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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 [email protected]