1. C ANCER M OLECULAR E PIDEMIOLOGY Epidemiology 242 2009

2. N UMBERS OF P APERS /Y EAR P UBLISHED WITH S UBJECT W ORDS “M OLECULAR E PIDEMIOLOGY ” U SING P UBMED S EARCH

3. E VOLUTION OF E PIDEMIOLOGY IN H ISTORY Systematic collection and analysis of vital statistics Defined triad of agent-host-vector for both infectious and chronic disease Refine exposure assessment such as job- exposure matrix, dietary and nutritional analysis Defined study design such as case-control and cohort study Use of the advance of statistical and computational capacities (MLE, Logistic regression, poission regression)

4. E VOLUTION OF E PIDEMIOLOGY Now, it is the time to add biological variables (physiologic, cellular, subcellular, molecular levels), which can be assayed by technically powerful biological methods Molecular epidemiology is the use of these biological markers in epidemiology research.

5. E PIDEMIOLOGY AND M OLECULAR S CIENCES E PIDEMIOLOGY M OLECULAR S CIENCES Health effects in grouped people Observation and inference of association between variables Macro Assessment of the individual at the component level Experimental proof of cause and effects Micro

6. M OLECULAR E PIDEMIOLOGY AND T RADITIONAL E PIDEMIOLOGY These capacities provide additional tool for epidemiologists studying questions on etiology, prevention and control of diseases Although molecular epidemiology can be viewed as an evolution step of epidemiology, it generally dose not represent a shift in the basic paradigm of epidemiology

7. T RADITIONAL AND M OLECULAR E PIDEMIOLOGY T RADITIONAL M OLECULAR Association High exposure and single outcome Prevention through control of exposure is feasible without understanding cellular process Mechanisms Smaller and mixed exposures; multicausal Intervention through cellular process has the need to understand mechanisms of the process

8. B ASICS OF M OLECULAR E PIDEMIOLOGY The term of molecular epidemiology indicates the incorporation of molecular, cellular, and other biological measurements into epidemiologic studies

9. M OLECULAR E PIDEMIOLOGY studies utilizing biological markers of exposure, disease and susceptibility studies which apply current and future generations of biomarkers in epidemiologic research.

10. F UNCTIONAL D EFINITION OF M OLECULAR E PIDEMIOLOGY The use of biologic markers or biologic measurements in epidemiologic research. Biological markers (or biomarkers) generally include biochemical, molecular, genetic, immunologic, or physiologic signals of events in biologic system.

11. M OLECULAR E PIDEMIOLOGY The goal of molecular epidemiology is to supplement and integrate, not to replace, existing methods Molecular epidemiology can be utilized to enhance capacity of epidemiology to understand disease in terms of the interaction of the environment and heredity.

12. C APACITIES OF M OLECULAR E PIDEMIOLOGY Identification of Exposure at the smaller scale Identification of events earlier in the nature history of disease Evaluation of gene-environment interaction In addition, it can be used to reduce misclassification, to indicate mechanisms, and enhance risk assessment

14. S TUDY OF B LACK B OX The concept of a continuum of events between exposure and disease provide opportunities To ensure that epidemiologic research has a biological basis for hypothesis To provide the analysis to test these ideas To generate new epidemiological methods to deal with new challenges

15. Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

18. M EASUREMENT OF B IOMARKERS Biomarkers can be measured quantitatively or qualitatively by biochemical, immunochemical, cytogentic, molecular and genetic techniques.

19. M ATERIALS FOR B IOMARKER M EASUREMENT Biomarkers can be measured in human biological materials including normal and tumor tissues, blood and urine sample, etc.. Their biological nature can be DNA, RNA, and protein, etc.

20. S TUDY Q UESTIONS : E XPOSURE M ARKERS How reliable are the exposure data obtained by questionnaire and what type of misclassification bias result? How are the carcinogens metabolized? What are the dynamics and distribution of carcinogen metabolization? What is the concentration of carcinogens in peripheral blood? What is the exposure level in the target tissue? Can we employ the exposure markers measured in peripheral blood to predict the concentrations of exposure at the target tissue?

21. E XPOSURE M EASUREMENTS The powerful tools of molecular biology, analytical chemistry, and related disciplines allow measure smaller amounts of exposures (10 -18 -10 -21 ) Reconstruct past exposure doses using molecular measurements (biologic dosimetry)

22. E XPOSURE B IOMARKERS Mutagenesis vol. 24,117–125, 2009

23. E XPOSURE M ARKERS : DNA A DDUCTS Exposure markers are a group of biomarkers, which can indicate the environmental exposures and can be measured in tumor tissues, or blood or urine specimens. The presence or concentration of specific environmental carcinogens or other agents can be measured in biological specimens, for example, blood levels of cotinine, polycyclic aromatic hydrocarbon (PAH) -DNA adducts, 4-aminobiphenyl (4- ABP) hemoglobin adducts.

24. E XPOSURE M ARKERS : DNA A DDUCTS exposure markers measure biological effective dose, that is, the amount of carcinogens bound to DNA in the target tissue such as DNA-adducts, or surrogate measurements which can represent the exposure levels of the target tissue such as hemoglobin adducts

25. E XPOSURE M ARKERS Aromatic Amines and 4-ABP DNA- Adducts. The human bladder carcinogens 2-naphtylamine and 4-aminobiphenyl, as well as the suspected carcinogen o- toluidine, are present in tobacco and certain occupational exposures. DNA adducts of 4-aminobiphenyl were found in tumor samples from smokers indicating that this agent may account for some of the carcinogenicity of tobacco smoke

26. E XPOSURE M ARKERS Polycyclic Aromatic Hydrocarbons (PAH) and PAH DNA-Adducts. PAHs are produced by incomplete combustion of organic materials and the sources of environmental PAH include industrial and domestic furnaces, gasoline and diesel engines and tobacco smoke. PAHs are carcinogens requiring metabolic activation to react with cellular macromolecules, the initial step in tumorigenesis

27. PHIP DNA Adducts

28. P32 postlabel ing

30. L IMITATIONS OF E XPOSURE M ARKERS These markers have to be measured in biological materials, which requires the collection of biological specimens; Some of exposure markers such as hemoglobin-adducts and blood level of cotinine only represent the current exposure status; The costs for measurement of exposure markers are generally more expensive than that of questionnaire data.

31. S TUDY Q UESTIONS : S USCEPTIBILITY G ENES Which gene or enzymes are involved? Is there any metabolic phenotype related to the risk of cancer? Are there any high risk individuals who are susceptible to cancer and how can we identify them?

32. S USCEPTIBILITY M ARKERS Susceptibility markers represent a group of tumor markers, which may make an individual susceptible to cancer. These markers may be genetically inherited or determined. They are independent of environmental exposures.

33. S USCEPTIBILITY M ARKERS Tumor susceptibility markers such as P450s, GSTs, and NATs, act in enzymatic pathways related to metabolizing and eliminating carcinogens.

34. S USCEPTIBILITY M ARKERS The phase I enzymes such as p450 enzyme superfamily metabolize exogenous or endogenous agents or carcinogens to intermediates, which can result in DNA damages and act as risk factors for cancer. The phase II enzymes such as glutathione S-transferase (GST) system are dealing with detoxification of oxygenated intermediates by conjugation process, acting as a protective factors for cancer.

35. Figure. GSTM1 and GSTT1 genotyping from buccal cell DNA. Case 5 is null for the GSTT1 genotype. Case 2 is null for the GSTM1 genotype GST T1 GST M1 beta-globin Case 1 Case 2 Case 3 Case 4 Case 5

36. Figure. GSTP1 polymorphism Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 ile/val ile/val ile/ile val/val ile/val ile/ile ile/val ile/ile

37. 14 13 12 11 10 9 8 7 6 5 4 3 2 1 PCR P450 2E1 after Using Pst1 RFLP

38. Case 1 Case 2 Case 3 Case 4 Case 5 Figure. P53 polymorphism at codon 72 from buccal cell DNA. Arg/Arg Arg/Arg Pro/Pro Arg/Arg Arg/Pro

39. Interactions between smoking and GST M1 (odds ratios* and 95% confidence intervals) *Adjusted for age, sex, race, and level of education 1.00 1.13 (0.32, 3.95) 2.79 (0.97, 7.99) 5.29 (1.81, 15.4)

40. Kingsmore, 2008

41. I SSUES IN GWAS S TUDIES False positive (multiple comparison) False negative (very small p-value) Population stratification Gene-Environmental Interaction

42. B ACKGROUND In 2006 and 2007 GWAS studies identified associations between SNPs in the 8q24 region and prostate cancer among Icelandic, Swedish, European American, African American, and the Multiethnic Cohort populations.

44. R ESEARCH Q UESTIONS : G ENETIC AND M OLECULAR A LTERATIONS What kinds of damages do the carcinogens make, and is the damage specific? Does the DNA repair capacity affect risk and how can we measure it? Is there any gene-gene interaction and is there any gene-environment interaction?

45. I DENTIFICATION OF E ARLIER E VENTS Identification of the patients at a very early stage - for better treatment and prognosis to improve the survival of cancer Identification of pre-malignant lesions - for intervention and early treatment to reduce the incidence of cancer

46. E ARLY B IOLOGICAL R ESPONSE : M OLECULAR G ENETIC A LTERATIONS Molecular genetic markers are defined as a group of markers which can be induced by certain carcinogens or by some intermediate end-point

47. E ARLY B IOLOGICAL R ESPONSE : M OLECULAR G ENETIC A LTERATIONS cytogenetic markers such as chromosome abnormalities by karyotyping; oncogenes such as RAS family; tumor suppressor genes such as TP53 and p16 genes.

48. P53 G ENE M UTATIONS TP53 Mutations as DNA Fingerprints of Environmental Exposures. The wide range of involvement of TP53 in human tumors and the broad spectrum of mutations make this gene a good candidate for molecular epidemiological studies

53. Case 607 Exon 8 1 2 3 Case 644 Exon 7 G A T C G A T CG A T C A C/G A A G A Thr Arg GlySer C G A/G G G C MutantWild TypeMutantWild Type A C/G A A/G G C Codon 280Codon 244

54. Figure 8-1. IHC Analysis of p53, p21, and mdm2

55. Figure 11. GST P1 methylation from lung cancer tissue. (U=unmethylated, M=methylated) Case 3 is unmethylated. Case 1 Case 2 Case 3 (unmethylated) (unmethylated) (methylated)

56. A GE AND TP53 M UTATIONS AgeP53+ No. (%) P53- No. (%) Total No. (%) <506 (8.7)11 (10.0)17 (9.5) 50-5916 (23.2)18 (16.4)34 (19.0) 60+47 (68.1)81 (73.6)128 (71.5)

57. G ENDER AND TP53 M UTATIONS GenderTP53+ No (%) TP53- No (%) Total No (%) Male47 (71.2)89 (81.7)136 (77.7) Female19 (28.8)20 (18.4)39 (22.3)

58. R ACE AND TP53 M UTATIONS RaceTP53+ No (%) TP53- No (%) Total No. (%) White60 (87.0)100 (90.9)160 (89.4) Non-White9 (13.0)10 (9.1)19 (10.6)

59. E DUCATION AND TP53 M UTATIONS Education (years) TP53+ No. (%) TP53- No. (%) Total No. (%) <122 (2.9)4 (3.6)6 (3.4) 12-1658 (84.1)76 (69.1)134 (74.9) >169 (13.0)30 (27.3)39 (21.8)

60. TP53 M UTATIONS IN B LADDER C ANCER BP changesReported, n=200Current study Transitions GC  AT 41.0%37.5% (at CpG)14.0%12.5% AT  GC 10.0%15.0% Transversions GC  TA 13.0%12.5% GC  CG 19.0%10.0% AT  TA 3.0%0.0% AT  CG 2.0%2.5% Deletion/Insert.12.0%10.0%

61. S MOKING AND TP53 M UTATIONS IN B LADDER C ANCER SmokingTP53+TP53-OR95%CI No8241.00 Yes58836.271.29-30.2 Adjusted for age, gender, and education

62. C IGARETTES / DAY AND TP53 M UTATIONS IN B LADDER C ANCER Cig/dayTP53+TP53-OR95%CI No8241.00 1-208212.070.22-19.9 21-4036475.501.08-28.2 >40171810.41.90-56.8 TrendP=0.003 Adjusted for age, gender, and education

63. Y EARS OF S MOKING AND TP53 M UTATIONS IN B LADDER C ANCER Years of smoking TP53+TP53-OR95%CI No8241.00 1-205105.640.82-38.7 21-4042586.451.24-33.4 >4014186.201.17-32.8 TrendP=0.041 Adjusted for age, gender and education

64. R EDUCTION OF M ISCLASSIFICATIONS Better classification of exposures by using markers of internal and biological effective doses. More homogeneous disease grouping by using marker of effect such as specific mutations. Reduced misclassification may lead to increased validity and precision of point estimates

65. I NDICATION OF M ECHANISMS Test association between mechanistic events in a defined continuum Knowledge of the mechanisms can guide future research and intervention applications

66. V ARIABILITY AND E FFECT M ODIFICATION Individual variability of susceptibility may be related to host factors such as genetic factors Effect modification can be evaluated between genetic susceptibility markers and exposure on the risk of cancer

67. E NHANCED I NDIVIDUAL AND G ROUP R ISK A SSESSMENT Providing more person-specific information allowing extrapolation of risk from one group to another, from animal species to humans, and from one group to individuals

69. E XAMPLE : S MOKING AND L UNG C ANCER Internal Dose (ID). The amount of a xenobiotic substance or its metabolites found in a biologic medium: e.g., Serum cotinine as an indicator of nicotine. Biologic Effective Dose (BED). The integration of exposure and effect modification by the host: e.g., DNA adducts of PAH in lung tissue.

70. E XAMPLE : S MOKING AND L UNG C ANCER Early Biologic Effect (or biological response) are biological or biochemical changes in target cells or tissues that result from the action of the chemical and are thought to be a step in the pathologic process toward disease, e.g., tumor suppressor gene TP53 mutations in lung cancer.