Transcript multilevel approach: contributions of molecular dosimetry

Paolo Vineis
University of Torino
and ISI Foundation
EPIC: Molecular markers of
carcinogenesis in a large
prospective study
EPIC is a prospective study on more than 500,000
Europeans (aged 45-70) in 10 countries
Two questionnaires (diet+other lifestyle factors)
and blood samples in liquid nitrogen
24-hor recall from 10%
“GENAIR”
Nested case-control study among the 500,000 EPIC
volunteers: cancers of lung, bladder, larynx,
pharynx, leukemias, COPD, emphysema
Follow-up until 2002:
1104 cases and 2983 controls
(MATCH 1:3)
Non smokers+ex-smokers (since at least 10 yrs),
matched by smoking habits, age, gender, time since
blood drawing, country
CASES:
BLADDER CANCER
LEUKEMIA
LUNG
ORAL
LARYNX AND PHARYNX
RESPIRATORY DEATHS
241
319
275
73
63
133
EXPOSURE ASSESSMENT (HOEK) ALMOST
COMPLETED
DETAILS IN THIRD TECHNICAL REPORT (MAY 2003)
IN www.isi.it
827 CASES AND 1562 CONTROLS (1:2 MATCH) HAVE
BIOLOGICAL SAMPLES
ANALYSES UNDER WAY, ALMOST COMPLETED FOR
DNA ADDUCTS AND POLYMORPHISMS, N=1800
Only a subsample analyzed for more complex markers such
as p53 mutations in plasma and for 4-ABP hemoglobin
adducts (N=458)
Exposure assessment for air pollution (G Hoek, M
Krzyzanowski, Bilthoven)
Bulky (aromatic) DNA adducts in WBC (M
Peluso, Genova)
Hemoglobin adducts (4-ABP, benzopyrene) (L
Airoldi, Milano)
Cotinine and antioxidants in plasma (L Airoldi,
Milano; E Riboli, Lyon)
DNA repair polymorphisms (G. Matullo, Torino;
A. Dunning, Cambridge)
Metabolic polymorphisms (C. Malaveille, Lyon; H
Autrup, Copenhagen; S Garte, Milano)
Mutations in p53 and ras in plasma DNA (P
Hainaut, Lyon)
Mathematical models (F Veglia, Torino)
Advantage of prospective study:
markers are measured in blood drawn years
before the onset of disease, i.e. the measurement is
not influenced by the presence of disease
(metabolic alterations)
Blood is stored at - 196° C in liquid nitrogen
Exposure assessment for air pollution: contrasts
population
PM10 (a)
Italy (Florence, Varese, Torino)
36,177
>40
Several locations in France
71,951
22
Oxford
56,453
24
Cambridge
28,904
24
Bilthoven
21,635
36
Utrecht
16,584
36
Denmark (Copenhagen, Aarhus)
55,259
24
Umea
24,590
<10
(a) microg/m3
Apoptosis
Detoxification
DNA repair
Silent mutation
Exposure
Metabolism
DNA damage
Cancer cell
• Environment
• Gene expression
• Carcinogen -
• Cancer risk
• Occupation
• Enzyme activity
DNA adducts
• Tobacco
• Gene
polymorphism
• DNA strand
breaks
• Diet
• Medicines
• Hormones
• Cosmetics, hair
dyes etc.
ADDUCTS PRELUDE TO MUTATIONS?
DENISSENKO ET AL (1996) HAVE SHOWN
THAT THERE WAS A STRONG SELECTIVE
FORMATION OF ADDUCTS BY 7,8,9,10tetrahydrobenzo(a)pyrene AT GUANINES IN CpG
SEQUENCES OF CODONS 157, 248
AND 273 OF P53 GENE, THE MAJOR
MUTATIONAL HOTSPOTS IN LUNG CANCER
ROLE OF POLYMORPHISMS FOR DNA REPAIR:
XRCC1, XRCC3, XPD (RARE ALLELES) AND
THEIR COMBINATION - MODULATION OF DNA
ADDUCTS IN EPIC ITALY
(Matullo et al, CEBP, 2003)
20
18
16
14
12
10
8
6
4
2
0
N=
32
125
195
174
85
15
0
1
2
3
4
5
NUMBER OF RISK ALLELES
Some theoretical considerations:
What is susceptibility on a population
scale?
Burnet NG, Johansen J, Turesson I, Nyman J, Peacock
JH. Describing patients’ normal tissue reactions
concerning the possibility of individualising
radiotherapy dose prescriptions based on potential
predictive assays of normal tissue radiosensitivity. Int. J.
Cancer 1998; 79: 606-613
HYPOTHESES:
1. GENETIC SUSCEPTIBILITY HAS A CONTINUOUS
DISTRIBUTION, WITH HIGLY PENETRANT GENES
THAT CONFER EXCEPTIONALLY HIGH RISKS OF
DISEASE, AND LOW-PENETRANT GENES THAT
MODULATE THE RESPONSES
2. THE COMBINATION OF GENES IS MORE
IMPORTANT THAN SINGLE GENES
3. LOW-PENETRANT GENES ARE MORE
IMPORTANT AT LOW DOSES (I.E. A LOW DOSE IS
SUFFICIENT TO INDUCE THE DISEASE IN
SUSCEPTIBLE PERSONS)
SHAPE OF DOSE-RESPONSE RELATIONSHIPS
IN PRESENCE OF MODULATION FROM
POLIMORPHIC GENES:
1. EXAMPLE OF CYP1A1 MSPI (Vineis et al, Int. J
Cancer 2003; 104: 650): the dose effect is greater in
polymorphic individuals
2. EXAMPLE OF NAT2 (Vineis, Alavanja, Garte, Int
J Cancer 2003 in press): the effect of polymorphism is
greater at low doses
Odds Ratio
Caucasians - Ever smokers
20
18
16
14
12
10
8
6
4
2
0
w ildtype
heterozygotes+homozygotes
1
2
3
Quartiles of duration
4
LOW DOSE EFFECT
Y
2
1
0
0
0,2
0,4
0,6
0,8
1
1,2
v/Vmax
Figure 1: Hypothetical example: the graph is a plot of rate/Vmax (which is a function of the dose)
vs. Y (the extent of the low dose effect)(see text).
Genetic alterations in plasma DNA
* Useful when tumours not available
* Good concordance between tumour and plasma mutations
* When does tumour DNA appear in the blood?
* Can plasma DNA be used as a biomarker for genotoxic exposure?
DNA concentration sorted by EPIC
number (origin)
6702
6478
6841
5960
5974
7413
5297
3687
5521
4821
4555
3505
3239
2875
2637
2357
5171
3939
7313
1600
1400
1200
1000
800
600
400
200
0
3967
DNA concentration (ng/ml)
GENAIR DNA concentration
MOC number
Cambridge
Oxford
Utrecht
Distribution of plasma DNA amount by type of tumours and controls
(N=1151 total observations). Values are ng/100 ml.
N
Mean
Std. Deviation
p-value (a)
Controls
778 6.7
40.5
Deaths (COPD)
49
8.5
13.4
0.005
Bladder cancer
89
7.3
18.6
0.31
Leukemia
129 7.2
12.7
0.008
Lung
82
6.5
14.3
0.64
Oral
28
6.2
10.4
0.42
Pharynx-larynx
30
8.9
28.1
0.57
(a) (comparison with controls)
Genetic alterations in GENAIR
plasma
DNA
* TP53 mutations and CDKN2a hypermethylation
* Mutations K-ras codon 12: Mutant Enriched PCR
Distribution of cases and controls according to p53
mutations (WT=wildtype).
Controls All cancers
Mutated
WT
3
243
Odds ratio
(95% CI)
8
151
4.3 (1.1-16.4)
p=0.02
Distribution of cases+controls according to p53 mutations
(WT=wildtype) and presence or absence of P32postlabelling DNA adducts.
Mutated
ADDUCTS
yes
no
10
1
Odds ratio
(95% CI)
4.4 (0.6-35)
WT
262
115
Distribution of 6 mutated incident cases according to
time between p53 mutation and cancer onset (months)
bladder
bladder
bladder
leukemia
lung
lung
months
1.8
6.3
32.2
8.6
18.1
19.1
smoking
never
former
never
former
never
former
Distribution of cases+controls according to p53 mutations
(WT=wildtype) and genotype for XRCC1 (polymorphism
in codon 28152).
Mutated
WT
OR
p=0.006
Cases only
Mutated
WT
OR
p=0.02
AA
AG GG
4
43
13.5
3
148
3.0
3
15
10.3
1
50
1.l
1
147
1.0
1
55
1.0
THE END
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