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Paolo VineisUniversity of Torinoand ISI FoundationEPIC: 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 countriesTwo questionnaires (diet+other lifestyle factors) and blood samples in liquid nitrogen24-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 133EXPOSURE ASSESSMENT (HOEK) ALMOST COMPLETEDDETAILS 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=1800Only 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 >40Several locations in France 71,951 22Oxford 56,453 24Cambridge 28,904 24Bilthoven 21,635 36Utrecht 16,584 36Denmark (Copenhagen, Aarhus) 55,259 24Umea 24,590 <10(a) microg/m3
Apoptosis Detoxification DNA repair Silent mutation • Exposure • Environment • Occupation • Tobacco • Diet • Medicines • Hormones • Cosmetics, hair dyes etc. • Metabolism • Gene expression • Enzyme activity • Gene polymorphism • DNA damage • Carcinogen - DNA adducts • DNA strand breaks • Cancer cell • Cancer risk
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)
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 GENES3. 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 individuals2. EXAMPLE OF NAT2 (Vineis, Alavanja, Garte, Int J Cancer 2003 in press): the effect of polymorphism is greater at low doses
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?
Cambridge Oxford Utrecht DNA concentration sorted by EPIC number (origin)
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 8 4.3 (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 1 4.4 (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 43 148 147 OR 13.5 3.0 1.0p=0.006Cases onlyMutated 3 1 1WT 15 50 55 OR 10.3 1.l 1.0p=0.02