We thank Kejing Xu, Jianzhong He, and Min Zhao for laboratory assistance.
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
Nonhomologous end joining (NHEJ) is a pathway that repairs DNA double-strand breaks (DSBs) to maintain genomic stability in response to irradiation. The authors hypothesized that single nucleotide polymorphisms (SNPs) in NHEJ repair genes may affect clinical outcomes in patients with nonsmall cell lung cancer (NSCLC) who receive definitive radio(chemo)therapy.
The authors genotyped 5 potentially functional SNPs—x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) reference SNP (rs) number rs6869366 (−1394 guanine to thymine [−1394G→T] change) and rs28360071 (intron 3, deletion/insertion), XRCC5 rs3835 (guanine to adenine [G→A] change at nucleotide 2408), XRCC6 rs2267437 (−1310 cytosine to guanine [C→G) change], and DNA ligase IV (LIG4) rs1805388 (threonine-to-isoleucine change at codon 9 [T9I])—and estimated their associations with severe radiation pneumonitis (RP) (grade ≥3) in 195 patients with NSCLC.
A predictive role in radiation pneumonitis (RP) development was observed for the LIG4 SNP rs1805388 (adjusted hazard ratio, 2.08; 95% confidence interval, 1.04-4.12; P = .037 for the CT/TT genotype vs the CC genotype). In addition, men with the TT genotype of the XRCC4 rs6869366 SNP and women with AG + AA genotypes of the XRCC5 rs3835 SNP also were at increased risk of developing severe RP.
Radiation therapy is 1 of the major treatment modalities for unresectable nonsmall cell lung cancer (NSCLC) and has demonstrated the ability to improve local control and overall survival. However, acute radiation treatment-related pulmonary toxicity, known as radiation pneumonitis (RP), is the most common dose-limiting complication of thoracic radiation, and 10% to 20% of patients experience severe RP.1 Some therapeutic and patient-related factors have been associated the risk of RP, including patient performance status, dosimetric parameters, and plasma inflammatory cytokine levels.2 In addition, genetically determined cellular responses to ionizing radiation also have been suggested, because patients receiving similar treatment may have different responses to radiotherapy. Recently, we demonstrated that a single nucleotide polymorphism (SNP) of the transforming growth factor-β1 (TGFβ1) gene (reference SNP [rs] number rs1982073) independently predicted RP risk in patients with NSCLC.3 Considering the relatively sparse reports of RP investigation from a genetic perspective, we were interested in identifying additional genetic factors that are likely to predict RP susceptibility.
Ionizing radiation can produce a wide variety of lesions in cellular DNA, among which DNA double-strand breaks (DSBs) are the principle genotoxic lesions that pose major threats to genomic integrity. It is estimated that a dose of approximately 1 gray (Gy) of x-rays produces about 50 to 100 DSBs in the DNA of a typical mammalian cell, leading to 50% cell death.4, 5 There are 2 main pathways for DNA DSB repair: homologous recombination and nonhomologous end joining (NHEJ). In NHEJ, relatively small numbers of essential repair proteins mediate the DSB repair, including x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4), XRCC5 (Ku80), XRCC6 (Ku70), DNA ligase4, and NHEJ1 (XLF). The initial step involves recognition and signaling of DSB by the ring-shaped heterodimer Ku70/Ku80 (known as the Ku complex). Then, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interacts with Ku in a DNA-dependent manner to form a fully functional DNA-PK holoenzyme that functions in the synapsis of 2 broken DNA ends. Finally, XRCC4 and DNA ligase4 form a tight protein complex that catalyzes the rejoining of 2 compatible DNA ends. Another core NHEJ protein, XLF, may function by stimulating the basic NHEJ ligation reaction, but its precise function remains unknown.6
NHEJ is the major pathway to repair DSBs in mammalian species. Previous studies have demonstrated that loss of Ku or XRCC4/ligase4 function compromises the rejoining of radiation-induced DSBs and leads to increased radiosensitivity.7, 8 Patients with a rare congenital disorder, the ligase IV syndrome, are extremely sensitive to radiation because of mutations in the DNA ligase4 gene.9 Hence, it is reasonable to speculate that the interindividual variability in NHEJ may modulate phenotypes of radiosensitivity, hence affecting clinical outcomes of radiotherapy.
Because SNPs can contribute directly to disease predisposition by modifying a gene's function or can serve as genetic markers for nearby disease-causing variants through association or linkage disequilibrium, we hypothesized that functional SNPs of the NHEJ genes are biomarkers for predicting susceptibility to RP among patients with lung cancer who receive thoracic radiation. To test this hypothesis, we performed a case-only study, seeking associations between RP risk and common functional variants of XRCC4, XRCC5, XRCC6, and DNA ligase IV (LIG4) in patients with NSCLC, who received definitive radiation therapy with or without chemotherapy.
MATERIALS AND METHODS
We identified 261 patients who had DNA samples available from a dataset of 576 patients with NSCLC treated with definitive radiation at The University of Texas MD Anderson Cancer Center between 1999 and 2005. Among these 261 patients, 195 patients had documented information on RP with complete follow-up information and radiation dosimetric data. The median total radiation dose was 63 Gy (range, 50.4-84.0 Gy) given at 1.2 to 2.0 Gy per fraction. Among all patients, 20 patients received radiation only, and 175 patients received radiation in combination with chemotherapy. Details of the radiation treatment planning, follow-up schedule and tests, guidelines for RP scoring, and dosimetric data analysis have been described elsewhere.10, 11
Briefly, all patients were examined by their treating radiation oncologists weekly during concurrent chemoradiotherapy and 4 to 6 weeks after the completion of treatment. Then, the patients were followed every 3 months for the first 3 years and every 6 months thereafter unless they had symptoms that required immediate examination or intervention. Radiographic examinations by chest x-ray or computerized tomography were performed at each follow-up visit after the completion of chemoradiotherapy. For the current analysis, we reviewed all relevant dictated clinical notes by the treating physicians and all radiographic images for every patient. Radiation treatment-related pneumonitis was diagnosed by clinical presentation and any of the following radiographic abnormalities: ground-glass opacity, attenuation, or consolidation changes within the radiation field. Pneumonitis was assessed and scored using the Common Terminology Criteria for Adverse Events version 3.0.12 The time to the endpoint development was calculated from the beginning of radiation therapy; patients who did not experience the endpoint were censored at the last follow-up. This study was approved by the appropriate MD Anderson Cancer Center institutional review board.
We selected 5 common, well studied, and potentially functional variants of the XRCC4, XRCC5, XRCC6, and LIG4 genes involved in NHEJ: XRCC4 rs6869366 (−1394 guanine to thymine [−1394G→T]) and rs28360071 (intron 3, deletion/insertion from nucleotide 37403 to 37432), XRCC5 rs3835 (guanine-to-adenine change at nucleotide 2408 [2408G→A]), XRCC6 rs2267437 (−1310 cytosine to guanine [−1310C→G]), and LIG4 rs1805388 (threonine-to-isoleucine change at codon 9 [T9I]). These SNPs were selected, because they met at least 2 of 3 criteria: 1) the variant had a minor allele frequency of at least 5% in Caucasians, 2) the variant was located in the promoter region or splicing site or caused amino acid changes, 3) the variant was associated previously with cancer risk or survival.13-17 Although the XLF gene is also important in the NHEJ response to DSB,6 to our knowledge, no functional SNPs have been reported to date; therefore, this gene was not included in the current study. Genomic DNA was extracted from the buffy coat fraction of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, Calif) according to the manufacturer's instructions. DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 nm and 280 nm by using an ultraviolet spectrophotometer (Nano Drop Technologies, Inc., Wilmington, Del). Genotypes were generated by using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. Two research assistants independently read the gel pictures, and the genotyping was repeated if there was a disagreement regarding the result. We also selected 10% of the samples for replication, and the results were 100% concordant. The primer sequences, restriction enzymes, and PCR conditions that were used for these experiments are listed in Table 1.
Table 1. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Conditions for Selected Single Nucleotide Polymorphisms
Abbreviations: A, adenine; C, cytosine; G, guanine; LIG4, DNA ligase IV; rs, reference single nucleotide polymorphism; SNP, single nucleotide polymorphism; T, thymine; XRCC4-XRCC6, x-ray repair complementing defective repair in Chinese hamster cells 4-6, respectively.
Patients were grouped according to their genotypes. Statistical analysis was performed using the SAS 9.2 statistical software package (SAS Inc., Chicago, Ill). Cox proportional hazards analysis was performed to evaluate the effect of genotypes on RP risk by calculating the hazard ratios (HRs) and 95% confidence intervals (CIs). Kaplan-Meier analysis was performed to estimate the cumulative RP probability. In addition, a multivariate Cox regression analysis was performed to adjust for other covariates. Likelihood ratio tests also were performed for each multivariate Cox regression model to assess the goodness of fit. A 2-sided test with a P value ≤.05 was considered statistically significant.
Finally, we used classification and regression tree (CART) analysis to identify higher order interactions between clinical factors and genetic variants using the Rpart package in S-PLUS Version 8.0.4 (TIBCO, Palo Alto, Calif). CART creates a decision tree to identify how well each genotype or environmental factor predicts the class (eg, RP). Specifically, the recursive procedure starts at the root node and uses a log-rank statistic to determine the first optimal split and each subsequent split of the dataset. This process continues until the terminal nodes have no subsequent statistically significant splits or the terminal nodes reach a prespecified minimum size (>10).
Patient Characteristics and Association With RP
Table 2 lists the characteristics of the 195 patients (112 men and 83 women; median age, 63 years; age range, 35-88 years), of whom 71.3% were white, 85.1% had stage III/IV disease, 89.7% received treatment with a combination of chemotherapy and radiation therapy, and 96.8% received radiation doses between 60 Gy and 70 Gy. To determine whether any confounding factors were influencing the risk of RP, we analyzed the associations between RP and clinicopathologic characteristics, including age, sex, race, Karnofsky performance score (KPS), disease stage, tumor histology, smoking history, receipt of chemotherapy, radiation dose, and mean lung dose (MLD). The median follow-up for patients who had RP of any grade after radiation therapy, with or without chemotherapy, was 21 months, and the median time to the development of RP (grade ≥3) was 3.5 months (95% CI, 3.3-4.5 months). We observed that sex and MLD were associated significantly with severe RP (grade ≥3) in univariate analysis (men vs women: HR, 2.28; 95% CI, 1.07-4.86; P = .034; MLD ≥19.7 Gy vs MLD <19.7 Gy: HR, 2.16; 95% CI, 1.04-4.50; P = .040). No other clinicopathologic characteristics were associated with the risk of developing severe RP in this study population.
Table 2. Patient Demographics and Association With Radiation Pneumonitis Grade ≥3 (N = 195)
Table 3 provides the results from univariate and multivariate analyses of the associations between genetic polymorphisms and grade ≥3 RP using Cox proportional hazards analysis. In the univariate analysis, the G allele of XRCC4 rs6869366 indicated a tendency toward a decreasing risk of severe RP (GT vs TT: HR, 0.51; 95% CI, 0.12-2.13; P = .356), whereas the LIG4 rs1805388 T allele was associated with a significantly increased risk of severe RP (CT + TT vs CC: HR, 1.96; 95% CI, 1.00-3.85; P = .048). Similar results were observed in multivariate analyses that were adjusted for sex, KPS, TNM stage, and MLD (Table 3), which were potential confounding factors for RP, as suggested by the current and previous studies.2, 3 Figure 1 plots the incidence of RP grade ≥3 as a function of time after radiation therapy according to selected clinical factors and genetic polymorphisms. We also assessed these associations in white patients only (n = 139) and observed similar associations (data not shown).
Table 3. Univariate and Multivariate Analyses of Different Genotypes and Radiation Pneumonitis Grade ≥3 in Patients With Nonsmall Cell Lung Cancer
Abbreviations: A, adenine; C, cytosine; CI, confidence interval; del, deletion; G, guanine; HR, hazard ratio; ins, insertion; LIG4, DNA ligase IV; rs, reference single nucleotide polymorphism; N/A, not applicable; SNP, single nucleotide polymorphism; T, thymine; T9I, threonine-to-isoleucine change at codon 9; XRCC4-XRCC6, x-ray repair complementing defective repair in Chinese hamster cells 4-6, respectively.
P values were calculated using a Cox proportional model for univariate analysis.
P values were calculated adjusting for sex, Karnofsky performance status score, tumor stage, and mean lung dose.
XRCC4 (rs6869366 -1394G→T)
XRCC4 (rs28360071 del/ins)
XRCC5 (rs3835 2408G→A)
XRCC6 (rs2267437 −1310C>G)
LIG4 (rs1805388 T9I)
Association of Multiple Factor Interaction With RP Risk (CART Analysis)
To assess potential interactions on the risk of developing RP, we built a decision tree using clinicopathologic variables (sex, disease stage, KPS, and MLD) and all SNPs included in the current study. In the CART analysis, the first split in the decision tree was MLD, indicating that MLD was the strongest predictor for RP among the factors considered. Further inspection of the tree structure revealed distinct patterns between the low MLD group (<19.7Gy) and the high MLD group (≥19.7Gy) (Fig. 2). In the low MLD group, compared with patients who carried the wild-type LIG4 CC genotype, patients with CT/TT genotypes had a significantly higher risk of developing severe RP (HR, 6.34; 95% CI, 1.64-24.51). In the high MLD group, sex was the second split in the decision tree. Men with the TT genotype of XRCC4 rs6869366 and women with AG/AA genotypes of XRCC5 rs3835 had a significantly higher risk of developing severe RP compared with the reference group (HR, 8.67 [95% CI, 2.55-29.47] and 4.69 [95% CI, 1.05-20.96], respectively) (Fig. 2).
To the best of our knowledge, this is the first report of an association between potentially functional SNPs of the NHEJ genes and the risk of developing severe RP in a reasonable sample size (N = 195) of patients with lung cancer who received radiotherapy. Our results suggest some statistical evidence that LIG4 rs1805388 may independently modulate the risk of developing severe RP in patients who receive radiotherapy; and, if this finding is validated, then such genetic variants may be useful as biomarkers for susceptibility to RP in patients with NSCLC who are under consideration for radiotherapy.
The exposure of cells to ionizing radiation leads to the generation of DNA DSBs, which cause disastrous consequences if left unrepaired or misrepaired. Two main pathways are involved in the repair of DNA DSBs in eukaryotic or mammalian cells, including homologous recombination and NHEJ. Different from homologous recombination, which requires extensive homology, and the RAD52 epistasis group of genes, NHEJ does not require homology but is greatly facilitated by the DNA-PKc and the Ligase4/XRCC4 complex. Although it has been demonstrated that both pathways are active from yeast to humans, their significance varies substantially between higher and lower eukaryotes. In yeast, the majority of radiation-induced DNA DSBs are removed by homologous recombination; whereas, in humans, NHEJ appears to be the dominant pathway of removing radiation-induced DNA DSBs.18
The classic model of NHEJ-mediated DSB repair suggests that the progression of NHEJ reaction from 1 event to the next is carried out by sequential recruitment of the core proteins that play specific roles. However, the most recent investigation suggests that NHEJ proceeds in the presence of all core proteins that are preassembled into a functional machinery at the site of DSBs.6 Despite all the uncertainties, it is believed that some core proteins have more direct physical interactions, as detected by biochemical methods, including the XRCC5/XRCC6 (Ku) complex with a high affinity for DSB and the Ligase4/XRCC4 complex responsible for the ligation.
To date, the 5 NHEJ SNPs included in the current study have been associated with different types of cancer susceptibility, including cancers of the lung (XRCC4 rs6869366),14 breast (XRCC6 rs2267437 and XRCC5 rs3835),15 colorectum (XRCC4 rs6869366),16 and oral cavity (XRCC4 rs2836071).17 However, their associations with radiation treatment-related toxicities rarely have been investigated. In our current study, LIG4 rs1805388 had a significant association with the development of severe RP in patients who received thoracic irradiation for NSCLC independent of patient-related, tumor-related, and therapy-related factors. This SNP causes a nonsynonymous amino acid change from threonine to isoleucine at the N-terminal of the LIG4 protein that is essential for its activity.19 A previous study indicated that the substitution led to an approximately 50% decrease in the adenylation and double-strand ligation activity of ligase IV,20, 21 which may result in increased radiosensitivity, as we observed in the current study. In addition, the XRCC4 rs6869366 SNP, which is located in the promoter region of that gene, also had a tendency to confer RP susceptibility, although statistical significance was not reached, probably because of the reduced power of the study. Considering the functional relevance of these 2 proteins, the subsequent changes in the activity of the Ligase4/XRCC4 complex resulting from a functional change caused by SNPs may pose a substantial influence on the whole NHEJ machinery. Indeed, although LIG4 primarily interacts with XRCC4 through the region located between the breast cancer 1 (BRCA1) and BRCA1 C-terminal repeats (BRCT) domain,19 previous investigators reported a significant gene-gene interaction between LIG4 and XRCC4 in the modulation of lung cancer risk mediated by LIG4 rs1805388 SNP.13 Taken together, these results indicated that the LIG4 rs1805388 SNP may influence the capacity of NHEJ by regulating both its own activity and interactions with the XRCC4 protein. However, the current study did not have the statistical power to evaluate such an interaction, and additional mechanistic investigations are needed if our findings are validated in larger studies.
Our CART analysis was an explorative, nonparametric approach, which required no assumption of a genetic model. Through this decision tree-based data mining, we confirmed that MLD, sex, and LIG4 rs1805388 were the most important risk factors for developing severe RP in our study population, because those variables were the first and second splits in the decision tree. The XRCC4 rs6869366 TT genotype with an MLD ≥19.7 Gy in men, XRCC5 rs3835 AG/AA genotypes with an MLD ≥19.7 Gy in women, and LIG4 rs1805388 CT/TT genotypes with an MLD <19.7 Gy were most likely to be associated with severe RP. These findings suggest that patients who have such genotypes may require close surveillance after thoracic radiotherapy. Once again, these findings need to be validated in larger studies.
Despite these positive findings, our current study has some limitations. First, we were not able to explore the mechanism by which the NHEJ genetic polymorphisms lead to RP in patients with lung cancer. Second, we used a candidate polymorphism approach, which allowed us to focus on potentially functional SNPs reported in the literatures but did not comprehensively cover all SNPs in the entire gene. Some important but rare SNPs may have been missed, or the observed association may have resulted from genetic linkages with other untyped SNPs. These warrant additional investigations of the tagging SNPs in the future, which may help identify the underlying disease-causing variants. Third, we did not adjust P values for multiple tests, simply because this was an exploratory study with limited study power. We plan to confirm the current findings in our ongoing, prospective expansion studies under more stringent conditions with larger sample sizes.
In summary, the current study indicated that polymorphisms of the NHEJ gene LIG4 had a significant effect on the risk of developing severe RP in patients who received definitive radiation therapy for NSCLC and that XRCC4 rs6869366 and XRCC5 rs3835 may be involved in interactions in the risk of developing RP. Once validated, LIG4 SNPs may be useful biomarkers for susceptibility to RP. Therefore, larger independent studies are needed to validate these findings.
This study was supported in part by National Institutes of Health Grants R01 ES011740 and R01 CA131274 (to Q.W.) and by Cancer Center Core Grant P30 CA016672 to The University of Texas MD Anderson Cancer Center.