Prospects of genomics technologies in cancer molecular epidemiology Soterios A. Kyrtopoulos National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Athens, Greece

Pros and cons of “1st generation biomarkers” Advantages - highly sensitive and chemical-specific biomarkers of exposure - information on specific stages of carcinogenesis (e.g. gene or chromosome mutagenesis) Disadvantages - collect information one item at a time - different assays/technologies for different types of endpoints - limited mechanistic information

Potential advantages of –omics biomarkers Genomics Epigenomics Transcriptomics Metabonomics Proteomics - can be derived from global, untargeted searches – enormous dataset in which to look for biomarkers; no prior hypothesis - use generic technology regardless of disease or exposure of interest - provide mechanistic information on multiple endpoints - combined use of multiple –omics technologies, plus bioinformatics, can integrate multi-level information and provide a systems biology approach to biomarker discovery

1.Genomics: widespread use in GWAS studies Massive SNP analysis and search for association with disease risk Case-control studies, some hundreds to a few thousands of subjects ORs for individual alleles tend to be rather small (<1.5) Current evidence of potential of –omics in environmental health research

2. Εpigenomics Bulk hypomethylation, promoter hypermethylation - Most studies targeted on candidate genes (p16, MGMT, RASSF1A etc) or on surrogate sequences (alu, LINES) -Few genome-wide studies, mostly in relation with diseased states

 Perera et al., PLOS One 2009, e4488 Cord blood, children with maternal high/low PAH exposure; ACSL3 CpG island  Widschwendter et al., PLoS One 2008, e2656 DNA methylation of peripheral blood cell DNA provides good prediction of breast cancer risk in a nested case-control study

 Fry et al., PLoS Genetics 3(11) 2007, e207 Gene expression signatures (11 genes) in cord blood of women exposed to arsenic; could predict exposure of 2 nd set of subjects with 79% accuracy; activation of NFκB inflammation, cell proliferation, stress & apoptosis pathways  Forrest et al., Environ Health Perspect. 113 (2005), gene expression in PBMC in benzene-exposed workers 6 exposed & 6 controls Recent unpublished from same group suggest effects at very low benzene doses 3. Transcriptomics: Global analysis of gene expression, search for association with exposure or early effects Few, rather small studies to date

4. Proteomics - Adsorption on protein chips / SELDI-TOF MS - Multiplex ELISAs Few studies, limited to a few tens of subjects Luminex LabMAP technology  Vermeulen et al., PNAS 102 (2005 ), exposed & 10 controls Decreased levels of CXC-chemokines in serum of benzene- exposed workers identified by array-based proteomics

5. Μetabolomics full-profile analysis of serum/urine by NMR OR HPLC/MS/MS Studies so far mostly limited to diseased states Holmes et al., Nature 453 (2008) Human metabolic phenotype diversity and its association with diet and blood pressure ΙΝΤΕΡΜΑP project: metabolomic profiles in relation to blood pressure & influence of diet & other factors 4,630 subjects, 17 populations Urine analysis using NMR

Conclusion so far: Even with small studies, distinct –omics profiles, with biological meaning, have been identified, implying the potential to serve as biomarkers of toxic exposure or disease risk Questions:  existing studies based on very small populations  limited information on exposure  validation: between study reproducibility / intra-individual variability /...  uncertainty regarding technology applicability with samples already stored in existing biobanks

Genomics biomarkers of environmental health (EnviroGenomarkers; FP7 Collaborative Project; start March 2009, 4 yrs 11 partners incl. 5 ECNIS-associated groups Main aims: -Application of wide range of –omics technologies, in combination with advanced bioinformatics, in the context of molecular epidemiology studies, for the discovery of new biomarkers of exposure to toxic environmental agents, new biomarkers of disease risk, and exploration of their relationships - Exploration of technical potential and problems in the application of –omics technologies in large-scale population studies using biosamples in long-term

exposure biomarkers of exposure intermediate –omics biomarkers of early effects disease prospective study Epidemiologic design: case-control nested within prospective cohorts risk-predictive biomarkers relationship of environmental exposures vs risk biomarkers 1.Biomarkers with risk predictivity: Intermediate biomarkers vs disease 2.Εxposure-disease relastionships: biomarkers of risk vs exposure 3. Early effects of exposure: exposure vs intermediate biomarkers

The EnviroGenomarkers project cohorts

Diseases and exposures Case-control nested within EPIC Italy and NSHDS breast cancer vs PCBs PAHs cadmium B-cell lymphoma vs PCBs Prospective, Rhea cohort (Crete) chronic diseases of the nervous and immune system & allergies establishing themselves in early childhood vs early life exposure to endocrine disruptors (PCBs, PAHs, phthalates, polybrominated diphenyl ethers)

 Start off with pilot study to technically validate applicability of –omics technologies to existing biobank samples  Mimic treatment of biobank samples at the time of their collection Biomarker analyses

Biomarker analyses In total, approx. 2,700 blood samples will be analysed Intermediate biomarkers Transcriptomics Epigenomics Metabonomics Targeted proteomics (Luminex platform) [Genome-wide SNP data already available for many subjects] 2-phase approach will mostly be employed: a) discovery phase (genome-wide); 10-20% of samples b) validation phase (targeted on limited number of candidates): all samples Full cell metabolomics (HPLC/MS) on all samples

Biomarkers of exposure  PCBs: plasma concentrations of selected congeners, using GC/MS  cadmium: erythrocyte levels, using inductively-coupled plasma mass spectrometry  phthalates: urine metabolites, using HPLC/MS  BDPEs: plasma concentrations of selected congeners, using HPLC/MS  PAHs: DNA adducts (ELISA) Additional data on biomarkers of carcinogen exposure available from other projects Exposure assessment Other exposure information  FFQs and environmental exposure questionnaires  Data on exposure to air, water and food toxicants available from other projects  GIS

adapted from Butcher & Beck, 2008) controls transcriptomics epigenomics genomics (SNPs) proteomics metabolomics individual genomic profile diseased combined risk biomarker

Prospects and challenges 1.Omics technologies have high sensitivity, high throughput and allow untargeted searches for genomic profiles which can serve as biomarkers of exposure or early effect 2.Information obtained has mechanistic value 3.Combine with traditional biomarkers for phenotypic anchoring 4.Acculate exposure assessment of utmost importance; good exposure biomarkers needed 5.Prospect of combined analysis of data from multiple levels of cellular function 6.Systems-level bioinformatics analysis 7.Suitability of old biobank samples for certain –omics analysis (e.g. transcriptomics) 8.Detection of risk biomarkers in surrogate tissues (PBLs) 9.Validation, validation, validation

Funded by the European Union FP7, Theme: Environment (including climate change) (Grant no ) Nature, Vol. 458 (9 April 2009), p. 458