1. Molecular Epidemiology Studies of Indoor Air Pollution and Lung Cancer Qing Lan, M.D., Ph.D. Occupational Environmental Epidemiology Branch Division of Cancer Epidemiology and Genetics National Cancer Institute

2. Background: Indoor Air Pollution to Solid Fuels  Half of the world’s population is exposed to smoke from cooking or heating with solid fuels  Indoor air pollution from solid fuel use  the eighth largest risk factor for global disease  Coal is used extensively in China for cooking and heating, and is being increasingly utilized to generate electricity in many countries, including the United States

3. County-specific female lung cancer mortality rates (per 100,000, 1973-75) Lung Cancer Mortality Rates in Xuanwei are among the Highest in China Xuan Wei County

4. Xuanwei  Xuan Wei - rural county  Semi-mountainous region of Yuan Nan Province  >90% of the population are farmers  Very stable population  < 0.1% of females smoke  > 70% of males smoke

5. Firepit Used in Xuanwei

9. Average Age of Lung Cancer Patients at Death MaleFemale China60 Shanghai6162 Xuan Wei54 He et al., 1987

10. Annual Lung Cancer Mortality in the 1970s (1/100,000) (age Adjusted to 1964 Chinese Population) among Chinese Population, Xuanwei County, and Central Xuanwei (1973-79)

11. Unique Opportunity for Investigating Lung Cancer and Indoor Air Pollution  Wide range of lung cancer mortality rates cross Xuanwei communes  Very stable population with life-time exposure to smoky coal

12. Indoor Air Pollution to Solid fuels, Genetic Susceptibility, and Lung Cancer in Xuanwei, China  Initial work A.Smoky coal and lung cancer (cohort study) B.Coal type and lung cancer (case-control study) C.Molecular epidemiology and lung cancer (case-control study)  Ongoing/future work

13. A. Smoky Coal and Lung Cancer Association  Some homes replaced fire pits with stoves with chimneys  reduced exposure to coal combustion  Reduced exposure to coal combustion reduced risk of lung cancer and chronic obstructive pulmonary disease (COPD)?

14. Retrospective Cohort Study Goal: To test whether the incidence rates of lung cancer and COPD decreased in subjects who changed from a firepit to a stove with chimney among subjects used Lai Bin coal Birth periodFollow-up period 1917 1951 19761992 43,000 subjects 600,000 person-years 1,900 lung cancer cases 2,700 COPD cases | | | |

15. Stratified Cox Model Risk Ratios for Lung Cancer Incidence Rate, by Sex, in Xuanwei, 1976-1992 Stove improvement Males RR (95% CI) P Value Females RR (95% CI) P Value No stove improvement 1.00---1.00--- Changed to stove with chimney 0.59(0.49-0.71)<.0010.54(0.44-0.65)<.001 Lan et al., 2002 J Natl Cancer Inst.

16. Stove Improvement and Lung Cancer in Xuanwei, China Product-limit survival plot – Probability of not having lung cancer Males Females SC: with chimney NSI: without chimney Lan et al., 2002 J Natl Cancer Inst. P < 0.001

17. Chapman et al., 2005 BMJ Stove Improvement and COPD in Xuanwei, China Product-limit survival plot – Probability of not having COPD Females Males SC: with chimney NS: without chimney P < 0.001

18. Indoor Airborne Concentrations of PM10 and BaP in Study Area Homes, by Stove Type, Xuanwei Lan et al., 2002 J Natl Cancer Inst. PM10 BaP 2080 ug/m 3 710 ug/m 3 250 ng/m 3 1660 ng/m 3 BaP

19. Conclusion  Provided strong evidence of a causal association between smoky coal exposure and lung cancer and COPD risk  First evidence on the health benefits of stove improvement in households using coal or biomass fuel

20. B. Coal Types and Lung Cancer  The cohort study evaluated lung cancer risk from only one type coal, Lai Bin coal  Many other coal types are used as well  Association with lung cancer with other coal types not quantified

21. B. Coal Types and Lung Cancer  Goal: to examine the relationship between coal type and lung cancer risk  Population-based study, carried out 1985-1990  498 incident cases, 498 individually-matched on age, sex to controls  Participation rates > 95% for both cases and controls  Biologic samples were not collected

22. Odds Ratios (OR) of Lung Cancer in Relation to Coal Type Lan et al., Submitted P heterogeneity = 5.2x10 -10 27.1 4.8 1.3 Coal types

23. Odds Ratios (OR) of Lung Cancer in Relation to Coal Type, Stratified by Sex Lan et al., Submitted Coal types Male Female Coal types

24. 2.0 (0.9-4.6) 3775 >120 kg 1.1 (0.5-2.4) 3736 <=120 kg 1.0 (0.4-2.4) 3621 <=42.5 kg 13426 Never smokers Cigarette OR (95% CI) ControlsCases Tobacco and Lung Cancer Risk in Xuanwei in Men Adjusted for age, literacy, lung cancer in first-degree relatives, hours spent at home per day, lung disease history, coal mine work history, smoking, and coal type at birth

25. Conclusion  First etiologic study to report an example where lung cancer risk varies markedly by coal type.  Limited data show that PAH levels vary in emissions from these coals as well

26.  Evidence indicating PAHs are the key agents in the pathogenesis of lung cancer in this population  Substantial genetic variation in genes that activate and detoxify PAHs and repair DNA damage  Xuanwei provides a unique model of PAH exposure and lung cancer where tobacco does not play a large role C. Molecular Epidemiology Case-Control Study -- A Pilot Study

27.  Population-based case-control study, carried out in 1995-96  122 incident lung cancer cases and 122 controls individually matched on age and sex  Participation rates > 95% in cases and controls  Collected buccal cell and sputum samples  First epidemiologic study collected biologic samples in this region Molecular Epidemiology Case-Control Study -- A Pilot Study

28. Benzo(a)pyrene B(a)P epoxide B(a)P diol B(a)P diolepoxide CYP1A1, 1A2, 1B1, 3A4, 2C mEH CYP1A1, 1A2,1B1; MPO GSTM1 Detoxification DNA Adduct, repaired by NER pathway Catechol Quinones AKR1C3 O2O2 ROSNQO1 UGT1A6 S-Q UGT2B7 Detoxification Causes oxidative damage, repaired by BER, DSB pathways Detoxification OGG1 Key Pathways in B(a)P Metabolism

29. GSTM1 null Genotype  GSTM1 null genotype  lack of GSTM1 enzyme activity  Results in decreased detoxification of PAH metabolites

30. a ORs and 95% CIs adjusted for total smoky coal use without ventilation, pack -years, COPD, and family history of lung cancer by multiple conditional logistic regression. GSTM1 Genotype and Lung Cancer Risk GSTM1Case N (%) Control N (%) OR a (95%CI) Positive40(32.8)62(49.2) 1.0 Null82(67.2)60(50.8) 2.3 (1.3-4.2) Lan et al., 2000 CEBP

31. GSTM1 Genotype and Lung Cancer Risk, Stratified by Sex GSTM1 Genotype Case N (%) Control N (%) OR (95%CI) Males Positive27 (34.18)38 (48.10) Null52 (65.82)41 (51.90)1.8 (0.9-3.5) Females Positive13 (30.23)24 (55.81) Null30 (69.77)19 (44.19)3.3 (1.3-8.4)

32. Lan et al., 2000 CEBP Smoky coal use (tons) GSTM1 Positive Null <1301.0 (ref) 1.8 (0.8-4.1)  130 1.8 (0.7-4.6) 5.2 (2.1-12.6) Interaction between GSTM1 Genotype and Smoky Coal Use and Lung Cancer Risk

33. Interaction between GSTM1 Null Genotype and Smoky Coal Exposure  GSTM1-positive subjects: the risk of lung cancer increased by 1.2-fold per 100 tons life-time use  GSTM1-null genotype subjects: risk of lung cancer increased by 2.4-fold per 100 tons (test for multiplicative interaction, P = 0.05)

34. Benzo(a)pyrene B(a)P epoxide B(a)P diol B(a)P diolepoxide CYP1A1, 1A2, 1B1, 3A4, 2C mEH CYP1A1, 1A2,1B1; MPO Detoxification DNA Adduct, repaired by NER pathway Catechol Quinones AKR1C3 O2O2 ROSNQO1 UGT1A6 S-Q UGT2B7 Detoxification Causes oxidative damage, repaired by BER, DSB pathways Detoxification Key Pathways in B(a)P Metabolism

35. Aldo-keto Reductase Family 1, Member C3 (AKR1C3 Gln5His)  AKR1C3-Gln5His polymorphism (Exon 1)  Produces a change from glutamine  histidine  A shift from a neutral to a basic amino acid

36. AKR1C3-Gln5His Polymorphism and Lung Cancer Risk Lan et al., 2004 Carcinogenesis GenotypeCaseControlsOR (95%CI) N (%) His/His + His/Gln22 (19)33 (29)1.0 Gln/Gln94 (81)79 (71)1.8 (1.0-3.5)

37. AKR1C3 Genotype and Lung Cancer Risk, Stratified by Sex and Smoky Coal Use ORs and 95%CIs obtained by logistic regression analysis adjusted for age, sex, pack-year of smoking. 0.9 (0.2-5.0)1.5 (0.5-4.4) 1.0 Males His/His+His/Gln Gln/Gln 13.2 (2.3-109.2) 1.00 (0.2-5.8)Gln/Gln 1.0 His/His+His/Gln Females >=130 tons<130 tons Genotype Smoky Coal Use (tons) OR (95% CI) Lan et al., 2004 Carcinogenesis

38. Benzo(a)pyrene B(a)P epoxide B(a)P diol B(a)P diolepoxide CYP1A1, 1A2, 1B1, 3A4, 2C mEH CYP1A1, 1A2,1B1; MPO Detoxification DNA Adduct, repaired by NER pathway Catechol Quinones O2O2 ROSNQO1 UGT1A6 S-Q UGT2B7 Detoxification Causes oxidative damage, repaired by BER, DSB pathways Detoxification OGG1 Key Pathways in B(a)P Metabolism

39. OGG1 (Oxoguanine glycosylase 1) Ser326Cys polymorphism OGG1 repairs 8-oxo-7,8-dihydroguanine (8oxoG) OGG1 Ser326Cys polymorphism (Exon 6) Repair activity of OGG1-Ser326 has been shown to be > OGG1-Cys326

40. OGG1 Ser326Cys Polymorphism and Lung Cancer Risk ORs and 95%CIs obtained by logistic regression analysis adjusted for age, sex, pack-year of smoking. Lan et al., 2004 Carcinogenesis OGG1 Genotype Case N (%) Control N (%) OR (95%CI) Ser/Ser37 (31)51 (47)1.0 Ser/Cys +Cys/Cys 81 (69)58 (53)1.9 (1.1-3.3)

41. OGG1 Genotype and Lung Cancer Risk, Stratified by Sex and Smoky Coal Use ORs and 95%CIs obtained by logistic regression analysis adjusted for age, sex, pack-year of smoking. 2.0 (0.7-5.6)1.2 (0.5-2.9) 1.0 Males Ser/Ser Gln/Gln + Cys/Cys 5.8 (1.1-34.8) 1.3 (0.3-5.3)Gln/Gln + Cys/Cys 1.0 Ser/Ser Females >=130<130 Genotype Smoky Coal Use (tons) OR (95% CI) Lan et al., 2004 Carcinogenesis

42. Limitations  Small sample size can lead to false positive findings  Critical to replicate and extend findings

43. GSTM1 Genotype and Lung Cancer in Asian Populations with Indoor Air Pollution Exposure to Coal Combustion Products: a Meta-Analysis Hosgood et al., 2007 Mutat Res. 1.64 (1.25-2.14) 560 cases and 635 controls Study – Lan, 2000 Chen, 2006 Wang, 2003 Yang, 2004 Overall (95% CI)

44. Recent Meta-analysis of the GSTM1 Null Genotype and Lung Cancer in General Population  General population 130 studies, 23,452 lung cancer cases and 30,397 controls: OR = 1.18 (95% CI: 1.14–1.23)  Sample size > 500: OR = 1.04 (95% CI: 0.95–1.14)  Asian Populations with Indoor Air Pollution Exposure to Coal Combustion Products: OR = 1.64 (1.25–2.14)  GSTM1 null genotype may influence risk of lung cancer in populations exposed to high levels of indoor combustion products containing PAHs.

45. Conclusions of Initial Xuanwei Lung Cancer Studies  Smoky coal with high levels of PAH is the main cause of lung cancer  Variants in genes involved in activation and detoxification of PAH may modify the smoky coal-lung cancer association, particularly in women

46. Impact of Research Findings  IARC monograph, “Carcinogenicity of household use of solid fuels and high temperature frying ” classified indoor emissions from household combustion of coal as “carcinogenic to humans - Group 1” (October 16, 2006)

47. Unanswered Questions  What is the dose-response relationship between PAH exposure and lung cancer risk?  What is the role of genetic susceptibility for lung cancer risk overall and as a modifier of the PAH association?  What are the unique characteristics of tumors in this population?

48. Initiation of a New Case-Control of Lung Cancer among Nonsmoking Women in Xuan Wei (2006-2008)  Enroll 750 non-smoking female cases and 750 female controls  Biological sample collection: collect blood, sputum, buccal cells, and tumor tissue  Questionnaire collects extensive information on lifetime exposure to smoky coal, potential confounders  Detailed exposure assessment including personal and stationary air monitoring Yunnan Province

49. Advantages of the New Study  Enhanced diagnostic accuracy (special cytologic methods)  Increased sample size with adequate power to detect magnitude of effects observed previously  Extensive collection and processing of biologic samples  Will provide new mechanistic insights into the pathogenesis of lung cancer  Will provide important quantitative risk estimates from coal combustion and PAH exposure (US EPA)

50.  About 300 cases and 300 controls have been enrolled to date  Subject enrollment will be completed in 18 months  Participation rates have been > 90% for both cases and controls Accomplishments to Date of New Lung Cancer Study

51. Future Directions  Genetic analysis in case-control study of nonsmoking women in XW  Tag SNP and functional variants in candidate genes important for PAH  Also include SNPs in genes identified from ongoing genome-wide association  studies of lung cancer  Analyze main effects and interaction with PAH exposure  Detailed exposure assessment  International Lung Cancer Consortium (ILCCO)  Co-chair of working group on non-smoking lung cancer  Planning pooled analyses to study risk factors for lung cancer in various non- smoking populations

52. Collaborators China EPA: Fusheng Wei China CDC: Xingzhou He U.S. EPA: Robert Chapman, Judy Mumford University of Utrecht: Roel Vermeulen IARC: Pierre Hainaut UC Berkeley: Kathy Hammond University of Utah: Richard Cawthon Chinese University of Hong Kong: Linwei Tian U.S. NCI: Min Shen, Roel Vermeulen, Nat Rothman, Neil Caporaso, Aaron Blair, Jay Lubin, Michael Alavanja, Stephen Chanock

55. Correlation between BaP Exposures from a Specific Coal Types and Lung Cancer Risk