/Arg/Gln) haplotype solely yielded a borderline significant association with risk of clinical radiosensitivity in normal-weight women (HR, 0.55; 95% CI, 0.29-1.05) but not in overweight women (HR, 1.14; 95% CI, 0.76-1.72; Pinteraction = 0.12). None of the four XPD haplotypes showed clear association with development of clinical radiosensitivity.


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  		Table 4. Association of XRCC1 and XPD haplotypes with risk of acute skin reaction after radiotherapy in breast cancer patients stratified by BMI

 
Both XRCC1 and APE1 are involved in the BER pathway. We therefore explored the effects of combined genotypes of XRCC1 399Gln and APE1 148Glu (Table 5). Compared with homozygote carriers of the wild-type allele in both genes, carrying either the XRCC1 399Gln or the APE1 148Glu allele was associated with a significantly decreased risk of development of clinical radiosensitivity in patients with normal weight but not those who were overweight. Carriers of both variant alleles had the greatest risk reduction (HR, 0.19; 95% CI, 0.06-0.56), and this was also significantly different from the HR of 1.39 (95% CI, 0.56-3.45) among overweight patients (Pinteraction = 0.009).


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  		Table 5. The combined genotypes and clinical radiosensitivity investigated by Cox proportional hazard model stratified by BMI

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we evaluated possible associations between variants of the DNA repair genes, XRCC1, APE1, and XPD, and the risk of developing acute clinical side effects after radiotherapy, defined as moist desquamation of the skin. We observed no main effect of any of the variants in the breast cancer patients as a whole. Among patients with normal weight, however, carriers of the XRCC1 399Gln variant and the APE1 148Glu variant had a decreased risk of acute skin reactions after radiotherapy (HR, 0.51 and 0.49, respectively). This protective effect was not found among overweight or obese patients. The XRCC1 haplotype analysis provided no evidence for the involvement of a risk haplotype but confirmed the genotype results. There was however clear evidence of joint effects of the XRCC1 399Gln and the APE1 148Glu variant alleles, which were differential for normal and overweight women. Among normal-weight women, we found that the carriers of either one or both of the XRCC1 399Gln and APE1 148Glu variant alleles were protected against the development of clinical radiosensitivity when compared with those carrying neither the XRCC1 399Gln nor the APE1 variant allele.

We used a prospective study design to ensure standardized data collection on side effects at defined points during radiotherapy and the use of a predefined classification system comparable with other studies. By choosing grade 2c (moist desquamation of the skin or interruption of radiotherapy because of side effects) as an indicator of increased acute toxicity, we selected an indicator that is less prone to variability in classification than radiation-induced erythema (26). However, we cannot exclude the possibility that there may still have been interobserver variability in the evaluation of the degree of acute skin reaction. In addition, we tried to minimize sources of bias in design and analysis of our study by restriction on tumor type and type of side effects and we accounted for confounding by treatment-related or patient-related characteristics in the data analysis. Using Cox regression to model the association between risk factors and the occurrence of clinical radiosensitivity, we were able to account for differences in radiation dose received until end of follow-up as well as the dose at first occurrence of severe acute skin reactions for each individual patient. Therefore, our results are not likely to be biased.

To our knowledge, there are no published reports regarding the association between XPD gene polymorphism and increased acute toxicity. The only other study that investigated the association between the XRCC1 polymorphisms and acute toxicity reported a nonsignificantly increased risk of developing an adverse response to radiotherapy associated with the XRCC1 194Trp allele (12). Although as in our study, they found no association with haplotype analysis, they observed one particular genotype combination involving this allele and the XRCC1 399Gln allele to be associated with significantly increased risk of clinical radiosensitivity. Our results may differ because of the different definitions of clinical radiosensitivity used in the two studies. We included only the occurrence of acute skin reactions during and immediately after radiotherapy, whereas they included both acute and late skin reactions occurring within 2 years of follow-up. Therefore, their observations may be predominantly driven by the effect of inefficient DNA repair related to late reaction of radiotherapy. Indeed, in human fibroblasts, reduced DNA repair as an indicator of cellular radiosensitivity has been found associated with various late rather than acute normal tissue responses after radiotherapy (38–41). Early and late reactions are not necessarily related and they may be influenced differently by genetic predisposition (42).

Several reports indicate that the variant alleles of the repair polymorphisms examined may truly affect DNA repair function. The presence of the XRCC1 194Arg and 399Gln alleles was associated with increased mutagen sensitivity after bleomycin treatment (11) and the variant alleles in XRCC1 399Gln and/or APE1 148Glu were associated with a prolonged cell cycle G2 delay in response to ionizing radiation (10). The XPD 751Lys/Lys genotype was reported to be significantly associated with decreased DNA repair proficiency (43). It is therefore not easy to explain our findings of a genotype, which is associated with reduced DNA repair activity, to be protective against the development of acute side effect. However, our observations are supported by studies of the development of therapy-related acute myeloblastic leukemia, in which a protective effect of the XRCC1 399Gln allele was also observed (44). It is evident that the completeness of DNA repair after exposure to ionizing radiation is not only dependent upon the level of damage. The efficiency of the repair machinery depends also on the time out for a damaged cell (i.e., through cell cycle arrest) to ensure effective repair; otherwise, the damaged cell will be eliminated by cell death. Although the interaction between impaired recovery from ionizing radiation–induced DNA damage and tissue response is complex and still not well understood, there might be an increase in cell death in insufficiently repaired cells thus reducing the risk of adverse reactions. To confirm our findings, further functional studies of the XRCC1 and APE1 polymorphisms will be necessary.

The significant association between genotype and clinical radiosensitivity was found only in patients with normal weight and not in overweight or obese patients. In our study population, the risk of developing skin side effects after radiotherapy increased with increasing BMI (26). This relationship is clinically recognized and is likely due to the association of a high BMI with a large breast size. Radiotherapy of large breasts requires a special radiation protocol with tangential radiation fields, which often results in an increased maximum radiation dose at the surface. However, BMI could also be an indicator of as yet unknown individual factors that influence radiosensitivity. Thus, the strong association of high BMI with clinical radiosensitivity may override the effect of genotypes on the development of clinical radiosensitivity in overweight patients. The significant interaction of DNA repair gene polymorphisms with BMI and their association with the development of clinical radiosensitivity seems consistent with this hypothesis. Further clinical data on the influence of BMI and radiation-induced complications are unfortunately sparse (45). Therefore, additional studies including larger numbers of patients are needed to confirm our findings.

Ionizing radiation induces many types of damage to DNA, requiring multiple repair pathways to restore genomic integrity. Other important genes/pathways, especially those for DNA double-strand break repair, may also play a role and should be further investigated. Furthermore, polymorphisms leading to inefficient DNA repair might also be associated with late reactions to radiotherapy. We are presently conducting a follow-up of the patients for the occurrence of late side effects and shall in future be able to compare the effects of the DNA repair gene polymorphisms on acute and late skin reaction.


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


   Footnotes
 
Grant support: German Office for Radiation Protection, project 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 12/28/04; revised 3/ 3/05; accepted 4/14/05.


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