1. Molecular Epidemiology of Lung Cancer in Chinese Population Hongbing Shen, M.D., Ph.D Professor of Epidemiology Department of Epidemiology & Biostatistics Nanjing Medical University School of Public Health Tel: 86-25-86862756; Email: hbshen@njmu.edu.cn Alper-JCCC Molecular Epidemiology Symposium

2. Age-standardized Incidence Rates for Lung Cancer 2002 (per 100,000) Parkin et al. CA Cancer J Clin. 2005

3. Time trends for lung cancer mortality rates during 1987– 1999 in China, using age- standardized rate (ASR) by sex and area Rural Urban Men Woman Yang et al. Int J Cancer 2003

4. Yang et al. JAMA 1999 Sales Volume of Cigarettes in China, 1981-1995 +100%

5. Age-Specific Prevalence Rates of Current and Former Smoking in Men and Women in China 6.9 % 60.2 % 2001 35-74 Yang et al. JAMA 1999; Gu et al. Am J Public Health 2004 10.6 %

6. Prevalence Rates of Smoking in a Rural Area of South Jiangsu Province, China (2004-2005) Age MaleFemaleTotal No Smk Rate* （ % ） < 30 606 45.7 856 0.51462 19.4 30 ～ 1530 70.8 2388 0.4391828.4 40 ～ 2176 75.4 2865 0.7504133.4 50 ～ 2405 73.6 3050 1.0545533.5 60 ～ 1302 64.9 1611 0.72913 29.8 70 ～ 762 51.8 1183 2.51945 22.7 Total 8781 68.4 11953 1.720734 30.0 * Including former smokers Unpublished data

7. Prevalence of environmental tobacco smoke (ETS) exposure among nonsmokers by gender and age group in China, 2000-2001 Men Woman Gu et al. Am J Public Health. 2004

8. Background Lung cancer incidence rate has been increasing significantly in the last two decades in China. More than 80% lung cancer can be attributed to smoking, yet only <10% smokers develop lung cancer. Lung cancer is an excellent model for the research of gene-environment interactions.

9. Multi-step Carcinogenic Process of Lung Cancer Nicotine Dependent DNAdamages Cancer Apoptosis CarcinogenuptakeMetabolicactivationMetabolicdetoxification Excretion NormalDNA DNA Repair Persistencymiscoding Mutationaccumulation Exposure

10. Smoking Caused DNA Damage and Related Repair Pathways

11. DNA repair gene SNPs （genotypes） DRC （Phenotype） Lung Cancer Risk Association ？ Hypothesis: The genetic polymorphisms of the DNA repair genes are associated, independently or coordinately, with increased risk of lung cancer Gene-environment interaction?

12. Subjects & Methods  Hospital-based case-control studies of lung cancer  Inclusion Criteria Cases: Incident lung cancer patients Newly diagnosed, histopathologically confirmed No previous radiotherapy or chemotherapy Controls: Cancer-free subjects Recruited at the same time period as cases and frequented matched to the cases on age, sex All the subjects are Han Chinese (Nanjing).

13. ‘Sequence-based’ candidate SNPs approach Potentially functional SNPs of DNA repair gene XRCC1 and risk of lung cancer Hu Z, … Shen H. Pharmacogenetics & Genomics 2005 15(7): 457-64. A case-control study of 710 lung cancer cases and 710 cancer-free controls

14. Functional polymorphisms of DNA repair gene XRCC1: Promoter: T-77C (2004) Coding region: Arg194Trp (C>T) (DNA pol , PARP domain ) Arg399Gln (G>A) (PARP domain) Case-control study of 710 lung cancer cases and 710 cancer-free controls

15. EXON 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 XRCC1 5’UTR ATG 3’UTR -77T>C Exon6 Arg194Trp Exon10 Arg399Gln +1 -77T XRCC1 promoter -1152+93 Luciferase XRCC1 promoter -1152+93 Luciferase pGL3 -77C pGL3p77T p77C KpnI NheI Chromosome: 19q13.2 1246-bp 57–64%

16. Variable Cases (n=710)Controls (n=710) P value No.% % Age (years)0.7100 ≤ 6037452.736651.5 > 6033647.334448.5 Sex0.7207 Male52073.251372.3 Female19026.819727.7 Smoking status<0.0001 Non-smokers23132.537052.1 Ever-Smokers47967.534047.9 XRCC1 T-77C0.0003 T allele119884.4126489.0 C allele22215.615611.0 XRCC1 C194T0.8389 C allele98169.198669.4 T allele43930.943430.6 XRCC1 G399A0.4488 G allele104073.2102272.0 A allele38026.839828.0 Distribution of Select Variables and XRCC1 Variant Alleles in Lung Cancer Cases and Cancer-free Controls

17. XRCC1 T-77C Polymorphism and Lung Cancer Risk XRCC1 Genotype Cases(710)Controls(710) Adjusted OR (95%CI) No % XRCC1 T-77C TT (ref.)500 70.4558 78.61.00 CT198 27.9148 20.91.51 (1.17-1.94) CC 12 1.7 4 0.62.98 (0.93-9.59) CT/CC 210 29.6152 21.41.55 (1.21-1.98) C allele222 15.6156 11.0P = 0.0003 Hu Z, … Shen H. Pharmacogenetics & Genomics 2005 15(7): 457-64.

18. XRCC1 T-77C and Cumulative Smoking Pack-years of smoking -77TT -77CT/CC Adjusted OR Hu Z, … Shen H. Pharmacogenetics & genomics 2005 15(7): 457-64. P value for the test of additive gene-smoking interaction: 0.027

19. This significant association with lung cancer was validated in another independent case-control study in a Chinese population Hao et al. 2006 Oncogene

20. ‘block-free’ linkage disequilibrium-based SNPs approach Polymorphisms of NER pathway core genes (XP) and susceptibility of lung cancer Hu Z, … Shen H. Carcinogenesis 2006; Jul;27(7):1475-1480 Hu Z, … Shen H. CEBP 2006; 15(7):1336-40. A case-control study of 1010 lung cancer cases and 1011 cancer-free controls

21. NER Core Genes 1 From NIEHS SNPs database Modified from James E. Cleaver, Nature Review Cancer 2005

22. 8 candidate core genes in NER pathway 41 Tagging SNPs were selected / 32 were used in the analyses Based on the resequencing data of 90 individuals in the Environmental Genome Project (EGP) database (designed before the release of HapMap data) Based on the calculation of pairwise linkage disequilibrium (LD). LD parameter r 2 threshold 0.5 (Carlson et al.). Linkage disequilibrium-based tagSNPs selection

23. 5 ’ -nuclease (TaqMan) assay ABI PRISM 7900HT Sequence Detection System 384-well format Chinese National Human Genome Center at Shanghai, Quality control: Two blank control (water) and two duplicated samples in each 384-well format The intensity of each SNP should meet the criteria of three clear clusters in two scales generated by SDS software (ABI). Laboratory Assays

24. TaqMan assay results output: 5’-nuclease (TaqMan) assay in 384-well format

25. TaqMan Assay Results Output:

26. Chi-square test, Logistic regression Single locus analysis (ORs and 95% CIs) Multivariate logistic regression model Haplotype inference: We used the PHASE 2.0 program to infer haplotype frequencies based on the observed genotypes of each gene. Statistical analyses

27. Single SNPs Analyses in the Dominant (Blue Line) and Recessive Model (Green Line)

28. DDB2 Genotype CasesControls Crude OR (95%CI) Adjusted OR (95%CI) a No.(%)No.(%) rs830083961960 CC372(38.7)429(44.7)1.00 CG483(50.3)426(44.4)1.31 (1.08-1.58)1.31 (1.08-1.60) GG106(11.0)105(10.9)1.16 (0.86-1.58)1.22 (0.89-1.67) CG/GG589(61.3)531(55.3)1.28 (1.07-1.53)1.30 (1.08-1.56) rs37816203967978 CC374(38.7)429(43.9)1.00 CG491(50.8)437(44.7)1.29 (1.07-1.56)1.28 (1.06-1.56) GG102(10.6)112(11.5)1.04 (0.77-1.41)1.11 (0.81-1.51) CG/GG593(61.4)549(56.2)1.24 (1.03-1.48)1.25 (1.04-1.50) Analysis of association between the DDB2(XPE) polymorphisms and risk of lung cancer Hu … Shen et al, Carcinogenesis, 2006 Jul;27(7):1475-1480

29. Stratified analyses between the combined DDB2 rs830083 genotypes and lung cancer risk DDB2 rs830083 Cases (n=961)Controls (n=960) Adjusted OR (95% CI) CCCG/GGCCCG/GG CCCG/GG N (%) Age (years) ≤ 60175 (37.2)296 (62.9)206 (44.4)258 (55.6)1.001.40 (1.07-1.83) > 60197 (40.2)293 (59.8)223 (45.0)273 (55.0)1.001.20 (0.92-1.56) Pack-years of smoking 0112 (38.0)183 (62.0)207 (45.1)252 (54.9)1.001.29 (0.96-1.75) 1-2995 (39.8)144 (60.3)105 (40.4)155 (59.6)1.001.01 (0.70-1.45) >29165 (38.6)262 (61.4)117 (48.5)124 (51.5)1.001.48 (1.07-2.04) Family history of cancer No313 (39.5)480 (60.5)371 (44.3)467 (55.7)1.001.24 (1.01-1.51) Yes59 (35.1)109 (64.9)58 (47.5)64 (52.5)1.001.70 (1.03-2.80)

30. ERCC2 and ERCC3 polymorphisms and lung cancer risk Genotype CasesControls Adjusted OR (95%CI) No.(%)No.(%) ERCC2 rs1618536 975985 AA17718.219920.21.00 AG49250.548849.51.10 (0.86-1.40) GG30631.429830.31.15 (0.89-1.51) AG/AA79881.978679.81.12 (0.89-1.41) ERCC2 rs1799786 965986 CC84487.587789.01.00 CT11712.110811.01.12 (0.84-1.49) TT40.410.16.18 (0.68-56.52) CT/TT12112.510911.11.16 (0.87-1.54) ERCC2 rs1799793 970986 GG85087.687488.61.00 AG11612.011111.31.06 (0.80-1.41) AA40.410.16.13 (0.67-56.11) AG/AA12012.411211.41.10 (0.83-1.46) ERCC2 rs3916823 986987 AAAA/AAAA74475.575076.01.00 AAAA/--22522.822723.01.01 (0.81-1.25) --/--171.7101.01.88 (0.84-4.21) AAAA/-- + --/--24224.523724.01.04 (0.84-1.29) Hu … Shen et al, Cancer Epidemiology Biomarkers & Prevention 2006, 15(7):1336-40.

31. ERCC2 rs13181 975997 AA82784.886586.81.00 AC14114.512712.71.16 (0.89-1.51) CC70.750.51.64 (0.50-5.36) AC/CC14815.213213.21.18 (0.91-1.53) ERCC3 rs2271026 1002 1000 TT77477.378778.71.00 CT21421.420020.01.07 (0.86-1.34) CC141.4131.31.02 (0.46-2.24) CT/CC22822.821321.31.07 (0.86-1.33) ERCC3 rs4150441 952 975 AA14915.717818.21.00 AG47449.847548.81.20 (0.93-1.56) GG32934.632233.11.28 (0.97-1.69) AG/GG80384.479781.91.24 (0.97-1.58) Combined genotypes847899 0-1 ‘ at risk ’ locus 13616.118620.71.00 2 ‘ at risk ’ loci 33139.134938.81.29 (0.98-1.70) 3 ‘ at risk ’ loci 23127.323025.61.38 (1.02-1.85) >=4 ‘ at risk ’ loci 14917.613414.91.51 (1.09-2.10) P for trend: 0.02

32. ERCC1 genotypes and lung cancer risk GenotyopeCases (n=1010) Controls (n =1011) PAdjusted OR (95%CI) No.% % rs3212948992986 GG59459.952152.81.00 CG33834.140340.90.0050.73 (0.60-0.88) CC606.1626.30.96 (0.65-1.41) CG/CC39840.246547.20.0030.76 (0.63-0.91) rs1007616835908 CC51361.449354.31.00 CT27032.335439.00.0090.72(0.59-0.89) TT526.2616.70.90(0.61-1.35) CT/TT32238.541545.70.0040.75(0.62-0.91) Ma et al, Pharmacogenetics and Genomics, 2007. in press

33. Combined Effects of NER Core Genes and Lung Cancer * Six gene in dominant model and two in recessive model Unpublished data

34. Stratified Analysis of Dichotomized NER Combined Diplotypes by Cumulative Smoking Pack-years of smoking Adjusted OR P value for the test of gene-smoking interaction (multiplicative): 0.004 Unpublished data

35. Two Factor Gene-gene (Diplotype) Interaction and Gene-environment Interaction Unpublished data

36. ‘block-based’ linkage disequilibrium mapping Haplotyope-Tagging SNPs in MGMT (O6-alkylguanine- DNA alkyltransferase) and lung cancer susceptibility Hu Z, … Shen H. Human Mutation 2007 published online. Case-control study of 500 lung cancer cases and 517 cancer-free controls

37. MGMT Gene Structure and Haplotype Block for Beijing Han Chinese From HapMap 4 14 7 1 1 3 1 1 1 3 1 1 1 No. of SNPs:

38. Illumina High Throughout Genotyping Platform (CHGC-Shanghai)

39. MGMT Tagging SNPs Based on the Gene Blocks ( by Haploview program ) 25 informative SNPs (MAF>0.05) in our controls 10 htSNPs from the 6 blocks 1 1 2 2 2 2 htSNPs Gabriel et al. Stram et al. Rh2 > 0.80

40. Single variant analysis: no significant main effects was observed for each single genetic variant in MGMT on lung cancer risk. Haplotype analysis: no significant difference was observed for frequencies of haplotypes in different blocks between cases and controls. Diplotype analysis: only the diplotype carrying 1 variant copy of the block 3 was associated with a significantly decreased lung cancer risk. Stratification: The frequencies of haplotypes in blocks 1 and 2, block 4, 5 were significantly different between cases and controls in different stratum of smoking dose. Summary of the findings: Hu Z, … Shen H. Human Mutation 2007

41. MDR Models of Selected Gene Regions and Co-variables Best models CVC * Avg. Testing Accuracy Sign Test P Value One Factor: Pack-years of smoking 100/1000.5827 0.0009 0.0330 Two Factors: Pack-years of smoking; block 3 99/1000.5992 0.0018 0.0344 Three Factors: Pack-years of smoking; block 5; rs1625649:C>A 100/1000.6145 0.0000 0.0453 Four Factors: Pack-years of smoking; block 3; block 5; rs1625649:C>A (pre-block SNP) 100/1000.6374 0.0000 0.0146 Five Factors: Pack-years of smoking; block 3; block 5; block 6; rs1625649:C>A 68/1000.5349 0.5398 0.3194 * CVC: cross-validation consistency

42. Conclusions DNA repair gene polymorphisms do contribute to lung cancer risk and large prospective studies are needed to validate the findings. The candidate gene and sequence-based SNPs selection approach have a high priority to find a biological relevant association but our knowledge on the function of different kind of SNPs are still limited. The candidate gene and Mapping-based SNPs selection approach may have an increased power to detect a haplotype-based disease risk, however, multiple testing is a big issue when analyzing many SNPs and interactions using different combinations.

43. The resources for molecular epidemiological studies in Nanjing, China  Lung Cancer Case-control Study >1200 lung cancer cases and matched controls >500 lung cancer cases with survival information  Breast Cancer Case-control Study >1000 breast cancer cases and matched controls  Gastric Cancer Case-control Study >800 gastric cancer cases and matched controls  Changzhou population-based cohort study Established baseline information and blood samples for 21,000 participants (30-75yrs) in the suburb of Changzhou city, Jiangsu province. Expect to recruit more than 40,000 in the region.

44. Nanjing Medical University Jiangsu Cancer Hospital First Affiliated Hospital of Nanjing Medical University Shanghai Cancer Hospital Wuhan Zhongnan Hospital Acknowledgement Dr. Qinyi Wei U T M.D. Anderson Cancer Center Dr. Dongxin Lin Chinese Academy of Medical Science Drs. Daru Lu; Li Jin Fudan University Dr. Wei Huang CHGC, Shanghai Dr. Tangchun Wu Huazhong University of Science & Technology The Molecular Epi Lab in NJMU

45. Thank you!