Strategies for Prevention of

HCC
 Molecular Epidemiology
• Identify risk factors and outcome
• Biomarkers
• Carcinogen-macromolecular adducts
• Normal DNA sequence variants
• Mutations in target genes
• Measure in urine, serum or tissue
• Immunoassays
• GC/MS, LC/MS
• Florescence spectometry

May 2nd, 2005 Megan McBee-BE.450 2

 HCC Epidemiology
• Annual new cases ~600,000
• ~600,000 annual deaths
– 80% burden in Asia and sub-
saharan Africa
– 300,000+ cases in People’s
Republic of China
• High risk areas early
age of onset 20’s
• Low risk areas early
age of onset 50’s

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 HCC Epidemiology
• Main causes in high risk areas
– HBV infection
– Aflatoxins in diet
• Synergism leading to increased risk

Impact on HCC incidence

HBV vaccination aflatoxin exposure

May 2nd, 2005 Megan McBee-BE.450 4

HCC Epidemiology: Aflatoxin Studies

• Taiwan1: BsAg+ males with HCC

compared to control subjects
• OR = 2.8 detectable vs. nondetectable aflatoxin
metabolites
• OR = 5.5 high vs. low urinary metabolite levels
• Shanghai2: relative risk for HCC with
presence aflatoxin metabolites = 3.8

1. Wang LY et al. Int J Cancer. 1996 Sep 4;67(5):620-5.

2. Ross RK et al. Lancet. 1992 Apr 18;339(8799):943-6.

May 2nd, 2005 Megan McBee-BE.450 5

 Aflatoxins
O O

O
H

• Produced by fungi O
– 1960 outbreak of “Turkey H O OCH3
‘X’ disease” in UK Aflatoxin B1
– Aspergillus flavus
• Common in corn, peanuts, Cytochrome
P450
fermented soy products
O O
O
O
H

H O OCH3

Figure by MIT OCW.

Active DNA-modifying agent

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HCC Prevention/Intervention
• Primary
– Vaccination
– Reduced contamination
• Secondary
– Pharmaceuticals
– Natural products

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 HCC Prevention/Intervention
Hepatitis B Virus Aflatoxin B1
vaccination chlorophyllin
HBsAg (serum)

Insertional Mutagenesis Reactive Intermediates Aflatoxin-Mercapturic

Acid (urine)
oltipraz, ITCs
1762T/1764A X-Gene Mutations Promutagenic DNA Lesions Aflatoxin-N7-guanine
Double Mutation (urine)
(serum) antioxidants

Chronic Inflammation p53 Gene Mutations

249ser Mutations
(G:T-T:A transversions)
(serum)
antiviral drugs

Selective Clonal Expansion

green tea polyphenols,
and p53 Allelic Deletion
COX-2 inhibitors, ITCs,
vitamin K analogs,
retinoids

Cell Proliferation Chronic Hepatitis and/or

Cirrhosis
green tea polyphenols,
COX-2 inhibitors, ITCs, oltipraz
vitamin K analogs,
retinoids
Hepatocellular
Carcinoma

Figure by MIT OCW.

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 Primary Interventions
• HBV vaccination
– Taiwan HCC cases in 6-14 year olds
• Born 1981-1986 = 0.70
• Born 1986-1990 = 0.57
• Born 1990-1994 = 0.36
• Reduction of aflatoxins in food

Chang MH et al. N Engl J Med. 1997 Jun 26;336(26):1855-9.

May 2nd, 2005 Megan McBee-BE.450 9

 Secondary Intervention
Oltipraz Chlorophyllin
H 3C C 2H 5 H 3C C 2H 5

N
H2C=HC N CH3 H2C=HC N CH3
N Cu N N Cu N
N S
N COO-Na+ N
S H 3C H 3C
CH2COO-Na+ CH2COO-Na+
H3C H 3C CH2CH2COO-Na+ H 3C CH2CH2COO-Na+
S
Trisodium Copper Chlorin e6 Disodium Copper Chlorin e4

Isothiocyanates Polyphenols
R R
OH R+
3+
2+ 4+
HO O B 5+ HO O
7 8 1 2 R+
6 A C
5 43
OH
R N C S OH O
OH
R
OH O
R
OH OH
+
HO O HO O
R+ R+

OH
OH O OH
May 2nd, 2005 Megan McBee-BE.450 Figures by MIT OCW. 10
Secondary Intervention: Oltipraz

• Oltipraz
– Induces phase 2 enzymes
– Inhibits phase 1 enzymes
• Higher doses (500mg+) not more
effective at induction or inhibition
than lower doses (125mg and
250mg)

May 2nd, 2005 Megan McBee-BE.450 11

 Mechanism of Oltipraz

Source: Kensler,T. W. “Chemoprevention by inducers of carcinogen detoxication enzymes.” Environmental Health Perspective 105,
Supplement 4 (1997): 965-970. Reproduced with permission from Environmental Health Perspectives.

May 2nd, 2005 Megan McBee-BE.450 12

Secondary Intervention: Oltipraz

• Phase IIa intervention trial

– Feasibility of biomarker measurements
– Dose response
– Tolerance/effectiveness longer term
exposure
– Chronic toxicity
Source: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.

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Secondary Intervention: Oltipraz
Location: Dazin Township, Qidong,
People’s Republic of China
– Randomized, placebo-controlled, double
blind
– 240 adults without history of chronic disease
– Detectable serum aflatoxin-albumin adducts
– 3 intervention groups
1) Placebo
2) 125mg once daily
3) 500mg once weekly
Source: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.

May 2nd, 2005 Megan McBee-BE.450 14

Secondary Intervention: Oltipraz
• 500mg weekly after 1 month
– 51% decrease median levels aflatoxin M1
excretion
– No effect on aflatoxin-mercapturic acid
– Inhibits activation
• 250mg daily after 1 month
– 2.6-fold increase in median levels of aflatoxin-
mercapturic acid
– Modest effect on aflatoxin M1 levels
– Increase phase 2 conjugation
Source: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.

May 2nd, 2005 Megan McBee-BE.450 15

Secondary Intervention: Oltipraz

• Ongoing follow-up phase IIb trial

– Sustained expression enhancement of
aflatoxin detoxification enzymes
– 250mg versus 500mg once weekly for 1
year
– Measuring multiple biomarkers for
mechanisms of action

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Secondary Intervention: Chlorophyllin

• Mixture of sodium-copper salts

of chlorophyll
• OTC drug
– Wound healing accelerant
– Controls body, fecal and urinary odor
• In vitro and in vivo antimutagen in
short-term genotoxicity assays

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Secondary Intervention: Chlorophyllin

• Complexes with aflatoxin B1

• Reduction in bioavailability
• Needs molar excesses to
carcinogen for efficacy
• In vitro inhibitor of cytochrome P450
enzymes
• Antioxidant-reduction in lipid
peroxidation
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Secondary Intervention: Chlorophyllin

• Chemoprevention study in Qidong

– 180 healthy adults
– 100mg chlorophyllin or placebo 3-times
daily for 4 months
– Endpoint of modulated aflatoxin-N7-
guanine adducts in urine after 3
months
• Resulted in 55% decrease in median
urinary adduct levels
Source: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450 19
 Secondary Intervention:
Isothiocynates
Source: Family Cruciferae (mustards),
Genus Brassica (cauliflower, Brussels
sprouts, broccoli, cabbage)
– Lower cancer rates in individuals consuming
high levels of yellow and green vegetables
• Isothiocyanates
• Particularly glucosinolate precursors
• Sulforaphane induces phase 2 enzymes in rats
(glucoraphanin is precursor)

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 Secondary Intervention:
Polyphenols
Source: Green tea
– Inverse association of consumption
versus risk and development of cancer
– Green tea-derived polyphenols
(ongoing study)
• Reduce aflatoxin M2 excretion
• Increase aflatoxin-mercapturic acid
excretion
• Reduced 8-oxo-deoxyguanosine

May 2nd, 2005 Megan McBee-BE.450 21

Outlook: Primary Interventions
• HBV vaccination
– Only benefits younger generations
– Vertical transmission not prevented
• Reduced food contamination
– Requires infrastructure for production,
processing and distribution
– Monitoring mycotoxins $$
– Not feasible in developing countries

May 2nd, 2005 Megan McBee-BE.450 22

 Outlook: Pharmaceutical
Chemoprevention
• Not practical for populations at highest
risk
– SE Asia, China, Africa
• First-generation (oltipraz) expensive
• 2nd and 3rd generation dithiolethiones
– Cheaper
– 10-fold increase in potency over oltipraz
– Ongoing safety evaluations
• Long-term costs potentially high, chronic
treatment

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 Outlook: Natural Products
• Practical for populations at highest risk
– SE Asia, China, Africa
• Inexpensive, diet-based
• Long-term compliance better
• Immediate impact

May 2nd, 2005 Megan McBee-BE.450 24

 Potential Impact
• Reduction of aflatoxin-N7-
guanine
– Reduced risk HCC in animals
– Increased latency period Image removed due to copyright reasons.

• Decreased aflatoxin
exposure in Bejing
correlated with
later onset of HCC

Source: Kensler, T.W., et al. “Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas.”
Gastroenterology Review 127, no. 5, Supplement 1 (2004): S310-318.

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 Questions?

May 2nd, 2005 Megan McBee-BE.450 26