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Biological Markers for Exposures
Epidemiology 243
Molecular Epidemiology of Cancer
Early Studies
• MacMahon: Geographical correlation of urinary
estrogen concentrations with cancer of the breast
(1974)
• Cole & MacMahon: Urinary and blood estrogens
and breast cancer in case-control studies
(1969,1982, 1983)
• McMichael: The relationship between cancer
mortality and serum cholesterol concentrations
(1984)
Recent Studies
• Aflatoxins and hepatitis B on liver cancer in
a cohort study, including measurements of
urinary metabolites and nucleic acid adducts
of aflatoxin (Ross, 1992, 1994)
• The relationship between HPV and cervical
cancer (Munoz, 1992, Bosch, 1995)
Application of biomarkers in
Epidemiology
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Molecular markers can be applied to increase the
accuracy of measurements
of genetic and other acquired susceptibility to
disease;
of exposures that may cause or prevent disease;
of exposures that confound or modify the
associations between risk and other exposures
of disease itself
of factors that may determine the outcome of the
disease such as precursors and stages
Application of biomarkers in
Epidemiology
Biomarkers may also be used
• to reduce the time interval between the relevant
exposure and measurement of the putative effect
• To increase the yield of information on disease
pathogenesis
• To increase the cost-effectiveness of
epidemiological studies. More information is
gained per unit cost.
Biomarker in Epidemiology:
Biomarkers of Biological Agents
• Biological agents associated with chronic
infection and subsequent development of
cancer are measured using serological or
nucleic acid markers.
Biomarker in Epidemiology:
Biomarkers of Biological Agents
• HPV DNA by PCR-based assays
HPV infection is often transient, especially
in young women so that repeated sampling
is required to assess persistent HPV
infections
Classification of Cervical Squamous Neoplasia
Dysplasia
Normal
Infla. Atypia
Koilocyt.Atypia
Mild dysplasia
Moderate dysp.
Severe dysp.
Ca. in situ
Invasive ca.
PapS.
1
2a
2b
3
3
3
4
5
CIN scale
Normal
Infla. atypia
Koilocyto a.
CIN1
CIN2
CIN3
CIN3
Invasive ca
Bethesda
Normal
Normal
LG SIL
LG SIL
HG SIL
HG SIL
HG SIL
Invasive ca
HPV Testing and Typing
• HPV infection is the main cause of cervical
cancer. Transient in women. Only 10-20%
persistent infections are at risk of neoplasia
• About 70 subtypes, of which 25 are tropic
for genital tract. Those are subdivided into
three categories:
HPV Testing and Typing
• HPV can be tested and typed by dot blot
hybridization, southern blot hybridization,
Hybrid Capture and PCR
• High sensitivity but relatively low
specificity, particular among young women
• HPV typing has great potential as a primary
screening tool for cervical cancer.
Biomarker in Epidemiology:
Biomarkers of Biological Agents
HBV infection by serological assays.
• There are serological markers that
distinguish between past and persistent
infections. HBV DNA detection in sera
further refines the assessment of exposure.
Gender distribution among cases and controls
Gender
Case
N
%
Control
N
%
Male
159 (77.94 )
287(69.16)
Female
45 (22.06)
128(30.84)
204
415
P Value
0.0221
Self-reported hepatitis virus infection type
Hepatitis History
Case
N
No
%
108(60.34)
Control
N
%
354(90.08)
Crude OR
(95%CI)
Adjusted OR
(95% CI)
1
1
HAV
16(8.94)
19(4.84)
2.77(1.38~5.57)
2.67(1.27~5.60)
HBV
55(30.73)
19(4.40)
11.30(6.22~20.5)
14.52(7.38~28.6)
HDV
0 (0)
1(0.26)
The relationship between liver cirrhosis and liver cancer
Liver cirrhosis
Case
N
No
Yes
Control
N
%
%
149(86.6)
23 (13.4)
Crude OR
(95%CI)
Adjusted OR
(95% CI)
18.3(5.40~61.8)
22.1(6.11~79.9)
355(99.2)
3
(0.8)
The relationship between HBV vaccine and liver cancer
HBV vaccine
Case
N
%
No
157(96.32)
Yes
6(3.68)
Control
N
%
Crude OR
(95%CI)
293(86.14)
1
47(13.9)
0.24(0.10~0.57)
Adjusted OR
(95% CI)
1
0.24(0.10~0.60)
The distribution of HBsAg among cases and controls
HBsAg
Case
N
%
Control
N
%
Negative
72(35.29)
312(75.36)
Positive
132(64.71)
102(24.64)
Crude OR
(95%CI)
1
5.59(3.95~8.18)
Adjusted OR
(95% CI)
1
5.06(3.45~7.43)
The distribution of anti-HCV among cases and controls
HBsAg
Case
%
Control
N
%
Negative
183(91.04)
403(97.11)
Positive
18 (8.96)
12 (2.89)
N
Crude OR
(95%CI)
1
3.49(1.66~7.33)
Adjusted OR
(95% CI)
1
3.21(1.46~7.06)
Most frequent HBV infection spectrum in cases and controls
TYPE
HBsAg
HBsAb
HBeAg
HBeAb
HBcAb
Crude OR
(95%CI)
Adjusteda
(95%CI)
1.00
2
-
-
-
-
-
1.00
1
-
+
-
-
-
0.24 (0.09~0.63)*
0.23 (0.09~0.61)*
3
-
-
-
-
+
1.00 (0.49~2.03)
1.02 (0.49~2.11)
1
+
-
-
+
+
4.74 (2.48~9.06)*
3.91 (1.99~7.66)*
2
+
-
-
-
+
8.9 (4.00~19.73)*
7.68 (3.23~18.31)*
3
+
-
+
-
+
12.50 (4.78~32.73)*
11.55 (4.18~31.90)*
The possible interaction between GSTM1 and mildewed food
Mildewed
food intake
GSTM1
Case
N
Controls
%
N
Crude OR(95%
CI)
Adjusted
OR(95%CI)
Further adjusted
OR(95%CI)
%
No
Normal
45(25.6)
126(32.9)
1
1
1
Yes
Normal
17(9.7)
29(7.6)
1.64(0.82~3.27)
1.81(0.87~3.76) 2.69(1.15~6.30)
No
Null
86(48.9)
193(50.4)
1.25(0.82~1.91)
1.20(0.77~1.87) 1.38(0.84~2.25)
Yes
Null
28(15.9)
35(9.1)
2.24(1.23~4.09)
2.67(1.36~5.23) 4.13(1.85~9.24)
The possible interaction between GSTT1 and HBV
HBsAg
GSTT1
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
Further
adjusted
OR(95%CI)
1
1
%
No
Normal
36(19.0)
146(37.1)
1
Yes
Normal
67(35.3)
54(13.7)
5.03(3.02~8.39)
4.49(2.63~7.67)
4.48(2.62~7.67)
No
Null
27(14.2)
150(38.1)
0.73(0.42~1.26)
0.73(0.41~1.29)
0.68(0.38~1.22)
Yes
Null
60(31.6)
44(11.2)
5.53(3.25~9.43)
4.91(2.81~8.60)
5.04(2.85~13.9)
The interaction between HBsAg and raw water drinking
Raw water
drinking
HBsAg
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
%
No
Negative
32(17.30)
184(51.40)
1
1
Yes
Negative
30(16.22)
83(23.18)
2.08(1.19~3.64)
1.84(1.03~3.28)
No
Positive
63(34.05)
63(17.60)
5.75(3.44~9.60)
5.25(3.04~9.06)
Yes
Positive
60(32.43)
28(7.82)
12.32(6.86~22.11)
9.66(5.22~17.89)
The interaction between HBsAg and mildewed food intake
Mildew food
intake
HBsAg
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
%
No
Negative
46(24.08)
151(62.13)
1
1
Yes
Negative
21(10.99)
52(12.87)
2.20(1.21~4.00)
2.47(1.32~4.63)
No
Positive
97(21.53)
87(21.53)
6.08(3.97~9.33)
5.41(3.46~8.45)
Yes
Positive
27(14.14)
14(3.47)
10.52(5.13~21.58)
10.82(5.09~22.98)
The interaction between HBsAg and alcohol drinking
Alcohol
drinking
HBsAg
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
%
No
Negative
34(17.71)
157(38.20)
1
1
Yes
Negative
35(18.23)
153(37.23)
1.06(0.63~1.78)
1.06(0.56~2.00)
No
Positive
53(27.60)
70(36.46)
5.00(2.92~8.54)
4.36(2.47~7.68)
Yes
Positive
49(11.92)
52(12.65)
6.22(3.71~10.41)
6.19(3.13~12.25).
The interaction between HBsAg and anti-HCV
Anti-HCV
HBsAg
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
%
No
Negative
63(31.19)
304(73.08)
1
1
Yes
Negative
6(2.97)
10(2.40)
2.90(1.02~8.26)
2.63(0.88~7.85)
No
Positive
120(59.41)
100(24.04)
5.79(3.96~8.46)
5.20(3.49~7.76)
Yes
Positive
13(6.44)
2(0.48)
31.37(6.91~ 42.44) 23.99(5.09~ 13.12)
The interaction between HBsAg and family history of liver cancer
Anti-HCV
HBsAg
Case
N
Control
%
N
Crude OR
(95% CI)
Adjusted
OR (95%CI)
%
No
Negative
58(28.29)
284(68.27)
1
1
Yes
Negative
14(6.83)
30(7.21)
2.285(1.41~4.58)
2.33(1.13~4.81)
No
Positive
94(45.85)
93(22.36)
4.95(3.31~7.40)
4.52(2.96~6.92)
Yes
Positive
39(19.02)
9(2.16)
21.22(9.75~46.19)
24.14(10.34~ 6.35)
Multivariate Logistic Regression Analysis
Selected Variables
OR ( 95%CI )
Age
0.95 (0.93~0.98)
Gender
1.71 (0.80~3.65)
Education
0.56 (0.38~0.82)
Mildewed food intake
2.64 (1.39~4.99)
Refrigerator using at home
0.30 (0.13~0.68)
Raw water drinking
1.19 (0.65~2.19)
Pack-year of smoking
1.00 (0.98~1.03)
Alcohol drinking
1.38 (0.72~2.62)
Characteristics
optimistic
0.40 (0.23~0.69)
Depressed
4.35 (1.41~13.45)
Family history of liver cancer
4.81 (2.41~9.60)
HBsAg
6.93 (4.07~11.78)
Anti-HCV
3.49(1.10~11.07)
GSTM1
1.87 (1.09~3.21)
GSTT1
0.72 (0.43~1.21)
Major Risk Factors for Stomach
Cancer in Chinese Population
• Helicobacter pylori was the first bacterium
to be officially recognized as a cancercausing agent.
Major Risk Factors for Stomach
Cancer in Chinese Population
• Helicobacter pylori Infection. Nitrates and
nitrites are substances commonly found in
cured meats, some drinking water, and
certain vegetables, that can be converted by
Helicobacter pylori, into compounds that
have been found to cause stomach cancer in
animals.
Figure 1a: H.pylori and gastric cancer - Prospective studies: meta-analysis of non cardia cancer cases.
Figure 1b: H.pylori and gastric cancer - Prospective studies: meta-analysis of cardia cancer cases.
H. Pylori Infection and Stomach
Cancer in Whites at MSKCC
H. Pylori case/control
no
69/54
yes
67/15
OR
1.00
3.50 (1.80-6.79)
Infection rates:
21.7% in controls
49.3% in cases
Biomarker in Epidemiology:
Biomarkers of Internal Dose
• Biomarker of internal dose of external
chemical exposures are measurements of a
parent compound or its metabolites in an
accessible biological matrix, such as serum
or urine
Biomarker in Epidemiology:
Biomarkers of Internal Dose
• The half-life of the external agent or its
metabolites in the body
• The pattern of the exposure it is measuring
(regular exposure or infrequent exposure)
• Whether the secular trends have occurred in
that exposure (e.g., smoking cessation)
• Direct or indirect influence of the disease
Urine Test Kit for Tobacco Use
Marker for Internal Dose
Fat-soluble substance such as DDT
metabolites
• Persist over time
• Will not be affected by disease status
DDT
• DDT (dichlorodiphenyltrichloroethane) is
a commercial organochlorine insecticide
that has been used in countries around
the world. It has been used widely on
agricultural crops as well as for "vector
control" - the control of insects that
carry such diseases as malaria and
typhus
DDT
DDT
• This organochlorine insecticide can be
considered as the pesticide of the greatest
historical significance, due to its effect on
the environment, agriculture, and human
health.
• First synthesized by a German graduate
student in 1873, it was rediscovered by Dr.
Paul Mueller, a Swiss entomologist, in
1939 while searching for a long-lasting
insecticide for the clothes moth.
DDT
• DDT subsequently proved to be extremely
effective against flies and mosquitoes, ultimately
leading to the award of the Nobel Prize in
medicine for Dr. Mueller in 1948.
• Effective January 1, 1973 the Environmental
Protection Agency (EPA) officially canceled all
uses of DDT, but not before more than 1 billion
kilograms of DDT had been introduced into the
United States.
Biomarker of Dietary Intake
• Whether it is a good indicator of intake
• Whether it is a long- or short-term marker
• Whether there is a need for multiple
measurements
• Whether it is acceptable for researcher and
the subject
• Whether it is compatible with study design
Main component of green Tea Catechins:
(-)-Epigallocatechin gallate ((-)EGCg)
Adduct as Biomarker
• Chemicals can bind covalently to cellular
macromolecules such as nucleic acids and
protein. The product of this addition of a
chemical moiety to a macromolecule is
termed an “adduct”
Adduct as Biomarker
• Adduct may be highly specific for
carcinogen of interest, but not necessarily
specific for a given exposure because of
multiple sources of carcinogen with
environment
• Adduct formation normally occurs after the
metabolic activation of the carcinogen;
DNA repair may follow adduct formation
Adduct as Biomarker
The persistent of the adducts is determined by
• Chemical stability of the adduct itself
• The turnover of macromolecule to which
the chemical is bound
Adduct as Biomarker
• A half-life of adducts on proteins (HB and
albumin): a few weeks to months
• A half-life of DNA adducts: a few hours to
several years depending on cell type
concerned
Adduct as Biomarker
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•
•
•
•
Adducts can be measured at
Blood
Exfoliated cells
Tissue
Urine (metabolites of adducts)
Group 1: Carcinogenic to Humans
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Tobacco Smoking
Tobacco Products, Smokeless
4-Aminobiphenyl (4-ABP)
Benzene
Carmium
Chromium
2-Naphthylamine (2-NA)
Nickel
Polonium-210 (Radon)
Vinyl Chloride
Group 2A: Probably
Carcinogenic to Humans
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Acrylonitrile
Benzo[a]pyrene
Benzo[a]anthracene
1,3-Butadiene
Dibenz(a,h)anthracene
Formaldehyde
N-Nitrosodiethylamine
N-Nitrosodimethylamine
P32
postlabel
ing