Association between the Risk for Lung Adenocarcinoma and a (−4) G-to-A Polymorphism in the XPA Gene

Nucleotide excision repair (NER) is a versatile DNA repair mechanism removing DNA lesions induced by UV irradiation, tobacco smoke, and environmental pollutants (1). Attenuated or abolished functioning of NER, due to inherited mutations in genes coding for protein components of NER, is associated with a very rare, cancer-prone syndrome (xeroderma pigmentosum), characterized by an extremely high risk of skin cancer (2). Xeroderma pigmentosum is caused by mutant alleles of one of eight repair genes (XPA-XPG and XP-V) involved in NER. Hereditary mutations in the XPA gene are associated with the xeroderma pigmentosum complementation group A (XP-A), which represents the most severe form of xeroderma pigmentosum (3).

Recently, common polymorphisms of NER genes, as well as other DNA repair genes, were found to modulate levels of DNA damage, individual DNA repair capacity, and cancer risk (4-7). In the XPA gene, a polymorphic site was identified that was in the 5′ untranslated region of the gene and consisted of a G-to-A substitution in the fourth nucleotide before the ATG start codon (8, 9). The polymorphism, termed XPA (−4) G-to-A, is in the Kozak sequence near the start codon and thus may affect XPA protein levels in cells (10). A functional association between XPA (−4) polymorphism and DNA repair capacity was reported (11), which showed significantly higher repair proficiency in healthy subjects with at least one G allele. An effect of the XPA polymorphism on lung cancer risk was also reported in Koreans and Americans (11, 12). As these studies showed considerable differences in allele frequencies, further studies, including European Caucasian populations, are necessary.

Here, we investigated the influence of the XPA (−4) G-to-A polymorphism on primary lung cancer risk overall and separately for adenocarcinoma and squamous cell carcinoma (SCC) in a European population from Germany. In addition, the influence of tobacco smoking or occupational exposures (i.e., lung carcinogens associated with specific jobs) on the XPA polymorphism associated lung cancer risk was evaluated.

For this case-control study, lung cancer cases and hospital controls, all Caucasians, were recruited from the surgery department of the Thoraxklinik Heidelberg between 1996 and mid-2000 (see also ref.13). Cases were newly diagnosed patients with histopathologically confirmed primary lung cancer who were all surgically treated. There were no age or sex restrictions, but patients with prior history of cancers were excluded. Controls had no malignant disease. This study was approved by the ethical committee of the University of Heidelberg (reference no. 201/98). All study participants signed an informed consent form and completed a questionnaire with detailed information on his or her personal history, smoking habits (never smoker, current smoker, ex-smoker, number of pack-years smoked), and occupational exposures. Occupational exposure status was established based on information given by the study participants in self-administered questionnaires. The questionnaire recorded data about job title (e.g., welders, drivers, mechanics, industry workers, and painters) or exposure to carcinogens (asbestos and mineral fibers, metals, all kinds of dust and fumes, cement, petrol and coal products, diesel exhaust, solvents, pesticides, etc.; refs. 14, 15). The nonexposure group consisted of individuals claiming lack of occupational exposure and/or having had jobs with no such exposure (e.g., office workers, teachers, and housewives).

In this study, 463 histologically confirmed primary lung cancer cases and 460 randomly selected hospital controls were analyzed. The cases included 204 adenocarcinomas, 212 SCC cases, and 47 other tumors with a variety of different pathologies (including 25 large cell and 5 small cell carcinomas, 4 carcinoids, and 13 mixed types). Because patients were recruited from surgical wards until mid-2000, the case population consisted mainly of SCC and adenocarcinoma patients. These two histologic groups, SCC and adenocarcinoma, were large enough to allow a separate statistical analysis. The controls were noncancer patients suffering mainly from alveolitis, bronchitis, pneumonia, fibrosis, sarcoidosis, chronic obstructive pulmonary disease, and emphysema. There were significantly more males (76% versus 68%) and smokers (90% versus 66%) among cases than among controls (Table 1). Regarding occupational exposure, subjects were categorized according to probably not exposed (0), possibly exposed (1), and probably exposed (2). For the purpose of the analysis presented here, subjects classified as 0 were compared with those classified as 1 or 2. Individuals with possible or probable occupational exposure to lung carcinogens (71% versus 58%) were more frequent among all cases. The group of patients with adenocarcinoma, however, showed no significant difference from the control group regarding gender (P = 0.17) and occupational exposure (P = 0.15).

For all participants, genomic DNA was isolated from peripheral blood lymphocytes using a QIAamp DNA Blood Kit (Qiagen, Hilden, Germany).

Detection of the XPA (−4) G-to-A polymorphism was done by PCR and melting curve analysis using fluorescence labeled hybridization probes (LightCycler, Roche Diagnostics, Mannheim, Germany). The sensor probe was designed for a perfect match with the G allele sequence. Thus, for the A allele, one nucleotide mismatch between sensor and the target DNA sequence caused destabilization of the hybrid and a melting point (T_m) shift from 59.5°C to 51°C. The analysis was done in 10 μL volumes in glass capillaries (Roche Diagnostics): 1× PCR buffer, 2.5 mmol/L MgCl₂, 0.2 mmol/L deoxynucleotide triphosphates, 0.1% bovine serum albumin in DMSO, 0.5× Q solution, 0.5 unit Taq DNA polymerase (Qiagen), 1 μmol/L of each primer,0.15 μmol/L of each probe, and 10 ng of DNA. Primers and probes used were 5′-GCAGGCGCTCTCACTCAGAA-3′ (forward primer), 5′-TGCCGCTTCCGCTCGATA-3′ (reverse primer), 5′-CATCTCCGGCCCACTCC x (sensor, x = fluorescein), and 5′-LC Red640-GGACCTAGCTCCCAGCTCCACGC p (anchor; Tib Molbiol, Berlin, Germany). Reaction conditions were initial denaturation at 95°C for 3 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 62°C for 10 seconds, and elongation at 72°C for 12 seconds. Melting curve analysis was done with an initial denaturation at 95°C for 30 seconds and 15 seconds at 40°C, slow heating to 75°C with a ramping rate 0.2°C/s, and continuous fluorescence detection. Melting curves were evaluated by two independent observers. To verify the LightCycler results, 100 randomly selected samples were analyzed by conventional PCR-RFLP method and 100% concordance was found.

Amplification of a 233-bp product was carried out in a volume of 20 μL using 20 ng of DNA, 1× buffer, 2.5 mmol/L MgCl₂, 0.2 μmol/L deoxynucleotide triphosphates, 1× Q solution, 1 unit of Taq polymerase (Qiagen), and 10 pmol of each primer (5′-TCAGAAAGGCCGCTGGGT-3′ and 5′-CTGGCGCAGCATCAGTGC-3′; Tib Molbiol). Cycling conditions were initial denaturation at 94°C for 3 minutes and 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds followed by 1 cycle of final elongation at 72°C for 10 minutes. The PCR product was digested with MspI enzyme (New England BioLabs, Beverly, MA) and separated on a 3% agarose gel. Results of RFLP analysis were checked by sequencing.

The lung cancer group overall and the histologic subgroups of adenocarcinoma and SCC were compared with the controls for the basic population characteristics gender, age, smoking, and occupational exposure (Table 1). Both the population of cases and controls were tested as to whether they were in Hardy-Weinberg equilibrium using the χ² test of goodness-of-fit with 2 df with respect to the distribution of the XPA genotypes. Lung cancer risk was analyzed for association with the XPA polymorphism: crude odds ratios (OR) and their 95% confidence intervals (95% CI) were calculated and tested for statistical significance (null hypothesis H₀: OR = 1 versus the alternative H₁: OR ≠ 1; ref. 16). In addition, multiple, unconditional logistic regression was done to adjust for age, gender, smoking, and occupational exposure. Smoking was quantified by the degree of tobacco consumption categorized as pack-year groups, and age at diagnosis was categorized in four groups (see Table 1). Presence of gene-environment interaction, in particular for smoking and occupational exposure, was tested by calculating second-order interaction terms between the genetic polymorphism and the environmental factors smoking and occupational exposure in a multiple logistic regression analysis. Multiple logistic regression analysis and calculation of confidence intervals was done with SAS statistical evaluation system (SAS Institute, Inc., Cary, NC).

Table 2 shows the distribution of the XPA (−4) genotypes in controls and all cases as well as in the cases divided according to their histology. The G allele frequency was similar in cases (0.66) and controls (0.67) as well as in adenocarcinoma (0.65) and SCC (0.67) cases. In contrast to other studies (11, 12), the more common G allele was considered the reference genotype in our analysis, whereas the rare A allele was examined as the variant. Genotypes were distributed according to the Hardy-Weinberg equilibrium. No remarkable differences in the XPA genotype distribution could be found between all cases and controls (P = 0.5) and when histology types were considered (P = 0.9 for SCC and P = 0.2 for adenocarcinoma). In adenocarcinoma, slightly more AA homozygotes were found than in controls (14% versus 10%), whereas more GA heterozygotes were observed among controls. Crude OR analysis did not show an association between the genotype and cancer risk (Table 2).

As the study population groups differed in age, gender, smoking, and occupational exposure status, ORs were adjusted for those factors. The risk estimates for the AA genotype were 1.53 in all cases (95% CI, 0.94-2.50), 1.62 in adenocarcinoma patients (95% CI, 0.91-2.88), and 1.67 among SCC cases (95% CI, 0.86-3.26). No statistically significant interaction of genotype with the confounding factors age and gender was observed (data not shown).

Based on the observation that subjects with at least one G allele had higher repair proficiency (11), GA carriers were incorporated to the reference group. Increased ORs were calculated for the AA genotype when adjusted for age, gender, smoking, and occupational exposure for all cases (OR, 1.58; 95% CI, 0.99-2.51), for SCC (OR, 1.72; 95% CI, 0.91-3.23), and for adenocarcinoma (OR, 1.72; 95% CI, 1.00-2.96).

Interaction terms between polymorphism and smoking as well as occupational exposure were determined which yielded, in the adenocarcinoma group only, a statistically significant interaction for genotype with occupational exposure status (P = 0.036) and, to a lesser extent, with smoking. Therefore, adjusted ORs were calculated for the adenocarcinoma group subdivided into occupationally exposed and nonexposed individuals as well as in pack-year groups (Table 3). A nearly 3-fold increased risk of adenocarcinoma was associated with the XPA AA genotype for occupationally exposed individuals (OR, 2.95; 95% CI, 1.42-6.14) and for those subjects smoking >20 pack-years (OR, 2.52; 95% CI, 1.17-5.42). No genotype-dependent increase in OR was found for the nonexposed subgroup or for individuals smoking <20 pack-years. When this subgroup analysis was done for SCC, no significant increase in risk was found either for the occupationally exposed and heavy smoking cases or for the unexposed cases (data not shown).

In this study, we observed an increase in lung cancer risk among the XPA AA genotype carriers. Risk estimates were obtained using clinical controls with noncancer lung diseases. As some of these diseases themselves may be associated with the XPA polymorphism, ORs may differ from those derived from a population-based control group.

Our results on cancer risk overall agree with published reports (11, 12) on the protective effect of the G allele on lung cancer risk in Korean and American populations comprising different ethnicities. This corresponds in our study to the enhanced risk found for subjects with the AA genotype, which is, nevertheless, lower than one would deduce from the published values. This deviation might, in part, result from differences in allele frequencies. The data reported by the other studies for the control groups (Caucasians, 0.55; Mexican Americans, 0.61; African Americans, 0.70; and Koreans, 0.52; refs. 11, 12) did not match our data (0.67), indicating considerable differences in the investigated populations. In addition, there might be a variability in the populations of all three studies, regardless of ethnicity, due to the selection of hospital-based control populations.

The published studies assessed the risk effect predominantly for males and younger individuals. This was not confirmed by our study, although we considered the differences between cases and controls by adjusting our ORs for these variables. Both published studies investigated the impact of smoking on the XPA genotype-related risk but the influence of occupational exposure was not analyzed. Interestingly, Park et al. (12) showed a decreased risk for small cell lung carcinomas associated with the GG genotype but not for adenocarcinoma and SCC. In our study, the polymorphism did not affect significantly the risk for SCC, but a risk was evident for adenocarcinoma patients with a history of heavy smoking and likely occupational exposure (see Table 3).

Adenocarcinoma is more frequent among other histologic subtypes of lung cancer among women (smokers and nonsmokers) and nonsmoking men (17), but a gender-dependent correlation of XPA genotype and risk for adenocarcinoma was not found in our study.

Smoking increases the risk for all histologic types of lung cancer, although it is thought to be less strong for adenocarcinoma than for SCC and small cell carcinoma (18). Thus, it seems that adenocarcinoma has a somewhat different etiopathology than other histologic types: The increasing incidence of adenocarcinoma, observed in North America and Europe, has been associated with changes in types of cigarettes smoked (mainly with filter), smoking behavior, and tobacco composition (i.e., relatively high levels of tobacco-specific nitrosamines in low-tar cigarettes; refs. 17, 19). One of these N-nitroso compounds, 4-methylnitrosamino-1-3-pyridyl-1-butanone, causes lung adenocarcinoma in rodents (20).

In our study, one might consider that adenocarcinoma cases were environmentally exposed (by smoking and/or occupation) to agents that reach the more peripheral airways where adenocarcinoma usually develops. There, they produce various types of DNA damage, among which DNA adducts that require the XPA activity for their repair. Tissue- and organ-specific differences in mRNA levels were observed for various NER genes, including XPA, which may influence the repair rate in a given tissue (21, 22). Such differences in DNA repair capacity and/or expression of DNA repair proteins, as well as between cell types and their location, contribute to organ-specific carcinogenesis.

Certain occupational exposures are important risk factors for lung cancer. There is, however, no convincing evidence thus far to link particular occupational exposures and increased risk of adenocarcinoma. Nevertheless, a slightly increased risk for adenocarcinoma was reported after arsenic, asbestos, or polyvinyl chloride exposure, which was not confirmed by others (17). The analysis of our occupational exposure data did not allow to pinpoint any particular agents or sources of exposure that might be responsible for higher adenocarcinoma risk in AA genotype carriers, as a limitation of this study is that the exposure data gathered through questionnaires were not detailed enough. They were based on job titles and self-reported exposure data only. Further studies should explore more refined approaches for occupational exposures and confounders (14, 23). The observed gene-environment interaction supports, however, the notion that the XPA genotype affects the risk of adenocarcinoma only when related to external exposure by occupation and/or heavy smoking.

Our study had an adequate size for the analysis of the main effects related to cancer incidence and XPA genotypes. However, we cannot definitely exclude those factors as risk factors for SCC and adenocarcinoma, which showed no significant interaction with the XPA polymorphism. These factors should be tested in further studies about gene-environment interaction analysis. Nevertheless, the observed increases in adenocarcinoma lung cancer risk for occupationally exposed persons and heavy smokers are remarkable. Mechanisms and types of lung carcinogens that render the XPA AA homozygotes more vulnerable to adenocarcinoma of the lung remain to be determined.

We thank Birgit Jäger and Peter Waas (Division of Toxicology and Cancer Risk Factors, German Cancer Research Center) for their excellent technical assistance, Renate Rausch (Division of Biostatistics, German Cancer Research Center) for her substantial help with the statistical analysis, O. Landt (Tib Molbiol) for helping with the LightCycler probe design, George Zizka for critically reading the article, and the patients and staff involved in sample and data collection.