1 Population Studies and Prevention Program, 2 Center for Molecular Medicine and Genetics and 3 Molecular Biology and Human Genetics Program, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA
* To whom correspondence should be addressed Email: bockc{at}med.wayne.edu
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Abstract |
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Abbreviations: ETS, environmental tobacco smoke
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Introduction |
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Findings from studies examining the association between NQO1 genotype and lung cancer have been mixed. The initial study examining the relationship between NQO1 genotype and lung cancer reported an increase in lung cancer risk associated with the T allele (3); however, two additional studies suggested an increase in lung cancer risk associated with the C allele (4,5). Several others suggested that the T allele had a protective effect for lung cancer (68), and two studies found no association between NQO1 genotype and lung cancer (9,10). These studies evaluated lung cancer risk primarily in smoking populations; none had a large sample of never smokers.
This study was designed to examine the association between NQO1 genotype and lung cancer among never smokers from a population-based case-control study. Approximately 510% of all lung cancers occur among non-smokers (1113), and never smokers were targeted for this study because this understudied population is most likely very susceptible to lung carcinogens, even at low levels (12). Furthermore, lower levels of carcinogen exposure among never smokers may reveal small effects of variations in NQO1 genotype on lung cancer risk, which might otherwise be overwhelmed among smokers.
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Materials and methods |
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The overall interview response rate for cases was 66.1%. It was not possible to determine a response rate for controls because eligibility could not be determined in households refusing to answer the eligibility screening questions. Of those individuals answering the phone and providing a household census, 93% participated in the study. Biologic samples across both studies were available for 174 never smoking cases and 177 selected never smoking controls. Of these, no genotype could be determined for 13 cases and four controls, resulting in a final sample size of 161 cases and 173 controls.
Data collection
Information regarding personal medical history, family history of cancer, sociodemographic characteristics and environmental tobacco smoke (ETS) exposure was obtained using a structured interview administered over the telephone by trained interviewers. If the study subject was unable to participate in the interview due to illness or death, an attempt was made to obtain proxy data by interviewing someone familiar with the subject's history, such as a spouse, parent or child. Written, informed consent was provided for all study subjects, and the protocol was approved by the Wayne State University Human Investigation Committee.
Because of the rapidly fatal nature of lung cancer, it was necessary to collect 139 (86.3%) paraffin-embedded tumor blocks to obtain DNA from the deceased cases. The remainder of DNA samples in case subjects was extracted from either venous blood (10.6%) or buccal cells (3.1%). DNA from control subjects was collected via blood (53.2%) or buccal cells (46.8%).
DNA extraction and genotyping
DNA was isolated from whole blood with the Genomic DNA Purification System (Gentra Systems, Minneapolis, MN), from buccal swabs with the BuccalAmpTM DNA Extraction Kit (Epicentre Technologies, Madison, WI), or from paraffin-embedded tissue with the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturers' protocols. When multiple tissues were obtained from a study participant, DNA extracted from blood was used preferentially, followed by DNA extracted from buccal swabs and DNA extracted from paraffin blocks.
DNA isolated from buccal cells or paraffin-embedded tissue was pre-amplified using a nested PCR strategy. Pre-amplification (outer amplification) was carried out in a 25-µl reaction containing 2.5 mmol/l MgCl2, 0.5 µmol/l of the forward primer (5'-ACT GAG AAG CCC AGA CCA ACT T-3') and reverse primer (5'-CTG GAG TGT GCC CAA TGC TA-3'), 1.25 U AmpliTaq Gold polymerase, and 200 µmol/l of dATP, dCTP, dGTP and dTTP. The outer amplification mixture was denatured at 95°C for 10 min and amplification was achieved by 15 cycles of 94°C for 30 s, 63°C for 30 s and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The outer amplification was performed on a Mastercycler® Gradient thermocycler (Eppendorf, Westbury, NY).
The TaqMan 5'-nuclease assay (Applied Biosystems, Foster City, CA) was used to detect the NQO1 (C609T) polymorphism using DNA extracted from whole blood or pre-amplified DNA (see above). Primers, for amplification of the region of the NQO1 gene containing the functional polymorphism, and fluorescent probes for allelic discrimination, were designed using Primer Express software (Applied Biosystems). Standard TaqMan conditions were used in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems) for amplification and detection of both wild-type and mutant alleles. Briefly, 25 ng DNA, or 2 µl of pre-amplification reaction, was added to 1x Universal PCR mix (Applied Biosystems) containing forward (5'-CAG AGT GGC ATT CTG CAT TTC T-3') and reverse (5'-CTG GAG TGT GCC CAA TGC TA-3') primers and NQO1 wild-type (5'-6FAM-CTT AGA ACC TCA ACT GA-MGBNFQ-3') and mutant (5'-VIC-CTT AGA ATC TCA ACT GAC A-MGBNFQ-3') probes. Randomly, 5% of the products were sequenced and 10% of the samples were repeated.
Statistical methods
Allele frequencies among controls were calculated for both racial groups combined and by race, and the frequencies within African American controls and Caucasian controls were compared. HardyWeinberg equilibrium was assessed in all controls and within each racial group using the 2 test with 1 d.f.
The subjects were categorized into two age groups to separate early and late onset of lung cancer. There is not an established age definition for early onset lung cancer, so 50 years of age was chosen to define an early onset group with the highest potential for a genetic contribution to lung cancer risk. Approximately 6.7% of incident lung cancers between 1973 and 2001 were diagnosed at ages younger than 50 (14). This early age at diagnosis classification also is consistent with the definition used in several previous studies (1517).
Unconditional logistic regression was used to examine the effect of having at least one copy of the T allele (genotype of C/T or T/T) on disease risk, controlling for age, sex and race in the entire sample and within the early and late age at diagnosis groups. Associations between lung cancer and history of any obstructive lung disease, history of pneumonia, total years of ETS exposure and family history of lung cancer were each examined using unconditional logistic regression in the entire sample and by age at diagnosis category, controlling for age, sex and race.
The age variable used in all models was defined as the age at diagnosis of cases, and age at time of interview of controls. A history of obstructive lung disease was considered present if the subject reported a history of chronic obstructive pulmonary disorder, emphysema, chronic bronchitis or chronic breathing problems at least 1 year before diagnosis (cases) or interview (controls). Subjects were asked whether they had experienced at least 6 months of ETS exposure in their childhood, adult household and/or adult workplace, and if yes, for how many years and for how many hours per day. Total years of ETS exposure were included in the analysis because this was deemed a more reliable measure than total hours of ETS exposure. The final models include age, sex and race. The other variables were not statistically significant and therefore were not included. The effect of having at least one copy of the T allele, controlling for age, sex and race (where appropriate) was further examined after stratification by sex, race, ETS exposure, family history of lung cancer and presence of adenocarcinoma, within each age group and in the entire sample. Statistical significance of results was assessed at the = 0.05 level.
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Results |
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Allele frequencies of C and T among controls were, respectively, 0.79 and 0.21 in Caucasians, and 0.84 and 0.16 in African Americans, with no statistically significant difference in allele frequencies between the two racial groups [2(1) = 0.96, P = 0.33]. In all controls combined, frequencies of the C and T alleles, respectively, were 0.79 and 0.20. There was no significant difference in allele frequencies between the controls from each of the two age strata [
2(1) = 1.93, P = 0.17]. The distribution of genotypes by select characteristics in the entire sample and within each age group is shown in Table II. The NQO1 alleles are in HardyWeinberg equilibrium among all controls [
2(1) = 0.96, P = 0.96], Caucasian controls [
2(1) = 1.51, P = 0.22], and African American controls [
2(1) = 0.18, P = 0.67].
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The effects of different stratification criteria on the adjusted lung cancer odds ratio associated with having at least one copy of the T allele are shown in Table II. In the younger age group, the T allele is not statistically significantly associated with lung cancer risk in either sex, although cell sizes are small. Within the older age group, the T allele is associated with a decreased risk of disease in both men (OR = 0.28, 95% CI: 0.110.75) and women (OR = 0.66, 95% CI: 0.301.44), but the effect is not statistically significant in women.
When data were stratified by race, risk of lung cancer was 0.46 (95% CI: 0.240.86) times lower for individuals with at least one copy of the T allele compared with individuals with no T allele among the 186 Caucasian subjects in the older age group, controlling for age and sex. Among the 33 African American individuals in the older age group, controlling for age and sex, the decrease in risk of lung cancer associated with the T allele was also observed, but this finding was not statistically significant (OR: 0.48, 95% CI: 0.073.24, Table II).
After stratifying the sample on ETS exposure, results suggest that the protective effect of the T allele may be greater among those with ETS exposure (OR = 0.57, 95% CI: 0.321.03) than among those without (OR = 0.98, 95% CI: 0.412.38) controlling for age, sex and race (Table II), although neither association is statistically significant. The protective effect of the T allele is statistically significant among those exposed to ETS within the age at diagnosis 50 group (OR = 0.42, 95% CI: 0.200.89).
Inferences regarding the protective effect of the T allele remained the same as those from the entire sample when the subjects were stratified by family history of lung cancer and histologic type (data not shown).
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Discussion |
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Self-reported ETS exposure may not be completely accurate, and biologic confirmation of exposure, e.g. by plasma cotinine levels, was not obtained. However, plasma cotinine levels only indicate recent exposure, and do not provide information about lifetime ETS exposure. Cumulative lifetime years of exposure, rather than cumulative lifetime hours of exposure, were examined because this measure was believed to be more credible. The greater cumulative lifetime ETS exposure among the older age group (mean = 28.6 years) than among the younger group (mean = 18.7 years) may explain some of the observed protective effect of the T allele only in the older group. It is likely that there are other carcinogens not measured in this study, which also increase with age that are activated by NQO1. The ETS findings suggest that decreased phase I enzymatic activity of NAD(P)H:quinone oxidoreductase 1 is the mechanism responsible for the beneficial effect of this polymorphism. Within the younger age group, ETS and other carcinogen exposure levels may not be great enough in combination with the NQO1 genotype to significantly influence risk. Thus, the genetic mechanism involved in the early onset lung cancer among never smokers is likely different than that of later onset lung cancer among never smokers, and is less reliant upon NAD(P)H:quinone oxidoreductase 1 activity.
Our findings regarding the protective effect of the T allele are in concordance with several previous studies (48). Chen et al. found a significant decrease in lung cancer risk (OR = 0.3, 95% CI: 0.10.7) associated with having two copies of the T allele as compared with having two copies of the C allele in a Japanese population (n = 276), and a marginally significant association in a Hawaiian population (n = 185). Chen et al. also reported similar associations across histologic types, and did not observe a statistically significant interaction between genotype and smoking stratified on the median (28 pack-years) (6). Results from a Chinese study of 84 cases and 84 matched controls by Yin et al. were also in agreement with those from our study, but were not statistically significant. Among the 78 never smokers, those with the C/T or T/T genotype were at 0.79 (95% CI: 0.302.06) times lower risk of lung cancer compared with those with a C/C genotype, but among the 90 current smokers, there was no difference in lung cancer risk between those with and without a copy of the T allele (OR: 1.0, 95% CI: 0.422.40) (8). Similarly, Hamajima et al. found a statistically significant protective effect of having at least one copy of the T allele in a Japanese population when 192 cases were compared with 640 hospital controls; subjects with at least one copy of the T allele were at 0.71 (95% CI: 0.501.0) times lower risk of lung cancer as compared with individuals homozygous for the C allele, controlling for age and sex (7).
Two studies examined the inverse relationship, comparing the risk of lung cancer associated with having the C/C versus the C/T or T/T genotype. The first of these by Wiencke et al. found an increased risk among Mexican Americans and African Americans with the C/C genotype as compared with those with C/T or T/T genotype (OR = 1.80, 95% CI: 1.092.97) among 177 cases and 297 community volunteer controls. Risk was even higher among the 128 never smokers, with those with the C/C genotype having a 2.92 (95% CI: 0.5814.7) times higher lung cancer risk as compared with those with the C/T or T/T genotype (4). Similarly, Sunaga et al. used the risk associated with having the T/T genotype as the baseline, and found a non-significant increase in risk associated with the C/T genotype (OR = 1.49, 95% CI: 0.742.98) and a statistically significant increase in risk associated with the C/C genotype (OR = 2.15, 95% CI: 1.034.48), adjusted for gender, age and smoking habit. When the 198 Japanese adenocarcinoma cases and 152 hospital controls were stratified on smoking status, the increase in risk associated with the C/T and C/C genotypes appeared higher among the never smokers than among the smokers, although none of the odd ratios were statistically significant. Lung cancer risk associated with having either the C/T or C/C genotype was not calculated (5). Results from both of these studies provide essentially the same inferences as ours, as the estimated risk is associated with not having at least one copy of the T allele, which is the inverse of the risk estimated in our study.
An interesting interaction between genotype and smoking history was observed by Xu et al. in 814 Caucasian cases (any histology type) and 1123 unmatched Caucasian family and friend controls. The study reported a decrease in lung cancer risk associated with the T allele among long-term former smokers, which is similar to the observed effect in our study of never smokers; however, Xu et al. also observed an increased risk from the T allele among short-term current heavy smokers (19).
Lin et al. only found a statistically significant effect of the NQO1 genotype among male smoking cases with adenocarcinoma as compared with male smoking controls, where having the C/C genotype increased risk 2.49 (95% CI: 1.175.32) times the baseline risk of those who had the C/T or T/T genotype; no genotype effect was observed in the entire Taiwanese sample of 198 cases and 332 age and gender-matched controls (10). Results of our study would lead us to expect increased risk associated with the C/C genotype in non-smokers, regardless of sex or histological type. Lewis et al. reported no association between NQO1 genotype and risk of lung cancer in a Caucasian sample of 94 cases and 165 bronchoscopy clinic controls, except for an increase in risk of small-cell lung cancer associated with having at least one copy of the T allele (age and sex adjusted OR = 3.80, 95% CI: 1.1912.1). Their observed association was even stronger within the 15 heavy smokers where cases had NSCLC (age and sex adjusted OR = 12.5, 95% CI: 2.175.5) (9).
This is the largest study of never smokers to date, and is strengthened by being population-based, including large numbers of African Americans, and encompassing both early- and late-onset cases. It is also the only study to specifically examine genotype effects within age categories. In addition to the ETS measurement issues discussed above, there are several other limitations to this study, including small sample sizes within cells after stratification restricting inferences that could be made, lack of assessment of exposures to other environmental chemicals, vitamins and chemopreventive agents, the use of proxy environmental and behavioral data in a subset of subjects, and the age difference between cases and controls in the final sample. In general, there were more young controls available and eligible for matching to cases than there were older controls. The age difference is due to both the greater availability of younger controls and subsequent oversampling of controls in the early onset study (phase II), and the lower likelihood of older controls to provide biologic samples. Thus, the distribution of controls eligible for the current analyses had more younger and fewer older individuals. There was, however, no difference in genotype distribution between the 10-year age groups among the cases or the controls (data not shown), thus the age difference between cases and controls should not affect the conclusions regarding genotype and lung cancer risk.
Another potential limitation of this study is the use of tumor blocks as the DNA source for some of the cases. This could lead to bias if tumor tissue was used from the blocks, and if there is deletion of the genomic region (16q22.1) containing NQO1. Of the 83 genotypes obtained using tissue from blocks, 49 were from normal tissue, 34 were from tumor tissue and 56 were from mixed tumor and normal tissue. When the normal were compared with tumor (P = 0.72), mixed (P = 0.68) and tumor plus mixed (P = 0.79), there were no significant differences with respect to genotype using Fisher's exact test. We conclude that any bias from the use of DNA from tumor blocks is therefore minimal.
While this study was limited to subjects providing a biologic specimen, the observed T allele frequencies in Caucasians (0.21) and African Americans (0.16) in our study were similar to those reported in previous studies, with estimated T allele frequencies between 0.12 and 0.36 in Caucasians (3,6,9,19,20), and 0.22 in African Americans (4). These T allele frequencies are approximately half of those observed in Japanese (57), Taiwanese (10), Korean (21) and Chinese (8) populations.
Overall, having at least one copy of the NQO1 T allele (serine variant) may be associated with a decreased risk of lung cancer in never smokers, and this apparent protective effect is statistically significant in those diagnosed at ages 50 and older. This risk reduction may be attributable to the greatly reduced procarcinogenic activity of NAD(P)H:quinone oxidoreductase 1 in individuals with at least one copy of the variant allele. The observed protective effect of the NQO1 T allele is likely generalizable to other African American and Caucasian never smokers, who comprise 6.7% of all cases in Metropolitan Detroit (12).
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Acknowledgments |
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References |
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