NAD(P)H: Quinone Oxidoreductase 1 (NQO1) Pro187Ser Polymorphism and the Risk of Lung Cancer: A Meta-Analysis Chun Chao, Zuo-Feng Zhang, Julien Berthiller, Paolo Buffetta, Mia Hashibe Department of Epidemiology, UCLA International Agency for Research on Cancer

NQO1: an Antioxidant Enzyme NQO1 protects cells from oxidative damage by preventing the formation of free radical byproduct of quinone metabolism. NQO1 activates the antioxidant forms of vitamin E and ubiquinone after free radical attack. NQO1 stabilizes p53, especially in response to oxidative stress. NQO1 is highly expressed in tissues requiring high level of antioxidant protection E.g., Epithelial cells of lung and colon, vascular endothelium, corneal epithelium, etc.

NQO1 Catalyzes Two Electron Reduction Quinone o o R R R R OH R R R R NQO1 2 e- Hydroquinone Toxicity ↓ Toxicity ↑ Benzene Benzo(a)pyrene Nitro compounds Mitomycin C Semi-quinone Alkylating species Free radicals Phase I Enzymes 1e-

NQO1 Polymorphisms Over 80 SNPs in NQO1 identified Over 80 SNPs in NQO1 identified 2 nonsynonymous SNPs in exons have been described. 2 nonsynonymous SNPs in exons have been described. The 609 variant (Pro187Ser) results in reduced enzyme activity: The 609 variant (Pro187Ser) results in reduced enzyme activity: Heterozygous: 3-fold decrease Heterozygous: 3-fold decrease Homozygous variant allele: almost null activity Homozygous variant allele: almost null activity

Frequency of NQO1 609 Variant Genotype Asian CT/TT:48.3% TT: 20.3% Non-Hispanic White CT/TT: 39.5% TT: 4.4% African-American CT/TT: 33.8% TT: 5.2% Mexican-American CT/TT: 52.2% TT: 15.5% Ref: Kelsey KT et al. Br J Cancer 1997; 76:852-4.

Defected NQO1 Activity has been Link to Several Diseases Benzene toxicity Benzene toxicity Hematological toxicity (leukocytopenia, anemia, thrombocytopenia) Hematological toxicity (leukocytopenia, anemia, thrombocytopenia) Leukemia Leukemia Renal cell and urothelial cell carcinoma Renal cell and urothelial cell carcinoma Basal cell carcinoma Basal cell carcinoma Secondary myeloid leukemia among individuals receiving chemotherapy. Secondary myeloid leukemia among individuals receiving chemotherapy. Lung cancer risk?

Meta-analysis of NQO1 Pro187Ser polymorphism and lung cancer risk 21 case-control studies identified 19 studies included (6,980 cases/8,080 controls) Whites: 8 studies Asians: 7 studies Multiethnic: 4 studies Inclusion Criteria Disease histologically confirmed Genotype distribution in controls in H-W equilibrium Genotype frequency/precision estimate reported Identification of Studies PubMed and ISI web of knowledge database Reference search Unpublished data that we were aware of

Methods for Meta-Analysis Estimates combined using inverse variance weighting Estimates combined using inverse variance weighting Random effects model used if heterogeneity was detected. Random effects model used if heterogeneity was detected. Mantel-Haenszel test for heterogeneity Mantel-Haenszel test for heterogeneity Stratified analysis by gender and region to explore source of heterogeneity Stratified analysis by gender and region to explore source of heterogeneity Publication bias assessed using Egger’s test & Begg’s funnel plot Publication bias assessed using Egger’s test & Begg’s funnel plot Influence analysis Influence analysis 20% change in summary estimate as an indication for influential study. 20% change in summary estimate as an indication for influential study.

Results *Reference group are persons with the C/C genotype

Summary OR for Whites OR C/T vs. C/C: 1.07 ( ) OR T/T vs. C/C: 1.19 ( ) OR C/T+T/T vs. C/C: 1.04 ( ) P for heterogeneity: 0.87 P for heterogeneity: 0.44 P for heterogeneity: 0.30

Summary OR for Asians OR C/T vs. C/C: 1.00 ( ) OR T/T vs. C/C: 0.95 ( ) OR C/T + T/T vs. C/C: 0.99 ( ) P for heterogeneity: 0.01 P for heterogeneity: < 0.01 After excluding the outlier study: OR C/T vs. C/C: 0.85 ( ) OR T/T vs. C/C: 0.81 ( ) OR C/T+T/T vs. C/C: 0.87 ( )

Summary OR for Blacks OR C/T + T/T vs. C/C: 0.95 ( ) P for heterogeneity: 0.63

Adjusted vs. Unadjusted OR Summary adjusted OR are similar to the summary unadjusted OR. Summary adjusted OR are similar to the summary unadjusted OR. Analyses restricted to studies that reported adjusted OR. Most studies adjusted for age, sex, and cigarette smoking.

Summary OR by Smoking Status and Lung Cancer Cell Type *ORs are (C/T+T/T) vs. C/C 0

Discussion A positive association between the variant alleles and lung cancer risk was suggested in Whites. A positive association between the variant alleles and lung cancer risk was suggested in Whites. Low power to detect association for the T/T genotype Low power to detect association for the T/T genotype The variant alleles appeared to be inversely associated with lung cancer risk in Asians. The variant alleles appeared to be inversely associated with lung cancer risk in Asians. No association was observed in Blacks. No association was observed in Blacks. Increased risk was observed among white ever smokers. Increased risk was observed among white ever smokers. Increased risk was suggested for squamous cell carcinoma among both Whites and Asians. Increased risk was suggested for squamous cell carcinoma among both Whites and Asians. Inverse association between the variant alleles and adenocarcinoma was observed in Asians. Inverse association between the variant alleles and adenocarcinoma was observed in Asians.

Discussion (Cont.) The difference seen in Whites and Asians was consistent with results from bladder cancer and colorectal cancer meta-analysis results. The difference seen in Whites and Asians was consistent with results from bladder cancer and colorectal cancer meta-analysis results. Different results observed between ethnic groups may be due to: Different results observed between ethnic groups may be due to: Relevant environmental exposures in Asia may differ from Western countries. Relevant environmental exposures in Asia may differ from Western countries. There may exist other genetic mechanisms that compensate more effectively for the loss of the detoxifying activity of NQO1 in Asians. There may exist other genetic mechanisms that compensate more effectively for the loss of the detoxifying activity of NQO1 in Asians.

Copyright ©2001 American Association for Cancer Research Xu, L. L. et al. Cancer Epidemiol Biomarkers Prev 2001;10: OR of C/T vs. C/C in former smokers, ♦ light smoker, five cigarettes/day; ■, mild smoker, 20 cigarettes/day; ∆, heavy smoker, 40 cigarettes/day. NQO1 and Cigarette Smoking: Gene-Environment Interaction

Conclusions The effect of NQO1 Pro187Ser polymorphism may be different in each individual depending on his/her other genetic make up and environmental exposure. The effect of NQO1 Pro187Ser polymorphism may be different in each individual depending on his/her other genetic make up and environmental exposure. Multiple genes and smoking behaviors (or other exposures) should be considered if the genotype information were to be used to assess susceptibility. Multiple genes and smoking behaviors (or other exposures) should be considered if the genotype information were to be used to assess susceptibility.

Thank you! We thank the following individuals for providing unpublished genotype data for our analysis: Dr. Cathryn Bock, Dr. Karin Broberg, Dr. Nobuyuki Hamajima, Dr. Rayjean Hung, Dr. Pinpin Lin, and Dr. Mette Sorensen.