Abstract

Cytochrome P450 (CYP) enzymes, involved in metabolism of tobacco carcinogens, are also involved in estrogen metabolism and many are regulated by estrogens. These genes may thus be of relevance to gender-specific differences in lung cancer risk, particularly in early-onset lung cancer, where a high proportion of women is observed. We conducted a case–control study to investigate genetic polymorphisms in cytochromes that might modify the risk of developing early-onset lung cancer. In total, 638 Caucasian patients under the age of 51 with primary lung cancer and 1300 cancer-free control individuals, matched by age and sex, were included in this analysis. Thirteen polymorphisms in the CYP1A1 , CYP1B1 , CYP2A13 , CYP3A4 and CYP3A5 genes were analyzed. No significant association was found for any of the analyzed polymorphisms and lung cancer risk overall. However, among women, a significantly increased risk of early-onset lung cancer was observed for carriers of the minor allele of CYP1B1 SNP rs1056836 [odds ratio (OR) 1.97; 95% confidence interval (CI) 1.32–2.94; P  < 0.001]. Also, a non-significant increase in lung cancer risk was observed in the group of women carriers of the minor allele of CYP2A13 SNP rs1709084 (OR 1.64; 95% CI 1.00–2.70; P  = 0.05). The effect of these two polymorphisms was shown to be modified by smoking. Haplotype analysis was performed for CYP1B1 and CYP2A13 . No differences between cases and controls were observed for both genes ( P  = 0.63 and P  = 0.42 for CYP1B1 and CYP2A13 , respectively). Our results suggest that the CYP1B1 and the CYP2A13 genotypes may contribute to individual susceptibility to early-onset lung cancer in women.

Introduction

Due to the decrease in cigarette consumption over the past several decades, lung cancer incidence has slowly declined in men, but not in women. In Germany, lung cancer is still a major cause of cancer death for men and the second only after breast cancer in women ( 1 ). Numerous studies, including recent whole-genome association studies on lung cancer, have shown the contribution of genetic factors to the risk of lung cancer development ( 2–4 ). It is supposed that a particularly strong genetic component might exist in a group of individuals with early-onset lung cancer (under the age of 51). The early-onset lung cancer cases are characterized by a higher proportion of never smokers and higher prevalence of adenocarcinoma compared with other histological type, poor survival outcome, higher proportion of family history of lung cancer and, interestingly, a higher proportion of women ( 5–7 ). Therefore, molecular epidemiological investigation of early-onset lung cancer cases is a subject of particular interest allowing the detection of genetic effects that may be too weak to detect among all lung cancer patients overall.

Among the main enzymes involved in metabolism of tobacco compounds, and thus potentially involved in individual susceptibility to a range of carcinogens, is the cytochrome P450 (CYP) superfamily. The CYP enzymes play an important role in the metabolism of many exogenous carcinogens such as polycyclic aromatic hydrocarbons, aromatic and heterocyclic amines and endogenous substrates such as steroids and eicosanoids ( 8 ). Expression of CYPs can be induced by their substrates and has been shown in many tissues, including liver, lung tissue, intestine, etc. ( 9–11 ).

CYP1A1 is a typical representative of the CYP450 superfamily, which is involved both in metabolism of carcinogens ( 12 ) and in catechol estrogen formation, predominantly catalyzing 2-hydroxyestradiol formation in extrahepatic tissue ( 13 ). Another well-known member of CYP family is CYP1B1, which activates environmental procarcinogens and catalyzes the conversion of 17β-estradiol to genotoxic catechol estrogens, such as 4-hydroxy-17β-estradiol. The latter is implicated in the development of hormone-related cancers ( 14 , 15 ). CYP3A isozymes are involved both in tobacco carcinogen metabolism and steroid metabolism and are expressed in human lung tissue showing variation in expression and activity between individuals ( 11 , 16 ). The cytochrome CYP2A13 is also active in the metabolism of many xenobiotic compounds and, particularly, the major tobacco-specific procarcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( 17 ).

Relevance of known genetic polymorphisms in CYP450 on the risk of lung cancer has been investigated intensively over the last several decades. Weak or modest associations with the risk of lung cancer were observed for some of the CYP genes and polymorphisms ( 18–21 ). However, only few studies were devoted to the effect of the CYP polymorphisms on the risk of early-onset lung cancer ( 22 , 23 ).

Therefore, we conducted a case–control study to investigate the effect of CYP genotypes on early-onset lung cancer risk and its modification by gender and smoking.

Material and methods

Study population

A case–control study was conducted ( 24 , 25 ). In total, 638 Caucasian patients under the age of 51 with confirmed primary lung cancer were included in the current study from two existing studies: Lung Cancer in the Young (LUCY) and young cases from a case–control study of lung cancer carried out at the German Cancer Research Center, Heidelberg, in collaboration with the Thoraxklinik, Heidelberg ( 26 ). The LUCY study is a multicenter ongoing study with 31 participating clinics all over Germany. Newly diagnosed patients under the age of 51 with histologically or cytologically confirmed primary lung cancer were included in the study. Overall, 472 LUCY patients, recruited between 2000 and 2004, were included in the present study. The Heidelberg Lung Cancer Study is an ongoing hospital-based case–control study in collaboration with the Thoraxklinik, Heidelberg, Germany ( 26 ). More than 1000 lung cancer patients of Caucasian origin with primary lung cancer were recruited between January 1997 and December 2003. Of these lung cancer cases, 166 had onset of disease before age 51 and were included in the present study. The 1300 controls were from the Kooperative Gesundheitsforschung in der Region Augsburg [KORA (Cooperative Health Research in the Augsburg Region)] survey S3, recruited in 1994/1995 and survey S4, recruited in 1999/2000. Controls were frequency matched by age at diagnosis/interview (≤40, 41–45 and 46–50 years) and sex. The KORA Study is a population-based epidemiological survey of individuals living in or near the city of Augsburg, South Germany ( 27 , 28 ). These cross-sectional health surveys were performed in a population aged 25–74 with German nationality. Epidemiological data on tobacco and occupational exposure, educational and family status have been collected and blood samples were taken. Data from the KORA, LUCY and Heidelberg lung case–control studies were merged to create a common database for the present study.

Cases and controls were classified as never smokers, current smokers and former smokers. A non-smoker was defined as someone who had never smoked or who had smoked <1 pack-year (PY) by the date of diagnosis for cases or by the date of interview for controls. A current smoker was defined as someone who still smokes or who had quit smoking <1 year before the date of diagnosis/interview. We considered an individual as a former smoker if he/she had smoked and had quit for at least 1 year at the time of diagnosis/interview. Cases and controls were grouped into four categories according to the smoking exposure level: never and light smokers (0–10 PY), moderate smokers (11–20 PY), heavy smokers (21–30 PY) and very heavy smokers (≥31 PY). For subgroup analysis, subjects were grouped into strong smokers (>20 PY) and light and never smokers (≤20 PY) ( Table I ). Information about passive smoking was not available for all study subjects and therefore not considered in the analysis.

Table I.

Characteristics of the study population

  All cases ( N  = 638)
 
Cases from the Heidelberg Lung Cancer Study ( N  = 166)
 
Cases from the LUCY study ( N  = 472)
 
Controls ( N  = 1300)
 
Chi-square test
 
 n n n n P (between all cases and controls)  
Gender          
    Men 406 63.6 96 57.8 310 65.7 818 62.9  
    Women 232 36.4 70 42.2 162 34.3 482 37.1 0.7597 
   aP  = 0.0707     
Age          
    ≤40 91 14.3 23 13.9 68 14.4 199 15.3  
    41–45 199 31.2 49 29.5 150 31.8 395 30.4  
    46–50 348 54.5 94 56.6 254 53.8 706 54.3 0.8158 
   aP  = 0.8164     
Smoking status b          
    Non-smoker 43 6.8 16 9.6 27 5.7 450 34.6  
    Current 481 75.9 128 77.1 353 74.8 472 36.3  
    Former 110 17.3 22 13.3 88 18.6 377 29.0 <0.0001 
   aP  = 0.1051     
PYs c          
    ≤10 80 12.8 23 14.8 57 12.2 712 56.1  
    11–20 94 15.1 13 8.4 81 17.3 178 14.0  
    21–30 177 28.4 33 21.3 144 30.8 188 14.8  
    ≥31 272 43.7 86 55.5 186 39.7 192 15.1 <0.0001 
   aP  = 0.0007     
Histology          
    SCLC 153 24.0 30 18.1 123 26.1 —   
    NSCLC 443 69.4 130 81.3 313 71.8 —   
        AC 224 35.1 72 43.4 152 32.2 —   
        SCC 150 23.5 45 27.1 105 22.3 —   
        LCLC 22 3.4 10 6.0 12 2.5 —   
        Other NSCLC 47 7.4 1.8 44 9.3 —   
    Other lung cancer histology 42 6.6 3.6 36 7.6 —   
   aP  = 0.0005     
  All cases ( N  = 638)
 
Cases from the Heidelberg Lung Cancer Study ( N  = 166)
 
Cases from the LUCY study ( N  = 472)
 
Controls ( N  = 1300)
 
Chi-square test
 
 n n n n P (between all cases and controls)  
Gender          
    Men 406 63.6 96 57.8 310 65.7 818 62.9  
    Women 232 36.4 70 42.2 162 34.3 482 37.1 0.7597 
   aP  = 0.0707     
Age          
    ≤40 91 14.3 23 13.9 68 14.4 199 15.3  
    41–45 199 31.2 49 29.5 150 31.8 395 30.4  
    46–50 348 54.5 94 56.6 254 53.8 706 54.3 0.8158 
   aP  = 0.8164     
Smoking status b          
    Non-smoker 43 6.8 16 9.6 27 5.7 450 34.6  
    Current 481 75.9 128 77.1 353 74.8 472 36.3  
    Former 110 17.3 22 13.3 88 18.6 377 29.0 <0.0001 
   aP  = 0.1051     
PYs c          
    ≤10 80 12.8 23 14.8 57 12.2 712 56.1  
    11–20 94 15.1 13 8.4 81 17.3 178 14.0  
    21–30 177 28.4 33 21.3 144 30.8 188 14.8  
    ≥31 272 43.7 86 55.5 186 39.7 192 15.1 <0.0001 
   aP  = 0.0007     
Histology          
    SCLC 153 24.0 30 18.1 123 26.1 —   
    NSCLC 443 69.4 130 81.3 313 71.8 —   
        AC 224 35.1 72 43.4 152 32.2 —   
        SCC 150 23.5 45 27.1 105 22.3 —   
        LCLC 22 3.4 10 6.0 12 2.5 —   
        Other NSCLC 47 7.4 1.8 44 9.3 —   
    Other lung cancer histology 42 6.6 3.6 36 7.6 —   
   aP  = 0.0005     

AC, adenocarcinoma; LCLC, large cell lung carcinoma; SCC, squamous cell carcinoma.

a

P -value for chi-square test comparing the Heidelberg Lung Cancer Study and LUCY Study.

b

Data on smoking status are missing for four cases and one control.

c

Data on PYs are missing for 15 cases and 30 controls.

Histological subtypes of cancer were grouped into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Histology types different from NSCLC or SCLC, e.g. mixed type of cancer, were combined into the group ‘other types of lung cancer’. The NSCLC group consisted of patients with adenocarcinoma, squamous cell carcinoma, large cell lung carcinoma and other NSCLC histologies. The latter includes cases with adenosquamous carcinoma and cases for whom the particular NSCLC histology subtypes were not obtainable ( Table I ).

Informed consent was obtained from all study participants and the studies were approved by the ethics committee of the Bayerische Landesärztekammer, the corresponding local ethics committees of the participating clinics and the ethics committee of the University of Heidelberg (Ref. Nr. 182/96, 201/98 and 199/2001).

Single-nucleotide polymorphism selection and genotyping methods

DNA was extracted from ethylenediaminetetraacetic acid anticoagulated blood using a commercially available kit (Gentra, Minneapolis, MN) according to the manufacturer's protocol. For the Heidelberg Lung Cancer Study, buffy coats from 5 ml of venous blood in ethylenediaminetetraacetic acid were stored at −80°C, and human genomic DNA was isolated using the QIAamp DNA blood midi kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany).

Polymorphisms for the genotyping were selected based on a known or expected functional effect. Additionally, several tagging single-nucleotide polymorphisms (SNPs) (rs10916 and rs1709084) were added to the analysis if they fulfilled the following criteria: pairwise tagging of the HapMap Population of Utah residents with Northern and Western European ancestry (CEU) with r2 ≥0.8 (Data Rel 20/phase II) ( 29 ) and a minor allele frequency ≥5%. Finally, the following genes and SNPs were selected for the analysis: CYP1A1 —rs1048943, rs1799814 and rs2606345; CYP1B1 —rs10916, rs1800440, rs1056836, rs1056827 and rs2567206; CYP2A13 —rs8192784, rs8192789 and rs1709084; CYP3A4 —rs2740574 and CYP3A5 —rs776746 ( supplementary Table 1 is available at Carcinogenesis Online).

Genotyping of the CYP3A4 rs2740574, CYP3A5 rs776746 and CYP1B1 rs1056836 polymorphisms was carried out using a fluorescence-based melting curve analysis method on the LightCycler 480 (rs2740574 and rs776746) or LightTyper (rs1056836) (Roche, Mannheim, Germany). We modified the previously described melting curve analysis methods for detection of the CYP3A4 rs2740574 and CYP3A5 rs776746 polymorphisms ( 26 ) by performing them in a multiplex reaction in 96-well plates on the LightCycler 480 (Roche). The modified protocol for genotyping CYP3A4 rs2740574 and CYP3A5 rs776746 polymorphisms, as well as new developed protocol for genotyping CYP1B1 rs1056836 polymorphism on the LightCycler 480 is presented in the supplementary Methods (available at Carcinogenesis Online).

Genotyping of other CYP1B1 , CYP1A1 and CYP3A12 SNPs was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Mass Array; Sequenom, San Diego, CA) to detect allele-specific primer extension products as described elsewhere ( 30 ).

The primer sequences, length of the amplification products and melting temperature are presented in supplementary Table 2 (available at Carcinogenesis Online). The allele with the highest frequency in the control group was considered as the major allele. A random selection of 10% of all samples was genotyped twice for quality control with 99.7% concordance.

Statistical methods

The chi-square test was used to compare the distribution of potential risk factors between cases and controls and to assess deviation from Hardy–Weinberg equilibrium (HWE) in the control group. Power calculations were carried out using QUANTO version 1.2 for the main effect of genes and dominant model of inheritance for whole sample set ( 31 ).

Multivariate conditional logistic regression analysis was applied to assess odds ratios (ORs) and their 95% confidence intervals (CIs) using the LOGISTIC procedure of the statistical software package SAS (SAS Institute, Cary, NC). Dominant, codominant and recessive, when possible, modes of inheritance were considered. The analyses were stratified by 5 year age–gender groups and additionally adjusted for PY of smoking (0–10, 11–20, 21–30 and >30 PY). For gender subgroup analysis, the model was stratified by 5 year age groups only (≤40, 41–45, 46–50 years). Effect modification was assessed by testing for multiplicative interaction. An interaction term of the genotypes of interest and potential interaction variables (gender and smoking) was included in a fully adjusted model and statistical significance was evaluated with the likelihood ratio test.

A generalized linear model framework was applied to test for haplotype–trait association, adjusted for age, gender and smoking and to calculate ORs and 95% CI. Haplotype analysis was performed using the software package Haplo.stats in R.

The nominal and reported significance level is set to α  = 0.05. However, the Bonferroni correction for multiple testing was also applied. If a result is also significant after this correction, it is indicated as such.

Results

Selected baseline characteristics of the 638 early-onset lung cancer patients and 1300 controls are summarized in Table I . As cases were selected from two independent studies, LUCY and the Heidelberg Lung Cancer study, distribution of gender, age, smoking variables and histology is presented separately for the two study groups. The Heidelberg Lung Cancer Study had a higher proportion of women (42.2%) compared with the LUCY study (34.3%), but the differences were not statistically significant ( P  = 0.07). The distribution of 5 year age groups ( P  = 0.82) and smokers ( P  = 0.11) were similar in both studies ( Table I ). Statistically significant differences were observed in PY distribution ( P  < 0.001) and histology distribution ( P  < 0.001) between these two groups. These might be explained by the fact that the Heidelberg Lung Cancer Study included predominantly surgery patients in the early phase of sampling, and thus collected mainly cases with NSCLC, which is often treated surgically in early stages. For further analysis, the LUCY and Heidelberg Lung Cancer Study groups were combined into one case group. The mean age of the cancer patients was 45.1 (SD, ±4.3), and the mean age of control individuals was 45.0 (SD, ±4.4). Cases were more likely than controls to be current smokers. The mean amount of PYs was 31.4 (SD, ±22.4) among cases and 11.2 (SD, ±16.2) among controls.

Frequencies of the analyzed polymorphisms in the study populations are given in supplementary Table 1 (available at Carcinogenesis Online). No statistically significant differences in genotype distributions were observed between LUCY and the Heidelberg Lung Cancer Study. Among cases and controls, all SNPs were in HWE, except for CYP1A1 rs1048943 ( P  = 0.01) and CYP1B1 rs2567206 ( P  = 0.02) in the control group. The possible effect of CYP1A1 , CYP1B1 , CYP3A4 , CYP3A5 and CYP2A13 genotypes on early-onset lung cancer risk was analyzed separately for each SNP. No significant association was found for any of the analyzed polymorphisms and lung cancer risk overall ( Table II ).

Table II.

ORs for the risk of lung cancer overall and in women and men separately associated with the polymorphisms in CYP genes

SNP  All
 
Men
 
Women
 
 Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P 
CYP1A1 rs1048943          
    AA 578/1175 1 (ref)  369/732 1 (ref)  209/443 1 (ref)  
    AG + GG 41/89 0.96 (0.63–1.48) 0.8649 28/52 1.19 (0.69–2.30) 0.5371 13/37 0.68 (0.33–1.40) 0.2921 
CYP1A1 rs1799814          
    CC 549/1159 1 (ref)  349/726 1 (ref)  200/433 1 (ref)  
    CA + AA 44/96 1.11 (0.73–1.69) 0.6133 28/57 1.13 (0.67–1.92) 0.6434 16/39 1.10 (0.56–2.19) 0.7790 
CYP1A1 rs 2606345          
    AA 260/585 1 (ref)  173/383 1 (ref)  87/202   
    AC 284/545 1.25 (0.99–1.57) 0.0625 176/321 1.30 (0.98–1.74) 0.0691 108/224 1.15 (0.78–1.70) 0.4821 
    CC 61/117 1.14 (0.77–1.67) 0.5146 37/72 1.06 (0.66–1.72) 0.8007 24/45 1.28 (0.68–2.41) 0.4507 
    AC + CC 345/662 1.22 (0.98–1.53) 0.0711 213/393 1.26 (0.96–1.65) 0.1009 132/269 1.17 (0.81–1.70) 0.4056 
    CC versus AC + AA  1.02 (0.71–1.47) 0.9195  0.94 (0.59–1.49) 0.7857  1.19 (0.65–2.16) 0.5785 
CYP3A4 rs2740574          
    AA 570/1165 1 (ref)  366/730 1 (ref)  204/435 1 (ref)  
    AG + GG 44/85 1.12 (0.73–1.73) 0.5925 28/48 1.28 (0.74–2.22) 0.3800 16/37 0.93 (0.47–1.87) 0.8451 
CYP3A5 rs776746          
    GG 542/1100 1 (ref)  342/692 1 (ref)  200/408 1 (ref)  
    AG + AA 71/152 1.08 (0.77–1.52) 0.6447 50/86 1.28 (0.84–1.95) 0.2428 21/66 0.79 (0.44–1.42) 0.4348 
CYP1B1 rs10916          
    TT 360/762 1 (ref) 0.2447 241/487 1 (ref)  119/275 1 (ref)  
    TG 219/413 1.15 (0.91–1.45) 0.8727 131/252 1.04 (0.78–1.39) 0.7932 88/161 1.36 (0.92–2.00) 0.7155 
    GG 33/73 1.04 (0.64–1.69) 0.2717 21/38 1.19 (0.63–2.24) 0.5873 12/35 0.87 (0.40–1.89) 0.1218 
    TG + GG 252/486 1.13 (0.91–1.41) 0.9648 152/290 1.06 (0.80–1.40) 0.6919 100/196 1.27 (0.88–1.84) 0.2051 
    GG versus TT + TG  0.99 (0.61–1.60)   1.18 (0.63–2.19) 0.6120  0.77 (0.36–1.65) 0.4973 
CYP1B1 rs1800440          
    AA 409/845 1 (ref)  249/515 1 (ref)  160/330 1 (ref)  
    AG 184/376 1.01 (0.79–1.28) 0.9552 131/243 1.15 (0.86–1.54) 0.3487 53/133 0.78 (0.51–1.19) 0.2417 
    GG 24/47 1.06 (0.60–1.86) 0.8491 15/30 0.96 (0.48–1.92) 0.9051 9/17 1.32 (0.50–3.43) 0.5754 
    AG + GG 208/423 1.01 (0.81–1.27) 0.9155 146/273 1.13 (0.85–1.49) 0.4062 62/150 0.83 (0.56–1.24) 0.3609 
    GG versus AA + AG  1.05 (0.60–1.84) 0.8536  0.92 (0.46–1.82) 0.8028  1.41 (0.54–3.64) 0.4820 
CYP1B1 rs1056836          
    CC 183/424 1 (ref)  128/243 1 (ref)  55/181 1 (ref)  
    CG 303/604 1.06 (0.83–1.36) 0.6280 179/391 0.74 (0.55–1.02) 0.0631 124/213 2.00 ( 1.313.06) 0.0014
    GG 129/234 1.23 (0.90–1.68) 0.1884 83/149 0.95 (0.65–1.40) 0.7984 46/85 1.90 ( 1.113.24) 0.0189 
    CG + GG 432/838 1.11 (0.88–1.40) 0.3823 262/540 0.80 (0.60–1.07) 0.1354 170/298 1.97 ( 1.322.94) 0.0009
    GG versus CC + CG  1.19 (0.90–1.56) 0.2167  1.14 (0.81–1.59) 0.4509  1.25 (0.79–1.99) 0.3404 
CYP1B1 rs1056827          
    GG 300/613 1 (ref)  192/382 1 (ref)  108/231 1 (ref)  
    TG 238/543 0.84 (0.67–1.06) 0.1330 155/339 0.88 (0.66–1.17) 0.3781 83/204 0.76 (0.51–1.12) 0.1695 
    TT 62/99 1.22 (0.82–1.80) 0.3250 38/63 1.07 (0.66–1.74) 0.7911 24/36 1.52 (0.79–2.93) 0.2052 
    TG + TT 300/642 0.90 (0.72–1.11) 0.3215 193/402 0.91 (0.69–1.20) 0.4981 107/240 0.86 (0.59–1.25) 0.4247 
    TT versus GG + TG  1.32 (0.91–1.97) 0.1451  1.13 (0.71–1.81) 0.5980  1.73 (0.93–3.24) 0.0852 
CYP1B1 rs2567206          
    GG 316/616 1 (ref)  204/388 1 (ref)  112/228 1 (ref)  
    GA 241/556 0.78 (0.62–0.98) 0.0348 155/339 0.84 (0.63–1.11) 0.2185 86/217 0.69 (0.47–1.01) 0.0582 
    AA 59/95 1.19 (0.80–1.78) 0.3835 36/60 1.04 (0.63–1.72) 0.8700 23/35 1.51 (0.78–2.92) 0.2276 
    GA + AA 300/651 0.84 (0.68–1.04) 0.1145 191/399 0.87 (0.66–1.14) 0.3068 109/252 0.78 (0.54–1.13) 0.1924 
    AA versus GA + GG  1.34 (0.91–1.97) 0.1383  1.13 (0.70–1.83) 0.6177  1.79 (0.95–3.40) 0.0727 
CYP2A13 rs8192784          
    GG 603/1237 1 (ref)  385/772 1 (ref)  218/465 1 (ref)  
    GA + AA 17/31 0.95 (0.49–1.84) 0.8730 12/16 1.37 (0.58–3.20) 0.4718 5/15 0.52 (0.17–1.61) 0.2599 
CYP2A13 rs8192789          
    CC 594/1224 1 (ref)  378/761 1 (ref)  216/463 1 (ref)  
    CT + TT 20/33 1.04 (0.55–1.96) 0.9019 14/17 1.48 (0.66–3.36) 0.3436 6/16 0.59 (0.20–1.70) 0.3266 
CYP2A13 rs1709084          
    AA 515/1071 1 (ref)  335/651 1 (ref)  180/420 1 (ref)  
    AG + GG 100/184 1.16 (0.86–1.56) 0.3350 59/126 0.95 (0.66–1.39) 0.8006 41/58 1.64 ( 1.002.70) 0.0515 
SNP  All
 
Men
 
Women
 
 Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P 
CYP1A1 rs1048943          
    AA 578/1175 1 (ref)  369/732 1 (ref)  209/443 1 (ref)  
    AG + GG 41/89 0.96 (0.63–1.48) 0.8649 28/52 1.19 (0.69–2.30) 0.5371 13/37 0.68 (0.33–1.40) 0.2921 
CYP1A1 rs1799814          
    CC 549/1159 1 (ref)  349/726 1 (ref)  200/433 1 (ref)  
    CA + AA 44/96 1.11 (0.73–1.69) 0.6133 28/57 1.13 (0.67–1.92) 0.6434 16/39 1.10 (0.56–2.19) 0.7790 
CYP1A1 rs 2606345          
    AA 260/585 1 (ref)  173/383 1 (ref)  87/202   
    AC 284/545 1.25 (0.99–1.57) 0.0625 176/321 1.30 (0.98–1.74) 0.0691 108/224 1.15 (0.78–1.70) 0.4821 
    CC 61/117 1.14 (0.77–1.67) 0.5146 37/72 1.06 (0.66–1.72) 0.8007 24/45 1.28 (0.68–2.41) 0.4507 
    AC + CC 345/662 1.22 (0.98–1.53) 0.0711 213/393 1.26 (0.96–1.65) 0.1009 132/269 1.17 (0.81–1.70) 0.4056 
    CC versus AC + AA  1.02 (0.71–1.47) 0.9195  0.94 (0.59–1.49) 0.7857  1.19 (0.65–2.16) 0.5785 
CYP3A4 rs2740574          
    AA 570/1165 1 (ref)  366/730 1 (ref)  204/435 1 (ref)  
    AG + GG 44/85 1.12 (0.73–1.73) 0.5925 28/48 1.28 (0.74–2.22) 0.3800 16/37 0.93 (0.47–1.87) 0.8451 
CYP3A5 rs776746          
    GG 542/1100 1 (ref)  342/692 1 (ref)  200/408 1 (ref)  
    AG + AA 71/152 1.08 (0.77–1.52) 0.6447 50/86 1.28 (0.84–1.95) 0.2428 21/66 0.79 (0.44–1.42) 0.4348 
CYP1B1 rs10916          
    TT 360/762 1 (ref) 0.2447 241/487 1 (ref)  119/275 1 (ref)  
    TG 219/413 1.15 (0.91–1.45) 0.8727 131/252 1.04 (0.78–1.39) 0.7932 88/161 1.36 (0.92–2.00) 0.7155 
    GG 33/73 1.04 (0.64–1.69) 0.2717 21/38 1.19 (0.63–2.24) 0.5873 12/35 0.87 (0.40–1.89) 0.1218 
    TG + GG 252/486 1.13 (0.91–1.41) 0.9648 152/290 1.06 (0.80–1.40) 0.6919 100/196 1.27 (0.88–1.84) 0.2051 
    GG versus TT + TG  0.99 (0.61–1.60)   1.18 (0.63–2.19) 0.6120  0.77 (0.36–1.65) 0.4973 
CYP1B1 rs1800440          
    AA 409/845 1 (ref)  249/515 1 (ref)  160/330 1 (ref)  
    AG 184/376 1.01 (0.79–1.28) 0.9552 131/243 1.15 (0.86–1.54) 0.3487 53/133 0.78 (0.51–1.19) 0.2417 
    GG 24/47 1.06 (0.60–1.86) 0.8491 15/30 0.96 (0.48–1.92) 0.9051 9/17 1.32 (0.50–3.43) 0.5754 
    AG + GG 208/423 1.01 (0.81–1.27) 0.9155 146/273 1.13 (0.85–1.49) 0.4062 62/150 0.83 (0.56–1.24) 0.3609 
    GG versus AA + AG  1.05 (0.60–1.84) 0.8536  0.92 (0.46–1.82) 0.8028  1.41 (0.54–3.64) 0.4820 
CYP1B1 rs1056836          
    CC 183/424 1 (ref)  128/243 1 (ref)  55/181 1 (ref)  
    CG 303/604 1.06 (0.83–1.36) 0.6280 179/391 0.74 (0.55–1.02) 0.0631 124/213 2.00 ( 1.313.06) 0.0014
    GG 129/234 1.23 (0.90–1.68) 0.1884 83/149 0.95 (0.65–1.40) 0.7984 46/85 1.90 ( 1.113.24) 0.0189 
    CG + GG 432/838 1.11 (0.88–1.40) 0.3823 262/540 0.80 (0.60–1.07) 0.1354 170/298 1.97 ( 1.322.94) 0.0009
    GG versus CC + CG  1.19 (0.90–1.56) 0.2167  1.14 (0.81–1.59) 0.4509  1.25 (0.79–1.99) 0.3404 
CYP1B1 rs1056827          
    GG 300/613 1 (ref)  192/382 1 (ref)  108/231 1 (ref)  
    TG 238/543 0.84 (0.67–1.06) 0.1330 155/339 0.88 (0.66–1.17) 0.3781 83/204 0.76 (0.51–1.12) 0.1695 
    TT 62/99 1.22 (0.82–1.80) 0.3250 38/63 1.07 (0.66–1.74) 0.7911 24/36 1.52 (0.79–2.93) 0.2052 
    TG + TT 300/642 0.90 (0.72–1.11) 0.3215 193/402 0.91 (0.69–1.20) 0.4981 107/240 0.86 (0.59–1.25) 0.4247 
    TT versus GG + TG  1.32 (0.91–1.97) 0.1451  1.13 (0.71–1.81) 0.5980  1.73 (0.93–3.24) 0.0852 
CYP1B1 rs2567206          
    GG 316/616 1 (ref)  204/388 1 (ref)  112/228 1 (ref)  
    GA 241/556 0.78 (0.62–0.98) 0.0348 155/339 0.84 (0.63–1.11) 0.2185 86/217 0.69 (0.47–1.01) 0.0582 
    AA 59/95 1.19 (0.80–1.78) 0.3835 36/60 1.04 (0.63–1.72) 0.8700 23/35 1.51 (0.78–2.92) 0.2276 
    GA + AA 300/651 0.84 (0.68–1.04) 0.1145 191/399 0.87 (0.66–1.14) 0.3068 109/252 0.78 (0.54–1.13) 0.1924 
    AA versus GA + GG  1.34 (0.91–1.97) 0.1383  1.13 (0.70–1.83) 0.6177  1.79 (0.95–3.40) 0.0727 
CYP2A13 rs8192784          
    GG 603/1237 1 (ref)  385/772 1 (ref)  218/465 1 (ref)  
    GA + AA 17/31 0.95 (0.49–1.84) 0.8730 12/16 1.37 (0.58–3.20) 0.4718 5/15 0.52 (0.17–1.61) 0.2599 
CYP2A13 rs8192789          
    CC 594/1224 1 (ref)  378/761 1 (ref)  216/463 1 (ref)  
    CT + TT 20/33 1.04 (0.55–1.96) 0.9019 14/17 1.48 (0.66–3.36) 0.3436 6/16 0.59 (0.20–1.70) 0.3266 
CYP2A13 rs1709084          
    AA 515/1071 1 (ref)  335/651 1 (ref)  180/420 1 (ref)  
    AG + GG 100/184 1.16 (0.86–1.56) 0.3350 59/126 0.95 (0.66–1.39) 0.8006 41/58 1.64 ( 1.002.70) 0.0515 
a

P -value is significant after Bonferroni correction for 26 tests (Bonferroni adjusted level of significance α  = 0.0019).

However, a subgroup analysis separating men and women showed a significantly increased risk of early-onset lung cancer overall for carriers of the minor allele of CYP1B1 SNP rs1056836 among women (OR 1.97; 95% CI 1.32–2.94; P  < 0.001), but not among men (OR 0.80; 95% CI 0.60–1.07; P  = 0.14) ( Table II ). The interaction between gender and CYP1B1 rs1056836 was statistically significant ( Pinteraction  < 0.0001). The effect of this polymorphism in women was persistent and significant in all histological subtypes (data not presented). The effect of CYP1B1 rs1056836 on the risk of lung cancer in women was shown to be modified by smoking ( Pinteraction  = 0.03). Women carriers of the minor allele who smoked >20 PY had a significantly increased risk of early-onset lung cancer (OR 2.69; 95% CI 1.49–4.84; P  = 0.001). This risk-rising effect was not identified in the group of women who smoked ≤20 PY (OR 1.35; 95% CI 0.82–2.21; P  = 0.24) ( Table III ). The gene–smoking interaction was not observed in the group of men ( Table III ).

Table III.

ORs for the risk of lung cancer associated with the polymorphisms in CYP genes, stratified by gender and smoking

SNP  Men
 
Women
 
  Non-smoker and light smoker (≤20 PY)
 
Strong smoker (≥21 PY)
 
Non-smoker and light smoker (≤20 PY)
 
Strong smoker (≥21 PY)
 
 Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P 
CYP1A1 rs 1048943 
    AA 75/461 1 (ref)  294/271 1 (ref)  85/360 1 (ref)  124/83 1 (ref)  
    AG + GG 9/34 1.79 (0.82–3.92) 0.1442 19/18 0.93 (0.48–1.81) 0.8291 3/31 0.42 (0.13–1.41) 0.1593 10/6 1.11 (0.39–3.16) 0.8388 
CYP1A1 rs1799814             
    CC 74/455 1 (ref)  275/271 1 (ref)  75/355 1 (ref)  125/78 1 (ref)  
    CA + AA 5/39 0.77 (0.29–2.03) 0.5951 23/18 1.27 (0.67–2.40) 0.4666 9/30 1.41 (0.64–3.09) 0.3920 7/9 0.49 (0.18–1.36) 0.1701 
CYP1A1 rs2606345             
    AA 36/236 1 (ref)   1 (ref)   1 (ref)  54/38 1 (ref)  
    AC 41/209 1.30 (0.80–2.12) 0.2893 137/147 1.26 (0.72–2.18) 0.1598 33/164 1.12 (0.68–1.85) 0.6672 67/40 1.17 (0.67–2.07) 0.5806 
    CC 4/44 0.63 (0.21–1.85) 0.3987 135/112 1.28 (0.91–1.81) 0.4222 41/184 1.68 (0.79–3.59) 0.1803 12/10 0.84 (0.33–2.13) 0.7103 
    AC + CC 45/253 1.19 (0.74–1.91) 0.4784 33/28 1.28 (0.92–1.76) 0.1425 12/35 1.21 (0.75–1.95) 0.4432 79/50 1.11 (0.64–1.90) 0.7158 
    CC versus AA + AC  0.55 (0.19–1.58) 0.2651 168/14 1.12 (0.66–1.91) 0.6728 53/219 1.58 (0.78–3.21) 0.2027  0.77 (0.32–1.86) 0.5627 
CYP3A4 rs2740574             
    AA 79/456 1 (ref)  287/274 1 (ref)  81/354 1 (ref)  123/81 1 (ref)  
    AG + GG 4/34 0.71 (0.25–2.08) 0.5375 24/14 1.67 (0.85–3.30) 0.1399 6/31 0.84 (0.34–2.07) 0.6986 10/6 1.10 (0.39–3.11) 0.8652 
CYP3A5 rs776746             
    GG 73/432 1 (ref)  269/260 1 (ref)  78/330 1 (ref)  122/78 1 (ref)  
    AG + AA 10/59 1.01 (0.49–2.07) 0.9824 40/27 1.46 (0.87–2.45) 0.1519 10/57 0.75 (0.37–1.54) 0.4363 11/9 0.78 (0.31–1.95) 0.5926 
CYP1B1 rs10916             
    TT 47/308 1 (ref)  194/179 1 (ref)  48/220 1 (ref)  71/55 1 (ref)  
    TG 33/155 1.42 (0.87–2.31) 0.1605 98/97 0.92 (0.65–1.31) 0.6485 33/136 1.11 (0.68–1.82) 0.6765 55/25 1.73 (0.96–3.12) 0.0694 
    GG 4/26 1.02 (0.34–3.06) 0.9772 17/12 1.23 (0.57–2.66) 0.6001 7/28 1.13 (0.47–2.75) 0.7805 5/7 0.55 (0.17–1.81) 0.3243 
    TG + GG 37/181 1.36 (0.85–2.18) 0.2006 115/109 0.96 (0.69–1.33) 0.7916 40/164 1.12 (0.70–1.78) 0.6478 60/32 1.46 (0.84–2.55) 0.1783 
    GG versus TT + TG  0.89 (0.30–2.65) 0.8394  1.27 (0.59–2.71) 0.5454  1.09 (0.46–2.58) 0.8466  0.45 (0.14–1.46) 0.1831 
CYP1B1 rs1800440             
    AA 52/322 1 (ref)  197/193 1 (ref)  61/273 1 (ref)  99/57 1 (ref)  
    AG 29/157 1.18 (0.72–1.94) 0.5045 102/86 1.19 (0.84–1.69) 0.3246 22/106 0.94 (0.55–1.61) 0.8309 31/27 0.67 (0.36–1.22) 0.1885 
    GG 3/18 1.18 (0.33–4.18) 0.7974 12/12 1.00 (0.43–2.26) 0.9811 4/13 1.36 (0.43–4.31) 0.6011 5/4 0.73 (0.19–2.84) 0.6490 
    AG + GG 32/175 1.18 (0.73–1.92) 0.4921 114/98 1.17 (0.83–1.63) 0.3672 26/119 0.99 (0.60–1.64) 0.9688 36/31 0.67 (0.38–1.20) 0.1805 
    GG versus AA + AG  1.11 (0.32–3.89) 0.8681  0.94 (0.41–2.11) 0.8716  1.38 (0.44–4.34) 0.5797  0.82 (0.21–3.15) 0.7732 
CYP1B1 rs1056836             
    CC 32/163 1 (ref)  96/80 1 (ref)  27/144 1 (ref)  28/37 1 (ref)  
    CG 35/242 0.70 (0.41–1.18) 0.1747 144/149 0.81 (0.56–1.19) 0.2831 42/173 1.29 (0.76–2.20) 0.3416 82/40 2.69 ( 1.454.97) 0.0017 
    GG 17/91 1.01 (0.53–1.93) 0.9761 66/58 0.97 (0.61–1.54) 0.8899 21/73 1.47 (0.77–2.78) 0.2416 25/12 2.70 ( 1.156.36) 0.0227 
    CG + GG 52/333 0.78 (0.48–1.26) 0.3040 210/207 0.86 (0.60–1.22) 0.3937 63/246 1.35 (0.82–2.21) 0.2406 107/52 2.69 ( 1.494.84) 0.0010
    GG versus CC + CG  1.24 (0.69–2.22) 0.4797  1.10 (0.74–1.64) 0.6355  1.26 (0.72–2.20) 0.4100  1.47 (0.68–3.15) 0.3287 
CYP1B1 rs1056827             
    GG 42/246 1 (ref)  150/136 1 (ref)  49/187 1 (ref)  59/44 1 (ref)  
    TG 32/216 0.86 (0.53–1.42) 0.5657 123/123 0.90 (0.64–1.27) 0.5445 26/164 0.60 (0.36–1.01) 0.0533 57/40 1.08 (0.61–1.89) 0.7972 
    TT 7/34 1.31 (0.54–3.17) 0.5513 31/29 0.97 (0.55–1.70) 0.9090 9/33 1.08 (0.49–2.42) 0.8450 15/3 4.05 ( 1.0715.36) 0.0400 
    TG + TT 39/250 0.92 (0.57–1.48) 0.7326 154/152 0.91 (0.66–1.26) 0.5796 35/197 0.68 (0.42–1.09) 0.1102 72/43 1.25 (0.73–2.17) 0.4178 
    TT versus TG + TT  1.40 (0.59–3.29) 0.4445  1.02 (0.60–1.74) 0.9512  1.33 (0.61–2.90) 0.4732  3.89 ( 1.0614.32) 0.0406 
CYP1B1 rs2567206             
    GG 42/250 1 (ref)  162/138 1 (ref)  50/185 1 (ref)  62/43 1 (ref)  
    GA 34/213 0.95 (0.58–1.56) 0.8513 121/126 0.81 (0.57–1.13) 0.2083 28/173 0.59 ( 0.360.98) 0.0415 58/44 0.92 (0.53–1.61) 0.7801 
    AA 7/33 1.34 (0.55–3.24) 0.5212 29/27 0.90 (0.51–1.60) 0.7288 9/33 1.03 (0.46–2.29) 0.9432 14/2 5.06 ( 1.0723.94) 0.0408 
    GA + AA 41/246 1.00 (0.63–1.60) 0.9890 150/153 0.82 (0.60–1.13) 0.2317 37/206 0.66 (0.41–1.05) 0.0815 72/46 1.07 (0.63–1.87) 0.7658 
    AA versus GG + GA  1.37 (0.58–3.22) 0.4770  1.00 (0.57–1.73) 0.9940  1.28 (0.59–2.79) 0.5296  5.28 ( 1.1524.28) 0.0327 
CYP2A13 rs8192784             
    GG 80/490 1 (ref)  305/282 1 (ref)  87/381 1 (ref)  131/84 1 (ref)  
    GA + AA 4/7 3.84 ( 1.0913.53) 0.0361 8/9 0.80 (0.30–2.10) 0.6463 2/11 0.87 (0.19–4.03) 0.8623 3/4 0.48 (0.10–2.19) 0.3402 
CYP2A13 rs8192789             
    CC 80/487 1 (ref)  298/274 1 (ref)  86/379 1 (ref)  130/84 1 (ref)  
    CT + TT 4/8 3.40 (0.99–11.64) 0.0515 10/9 0.98 (0.39–2.46) 0.9720 2/12 0.82 (0.18–3.73) 0.7920 4/4 0.63 (0.15–2.60) 0.5245 
CYP2A13 rs1709084             
    AA 73/413 1 (ref)  262/238 1 (ref)  66/347 1 (ref)  114/73 1 (ref)  
    AG + GG 11/81 0.74 (0.38–1.47) 0.3938 48/45 0.99 (0.63–1.54) 0.9481 22/43 2.72 ( 1.534.85) 0.000719/15 0.82 (0.39–1.70) 0.5892 
SNP  Men
 
Women
 
  Non-smoker and light smoker (≤20 PY)
 
Strong smoker (≥21 PY)
 
Non-smoker and light smoker (≤20 PY)
 
Strong smoker (≥21 PY)
 
 Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P Cases/controls OR (95% CI) P 
CYP1A1 rs 1048943 
    AA 75/461 1 (ref)  294/271 1 (ref)  85/360 1 (ref)  124/83 1 (ref)  
    AG + GG 9/34 1.79 (0.82–3.92) 0.1442 19/18 0.93 (0.48–1.81) 0.8291 3/31 0.42 (0.13–1.41) 0.1593 10/6 1.11 (0.39–3.16) 0.8388 
CYP1A1 rs1799814             
    CC 74/455 1 (ref)  275/271 1 (ref)  75/355 1 (ref)  125/78 1 (ref)  
    CA + AA 5/39 0.77 (0.29–2.03) 0.5951 23/18 1.27 (0.67–2.40) 0.4666 9/30 1.41 (0.64–3.09) 0.3920 7/9 0.49 (0.18–1.36) 0.1701 
CYP1A1 rs2606345             
    AA 36/236 1 (ref)   1 (ref)   1 (ref)  54/38 1 (ref)  
    AC 41/209 1.30 (0.80–2.12) 0.2893 137/147 1.26 (0.72–2.18) 0.1598 33/164 1.12 (0.68–1.85) 0.6672 67/40 1.17 (0.67–2.07) 0.5806 
    CC 4/44 0.63 (0.21–1.85) 0.3987 135/112 1.28 (0.91–1.81) 0.4222 41/184 1.68 (0.79–3.59) 0.1803 12/10 0.84 (0.33–2.13) 0.7103 
    AC + CC 45/253 1.19 (0.74–1.91) 0.4784 33/28 1.28 (0.92–1.76) 0.1425 12/35 1.21 (0.75–1.95) 0.4432 79/50 1.11 (0.64–1.90) 0.7158 
    CC versus AA + AC  0.55 (0.19–1.58) 0.2651 168/14 1.12 (0.66–1.91) 0.6728 53/219 1.58 (0.78–3.21) 0.2027  0.77 (0.32–1.86) 0.5627 
CYP3A4 rs2740574             
    AA 79/456 1 (ref)  287/274 1 (ref)  81/354 1 (ref)  123/81 1 (ref)  
    AG + GG 4/34 0.71 (0.25–2.08) 0.5375 24/14 1.67 (0.85–3.30) 0.1399 6/31 0.84 (0.34–2.07) 0.6986 10/6 1.10 (0.39–3.11) 0.8652 
CYP3A5 rs776746             
    GG 73/432 1 (ref)  269/260 1 (ref)  78/330 1 (ref)  122/78 1 (ref)  
    AG + AA 10/59 1.01 (0.49–2.07) 0.9824 40/27 1.46 (0.87–2.45) 0.1519 10/57 0.75 (0.37–1.54) 0.4363 11/9 0.78 (0.31–1.95) 0.5926 
CYP1B1 rs10916             
    TT 47/308 1 (ref)  194/179 1 (ref)  48/220 1 (ref)  71/55 1 (ref)  
    TG 33/155 1.42 (0.87–2.31) 0.1605 98/97 0.92 (0.65–1.31) 0.6485 33/136 1.11 (0.68–1.82) 0.6765 55/25 1.73 (0.96–3.12) 0.0694 
    GG 4/26 1.02 (0.34–3.06) 0.9772 17/12 1.23 (0.57–2.66) 0.6001 7/28 1.13 (0.47–2.75) 0.7805 5/7 0.55 (0.17–1.81) 0.3243 
    TG + GG 37/181 1.36 (0.85–2.18) 0.2006 115/109 0.96 (0.69–1.33) 0.7916 40/164 1.12 (0.70–1.78) 0.6478 60/32 1.46 (0.84–2.55) 0.1783 
    GG versus TT + TG  0.89 (0.30–2.65) 0.8394  1.27 (0.59–2.71) 0.5454  1.09 (0.46–2.58) 0.8466  0.45 (0.14–1.46) 0.1831 
CYP1B1 rs1800440             
    AA 52/322 1 (ref)  197/193 1 (ref)  61/273 1 (ref)  99/57 1 (ref)  
    AG 29/157 1.18 (0.72–1.94) 0.5045 102/86 1.19 (0.84–1.69) 0.3246 22/106 0.94 (0.55–1.61) 0.8309 31/27 0.67 (0.36–1.22) 0.1885 
    GG 3/18 1.18 (0.33–4.18) 0.7974 12/12 1.00 (0.43–2.26) 0.9811 4/13 1.36 (0.43–4.31) 0.6011 5/4 0.73 (0.19–2.84) 0.6490 
    AG + GG 32/175 1.18 (0.73–1.92) 0.4921 114/98 1.17 (0.83–1.63) 0.3672 26/119 0.99 (0.60–1.64) 0.9688 36/31 0.67 (0.38–1.20) 0.1805 
    GG versus AA + AG  1.11 (0.32–3.89) 0.8681  0.94 (0.41–2.11) 0.8716  1.38 (0.44–4.34) 0.5797  0.82 (0.21–3.15) 0.7732 
CYP1B1 rs1056836             
    CC 32/163 1 (ref)  96/80 1 (ref)  27/144 1 (ref)  28/37 1 (ref)  
    CG 35/242 0.70 (0.41–1.18) 0.1747 144/149 0.81 (0.56–1.19) 0.2831 42/173 1.29 (0.76–2.20) 0.3416 82/40 2.69 ( 1.454.97) 0.0017 
    GG 17/91 1.01 (0.53–1.93) 0.9761 66/58 0.97 (0.61–1.54) 0.8899 21/73 1.47 (0.77–2.78) 0.2416 25/12 2.70 ( 1.156.36) 0.0227 
    CG + GG 52/333 0.78 (0.48–1.26) 0.3040 210/207 0.86 (0.60–1.22) 0.3937 63/246 1.35 (0.82–2.21) 0.2406 107/52 2.69 ( 1.494.84) 0.0010
    GG versus CC + CG  1.24 (0.69–2.22) 0.4797  1.10 (0.74–1.64) 0.6355  1.26 (0.72–2.20) 0.4100  1.47 (0.68–3.15) 0.3287 
CYP1B1 rs1056827             
    GG 42/246 1 (ref)  150/136 1 (ref)  49/187 1 (ref)  59/44 1 (ref)  
    TG 32/216 0.86 (0.53–1.42) 0.5657 123/123 0.90 (0.64–1.27) 0.5445 26/164 0.60 (0.36–1.01) 0.0533 57/40 1.08 (0.61–1.89) 0.7972 
    TT 7/34 1.31 (0.54–3.17) 0.5513 31/29 0.97 (0.55–1.70) 0.9090 9/33 1.08 (0.49–2.42) 0.8450 15/3 4.05 ( 1.0715.36) 0.0400 
    TG + TT 39/250 0.92 (0.57–1.48) 0.7326 154/152 0.91 (0.66–1.26) 0.5796 35/197 0.68 (0.42–1.09) 0.1102 72/43 1.25 (0.73–2.17) 0.4178 
    TT versus TG + TT  1.40 (0.59–3.29) 0.4445  1.02 (0.60–1.74) 0.9512  1.33 (0.61–2.90) 0.4732  3.89 ( 1.0614.32) 0.0406 
CYP1B1 rs2567206             
    GG 42/250 1 (ref)  162/138 1 (ref)  50/185 1 (ref)  62/43 1 (ref)  
    GA 34/213 0.95 (0.58–1.56) 0.8513 121/126 0.81 (0.57–1.13) 0.2083 28/173 0.59 ( 0.360.98) 0.0415 58/44 0.92 (0.53–1.61) 0.7801 
    AA 7/33 1.34 (0.55–3.24) 0.5212 29/27 0.90 (0.51–1.60) 0.7288 9/33 1.03 (0.46–2.29) 0.9432 14/2 5.06 ( 1.0723.94) 0.0408 
    GA + AA 41/246 1.00 (0.63–1.60) 0.9890 150/153 0.82 (0.60–1.13) 0.2317 37/206 0.66 (0.41–1.05) 0.0815 72/46 1.07 (0.63–1.87) 0.7658 
    AA versus GG + GA  1.37 (0.58–3.22) 0.4770  1.00 (0.57–1.73) 0.9940  1.28 (0.59–2.79) 0.5296  5.28 ( 1.1524.28) 0.0327 
CYP2A13 rs8192784             
    GG 80/490 1 (ref)  305/282 1 (ref)  87/381 1 (ref)  131/84 1 (ref)  
    GA + AA 4/7 3.84 ( 1.0913.53) 0.0361 8/9 0.80 (0.30–2.10) 0.6463 2/11 0.87 (0.19–4.03) 0.8623 3/4 0.48 (0.10–2.19) 0.3402 
CYP2A13 rs8192789             
    CC 80/487 1 (ref)  298/274 1 (ref)  86/379 1 (ref)  130/84 1 (ref)  
    CT + TT 4/8 3.40 (0.99–11.64) 0.0515 10/9 0.98 (0.39–2.46) 0.9720 2/12 0.82 (0.18–3.73) 0.7920 4/4 0.63 (0.15–2.60) 0.5245 
CYP2A13 rs1709084             
    AA 73/413 1 (ref)  262/238 1 (ref)  66/347 1 (ref)  114/73 1 (ref)  
    AG + GG 11/81 0.74 (0.38–1.47) 0.3938 48/45 0.99 (0.63–1.54) 0.9481 22/43 2.72 ( 1.534.85) 0.000719/15 0.82 (0.39–1.70) 0.5892 
a

P -value is borderline significant after Bonferroni correction for 52 tests (Bonferroni adjusted level of significance α  = 0.96 × 10 −3 ).

b

P -value is significant after Bonferroni correction for 52 tests (Bonferroni adjusted level of significance α  = 0.96 × 10 −3 ).

Another CYP polymorphism, which was associated with a non-significantly increased risk of lung cancer overall in women for carriers of the minor allele, was CYP2A13 SNP rs1709084 (OR 1.64; 95% CI 1.00–2.70; P  = 0.05 and OR 0.95; 95% CI 0.66–1.39; P=  0.80 for women and men, respectively, Table II ). However, the effect modification genotype effect by gender was not significant ( P  = 0.61). The effect of this polymorphism seems more pronounced in a group of women with SCLC only (OR 2.26; 95% CI 1.00–5.09; P  = 0.05 and OR 1.47; 95% CI 0.83–2.59; P  = 0.18 for women diagnosed with SCLC and NSCLC, respectively). Similar to the CYP1B1 rs1056836 polymorphism, the effect of the CYP2A13 rs1709084 polymorphism was different between the groups of strong smoking (>20 PY) and light-smoking women (≤20 PY) ( Table III ). The OR for women carriers of rs1709084 minor allele, who smoked >20 PY, was 0.82 (95% CI 0.39–1.70, P  = 0.59), whereas in light and never smokers (≤20 PY) the minor genotype was associated with an increased risk of lung cancer (OR 2.72; 95% CI 1.53–4.85; P  < 0.001). The three-way interaction between gender, smoking and CYP2A13 genotype was statistically significant ( Pinteraction  < 0.001).

A statistically significant effect of the minor allele of CYP2A13 rs8192784 polymorphism was observed for men who smoked ≤20 PY (OR 3.84; 95% CI 1.09–13.53; P  = 0.04). In the group of heavy-smoking men, this effect was not significant (OR 0.80; 95% CI 0.30–2.10; P  = 0.65) ( Table III ). The heterozygous genotype CYP1B1 rs2567206 was associated with a significantly decreased risk of lung cancer in women who smoked ≤20 PY (OR 0.59; 95% CI 0.36–0.98; P  = 0.04). No significant effect was observed for homozygous minor allele carriers of this polymorphism (OR 1.03; 95% CI 0.46–2.29; P  = 0.94) or carriers of at least one minor allele (dominant model) (OR 0.66; 95% CI 0.41–1.05; P  = 0.08; Table III ). The homozygous genotype for the same polymorphism in the group of heavy-smoking women was associated with an increased risk of early-onset lung cancer in both codominant (OR 5.06; 95% CI 1.07–23.94; P  = 0.04; Table III ) and recessive models (OR 5.28; 95% CI 1.15–24.28; P  = 0.03). None of these findings were confirmed by an interaction test.

Haplotype analysis was performed for CYP1B1 polymorphisms and CYP2A13 polymorphisms. SNP rs2567206 was excluded from the haplotype analysis of the CYP1B1 region, as this polymorphism showed deviation from the HWE in the control population ( P  = 0.02, supplementary Table 1 is available at Carcinogenesis Online). The combination of four analyzed CYP1B1 SNPs led to eight distinct haplotypes with a frequency >1% in the control population ( supplementary Table 3 is available at Carcinogenesis Online). None of the observed haplotypes showed an association with early-onset lung cancer (global score statistic = 8.95, global P  = 0.63). The haplotype frequencies for CYP2A13 were also estimated and three distinct haplotypes with a frequency of >1% in the control population were detected (data not presented). None of the observed haplotypes showed an association with early-onset lung cancer in the complete sample set (global score statistic = 2.83, global P  = 0.42).

Discussion

The current study did not support the hypothesis that polymorphisms in CYP1A1 , CYP1B1 , CYP3A4 , CYP3A5 and CYP2A13 are associated with the increased risk of early-onset lung cancer. However, our findings suggest that CYP1B1 rs1056836 and CYP2A13 rs1709084 polymorphisms are associated with the risk of early-onset lung cancer in women. The effect of these polymorphisms was shown to be modified by smoking intensity ( Table III ).

The CYP1B1 rs1056836 polymorphism ( CYP1B1*3 ) is characterized by the C4326G transition, which causes an amino acid substitution of leucine to valine at the position 432 (Leu432Val). The SNP is located in a heme-binding domain, which is important for the catalytic activity of the enzyme ( 32 ). Previously it was shown that the polymorphism is associated with changed enzyme activity toward the 4- and 2-hydroxylation of estradiol. The 432Val allele was found to display a higher 4-hydroxylase activity compared with the Leu432 allele, but slightly decreased activity toward an activation of procarcinogens, such as dihydrodiols, heterocyclic aryl amines and polycyclic aromatic hydrocarbons ( 33–35 ). Due to the involvement of CYP1B1 enzyme in 4-hydroxylation of estrogen and estrogen–quinone carcinogenesis, associations between hormone-related cancers and CYP1B1 genetic polymorphisms have been extensively studied ( 36 , 37 ). Thus, an association of the 432Val variant of CYP1B1 with the risk of breast cancer in Caucasians (OR = 1.5; 95% 1.1–2.1; P  = 0.05) was suggested on the basis of a recent pooled analysis ( 36 ). Negative associations between this polymorphism and the risk of lung cancer overall has been described by several authors ( 38–40 ). Cote et al. ( 22 ) investigated the effect of the Leu432Val polymorphism on the risk of early-onset lung cancer (<50 years) and did not observe an association. A non-significantly increased risk of lung cancer (OR = 1.57; 95% CI 0.90–2.73; P  = 0.12) was observed in non-smoking Korean women ( 41 ). This result is in line with our results, where we observed a slight non-significantly increased risk of lung cancer in never and light-smoking women (≤20 PY) (OR = 1.35; 95% CI 0.82–2.21; P  = 0.24). However, in the current study, the effect of this SNP was more pronounced in heavy-smoking women (OR = 2.69; 95% CI 1.49–4.84; P  = 0.001).

In view of known facts about function and regulation of the CYP1B1 enzyme and effect of this polymorphism, the observed findings look plausible. 4-Hydroxylation of estradiol, catalyzed by CYP1B1, leads to formation of carcinogenic estradiol-3,4-semiquinones and quinones. Expression of CYP1B1 might be activated by estrogens in women ( 42 ) and also is induced by exposure to smoke compound ( 10 ). Higher amount of enzymes induced by both smoking and estrogens leads to active transformation of tobacco procarcinogens into carcinogens. Additionally, production of carcinogenic estradiol-3,4-semiquinones and quinones causes higher risk of tumorigenesis in heavy-smoking women under the age of 51.

Although described previously as potential risk factors for lung cancer, CYP1B1 polymorphisms rs1800440 ( CYP1B1*4 ) and rs1056827 ( CYP1B1*2 ) did not show any association with the risk of early-onset lung cancer in our study. Both of these SNPs cause amino acid substitutions of asparagine to serine at the 453 position and of alanine to serine at the 119 position, respectively. In contrast to our results, a positive significant association of the Ala119Ser polymorphism with the incidence of lung cancer was detected by Watanabe et al. ( 43 ). However, another group observed no effect of Ala119Ser and Asp453Ser polymorphisms on the risk of lung cancer ( 38 ).

We also detected a significantly increased risk of lung cancer in heavy-smoking women for the homozygous carriers of the CYP1B1 rs2567206 variant allele and a decreased risk in light-smoking women for the heterozygous carriers of the CYP1B1 rs2567206 variant allele ( Table III ). However, this is probably to be a spurious finding as we observed a deviation from the HWE for this polymorphism in the control group ( P  = 0.02). We also believe that a significantly increased risk of lung tumors associated with the CYP2A13 rs8192784 variant allele in men who smoked ≤20 PY, as well as an increased risk for women with the CYP1B1 rs1056827 genotype TT who smoked >20 PY might be a chance finding due to the limited number of individuals in these subgroups ( Table III ).

The tagging SNP CYP2A13 rs1709084, which showed a significant effect on the risk of early-onset lung cancer in women in the current study, is in high linkage disequilibrium with several non-functional SNPs in the CYP2A13 and CYP2F gene's regions (HapMap Data Rel 20/phase II). To the authors’ knowledge, it has never been investigated with respect to cancer risk before. It is possible that there are other unknown functional polymorphisms linked with this marker (rs1709084), which can modulate function of the CYP2A13 enzyme and influence the individual susceptibility to carcinogens. However, the biological plausibility of the gender-specific effect detected for the CYP2A13 polymorphism is not so clear. This gene is not involved directly in the metabolism of estrogens and, to the authors’ knowledge, gender specificity in expression of the CYP2A13 enzyme has not yet been studied. The observation that the effect of SNP is strongly significant in the group of women who smoked ≤20 PY only might indicate stronger genetic component in this group of patients than in women who smoked >20 PY where lung cancer was caused mainly by smoking. In view of the observed results, in the future, focus should be done on investigation of gender-specific differences in function and expression of the CYP2A13 enzyme.

In disagreement with a previous finding ( 44 ), we did not detect any association of the CYP2A13 rs8192789 polymorphism and lung cancer risk. This SNP leads to an Arg257Cys amino acid change, which is ∼50% less active than the Arg-257 enzyme ( 45 ). This polymorphism is in linkage with another functional substitution in the exon 1 of the CYP2A13 gene, Arg25Gln (rs8192784) ( 46 ).

For the three studied CYP1A1 polymorphisms, we did not observe any significant effect. CYP1A1 rs1048943 polymorphism ( CYP1A1*2C ) in the coding region causes an amino acid substitution of isoleucine to valine at position 462 of the enzyme ( 47 ). The published results for the effect of this polymorphism on lung cancer risk are inconsistent. Positive ( 22 , 48 , 49 ) and negative ( 19 , 50 ) associations have been observed between Ile462Val polymorphism and lung cancer risk. Our results for CYP1A1 Ile462Val polymorphism have to be interpreted with caution as the frequency of this polymorphism in controls deviated from the HWE ( P  = 0.01). Another functional CYP1A1 polymorphism studied in the current project is a threonine to asparagine amino acid substitution (Thr461Asn, rs1799814). Previously, the Thr461Asn polymorphism has also been studied as a possible lung cancer risk factor. As in the case of the Ile462Val polymorphism, both negative for lung cancer overall ( 51 ) and positive for adenocarcinoma ( 52 ) or lung cancer in non-smokers ( 19 ) results have been reported.

We also did not observe any effect of CYP3A polymorphisms analyzed in the current study. The CYP3A4 rs2740574 polymorphism ( CYP3A4*1B ) is a single substitution of A > G in the promoter region and is in linkage disequilibrium with the CYP3A5 rs776746 polymorphism. Results of the present study were not able to reproduce previously published effect of CYP3A4 rs2740574 polymorphism in the group of heavy-smoking women with lung cancer ( 26 ).

Interestingly, our study observed the effect of two polymorphisms in CYP1B1 and CYP2A13 only in women. In fact, several authors have postulated that there are differences in susceptibility to carcinogen, the mechanism of lung cancer development and etiology of disease between genders, which can be explained by genetic and biological factors ( 53–55 ). From animal models, it is known that gender and sex hormones play an essential role both in normal lung development and pathological processes in lung tissue. Thus, it was shown previously that female mice are more susceptible to developing lung cancer induced by benzo[ a ]pyrene than their male counterparts ( 56 ).

Hence, an association between CYP1B1 and CYP2A13 polymorphisms and risk of early-onset lung cancer in women is biologically plausible. CYP can be regulated and activated by estrogen. In fact, expression of CYP1B1 and CYP1A1 has been shown to be higher in women than in men ( 42 ). The current study has the advantage of looking for the effect of genetic factors in early-onset patients (under the age of 51). It is realistic to assume that we have a higher proportion of premenopausal women in the study population than in general within lung cancer patients. In premenopausal women, a higher expression of estrogen can be expected. Estrogen by itself can be involved in carcinogenesis and additionally, it can stimulate expression of cytochromes in the female lung. Indeed, in non-tumor lung tissue, estrogen receptor β expression has been positively correlated with CYP1B1 expression in women ( 57 ).

The current study has several strengths, such as population-based design and the availability of biological and clinical material for all study participants. To the authors’ knowledge, it is the largest study evaluating effect of CYP polymorphisms on the risk of early-onset lung cancer. The present study had sufficient power to detect associations between genotypes and lung cancer risk of OR for the dominant model ranging from ≥1.32 to ≥2.08 ( supplementary Table 1 is available at Carcinogenesis Online).

However, our study has limitations that have to be considered. Retrospective case–control design may cause recall bias on exposure to tobacco smoke. We cannot exclude that a lung cancer diagnosis may change behavior habits of patients and lead to smoking cessation. The percentage of smokers and non-smokers in the present study did not differ significantly from those published by other authors for early-onset lung cancer cases in Germany ( 7 , 58 ). Therefore, it is unlikely that recall bias introduced significant bias, which may affect the results.

The current study combined individuals from different German populations. This fact raises a problem of possible population stratification. However, Steffens et al. ( 59 ) compared genetic differentiation between three population-based studies from different geographic areas of Germany and reported low population stratification in the German population. In agreement with this finding, we did not observe differences in genotype distribution between the two case groups from LUCY and the Heidelberg Lung Cancer Study. We do not believe that this design introduced selection bias, as distribution of smokers in controls, as well as gender and smoking distribution in cases, as mentioned above, were similar to those previously observed for German populations or for early-onset lung cancer cases from Germany ( 7 , 58 , 60 ). Additionally, analysis performed separately for the LUCY and The Heidelberg Lung Cancer Study revealed similar pattern of two main effects for CYP1B1 rs1056836 and CYP2A13 rs1709084 SNPs as the analysis for the combined group of patients (data not presented).

The present study failed to reproduce a range of previously published results, such as effects of CYP1B1 rs1800440, rs1056827, CYP2A13 rs8192789, etc. This might be explained through several factors, such as the limited power to detect weak effects, which, as we learn, are usually expected in the case of genetic factors. Although it is one of the biggest studies on early-onset lung cancer, it is limited by the number of cases, which is much lower than for studies focusing on lung cancer without age limit. Additionally, certain previous studies might also lack power and may have had design limitations. Therefore, it would be important to replicate our findings in another large study of early-onset lung cancer.

In view of the observed results, it might be of particular interest to study a possible modification by hormone status of CYP1B1 - and CYP2A13 polymorphism-associated risk of early-onset lung cancer in men and particularly women. Unfortunately, the presented study, as well as the majority of molecular epidemiological studies on lung cancer in general, lacks information about sex hormone status and some reproductive characteristics. Therefore, future studies on early-onset lung cancer should include the collection of information about reproductive characteristics and the hormone status of cases and controls.

In conclusion, we observed an increased risk for early-onset lung cancer in women associated with genetic polymorphisms of CYP1B1 and CYP2A13 . The effect of the CYP1B1 rs1056836 polymorphism in women and the effect of the CYP2A13 rs1709084 polymorphism in never and light-smoking women were significant even after Bonferroni correction for multiple testing. Smoking was identified as modifier of the association between CYP1B1 and CYP2A13 genotypes and early-onset lung cancer risk in women. Our findings suggest a differential by gender effect of CYP1B1 and CYP2A13 polymorphisms in lung carcinogenesis. To confirm our hypothesis of a gender-specific effect on lung cancer risk with respect to CYP1B1 and CYP2A13 genotypes, further large studies on early-onset lung cancer have to be conducted.

Supplementary material

Supplementary Methods and Tables 13 can be found at http://carcin.oxfordjournals.org/

Funding

Deutsche Krebshilfe to A.R. (70-2387 BaI, 70-2919 Ri) and H.B.; German Research Foundation DFG (BE 3906/2-1) to L.B.; Helmholtz-DAAD Fellowship to M.T. (A/07/97379).

Abbreviations

    Abbreviations
  • CI

    confidence interval

  • CYP

    cytochrome P450

  • HWE

    Hardy–Weinberg equilibrium

  • KORA

    Kooperative Gesundheitsforschung in der Region Augsburg

  • LUCY

    Lung Cancer in the Young

  • NSCLC

    non-small-cell lung cancer

  • OR

    odds ratio

  • PY

    pack-year

  • SCLC

    small-cell lung cancer

  • SNP

    single-nucleotide polymorphism

We thank all the participating hospitals (LUCY-Consortium): Aurich (Dr Heidi Kleen); Bad Berka (Dr med. R.Bonnet, Klinik für Pneumologie, Zentralklinik Bad Berka GmbH); Bonn (Prof. Ko, Dr Geisen, Innere Medizin I, Johanniter Krankenhaus); Bonn (Dr Stier, Medizinische Poliklinik, Universität Bonn); Bremen (Prof. Dr D.Ukena, Dr Penzl, Zentralkrankenhaus Bremen Ost, Pneumologische Klinik); Chemnitz (PD Dr Schmidt, OA Dr (Jielge, Klinikum Chemnitz, Abteilung Innere Medizin); Coswig (Prof. Höffken, Dr Schmidt, Fachkrankenhaus Coswig); Diekholzen (Dr Hamm, Kreiskrankenhaus Diekholzen, Klinik für Pneumologie); Donaustauf (Prof. Pfeifer, Dr v.Bültzingslöwen, Fr. Schneider, Fachklinik für Atemwegserkrankungen); Essen (Prof. Teschler, Dr Fischer, Ruhrlandklinik—Universitätsklinik, Abt. Pneumologie); Gauting (Prof. Häußinger, Prof. Thetter, Dr Düll, Dr Wagner, Pneumologische Klinik München-Gauting); Gera (CA MR Dr Heil, OÄ Dr Täuscher, OA Dr Lange, II. Medizinische Klinik, Wald-Klinikum Gera); Göttingen (Prof. Trümper, Prof. Griesinger, Dr Overbeck, Abteilung Onkologie, Hämatologie); Göttingen (Prof. Schöndube, Dr Danner, Abteilung Thorax-, Herz- und Gefäßchirurgie); Göttingen/Weende (Dr med. Fleischer, Ev. Krankenhaus Göttingen-Weende e.V., Abteilung Allgemeinchirurgie); Greifenstein (Prof. Morr, Dr M. Degen, Dr Matter, Pneumologische Klinik, Waldhof Elgershausen); Greifswald (Prof. Ewert, Dr Altesellmeier, Universitätsklinik Greifswald, Klinik für Innere Medizin B); Hannover (Prof. Schönhofer, Dr Kohlaußen, Klinikum Hannover Oststadt, Medizinische KlinikII, Pneumologie); Heidelberg (Prof. Drings, Dr Herrmann, Thoraxklinik gGmbH, Abt. Innere Medizin-Onkologie); Hildesheim (Prof. Kaiser, St Bernward Krankenhaus, Medizinische Klinik II); Homburg (Prof. Sybrecht, OA Dr Gröschel, Dr Mack, Uniklinik des Saarlandes, Innere Medizin V); Immenhausen (Prof. Andreas, Dr Rittmeyer, Fachklinik für Lungenerkrankungen); Köln (Priv. Doz. Dr Stölben, Kliniken der Stadt Köln, Lungenklinik Krankenhaus Merheim); Köln (Prof. Wolf, Dr Staratschek-Jox, Klinikum der Universität Köln, Klinik I für Innere Medizin); Leipzig (Prof. Gillisen, OA Dr Cebulla, Städt. Klinikum St Georg, Robert-Koch-Klinik); Leipzig (Kreymborg, Universitätsklinikum Leipzig, Medizinische Klinik I, Abteilung Pneumologie); Lenglern (Prof. Criée, Dr Körber, Dr Knaack, Ev. Krankenhaus Weende e.V., Standort Lenglern, Abt. Pneumologie; München (Prof. Huber, Dr Borgmeier, Klinikum der Ludwig-Maximilians-University-Innenstadt, Abt. Pneumologie); Neustadt a. Harz (Dr Keppler, Schäfer, Evangelisches Fachkrankenhaus für Atemwegserkrankungen); Rotenburg (Prof. Schaberg, Dr Struß, Diakoniekrankenhaus Rotenburg, Lungenklinik Unterstedt); St Pölten—Österreich (OA Dr M. Wiesholzer, Zentralklinikum St Pölten, I. Medizinische Klinik).

We thank Dr H.Dally, Mrs A.Seidel, Dr T.Muley, Dr S.Thiel, Dr H.Wikman, Ms U.von Seydlitz-Kurzbach, Ms D.Bodemer, Dr C.Klappenecker, Mr M.Hoffmann and Mr C.Beyon for help with sample and/or data collection and archiving for the Heidelberg lung study. We are grateful to all patients and staff at the Thoraxklinik, Heidelberg, who participated in the Heidelberg Lung Cancer Study.

We gratefully acknowledge the KORA Study group especially G.Fischer, H.Grallert, N.Klopp, C.Gieger and R.Holle, Institute of Epidemiology, Helmholtz Centre Munich, Neuherberg, Germany, and all individuals, who participated as cases or controls in this study and the KORA Study Center and their coworkers for organizing and conducting the data collection. We thank H.Jöshi for English editing.

Conflict of Interest Statement: None declared.

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