1. Paolo Vineis University of Torino and ISI Foundation EPIC: Molecular markers of carcinogenesis in a large prospective study

2. 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%

4. “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

5. CASES: BLADDER CANCER241 LEUKEMIA319 LUNG275 ORAL73 LARYNX AND PHARYNX 63 RESPIRATORY DEATHS133 EXPOSURE ASSESSMENT (HOEK) ALMOST COMPLETED DETAILS IN THIRD TECHNICAL REPORT (MAY 2003) IN www.isi.it

6. 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)

7. 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)

8. 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)

9. 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

10. Exposure assessment for air pollution: contrasts populationPM10 (a) Italy (Florence, Varese, Torino)36,177>40 Several locations in France71,95122 Oxford56,45324 Cambridge28,90424 Bilthoven21,63536 Utrecht16,58436 Denmark (Copenhagen, Aarhus)55,25924 Umea24,590<10 (a) microg/m 3

11. Exposure Environment Environment Occupation Occupation Tobacco Tobacco Diet Diet Medicines Medicines Hormones Hormones Cosmetics, hair dyes etc. Cosmetics, hair dyes etc.Metabolism Gene expression Gene expression Enzyme activity Enzyme activity Gene polymorphism Gene polymorphism DNA damage Carcinogen - DNA adducts Carcinogen - DNA adducts DNA strand breaks DNA strand breaks Cancer cell Cancer risk Cancer risk Detoxification DNA repair Apoptosis Silent mutation

12. 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

13. 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)

15. Some theoretical considerations: What is susceptibility on a population scale?

16. 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

18. 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)

19. 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

22. 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?

23. DNA concentration sorted by EPIC number (origin) Oxford Cambridge Utrecht

25. Genetic alterations in GENAIR plasma DNA * TP53 mutations and CDKN2a hypermethylation * Mutations K-ras codon 12: Mutant Enriched PCR

26. Distribution of cases and controls according to p53 mutations (WT=wildtype). Controls All cancersOdds ratio (95% CI) Mutated3 8 4.3 (1.1-16.4) WT243 151p=0.02

27. Distribution of cases+controls according to p53 mutations (WT=wildtype) and presence or absence of P 32 - postlabelling DNA adducts. ADDUCTSOdds ratio yesno(95% CI) Mutated10 1 4.4 (0.6-35) WT262 115

28. Distribution of 6 mutated incident cases according to time between p53 mutation and cancer onset (months) monthssmoking bladder1.8never bladder6.3former bladder32.2never leukemia8.6former lung18.1never lung19.1former

29. Distribution of cases+controls according to p53 mutations (WT=wildtype) and genotype for XRCC1 (polymorphism in codon 28152). AAAG GG Mutated431 WT43148 147 OR13.53.01.0 p=0.006 Cases only Mutated311 WT1550 55 OR10.31.l1.0 p=0.02

30. THE END paolo.vineis@unito.it