Binh Y. Goldstein, PhD Epidemiology 243 Aflatoxin B1, Hepatitis B, and IFNA17 on the Risk of Liver Cancer: An example of the application of exposure markers in cancer epidemiology Binh Y. Goldstein, PhD Epidemiology 243

Introduction

Background: Epidemiology of liver cancer Worldwide: Sixth most common new cancer Third most common cause of cancer death Rates among men 2-3x higher than women Over 80% of new cases occur in developing countries Low 5-year relative survival rates (<15%) Source: GLOBOCAN 2002

Age standardized incidence of liver cancer in world among men

Background: Epidemiology of liver cancer US: 19,200 new cases and 16,800 deaths Five year survival rate is 10% 8th leading cause of deaths from cancers in both sexes 6th for men 10th for women Source: American Cancer Society, 2007 China: 345,844 new cases and 321,851 deaths accounts for over 53% of all liver cancer cases and deaths worldwide 3rd most common cancer among men Most common cause of death from cancer among men Source: GLOBOCAN 2002

Background: Risk factors for liver cancer Hepatitis B virus: 350 million people chronically infected worldwide About two-thirds of liver cancers in China and Southeastern Asia are attributed to HBV infection Chronic carriers have a 20-fold increase in risk compared with non-carriers Major pathways by which HBV infection increases risk for liver cancer are: (1) viral DNA integration (2) oncogenic proteins (3) inflammation

Background: Risk factors for liver cancer Aflatoxin B1: Toxin found in mildewed grains and nuts Bioactivated intermediate AFB1-exo-8,9-epoxide has carcinogenic effect Adducts associated with increased risk of liver cancer Associated with a specific mutation in codon 249 of p53 tumor suppressor gene Potential multiplicative interaction with Hepatitis B viral infection (HBV)

Background: Metabolism of aflatoxin B1 AFB1 AFB1-exo-8,9-epoxide AFM1 AFQ1 AFB1-endo-8,9-epoxide dietary intake CYP3A4 (CYP1A2) DNA-adducts glutathione-AFB1 conjugate AFB1-8,9-dihydrodiol [phenolate resonance form] protein adducts excretion GST-μ, (GST-θ) + glutathione H2O (mEH) CYPs

Background: Other risk factors: Potential protective factors Hepatitis C virus Alcohol consumption Tobacco smoking Contaminated drinking water (microcystins) Potential protective factors Antioxidants Dietary nutrients Selenium

Background: IFNA17 Located at position 9p22 Encodes interferon, alpha 17 Has viral inhibitory and viral anti-proliferative effects Interferon, alpha investigated as a treatment for HBV and HCV infection and prevention of liver cancer among HBV infected individuals IFNA17 has a polymorphic site that results in either an arginine or isoleucine amino acid in codon 184 The 184Arg allele has a higher frequency in Chinese populations (~50%) than in other populations (<35%) Studied in cancers that have a viral component, including cervical cancer (papillomavirus) and nasopharyngeal cancer (Epstein-Barr virus)

Objectives Overall objective: Gain insight into the mechanism of interaction observed between aflatoxin and HBV infection Hypothesis: AFB1 may differentially suppress IFNA17 protein activity, thereby increasing a person’s susceptibility to the sequelae of HBV if chronically infected and increasing the risk for liver cancer Specific Aims: Assess the independent effects of IFNA17 Ile184Arg polymorphic site on liver cancer risk Explore the joint effects and three-way interaction among HBsAg-positivity, AFB1-albumin adduct level, and IFNA17 polymorphisms on the development of liver cancer

Research Design and Methods

Study Design Population-based case-control study with 204 incident liver cancer cases (57% of all cases) and 415 randomly selected healthy population controls (89% response rate) in Taixing City, China Collected epidemiologic data and blood specimens IFNA17 genotyped using PCR-RFLP Markers used to assess HBV chronic infection and aflatoxin B1 exposure (and HCV infection)

Taixing City, China Located on the east bank of the Yangtze River in middle of Jiangsu province Consists of 24 small villages, with estimated population of 1.28 million (660,000 males and 627,000 females), most of whom are farmers Has a high rate of alimentary cancer, among the highest in the world Has a tumor registry After esophageal cancer, liver cancer is the second largest cause of deaths from cancers In 2000, crude incidence rates for top three cancers - 65.2 (esophageal), 55.6 (liver), 54.8 (stomach) per 100,000 Incidence rate of liver cancer for males (84.6/100,000) is over three times the rate for females (25.0/100,000)

Taixing City, China Taixing City

Exposure Marker: HBV Different markers used to assess extent of infection with HBV Detection of HBV surface antigen (HBsAg) used to assess new and chronic infections Enzyme-linked immunosorbant assay (ELISA) used to detect HBsAg in serum

Exposure Marker: Aflatoxin B1 AFB1-exo-8,9-epoxide intermediate binds to DNA and proteins Aflatoxin-albumin adduct detection to assess aflatoxin exposure Competitive ELISA used to detect aflatoxin-albumin adducts in plasma

Statistical Analysis: Model Unconditional logistic regression model Complete analysis was used and missing data for independent variables were not imputed For potential confounders that were missing a large amount of data (>10%), like BMI, we imputed the median of controls by sex When aflatoxin B1 (~10% missing) was included as a potential confounder in a model, missing data was imputed using the median of controls

Results

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

Demographic data: Gender, education, and alcohol consumption

Main Effects of HBsAg, AFB1 levels, and IFNA17 on liver cancer development Variables Case Control Crude Age & Sex Adjusted Fully Adjusted**   N (%) OR (95%CI) HBsAg - 72 (35.3) 312 (75.4) 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) 247.1-388.8 46 (25.3) 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) 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 (17.4) (24.5) 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 IFNA17   HBsAg Case Control Crude Age & Sex Adjusted Fully Adjusted** N (%) OR (95%CI) AFB1 <247 - 12 (6.6) 69 (18.4) 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) + 21 (11.5) 25 4.83 (2.08-11.23) 4.61 (1.97-10.80) 6.43 (2.56-16.16) 27 (14.8) (7.2) 5.75 (2.55-12.96) 5.30 (2.34-12.02) 4.68 (1.92-11.38) 24 (6.4) 6.47 (2.84-14.74) 6.20 (2.70-14.21) 6.65 (2.72-16.25) 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) 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) 20 (10.5) (7.1) 3.76 (1.64-8.62) 3.49 (1.51-8.04) 3.99 (1.54-10.32) 107 (56.3) (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 IFNA17 stratified by HBsAg   HBsAg Crude Age & Sex Adjusted Fully Adjusted** 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 IFNA17 stratified by AFB1   AFB1 Crude Age & Sex Adjusted Fully Adjusted** 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 0.52 (0.20-1.34) 0.55 (0.21-1.44) 0.26 (0.07-0.92)   (RI&RR vs. II) 1.62 (0.66-3.99) 1.21 (0.45-3.21) 2.85 (0.82-9.96) 3.27 (1.05-10.19) 3.11 (0.99-9.84) 5.89 (1.16-29.87) 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 IFNA17 stratified by AFB1 Case Control Crude Age & Sex Adjusted Fully Adjusted**   N OR (95%CI) <388.9 - II 8 26 1 RI&RR 20 99 0.66 (0.26-1.66) 0.63 (0.24-1.62) 0.70 (0.24 + 9 13 2.25 (0.70-7.19) 2.04 (0.62-6.74) 2.07 (0.52-8.18) 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 5 34 25 104 1.63 (0.58-4.60) 1.62 (0.58-4.59) 2.09 (0.64-6.86) 11 8.31 (2.29-30.10) 8.07 (2.21-29.42) 9.22 (2.08-40.86) 57 27 14.35 (5.05-40.77) 13.88 (4.80-40.09) 21.80 (6.36-74.75) 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

Discussion

Findings summarized Main Effects: Possible Interactive Effects: Strong association between liver cancer and HBsAg Moderate association between liver cancer and aflatoxin B1 and IFNA17 R allele Possible Interactive Effects: HBV-AFB1 HBV-IFNA17 AFB1-IFNA17 HBV-AFB1-IFNA17

IFNA17 Genotype frequencies are similar to previous studies in Chinese populations No previous studies have evaluated association between IFNA17 and liver cancer 184Ile allele, the lower-risk allele for our study, was previously found to be positively associated with cervical and nasopharyngeal cancers Difference in risk alleles may be due to differences in their specific activities, i.e. Ile protein product may have more antiviral activity against HBV/HCV, whereas Arg protein product may have more against human papillomavirus and Epstein-Barr virus

IFNA17 Positive results may be due to: 1. False positive 2. Direct functional involvement with HCC development 3. Linkage disequilibrium with a nearby risk gene (like IFNA10 or p16)

Interpretation of HBV, AFB1, and IFNA17 Joint Effects AFB1 may negatively interact with IFNA17, leading to a differential decrease in protein function, resulting in a decreased resistance against HBV and increasing risk for the development of liver cancer

Limitations Recall bias Selection bias Reporting bias Subjects’ awareness of disease status may alter recall of past exposures Selection bias Selection of patients with less advanced and aggressive cancers Reporting bias Behaviors or habits carry social stigma, like smoking and alcohol drinking Confounding by indication Since blood samples were collected after diagnosis, cases may have altered their diet to contain less AFB1

Strengths Population-based study design Relatively large sample size controls were randomly selected from base population from which cases arose Relatively large sample size Detailed and extensive questionnaire dietary habits, smoking, alcohol Racially homogeneous population race would not be a potential confounder or effect modifier

Significance – Public Health Applications The associations and joint effects for IFNA17 have never been previously studied in liver cancer Early detection of liver cancer and identification of high-risk individuals for intervention Prevention strategies: HBV vaccine Control intake of foods that typically have higher levels of AFB1 and modify storage condition of food to prevent mold growth Chemoprevention of liver cancer: Interferon mixtures are currently under study to prevent liver cancer among HBV chronic carriers Oltipraz protects against AFB1-induced liver cancers by inhibiting phase I enzymes and increasing phase II enzymes

Acknowledgements Collaborators: Dr. Zuo-Feng Zhang Dr. Regina Santella Dr. Li-Na Mu Dr. Shun-Zhang Yu Dr. Qing-Wu Jiang Dr. Wei Cao Dr. Xue-Fu Zhou Dr. Bao-Guo Ding Dr. Ru-Hong Wang Dr. Jinkou Zhou Dr. Lin Cai Mr. John Garcia Laboratories at UCLA: Dr. Steven Dubinett Dr. Robert Lehrer Dr. John Timmerman Dr. Gang Zeng