Aflatoxin B1, Hepatitis B, and Aflatoxin B1, Hepatitis B, and IFNA17IFNA17 on the Risk of Liver on the Risk of Liver

Cancer:Cancer:

An example of the application An example of the application of exposure markers in cancer of exposure markers in cancer

epidemiologyepidemiology

Binh Y. Goldstein, PhDBinh Y. Goldstein, PhDEpidemiology 243Epidemiology 243

IntroductionIntroduction

Background:Background:Epidemiology of liver cancerEpidemiology of liver cancer

Worldwide:Worldwide:• Sixth most common new cancerSixth most common new cancer• Third most common cause of cancer Third most common cause of cancer

deathdeath• Rates among men 2-3x higher than Rates among men 2-3x higher than

womenwomen• Over 80% of new cases occur in Over 80% of new cases occur in

developing countries developing countries • Low 5-year relative survival rates (<15%)Low 5-year relative survival rates (<15%)

Source: GLOBOCAN 2002Source: GLOBOCAN 2002

Age standardized incidence of Age standardized incidence of liver cancer in world among liver cancer in world among

menmen

Background:Background:Epidemiology of liver cancerEpidemiology of liver cancer

US:US:

• 19,200 new cases and 19,200 new cases and 16,800 deaths 16,800 deaths

• Five year survival rate is Five year survival rate is 10% 10%

• 88thth leading cause of deaths leading cause of deaths from cancers in both sexesfrom cancers in both sexes– 66thth for men for men– 1010thth for women for women

Source: American Cancer Society, Source: American Cancer Society, 20072007

China:China:

• 345,844 new cases and 345,844 new cases and 321,851 deaths 321,851 deaths

• accounts for over 53% accounts for over 53% of all liver cancer cases of all liver cancer cases and deaths worldwide and deaths worldwide

• 3rd most common 3rd most common cancer among mencancer among men

• Most common cause of Most common cause of death from cancer death from cancer among men among men

Source: GLOBOCAN 2002Source: GLOBOCAN 2002

Background:Background:Risk factors for liver cancerRisk factors for liver cancer

Hepatitis B virus:Hepatitis B virus:• 350 million people chronically infected 350 million people chronically infected

worldwide worldwide • About two-thirds of liver cancers in China and About two-thirds of liver cancers in China and

Southeastern Asia are attributed to HBV Southeastern Asia are attributed to HBV infection infection

• Chronic carriers have a 20-fold increase in risk Chronic carriers have a 20-fold increase in risk compared with non-carriers compared with non-carriers

• Major pathways by which HBV infection Major pathways by which HBV infection increases risk for liver cancer are: increases risk for liver cancer are: (1) viral DNA integration (1) viral DNA integration (2) oncogenic proteins(2) oncogenic proteins(3) inflammation(3) inflammation

Background:Background:Risk factors for liver cancerRisk factors for liver cancer

Aflatoxin B1:Aflatoxin B1:• Toxin found in mildewed grains and nuts Toxin found in mildewed grains and nuts • Bioactivated intermediate AFB1-Bioactivated intermediate AFB1-exoexo-8,9--8,9-

epoxide has carcinogenic effectepoxide has carcinogenic effect• Adducts associated with increased risk of Adducts associated with increased risk of

liver cancer liver cancer • Associated with a specific mutation in Associated with a specific mutation in

codon 249 of p53 tumor suppressor genecodon 249 of p53 tumor suppressor gene• Potential multiplicative interaction with Potential multiplicative interaction with

Hepatitis B viral infection (HBV)Hepatitis B viral infection (HBV)

AFB1 AFB1-exo-8,9-epoxide

AFM1AFQ1AFB1-endo-8,9-epoxide

dietary intake

CYP3A4(CYP1A2

)

DNA-adducts

glutathione-AFB1 conjugate

AFB1-8,9-dihydrodiol

[phenolate resonance form]

protein adducts

excretion

excretion

GST-μ,(GST-θ)

+ glutathion

e

H2O(mEH)

CYPs

Background:Background:Metabolism of aflatoxin B1Metabolism of aflatoxin B1

Background:Background:

Other risk factors:Other risk factors:

• Hepatitis C virusHepatitis C virus

• Alcohol Alcohol consumptionconsumption

• Tobacco smokingTobacco smoking

• Contaminated Contaminated drinking water drinking water (microcystins)(microcystins)

Potential Potential protective protective factorsfactors

• AntioxidantsAntioxidants

• Dietary nutrientsDietary nutrients

• SeleniumSelenium

Background: Background: IFNA17IFNA17 • Located at position 9p22Located at position 9p22• Encodes interferon, alpha 17Encodes interferon, alpha 17• Has viral inhibitory and viral anti-proliferative Has viral inhibitory and viral anti-proliferative

effects effects • Interferon, alpha investigated as a treatment for Interferon, alpha investigated as a treatment for

HBV and HCV infection and prevention of liver HBV and HCV infection and prevention of liver cancer among HBV infected individualscancer among HBV infected individuals

• IFNA17IFNA17 has a polymorphic site that results in either has a polymorphic site that results in either an arginine or isoleucine amino acid in codon 184an arginine or isoleucine amino acid in codon 184

• The 184The 184ArgArg allele has a higher frequency in allele has a higher frequency in Chinese populations (~50%) than in other Chinese populations (~50%) than in other populations (<35%)populations (<35%)

• Studied in cancers that have a viral component, Studied in cancers that have a viral component, including cervical cancer (papillomavirus) and including cervical cancer (papillomavirus) and nasopharyngeal cancer (Epstein-Barr virus) nasopharyngeal cancer (Epstein-Barr virus)

ObjectivesObjectivesOverall objective:Overall objective:• Gain insight into the mechanism of interaction Gain insight into the mechanism of interaction

observed between aflatoxin and HBV infectionobserved between aflatoxin and HBV infectionHypothesis:Hypothesis:• AFB1 may differentially suppress IFNA17 protein AFB1 may differentially suppress IFNA17 protein

activity, thereby increasing a person’s activity, thereby increasing a person’s susceptibility to the sequelae of HBV if chronically susceptibility to the sequelae of HBV if chronically infected and increasing the risk for liver cancer infected and increasing the risk for liver cancer

Specific Aims:Specific Aims:• Assess the independent effects of Assess the independent effects of IFNA17IFNA17

Ile184ArgIle184Arg polymorphic site on liver cancer risk polymorphic site on liver cancer risk• Explore the joint effects and three-way interaction Explore the joint effects and three-way interaction

among HBsAg-positivity, AFB1-albumin adduct among HBsAg-positivity, AFB1-albumin adduct level, and level, and IFNA17 IFNA17 polymorphisms on the polymorphisms on the development of liver cancerdevelopment of liver cancer

Research Design and Research Design and MethodsMethods

Study DesignStudy Design

• Population-based case-control study with 204 Population-based case-control study with 204 incident liver cancer cases (57% of all cases) incident liver cancer cases (57% of all cases) and 415 randomly selected healthy and 415 randomly selected healthy population controls (89% response rate) in population controls (89% response rate) in Taixing City, ChinaTaixing City, China

• Collected epidemiologic data and blood Collected epidemiologic data and blood specimens specimens

• IFNA17IFNA17 genotyped using PCR-RFLP genotyped using PCR-RFLP

• Markers used to assess HBV chronic infection Markers used to assess HBV chronic infection and aflatoxin B1 exposure (and HCV and aflatoxin B1 exposure (and HCV infection) infection)

Taixing City, ChinaTaixing City, China• Located on the east bank of the Yangtze River in Located on the east bank of the Yangtze River in

middle of Jiangsu province middle of Jiangsu province • Consists of 24 small villages, with estimated Consists of 24 small villages, with estimated

population of 1.28 million (660,000 males and population of 1.28 million (660,000 males and 627,000 females), most of whom are farmers 627,000 females), most of whom are farmers

• Has a high rate of alimentary cancer, among the Has a high rate of alimentary cancer, among the highest in the world highest in the world

• Has a tumor registry Has a tumor registry • After esophageal cancer, liver cancer is the After esophageal cancer, liver cancer is the

second largest cause of deaths from cancers second largest cause of deaths from cancers • In 2000, crude incidence rates for top three In 2000, crude incidence rates for top three

cancers - 65.2 (esophageal), 55.6 (liver), 54.8 cancers - 65.2 (esophageal), 55.6 (liver), 54.8 (stomach) per 100,000 (stomach) per 100,000

• Incidence rate of liver cancer for males Incidence rate of liver cancer for males (84.6/100,000) is over three times the rate for (84.6/100,000) is over three times the rate for females (25.0/100,000) females (25.0/100,000)

Taixing City, ChinaTaixing City, China

Taixing City

Exposure Marker: HBVExposure Marker: HBV

• Different markers used to assess Different markers used to assess extent of infection with HBVextent of infection with HBV

• Detection of HBV surface antigen Detection of HBV surface antigen (HBsAg) used to assess new and (HBsAg) used to assess new and chronic infectionschronic infections

• Enzyme-linked immunosorbant assay Enzyme-linked immunosorbant assay (ELISA) used to detect HBsAg in (ELISA) used to detect HBsAg in serumserum

Exposure Marker: Aflatoxin Exposure Marker: Aflatoxin B1B1

• AFB1-AFB1-exoexo-8,9-epoxide intermediate -8,9-epoxide intermediate binds to DNA and proteinsbinds to DNA and proteins

• Aflatoxin-albumin adduct detection Aflatoxin-albumin adduct detection to assess aflatoxin exposureto assess aflatoxin exposure

• Competitive ELISA used to detect Competitive ELISA used to detect aflatoxin-albumin adducts in plasmaaflatoxin-albumin adducts in plasma

Statistical Analysis: ModelStatistical Analysis: Model

• Unconditional logistic regression modelUnconditional logistic regression model• Complete analysis was used and Complete analysis was used and

missing data for independent variables missing data for independent variables were not imputedwere not imputed– For potential confounders that were For potential confounders that were

missing a large amount of data (missing a large amount of data (>>10%), 10%), like BMI, we imputed the median of like BMI, we imputed the median of controls by sexcontrols by sex

– When aflatoxin B1 (~10% missing) was When aflatoxin B1 (~10% missing) was included as a potential confounder in a included as a potential confounder in a model, missing data was imputed using model, missing data was imputed using the median of controlsthe median of controls

ResultsResults

Demographic data: Average (SD) age, BMI, Demographic data: Average (SD) age, BMI, and smoking pack-years of cases and and smoking pack-years of cases and

controlscontrols

Demographic data: Gender, education, and Demographic data: Gender, education, and alcohol consumptionalcohol consumption

Main Effects of HBsAg, AFB1 levels, and Main Effects of HBsAg, AFB1 levels, and IFNA17IFNA17 on liver cancer development on liver cancer development

Variables Case Control Crude Age & Sex Adjusted Fully Adjusted**

  N (%) N (%) OR (95%CI) OR (95%CI) OR (95%CI)

HBsAg - 72 (35.3) 312 (75.4) 1 1 1

  + 132 (64.7) 102 (24.6) 5.61 (3.90-8.07) 5.21 (3.60-7.53) 5.68 (3.80-8.51)

AFB1 Mean (SD) 508.1 (328.7) 426.2 (250.4)      

  <247 33 (18.1) 94 (24.9) 1 1 1

  247.1-388.8 46 (25.3) 94 (24.9) 1.39 (0.82-2.37) 1.38 (0.81-2.37) 1.15 (0.61-2.14)

  388.9-545 42 (23.1) 95 (25.2) 1.26 (0.74-2.16) 1.27 (0.74-2.20) 1.19 (0.64-2.21)

  >545.1 61 (33.5) 94 (24.9) 1.85 (1.11-3.08) 1.75 (1.04-2.94) 1.63 (0.90-2.96)

p(trend)=0.031 p(trend)=0.055 p(trend)=0.109

IFNA17 II 33 (17.4) 94 (24.5) 1 1 1

RI 104 (54.7) 193 (50.4) 1.54 (0.97-2.44) 1.49 (0.93-2.38) 1.67 (0.95-2.93)

RR 53 (27.9) 96 (25.1) 1.57 (0.94-2.64) 1.58 (0.93-2.68) 1.99 (1.06-3.73)

    p(HW)=0.878 p(trend)=0.104 p(trend)=0.102 p(trend)=0.037

  RI&RR 157 (82.6)  289   (75.5)  1.55 (1.00-2.41) 1.52 (0.97-2.38) 1.77 (1.04-3.03)

**Model includes age, sex, BMI, education, alcohol consumption, tobacco smoking, HBsAg, imputed AFB1 levels, anti-HCV

Interaction between HBV and AFB1 and Interaction between HBV and AFB1 and IFNA17IFNA17  HBsAg Case Control Crude Age & Sex Adjusted Fully Adjusted**

      N (%) N (%) OR (95%CI) OR (95%CI) OR (95%CI)

AFB1                

  <247 - 12 (6.6) 69 (18.4) 1 1 1

  247.1-388.8 - 19 (10.4) 67 (17.8) 1.63 (0.74-3.62) 1.64 (0.73-3.65) 1.72 (0.73-4.08)

  388.9-545 - 15 (8.2) 71 (18.9) 1.22 (0.53-2.78) 1.22 (0.53-2.80) 1.34 (0.55-3.27)

  >545.1 - 17 (9.3) 77 (20.5) 1.27 (0.57-2.85) 1.26 (0.56-2.82) 1.15 (0.48-2.74)

  <247 + 21 (11.5) 25 (6.6) 4.83 (2.08-11.23) 4.61 (1.97-10.80) 6.43 (2.56-16.16)

  247.1-388.8 + 27 (14.8) 27 (7.2) 5.75 (2.55-12.96) 5.30 (2.34-12.02) 4.68 (1.92-11.38)

  388.9-545 + 27 (14.8) 24 (6.4) 6.47 (2.84-14.74) 6.20 (2.70-14.21) 6.65 (2.72-16.25)

  >545.1 + 44 (24.2) 16 (4.3)15.82 (6.84-

36.57)13.75 (5.90-32.06)

16.72 (6.60-42.38)

        1ORint (95%CI)= 0.73 (0.24-2.24) 0.70 (0.23-2.18) 0.42 (0.12-1.45)

        2ORint (95%CI)= 1.10 (0.35-3.49) 1.10 (.35-3.52) 0.77 (0.22-2.70)

        3ORint (95%CI)= 2.58 (0.82-8.12) 2.38 (0.75-7.55) 2.27 (0.65-7.92)

IFNA17              

  II - 13 (6.8) 66 (17.3) 1 1 1

  RI&RR - 50 (26.3) 220 (57.6) 1.15 (0.59-2.25) 1.14 (0.58-2.23) 1.34 (0.64-2.82)

  II + 20 (10.5) 27 (7.1) 3.76 (1.64-8.62) 3.49 (1.51-8.04) 3.99 (1.54-10.32)

  RI&RR + 107 (56.3) 69 (18.1) 7.87 (4.04-15.34) 7.17 (3.66-14.06) 9.18 (4.34-19.43)

        ORint (95%CI)= 1.81 (0.71-4.62) 1.81 (0.71-4.63) 1.71 (0.60-4.92)**Model includes age, sex, BMI, education, alcohol consumption, tobacco smoking, imputed AFB1 levels, anti-HCV; 1ORint for AFB1 (247.1-388.8 fmol/mg) and HBsAg; 2ORint for AFB1 (388.9-545 fmol/mg) and HBsAg; 3ORint for AFB1 >545.1 fmol/mg) and HBsAg

Effects of Effects of IFNA17IFNA17 stratified by HBsAg stratified by HBsAg   HBsAg Crude Age & Sex Adjusted Fully Adjusted**

    OR (95%CI) OR (95%CI) OR (95%CI)

IFNA17 - 1.15 (0.59-2.25) 1.11 (0.57-2.18) 1.35 (0.63-2.85)

 (RI&RR vs. II) + 2.09 (1.09-4.02) 2.08 (1.06-4.08) 2.19 (1.01-4.76)

**Model includes age, sex, BMI, education, alcohol consumption, tobacco smoking, imputed AFB1 levels, anti-HCV

Effects of HBV and Effects of HBV and IFNA17IFNA17 stratified by AFB1 stratified by AFB1   AFB1 Crude Age & Sex Adjusted Fully Adjusted**

    OR (95%CI) OR (95%CI) OR (95%CI)

HBsAg <247 4.83 (2.08-11.23) 4.72 (2.02-11.05) 7.65 (2.82-20.77)

 (Pos. vs. Neg.) 247.1-388.8 3.53 (1.69-7.37) 3.14 (1.42-6.96) 2.77 (1.16-6.66)

  388.9-545 5.33 (2.44-11.65) 5.27 (2.38-11.67) 5.89 (2.38-14.60)

  >545.1 12.46 (5.73-27.08) 12.24 (5.42-27.63) 18.34 (7.02-47.92)

IFNA17 <247 0.52 (0.20-1.34) 0.55 (0.21-1.44) 0.26 (0.07-0.92)

  (RI&RR vs. II) 247.1-388.8 1.62 (0.66-3.99) 1.21 (0.45-3.21) 2.85 (0.82-9.96)

  388.9-545 3.27 (1.05-10.19) 3.11 (0.99-9.84) 5.89 (1.16-29.87)

  >545.1 1.19 (0.53-2.67) 1.19 (0.53-2.70) 1.42 (0.43-4.71)

**Model includes age, sex, BMI, education, alcohol consumption, tobacco smoking, HBsAg, anti-HCV

Interaction between HBsAg and Interaction between HBsAg and IFNA17IFNA17 stratified by AFB1stratified by AFB1

AFB1 HBsAg IFNA17 Case Control Crude Age & Sex Adjusted Fully Adjusted**

      N N OR (95%CI) OR (95%CI) OR (95%CI)

               

<388.9 - II 8 26 1 1 1

  - RI&RR 20 99 0.66 (0.26-1.66) 0.63 (0.24-1.62) 0.70 (0.24

  + II 9 13 2.25 (0.70-7.19) 2.04 (0.62-6.74) 2.07 (0.52-8.18)

  + RI&RR 37 37 3.25 (1.30-8.11) 2.81 (1.10-7.19) 3.45 (1.21-9.83)

    ORint (95%CI)= 2.20 (0.58-8.38) 2.20 (0.56-8.70) 2.39 (0.50-11.45)

               

>388.9 - II 5 34 1 1 1

  - RI&RR 25 104 1.63 (0.58-4.60) 1.62 (0.58-4.59) 2.09 (0.64-6.86)

  + II 11 9 8.31 (2.29-30.10) 8.07 (2.21-29.42) 9.22 (2.08-40.86)

  + RI&RR 57 27 14.35 (5.05-40.77) 13.88 (4.80-40.09) 21.80 (6.36-74.75)

    ORint (95%CI)= 1.06 (0.25-4.44) 1.06 (0.25-4.45) 1.13 (0.22-5.81)

**Model includes age, sex, BMI, education, alcohol consumption, tobacco smoking, HCV

DiscussionDiscussion

Findings summarizedFindings summarized

Main Effects:Main Effects:• Strong association between liver cancer and HBsAgStrong association between liver cancer and HBsAg• Moderate association between liver cancer and Moderate association between liver cancer and

aflatoxin B1 and aflatoxin B1 and IFNA17IFNA17 R allele R allele

Possible Interactive Effects:Possible Interactive Effects:• HBV-AFB1HBV-AFB1• HBV-HBV-IFNA17IFNA17• AFB1-AFB1-IFNA17IFNA17• HBV-AFB1-HBV-AFB1-IFNA17IFNA17

IFNA17IFNA17• Genotype frequencies are similar to previous Genotype frequencies are similar to previous

studies in Chinese populationsstudies in Chinese populations• No previous studies have evaluated No previous studies have evaluated

association between association between IFNA17IFNA17 and liver cancer and liver cancer• 184184IleIle allele, the lower-risk allele for our allele, the lower-risk allele for our

study, was previously found to be positively study, was previously found to be positively associated with cervical and nasopharyngeal associated with cervical and nasopharyngeal cancerscancers

• Difference in risk alleles may be due to Difference in risk alleles may be due to differences in their specific activities, i.e. Ile differences in their specific activities, i.e. Ile protein product may have more antiviral protein product may have more antiviral activity against HBV/HCV, whereas Arg activity against HBV/HCV, whereas Arg protein product may have more against protein product may have more against human papillomavirus and Epstein-Barr virushuman papillomavirus and Epstein-Barr virus

IFNA17IFNA17

Positive results may be due to:Positive results may be due to:

1. False positive1. False positive

2. Direct functional involvement with 2. Direct functional involvement with HCC developmentHCC development

3. Linkage disequilibrium with a nearby 3. Linkage disequilibrium with a nearby risk gene (like risk gene (like IFNA10 IFNA10 or or p16p16))

Interpretation of HBV, AFB1, Interpretation of HBV, AFB1, and and IFNA17 IFNA17 Joint EffectsJoint Effects

• AFB1 may negatively interact with AFB1 may negatively interact with IFNA17IFNA17, leading to a differential , leading to a differential decrease in protein function, decrease in protein function, resulting in a decreased resistance resulting in a decreased resistance against HBV and increasing risk for against HBV and increasing risk for the development of liver cancerthe development of liver cancer

LimitationsLimitations• Recall bias Recall bias

– Subjects’ awareness of disease status may alter Subjects’ awareness of disease status may alter recall of past exposuresrecall of past exposures

• Selection biasSelection bias– Selection of patients with less advanced and Selection of patients with less advanced and

aggressive cancersaggressive cancers

• Reporting bias Reporting bias – Behaviors or habits carry social stigma, like Behaviors or habits carry social stigma, like

smoking and alcohol drinkingsmoking and alcohol drinking

• Confounding by indicationConfounding by indication– Since blood samples were collected after Since blood samples were collected after

diagnosis, cases may have altered their diet to diagnosis, cases may have altered their diet to contain less AFB1contain less AFB1

StrengthsStrengths

• Population-based study designPopulation-based study design– controls were randomly selected from controls were randomly selected from

base population from which cases arosebase population from which cases arose

• Relatively large sample sizeRelatively large sample size• Detailed and extensive questionnaire Detailed and extensive questionnaire

– dietary habits, smoking, alcohol dietary habits, smoking, alcohol

• Racially homogeneous populationRacially homogeneous population– race would not be a potential race would not be a potential

confounder or effect modifier confounder or effect modifier

Significance – Public Health Significance – Public Health ApplicationsApplications

• The associations and joint effects for The associations and joint effects for IFNA17IFNA17 have never been previously studied in liver have never been previously studied in liver cancercancer

• Early detection of liver cancer and Early detection of liver cancer and identification of high-risk individuals for identification of high-risk individuals for interventionintervention

• Prevention strategies:Prevention strategies:– HBV vaccineHBV vaccine– Control intake of foods that typically have higher Control intake of foods that typically have higher

levels of AFB1 and modify storage condition of food levels of AFB1 and modify storage condition of food to prevent mold growthto prevent mold growth

• Chemoprevention of liver cancer:Chemoprevention of liver cancer:– Interferon mixtures are currently under study to Interferon mixtures are currently under study to

prevent liver cancer among HBV chronic carriersprevent liver cancer among HBV chronic carriers– Oltipraz protects against AFB1-induced liver cancers Oltipraz protects against AFB1-induced liver cancers

by inhibiting phase I enzymes and increasing phase by inhibiting phase I enzymes and increasing phase II enzymesII enzymes

AcknowledgementsAcknowledgementsLaboratories at UCLA:Laboratories at UCLA:

Dr. Steven DubinettDr. Steven DubinettDr. Robert LehrerDr. Robert LehrerDr. John TimmermanDr. John TimmermanDr. Gang ZengDr. Gang Zeng

Collaborators:Collaborators:

Dr. Zuo-Feng ZhangDr. Zuo-Feng ZhangDr. Regina SantellaDr. Regina SantellaDr. Li-Na MuDr. Li-Na MuDr. Shun-Zhang YuDr. Shun-Zhang YuDr. Qing-Wu JiangDr. Qing-Wu JiangDr. Wei CaoDr. Wei CaoDr. Xue-Fu ZhouDr. Xue-Fu ZhouDr. Bao-Guo DingDr. Bao-Guo DingDr. Ru-Hong WangDr. Ru-Hong WangDr. Jinkou ZhouDr. Jinkou ZhouDr. Lin CaiDr. Lin Cai

Mr. John GarciaMr. John Garcia