Summary of Molecular Cancer Epidemiology

1. Summary of Molecular Cancer Epidemiology EPI243: Molecular Cancer Epidemiology Zuo-Feng Zhang,MD, PhD

2. Molecular Epidemiology • 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.

3. Molecular Epidemiology • studies utilizing biological markers of exposure, disease and susceptibility • studies which apply current and future generations of biomarkers in epidemiologic research.

5. Tasks for Molecular Epidemiologist The major tasks are • to reduce misclassification of exposure, • to assess effect of exposure on the target tissue, • to measure susceptibility/inherited predisposition to cancer, • to establish the link between environmental exposures and gene mutations, • to assess gene-environment interaction. • To set up prevention/intervention strategies.

6. High Throughput Techniques • Microarray technology • DNA chips • cDNA array format • in situ synthesized oligonucleotide format (Affymetrix) • Proteomics • Tissue arrays • These are powerful tools and high through put methods to study gene expression, but they are not the answers themselves • Individual targets/patterns identified need to be validated • In epidemiological studies, these methods can be used to identify specific exposure induced molecular changes, individual risk assessments, etc.

7. An example of our 9000 gene mouse-arrays using differential expression analysis with Cy3 and Cy5 fluorescent dyes.

8. Proteomics • Examine protein level expression in a high throughput manner • Used to identify protein markers/patterns associated with disease/function • Different formats: • SELDI-TOF (laser desorption ionization time-of-flight): the protein-chip arrays, the mass analyzer, and the data-analysis software • 2D Page coupled with MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) • Antibody based formats

10. Fig 1 3.5 3.5 3.5 3.5 3.5 3.5 4.5 4.5 4.5 4.5 4.5 4.5 5.1 5.1 5.1 5.1 5.1 5.1 5.5 5.5 5.5 5.5 5.5 5.5 6.0 6.0 6.0 6.0 6.0 6.0 7.0 7.0 7.0 7.0 7.0 7.0 8.4 8.4 8.4 8.4 8.4 8.4 9.5 9.5 9.5 9.5 9.5 9.5 217 217 116 116 98 98 55 55 37 37 30 30 20 20 A, GTE (20g/ml) pI 9 MW (kDa) 8 10 9 8 2 2 10 1 1 5 5 11 11 13 13 17 7 7 6 6 17 18 18 16 16 12 12 14 14 3 3 15 15 4 4 B, GTE (40g/ml) pI 20 19 MW (kDa) 1 1 10 5 10 5 11 11 13 13 17 17 18 12 18 16 16 12 14 14 15 15 4 Time: 48 hr 24 hr 48 hr - + + GTE:

11. Tissue Array • Provide a new high-throughput tool for the study of gene dosage and protein expression patterns in a large number of individual tissues for rapid and comprehensive molecular profiling of cancer and other diseases, without exhausting limited tissue resources. • A typical example of a tissue array application is in searching for oncogenes amplifications in vast tumor tissue panels. Large-scale studies involving tumors encompassing differing stages and grades of disease are necessary to more efficiently validate putative markers and ultimately correlate genotypes with phenotypes. • Also applicable to any medical research discipline in which paraffin-embedded tissues are utilized, including structural, developmental, and metabolic studies.

12. Bladder Array Gelsolin HE

13. DNA Methylation DNA methylation plays an important role in normal cellular processes, including X chromosome inactivation, imprinting control and transcriptional regulation of genes It predominantly found on cytosine residues in CpG dinucleotide, CpG island, to producing 5-Methylcytosine CpG islands frequently located in or around the transcription sites

14. DNA Methylation (Cont’d) Aberrant DNA methylation are one of the most common features of human neoplasia Two major potential mechanisms for aberrant DNA methylation in tumor carcinogenesis Silencing tumor suppressor genes (e.g. p16 gene) Point mutation: C to T transition (e.g. P53 gene) Source:Royal Society of Chemistry

15. Promoter-Region Methylation • Promoter-region CpG islands methylation • Is rare in normal cells • Occur virtually in every type of human neoplasm • Associate with inappropriate transcriptional silence • Early event in tumor progression • In tumor suppressor genes • Most of the tumor suppressor genes are under-methylated in normal cells but methylated in tumor cells. Methylation is often correlated with an decreasing level of gene expression and can be found in premalignant lesions

16. DNA methyltransferases DNMTs catalyze the transfer of a methyl group (CH3) from S-adenosylmethionine (SAM) to the carbon-5 position of cytosine producing the 5-methylcytosine There are several DNA methyltransferases had been discovered, including DNMT1, 3a, and 3b

17. NORMAL CIN 1 CIN 2 CIN 3 NORMAL LGSIL HG SIL HGSIL

18. Additional Molecular Event Exposure to Carcinogen Precancerous Intraepithelial Lesions, (PIN, CIN, PaIN..) Cancer Birth Surrogate End Point Markers Markers for Exposure Markers of Effect Tumor Markers Genetic Suscep. Marker CHEMOPREVENTION

22. Case-Control Studies • Disease end-point as a major interest • Clinical (Hospital)-based or population-based case-control studies • Inclusion of both questionnaire data and biological specimens • Biological markers can be measured and compared between cases and controls when other variables can be used as either confounding factors or effect modifiers

23. Prospective Cohort Studies • Exposure is measured before the outcome • The source population is defined • The participation rate is high if specimen are available for all subjects and follow-up is complete

24. Nested Case-Control Study • The biomarker can be measured in specimens matched on storage duration • The case-control set can be analyzed in the same laboratory batch, reducing the potential for bias introduced by sample degradation and laboratory drift

25. Case-Case Study Design • Case-only, Case-series, etc. • Studies with cases without using controls • Can be employed to evaluate the etiological heterogeneity when studying tumor markers and exposure • May be used to assess the statistical gene-environment or gene-gene interactions

26. Intervention Studies • In studies of smoking cessation intervention, we can measure either serum cotinine or protein or DNA adducts (exposure) or p53 mutation, dysplasia and cell proliferation (intermediate markers for disease) • Measure compliance with the intervention such as assaying serum b-carotene in a randomized trial of b-carotene.

27. Intervention Studies Susceptibility markers (GSTM1) can also be used to determine whether the randomization is successful (comparable intervention and control arms)

28. Family Studies • Does familial aggregation exist for a specific disease or characteristic? • Is the aggregation due to genetic factors or environmental factors, or both? • If a genetic component exists, how many genes are involved and what is their mode of inheritance? • What is the physical location of these genes and what is their function?

29. Sample Size and Power • False positive (alpha-level, or Type I error). The alpha-level used and accepted traditionally are 0.01 or 0.05. The smaller the level of alpha, the larger the sample size.

30. Power or Sample Size Estimate for Case-Control Studies • Alpha-level (false positive): 0.05 • Beta-level (false negative level; 1-beta=power): 0.20 • Delta-level: Proportion of exposure in controls and exposure in cases or expected odds ratio

31. Interaction Assessment

32. Sample Size Consideration for Interaction Assessment • Evaluation of interaction requires a substantial increase in study size. For example, in a case-control study involves comparing the sizes of the odds ratios (relating exposure and disease) in different strata of the effect modifier, rather than merely testing whether the overall odds ratio is different from the null value of 1.0.

33. Introduction • Sample Collection, such as handling, labeling, processing, aliquoting, storage, and transportation, may affect the results of the study • If case sample are handled differently from controls samples, differential misclassification may occur

34. Information linked to Sample • Time and date of collection • Recent diet and supplement use, • Reproductive information (menstrual cycle) • Recent smoking • current medication use • Recent medical illness • Storage conditions

35. Quality Assurance Systematic Application of optimum procedures to ensure valid, reproducible, and accurate results

36. -70 freezers

37. Types of Biospecimens: Blood The use of skilled technicians and precise procedures when perform phlebotomy are important because painful, prolonged or repeated attempts at venepuncture can cause patient discomfort or injury and result in less than optimum quality or quantity of sample.

38. Types of Biospecimens: Blood • Plasma • Serum • Lymphocytes • Erythrocytes • Platelets

39. Urine Collection Urine is an ultrafiltrate of the plasma. It can be used to evaluate and monitor body metabolic disease process, exposure to xenobiotic agents, mutagenicity, exfoliated cells, DNA adducts, etc.

40. Tissue Collections • Confirming clinical diagnosis by histological analysis • Examining tumor characteristics at chromosome and molecular level

41. Laboratory Techniques with Tissue tissue RT-PCR

42. Adipose Tissue • Adipose tissue may be quite feasible for subject and involve low risk. The tissue offers a relatively stable deposit of triglyceride and fat-soluble substances such as fat-soluble vitamins (vitamins A and D). It represents the greatest reservoir of carotenoids and reflect long-term dietary intake of essential fatty acids.

43. Bronchoalveolar Lavage (BAL) • BAL is used to assess and quantify asbestos exposures • Induced sputum sample and BALF can also provide sufficient DNA for PCR assays.

44. Exhaled Air • To evaluate exposure to different substances, particularly solvents such as benzene, styrene • To be used as a source of exposure and susceptibility markers (caffeine breath test for p4501A2 activity) • Breath urea (presence of urease positive organisms such as H. pylori)

45. Hair • Easy available biological tissue whose typical morphology may reflect disease conditions within the body • Provides permanent record of trace elements associated with normal and abnormal metabolism • A source for occupational and environmental exposure to toxic metals

46. Nail Clippings • Toenail or fingernail clippings are obtained in a very easy and comfortable way. • They do not require processing, storage and shipping condition and thus suitable for large epidemiological studies

47. Buccal cells • No invasive • Good for PCR-analysis • Can measure both germline and somatic mutations

48. Saliva • It is an efficient, painless and relatively inexpensive source of biological materials for certain assays • It provides a useful tool for measuring endogenous and xenobiotic compounds

49. Breast Milk • Measuring hormones, exposures to chemicals and biological contaminants (Aflatoxin), selenium levels • Cells of interests

50. Feaces • Certain cells of interest • Infectious markers • Oncogenes