From Biomarkers to Pathobiology: A Liver Cancer Template

From Biomarkers to Pathobiology: A Liver Cancer Template John D. Groopman, Ph.D. Anna M. Baetjer Professor and Chair Department of Environmental Health Sciences Johns Hopkins Bloomberg School of Public Health Sidney Kimmel Comprehensive Cancer Center

SUSPECT ETIOLOGY AND HUMAN DISEASE LINKAGE IDENTIFY AND DEVELOP METHODS FOR MEASURING SPECIFIC BIOMARKERS DETERMINE RELATION OF BIOMARKER TO EXPOSURE AND DISEASE IN EXPERIMENTAL MODELS CROSS-SECTIONAL STUDIES OF BIOMARKERS IN POPULATIONS LONGITUDINAL STUDY VALIDATED EXPOSURE/DISEASE MARKER MODULATION OF BIOMARKER/DISEASE RISK IN EXPERIMENTAL MODELS CASE-CONTROL COHORT STUDIES PREVENTION TRIALS VALIDATED RISK MARKER

Incidence of Liver Cancer in Males Incidence of Liver Cancer (Males)- 2002 Age-Standardized 600,000 Incidence deaths Rate in 2001 per 100,000

In 2006, we know that the etiology of liver cancer is multifactorial and varies across geographic regions n Hepatitis B Virus n Major cause of viral hepatitis in Africa and Asia n Hepatitis C Virus n Major cause of viral hepatitis in Japan, Europe and Asia n Emerging major issue in US n Environmental Factors n Aflatoxin in foods n Alcoholic beverages

25 years ago the etiology of liver cancer was thought to be less complex n Hepatitis B Virus n Relative risk for liver cancer in Taiwan is 223 in HBsAg carriers (Beasley et al. LANCET 2:1129, 1981) n Aflatoxin in foods n Linear relationship between aflatoxin in the diet and liver cancer in Asia and Africa (reviewed in Bosch and Munoz, IARC Publ. No. 89: 427, 1988) n Hepatitis C Virus n Not yet discovered n 10-fold range of liver cancer rates among populations with similar HBV prevalence

Aflatoxin biomarkers for molecular epidemiology last 10 years sensitivity of mass spectrometry increased 1000 fold higher throughput to thousands of samples per month

LIVER CANCER (MALE) Qidong 2 nd leading cause of cancer death in China 300,000 deaths per year

Cross-Sectional Study of Urinary Aflatoxin-N 7 -Guanine in Guangxi, P.R.C. TOTAL AFB-N 7 -GUANINE IN URINE (NG) TOTAL AFB-N 7 -GUANINE IN URINE GUANGXI PROVINCE, P.R.C. 4000 r=0.80 3000 2000 1000 0 0 200 400 600 800 1000 1200 TOTAL AFLATOXIN B 1 INTAKE (UG) Groopman et al Cancer Research 52:45-52, 1992

COHORT STUDY OF LIVER CANCER IN P.R.C.: Viral-Chemical n 18,244 urine and blood samples collected from healthy men age 45-65 n 50 liver cancer cases and 247 controls n Urinary aflatoxin biomarkers measured in blinded samples n HBV status determined for each subject Lancet 339: 943-946, 1992 and C.E.B.P. 3: 3-11, 1994 Interactions BIOMARKERS: HBsAg AND URINARY AFLATOXINS NO BIOMARKERS DETECTED HBV (YES) AFLATOXIN (NO) HBV (NO) AFLATOXIN (YES) HBV (YES) AFLATOXIN (YES) RELATIVE RISK FOR LIVER CANCER 1.0 7.3 3.4 60.0

Aflatoxin and HBV Infection in Liver Cancer Aflatoxin HBsAg Odds ratio 95% CI Negative Positive Negative Positive Low High Low High Negative Negative Positive Positive Shanghai Negative 1.0 Negative 3.4 Positive 7.3 Positive 59.4 Taiwan 1.0 1.7 22.8 111.9 1.1-10.0 2.2-24.4 16.6-212.0 0.3-10.8 3.6-143.4 13.8-905.0 (Lancet 339: 943-946, 1992; C.E.B.P. 3: 3-11, 1994; Int. J. Cancer 67: 620-625, 1996)

Prevalence of p53 codon 249 mutation 1 correlates with estimated aflatoxin exposure ser ser Prevalence of p53 Mutation % of Total HCC 70 60 50 40 30 20 10 0 USA/Europe (n=107) Japan (n=437) Low (>1-10) Monterrey, Mexico (n=14) Shanghai, PRC (n=52) Transkei, Natal (n=48) Thailand (n=15) Moderate (30-80) Senegal (n=15) Mozambique (n=15) Qidong and Quanxi, PRC (n=98) High (>81) Estimated Aflatoxin B Intake (ng/kg body weight/day) 1 Curtis Harris

Plasma concentrations of WT p53, and 249Sermutated p53 in aflatoxin-exposed groups (Median copies/ml plasma (IQR) using quantitative SOMA) Non-liver Diseased Controls Cirrhosis HCC WT plasma DNA level 15,000 (8,700 27,000) 21,000 (10,000 33,000) 25,000 (14,000 38,000) 249 Ser plasma DNA level 500 (250 2,000) 500 (500 2,600) 2,800 (500 11,000) Cancer Epidemiol Biomarkers Prev 14: 2956-62, 2005

Mechanistic basis for exposure and early effect biomarkers in HCC development Aflatoxin B 1 Reactive Intermediates Aflatoxin-Mercapturic Acid (urine) Aflatoxin- albumin adduct (serum) Hepatitis B Virus Promutagenic DNA Lesions Genomic Insertion P53 Gene Mutations (G:C-T:A) Transversions Aflatoxin- N 7 -guanine (urine) 249ser Mutations (serum) HBsAg (serum) X-Gene Mutations 1762T/1764 A Double mutation (serum) Chronic Inflammation Selective Clonal Expansion and p53 Allelic Deletions Cell Proliferation Chronic Hepatitis and/or Cirrhosis Hepatocellular Carcinoma

Mortality from Liver Cancer by Township: Jiangsu Province < 1 per 10 5 /yr > 50 per 10 5 /yr Median age of liver cancer death is 45-50 years Median survival from Dx is 6 months 25-fold change in HCC rate in 200 km Shanghai Qidong

Etiology of Liver Cancer in Qidong, P.R. China n Hepatitis B Virus (HBV) 70% of liver cancers attributed to HBV infection in early life Prevalence of Hepatitis B Surface Antigen (HBsAg) in Qidong general population is about 15% 75% of liver tumors in Qidong contain HBV 1762 T /1764 A double mutation (Kuang et al. PNAS: 101:3575-3580, 2004) Mutations in HBV genome are detected in plasma up to 14 years prior to liver cancer diagnosis n Aflatoxin B 1 n Also substantially contributes to risk for liver cancer in Qidong

Qidong Liver Cancer Cohort of 5581 HBsAg Positive Men (1989-2003) 667 Cases of Liver Cancer 536 Samples with sufficient serum volume (> 100 µl) 515 Cases deceased prior to 12-31-2003 355 (69%) DNA recovered from serum 295 (83%) Mutation at 1762 T /1764 A 83% of analyzable serum samples contained double mutation HBV DNA Chen et al. J. Med. Screen. 10:204-9, 2003.

93 82 88 78 86 92 90 83 88 74 80 85 P = 0.068 70 65 76 P = 0.012 P = 0.083 Guo et al, AACR, 2006 AGE <45 45-55 >55 < 1.5 1.5-3 3.01-5 5.01-9 >9 TIME to DEATH from SCREENING YEARS

STRATEGIES FOR PREVENTION OF LIVER CANCER SECONDARY PRIMARY Immunization with HBV vaccine Reduced aflatoxin consumption: - improve food storage; biocontrol - changes in dietary staples Chemopreventive interventions: e.g., oltipraz, chlorophyllin, broccoli sprouts

Oltipraz Phase II Trial in China 100% effective for chemoprotection against AFB1 induced HCC in rats (Canc. Res. 51:5501, 1991) 51% OLTIPRAZ Wang et al. J.N.C.I. 91: 347-353, 1999. 260%

CHLOROPHYLLIN non-prescription drug in U.S., food colorant (especially in Asia) antioxidant, anti-mutagen and anticarcinogen in a variety of experimental models safe, inexpensive Treatment N AFB-N 7 -guanine (pg /mg creat.) P-value Placebo, tid 82 0.20 (<LOD 0.38) a Chlorophyllin, tid 87 0.09 (<LOD 0.24) 55% 0.036 a Median (interquartile range) * In F344 rat model a 55% reduction in DNA adducts results in greater than 97% reduction in liver tumors PNAS 98:14601-6, 2001

Ind uc tion of Detoxic ation Enzym es b y Extr ac ts fr om 3 Fam ilies of Veg etab les (Paul Talalay - 1992) INDUCER ACTIVITY (U/g fr wt) 1600 1400 1200 1000 800 600 B r oc c oli B r us Sp r outs K ale Red Cab b ag e Gr een Onion 400 200 0 Tom ato Red Pep p er Gr een Pep p er Cauliflow er Gr een Cab b ag e Leek A sp ar ag us Solanaceae Cruciferae Liliaceae

O S OH O S OH N - OSO 3 GLUCORAPHANIN OH OH myrosinase H 2 O HSO 4 - D-glucose O S NCS SULFORAPHANE chewing sprouts intestinal microflora

Glucosinolate-rich Broccoli Sprouts Intervention Trial in a High-Risk Liver Cancer Population in Qidong, P.R.C. Kensler et al. C.E.B.P., 14: 2605-13, 2005.

Targets for intervention in HCC caused by AFB 1 and HBV Aflatoxin B 1 Chlorophyllin Reactive Intermediates Oltipraz, ITCs Promutagenic DNA Lesions Hepatitis B Virus Vaccination Insertional Mutagenesis X-Gene Mutations P53 Gene Mutations (G:C-T:A) Transversions Antioxidants Selective Clonal Expansion and p53 Allelic Deletions Chronic Inflammation Antiviral drugs Cell Proliferation Green tea polyphenols, COX-2 2 inhibitors, ITCs, Vitamin K analogs, Retinoids Chronic Hepatitis and/or Cirrhosis Hepatocellular Carcinoma

Mechanistic biomarkers for exposure, early effect and application to prevention and therapy Aflatoxin B 1 Chlorophyllin Reactive Intermediates Oltipraz, ITCs Aflatoxin-Mercapturic Acid (urine) Aflatoxin- albumin adduct (serum) Hepatitis B Virus HBsAg (serum) Vaccination Genomic Insertion X-Gene Mutations 1762T/1764 A Double mutation (serum) Promutagenic DNA Lesions Chronic Inflammation P53 Gene Mutations (G:C-T:A) Transversions Antioxidants Antiviral drugs Selective Clonal Expansion and p53 Allelic Deletions Cell Proliferation Green tea polyphenols, COX-2 2 inhibitors, ITCs, Vitamin K analogs, Retinoids Aflatoxin- N 7 -guanine (urine) 249ser Mutations (serum) Chronic Hepatitis and/or Cirrhosis Hepatocellular Carcinoma

45 years 70 years Data courtesy of Dr. Zong-Tang Sun, CAMS, Beijing, P.R.C.

Potential Public Health Benefit by Intervention Data courtesy of Dr. Zong-Tang Sun, CAMS, Beijing, P.R.C.

Hemoglobinomics

Hemoglobin Adducts in Human Non-Smokers COMPOUND fmole/g Hemoglobin HPB from NNK 29.3 ± 25.9 2-Aminonapthalene 40 ± 20 4-ethylaniline 99 ± 10 2,6-dimethylaniline 157 ± 50 4-Aminobiphenyl 166 ± 77 3,5-dimethylaniline 220 ± 20 o-toluidine 320 ± 90 p-toluidine 640 ± 370 m-toluidine 6400 ± 1900 N-(2-carbamoylethyl)valine 19000 ± 12000 Aniline 41000 ± 22000 N-(2-Hydroxyethyl)valine 58000 ± 25000 Modified from JNCI 96:1425, 2004; Int. J. Hyg. Env. Hlth 207: 534, 2004; Cancer Res. 50:5438, 1990.

Collaborators n Johns Hopkins: T. Kensler, M. Friesen, P. Egner, P. Scholl, L. Jacobson, S. Gange, D. Johnson, S. Iyer, A. Muñoz, S.K Kuang, K. Kinzler, B. Vogelstein, K. Brodovicz, K. Nelson n Chiang Mai University: S. Lewanvijit, N. Maneekarn, S. Thongsawat n Shanghai Cancer Institute: G-S Qian n Qidong Cancer Institute: Y-R Zhu, B-C Zhang, P-X Lu, J-B Wang