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

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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

THE END

paolo.vineis@unito.it