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Paolo VineisUniversity of Torinoand 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 241LEUKEMIA 319LUNG 275ORAL 73LARYNX AND PHARYNX 63RESPIRATORY DEATHS 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
ExposureExposure
• EnvironmentEnvironment
• OccupationOccupation
• TobaccoTobacco
• DietDiet
• MedicinesMedicines
• HormonesHormones
• Cosmetics, hair Cosmetics, hair dyes etcdyes etc..
MetabolismMetabolism
• Gene expressionGene expression
• Enzyme activityEnzyme activity
• Gene Gene polymorphismpolymorphism
DNA damageDNA damage
• Carcinogen - Carcinogen - DNA adductsDNA adducts
• DNA strand DNA strand breaksbreaks
Cancer cellCancer cell
• Cancer riskCancer risk
DetoxificationDetoxification DNA repairDNA repair
ApoptosisApoptosis
Silent mutationSilent mutation
ADDUCTS PRELUDE TO MUTATIONS?
DENISSENKO ET AL (1996) HAVE SHOWN
THAT THERE WAS A STRONG SELECTIVE
FORMATION OF ADDUCTS BY 7,8,9,10-
tetrahydrobenzo(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)
158517419512532N =
NUMBER OF RISK ALLELES
543210
RA
L (M
EA
N +
- S
E)
20
18
16
14
12
10
8
6
4
2
0
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
Caucasians - Ever smokers
0
2
4
6
8
10
12
14
16
18
20
1 2 3 4
Quartiles of duration
Odd
s R
atio
w ildtype
heterozygotes+homozygotes
F i g u r e 1 : H y p o t h e t i c a l e x a m p l e : t h e g r a p h i s a p l o t o f r a t e / V m a x ( w h i c h i s a f u n c t i o n o f t h e d o s e )
v s . Y ( t h e e x t e n t o f t h e l o w d o s e e f f e c t ) ( s e e t e x t ) .
L O W D O S E E F F E C T
0
1
2
0 0 ,2 0 ,4 0 ,6 0 ,8 1 1 ,2
v /V m a x
Y
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?
GENAIR DNA concentration
0200
400600
8001000
12001400
16003
96
7
73
13
39
39
51
71
23
57
26
37
28
75
32
39
35
05
45
55
48
21
55
21
36
87
52
97
74
13
59
74
59
60
68
41
64
78
67
02
MOC number
DN
A c
on
ce
ntr
ati
on
(n
g/m
l)
DNA concentration sorted by EPIC number (origin)
OxfordCambridge 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 Odds ratio (95% CI)
Mutated 3 84.3 (1.1-16.4)
WT 243 151 p=0.02
Distribution of cases+controls according to p53 mutations (WT=wildtype) and presence or absence of P32-postlabelling DNA adducts.
ADDUCTS Odds ratio yes no (95% CI)
Mutated 10 14.4 (0.6-35)
WT 262 115
Distribution of 6 mutated incident cases according to time between p53 mutation and cancer onset (months)
months smokingbladder 1.8 neverbladder 6.3 formerbladder 32.2 neverleukemia 8.6 formerlung 18.1 neverlung 19.1 former
Distribution of cases+controls according to p53 mutations (WT=wildtype) and genotype for XRCC1 (polymorphism in codon 28152).
AA AG GG
Mutated 4 3 1WT 43 148 147OR 13.5 3.0 1.0p=0.006
Cases onlyMutated 3 1 1WT 15 50 55OR 10.3 1.l 1.0p=0.02
