C ANCER M OLECULAR E PIDEMIOLOGY Epidemiology 242 2009

CANCER MOLECULAR EPIDEMIOLOGYEpidemiology 2422009

NUMBERS OF PAPERS/YEAR PUBLISHED WITH SUBJECT WORDS “MOLECULAR EPIDEMIOLOGY” USING PUBMED SEARCH

EVOLUTION OF EPIDEMIOLOGY IN HISTORY 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)

EVOLUTION OF EPIDEMIOLOGY

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.

EPIDEMIOLOGY AND MOLECULAR SCIENCES EPIDEMIOLOGY MOLECULAR SCIENCES

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

MOLECULAR EPIDEMIOLOGY AND TRADITIONAL EPIDEMIOLOGY

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

TRADITIONAL AND MOLECULAR EPIDEMIOLOGY

TRADITIONAL MOLECULAR

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

BASICS OF MOLECULAR EPIDEMIOLOGY

The term of molecular epidemiology indicates the incorporation of molecular, cellular, and other biological measurements into epidemiologic studies

MOLECULAR EPIDEMIOLOGY

studies utilizing biological markers of exposure, disease and susceptibility

studies which apply current and future generations of biomarkers in epidemiologic research.

FUNCTIONAL DEFINITION OF MOLECULAR EPIDEMIOLOGY 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.

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.

CAPACITIES OF MOLECULAR EPIDEMIOLOGY 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

STUDY OF BLACK BOX

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

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

MEASUREMENT OF BIOMARKERS

Biomarkers can be measured quantitatively or qualitatively by biochemical, immunochemical, cytogentic, molecular and genetic techniques.

MATERIALS FOR BIOMARKER MEASUREMENT

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.

STUDY QUESTIONS: EXPOSURE MARKERS 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?

EXPOSURE MEASUREMENTS

The powerful tools of molecular biology, analytical chemistry, and related disciplinesallow measure smaller amounts of exposures(10-18 -10-21)

Reconstruct past exposure doses using molecular measurements (biologic dosimetry)

EXPOSURE BIOMARKERS

Mutagenesis vol. 24,117–125, 2009

EXPOSURE MARKERS: DNA ADDUCTS

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.

EXPOSURE MARKERS: DNA ADDUCTS

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

EXPOSURE MARKERS

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

EXPOSURE MARKERS

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

PHIP DNA Adducts

P32 postlabeling

LIMITATIONS OF EXPOSURE MARKERS

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.

STUDY QUESTIONS: SUSCEPTIBILITY GENES 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?

SUSCEPTIBILITY MARKERS

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.

SUSCEPTIBILITY MARKERS

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

SUSCEPTIBILITY MARKERS

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.

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

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

14 13 12 11 10 9 8 7 6 5 4 3 2 1

PCR P450 2E1 after Using Pst1 RFLP

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

Interactions between smoking and GST M1(odds ratios* and 95% confidence intervals)

*Adjusted for age, sex, race, and level of education

0

1

2

3

4

5

6

Never/Positive

Never/ Null Ever/ Positive Ever/ Null

1.00 1.13 (0.32, 3.95)

2.79(0.97, 7.99)

5.29(1.81, 15.4)

ISSUES IN GWAS STUDIES

False positive (multiple comparison) False negative (very small p-value) Population stratification Gene-Environmental Interaction

BACKGROUND 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.

RESEARCH QUESTIONS: GENETIC AND MOLECULAR ALTERATIONS 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?

IDENTIFICATION OF EARLIER EVENTS

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

EARLY BIOLOGICAL RESPONSE: MOLECULAR GENETIC ALTERATIONS

Molecular genetic markers are defined as a group of markers which can be induced by certain carcinogens or by some intermediate end-point

EARLY BIOLOGICAL RESPONSE: MOLECULAR GENETIC ALTERATIONS

cytogenetic markers such as chromosome abnormalities by karyotyping;

oncogenes such as RAS family;

tumor suppressor genes such as TP53 and p16 genes.

P53 GENE MUTATIONS

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

Case 607 Exon 8

1 2 3 1 2 3

Case 644 Exon 7

G A T C G A T C G A T C G A T C

AC/G

A

AG

A

ThrArg Gly Ser

C

GA/GG

G

C

MutantWild Type MutantWild Type

A C/G A A/G G C

Codon 280 Codon 244

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

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)

AGE AND TP53 MUTATIONS

Age P53+

No. (%)

P53-

No. (%)

Total

No. (%)

<50 6 (8.7) 11 (10.0) 17 (9.5)

50-59 16 (23.2) 18 (16.4) 34 (19.0)

60+ 47 (68.1) 81 (73.6) 128 (71.5)

GENDER AND TP53 MUTATIONS

Gender TP53+

No (%)

TP53-

No (%)

Total

No (%)

Male 47 (71.2) 89 (81.7) 136 (77.7)

Female 19 (28.8) 20 (18.4) 39 (22.3)

RACE AND TP53 MUTATIONS

Race TP53+

No (%)

TP53-

No (%)

Total

No. (%)

White 60 (87.0) 100 (90.9) 160 (89.4)

Non-White 9 (13.0) 10 (9.1) 19 (10.6)

EDUCATION AND TP53 MUTATIONS

Education

(years)

TP53+

No. (%)

TP53-

No. (%)

Total

No. (%)

<12 2 (2.9) 4 (3.6) 6 (3.4)

12-16 58 (84.1) 76 (69.1) 134 (74.9)

>16 9 (13.0) 30 (27.3) 39 (21.8)

TP53 MUTATIONS IN BLADDER CANCER

BP changes Reported, n=200 Current study

Transitions

GC AT 41.0% 37.5%

(at CpG) 14.0% 12.5%

ATGC 10.0% 15.0%

Transversions

GCTA 13.0% 12.5%

GCCG 19.0% 10.0%

ATTA 3.0% 0.0%

ATCG 2.0% 2.5%

Deletion/Insert. 12.0% 10.0%

SMOKING AND TP53 MUTATIONS IN BLADDER CANCER

Smoking TP53+ TP53- OR 95%CI

No 8 24 1.00

Yes 58 83 6.27 1.29-30.2

Adjusted for age, gender, and education

CIGARETTES/DAY AND TP53 MUTATIONS IN BLADDER CANCER

Cig/day TP53+ TP53- OR 95%CI

No 8 24 1.00

1-20 8 21 2.07 0.22-19.9

21-40 36 47 5.50 1.08-28.2

>40 17 18 10.4 1.90-56.8

Trend P=0.003

Adjusted for age, gender, and education

YEARS OF SMOKING AND TP53 MUTATIONS IN BLADDER CANCER

Years of smoking

TP53+ TP53- OR 95%CI

No 8 24 1.00

1-20 5 10 5.64 0.82-38.7

21-40 42 58 6.45 1.24-33.4

>40 14 18 6.20 1.17-32.8

Trend P=0.041

Adjusted for age, gender and education

REDUCTION OF MISCLASSIFICATIONS 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

INDICATION OF MECHANISMS

Test association between mechanistic events in a defined continuum

Knowledge of the mechanisms can guide future research and intervention applications

VARIABILITY AND EFFECT MODIFICATION 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

ENHANCED INDIVIDUAL AND GROUP RISK ASSESSMENT

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

EXAMPLE: SMOKING AND LUNG CANCER 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.

EXAMPLE: SMOKING AND LUNG CANCER 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.