Environmental exposure assessment: collateral damage in the genomic revolution?

1. Environmental exposure assessment: collateral damage in the genomic revolution? Christopher P. Wild International Agency for Research on Cancer, Lyon, France

2. The role for IARC Cancer research with an international perspective • Inter-disciplinary (lab/epidemiology) • Collaborative • Low- and middle-income countries • Research to inform public health decisions on cancer prevention

3. Cancer Research Community National Cancer Institutes/Organisations The context for IARC International Agency for Research on Cancer World Health Organisation - prevention and control; research International Organisations - UICC – Global Control of Cancer

4. Current Participating States Governing Council sets strategy Scientific Council advises Director and evaluates performance

5. IARC – five core priorities • Describe cancer occurrence (Cancer incidence in Five continents; GLOBOCAN; childhood cancer registration; CANREG) • Establish cancer aetiology • Evaluate cancer risks (IARC Monographs) • Develop and evaluate prevention • Education and training (post-doctoral training fellowships, courses)

7. Comparison of visual inspection with acetic acid, HPV testing and conventional cytology in cervical cancer screening: randomized intervention trial in Osmanabed District, Maharashtra State, India Supported by the Bill & Melinda Gates Foundation through the ACCP

8. Hazard ratios (HR) of cervical cancer deaths rates Sankaranarayanan et al., N Engl J Med 2009;360:1385-1394

9. Growing global burden of cancer In 2008: 12.4 million new cases 7.6 million deaths worldwide IARC, GLOBOCAN 2002

10. Socio-demographic influences on cancer burden • Population growth- world population estimated to grow from 6.7 billion in 2008 to 8.3 billion by 2030; 4% growth in more developed regions, ~40% in more developing regions • Ageing- 10 per cent >65 years in 2000, projected to reach 21 per cent in 2050 • Changing lifestyle and exposures – 1% increase per year in incidence

11. 32.5 (100/29) 83.0 (54/2) 4.0 (44/15) 1.1 (5/5) 5.5 (11/7) 80.5 (11/2) 11.6 total (225/60) Cancer incidence data % population covered by cancer registries in IARC CI5 Vol. IX (number of registries/number of countries providing data)

12. Defining the “environment” “the physical, chemical and biological factors external to a person and all the related behaviours (WHO 2006)”

13. Major cancer risk factors • Tobacco (multiple tumour sites; 30% of cancers in high-resource countries; 1.3 billion people worldwide are smokers ) • Infections (15-20% of cancers worldwide; >25% in developing countries) • Diet –(relatively little understood concerning how specific nutrients or dietary patterns affect risk) • Obesity, overweight, physical inactivity -(estimated 1.5 billion people obese by 2015; up to 1/3 of cancers of colon, breast, endometrium, oesophagus and kidney) • Radiation (ionizing, sunlight) • Reproductive factors and hormones • Alcohol • Occupation • Environmental pollution

14. Importance of environmental exposure assessment • Most major common diseases have an environmental aetiology • Currently exposure measurement is problematic in many areas, leading to misclassification • Large prospective cohort studies (e.g. UK Biobank) are predicated on the availability of accurate exposure assessment • Exposure biomarkers can contribute to several areas in addition to elucidating disease aetiology

15. Uca Pugnax, the male Fiddler Crab Complementing the genome with an “exposome”: the outstanding challenge of environmental exposure measurement in molecular epidemiology • Wild CP (2005) Cancer Epidemiology, Biomarkers and Prevention, 14: 1847-1850. • Wild CP (2009) Mutagenesis 24: 117-125.

16. Challenges in characterising the “exposome” • Scale and complexity:characterisation of life-course environmental exposures, including lifestyle, nutrition, occupation etc., • Dynamic:Unlike the genome, the “exposome” changes over time – possibility of critical windows of exposure e.g. in early life • However, even partial characterisation can bring major benefits

17. Advances in exposure assessment • Biomarkers • Geographic information systems • Personal and environmental monitoring • Sophisticated questionnaires (e.g. for diet, occupation)

18. Exposure biomarkers in population studies – what do they promise? • Defining etiology • Improved exposure assessment – reduced misclassification • Identifying susceptible individuals or sub-groups • Contributing to biological plausibility

19. Exposure biomarkers in population studies – what do they promise? • Evaluating Interventions • Primary and secondary prevention • Bio-monitoring e.g. occupational setting • Hazard and Risk Assessment • Mechanistic data (e.g. IARC Monographs) • Extrapolation from animal to human • Pharmacokinetic-based models

20. HCC incidence correlated with aflatoxin ingestion in Africa and Asia Thailand, 1972; Kenya, 1973; Swaziland, 1976, 1987; Mozambique and Transkei, 1985 Bosch and Munoz, IARC Publ. No. 89: 427 (1988) Modified

21. Interaction between HBV infection and aflatoxins in hepatocellular carcinoma Relative Risk of hepatocellular carcinoma Aflatoxins (urinary biomarkers) HBV (HBsAg) HBVand Aflatoxins none adapted from Qian et al, CEBP 1994, following Ross et al., Lancet 1992

22. Validation and application • A plea for validation – difficult to find support for, but essential for progress • An integral part of method development should be the consideration of throughput, cost and applicability to biobank samples

23. Biomarkers and classification of carcinogenicity

24. Complementary emphasis in exposure biomarkers • First generation exposure biomarkers tended to focus on a classical mutagen – carcinogen model of carcinogenesis (metabolites, adducts, chromosomal alterations, somatic mutations)

25. Biomarkers in relation other mechanisms of carcinogenesis • Epigenetic changes (promoter methylation, histone acetylation, microRNA) • Altered gene, protein or metabolite levels Potential application to “exposures” such as obesity, physical activity, nutrition, complex mixtures

26. Epigenetic biomarkers – applicability to population studies 1 • Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids(Vaissiere et al., Epigenetics, 2009)

27. Epigenetic biomarkers – applicability to population studies 2 • Detection of stable miRNAs in plasma and serum – differences by disease status (Mitchell et al., PNAS 105: 10513, 2008; Chen et al., Cell Res., 18: 997, 2008) • Cell and tissue specific expression • Stable in biological fluids such as plasma and serum • PCR based assays available • Profiling a small number may provide discrimination • Genetic variations in miRNA processing genes and in miRNA binding sites may confer genetic susceptibility • Functional information is vital

28. Can “omics” help improve exposure assessment? • Do specific exposures, or categories of exposure, alter the expression of specific groups of genes, proteins or metabolites (“exposure fingerprint”)? • How do such alterations relate to dose? • How stable are the alterations over time? • How do potential confounding factors affect the association between exposure and “omics” biomarkers

29. Transcriptomics and exposure assessment (see Wild CP, Mutagenesis 24: 117-125, 2009) • Smoking – Lampe et al., CEBP, 13: 445-453, 2004; van Leeuwen et al., Carcinogenesis, 28: 691-697, 2007 • Benzene - Forrest et al., EHP 113: 801, 2005 • Arsenic – Fry et al., PLoS Genet., 3: 2180-2189, 2007; Wu et al., 111: 1429-1438, 2003 • Metal fumes – Wang et al., Env. Health Persp., 113: 233-241, 2005 • Air pollution – van Leeuwen et al., Mutat. Res., 600: 12-22, 2006

30. Metabonomics and population studies • Connects molecular events to those at the macro level • Applicable to blood and urine samples • LC-mass spectrometry methodology affordable and of requisite throughput • Demonstrated applicability to studies of diet (Solanky et al., Anal. Biochem., 323: 197-204, 2003; Holmes et al., Nature, 453: 396-400, 2008)

31. Problems in comparisons of “omics” data in poorly designed studies See Potter JD Trends in Genetics, 19: 690-695, 2003 • Unmeasured confounding by lack of information on age, sex and other exposures • Bias through differences in sample processing • Selection bias through sampling procedures • High costs leading to one-off or small-scale studies

32. Early life exposure and cancer risk • Observational studies linking early life exposures to disease later in life • Foetal programming; adaptive response - indications of alterations in the epigenome • Vulnerability of children to environmental exposures • Reported rise in childhood cancer rates (see Steliarova-Foucher et al., Lancet 364: 2097, 2004 from the Automated Childhood Cancer Information System (ACCIS Project)

33. Adult Peri-natal Adolescence Childhood Temporal application of exposure biomarkers in cancer epidemiology Exposure Disease Birth cohort Adult cohort Case-control study Carcinogen metabolites DNA/protein adducts Cytogenetic alterations Mutation spectra Antibodies Timing of exposure measurement

34. Early life exposure and cancer risk - opportunities • Mother:child birth cohorts – need for international cooperation • Mechanism-based biomarkers to relate exposure to disease – a necessity?

35. Activation of inflammation/NF-κB signalling in infants born to arsenic-exposed mothersFry et al., PLoS Genetics, 3: 2180-2189, 2007 • 32 pregnant women in Thailand in high and low areas of arsenic exposure • Toenail analysis of arsenic; cord blood for microarray gene expression • Expression signatures highly predictive of prenatal arsenic exposure; genes related to stress, inflammation, metal exposure and apoptosis

36. Sub-Saharan Africa • 4.5 million deaths in children under age 5 annually • 175 child deaths (<5 yrs) per 1000 live births (c.f. 6 per 1000 in industrialized nations) Under-nutrition and growth faltering is an underlying cause of 50% of deaths in children <5 years age (Black et al., Lancet, 2003)

37. Aflatoxin, weaning and effects on growth in young children in Benin and Togo • Sixteen villages in four agro-ecological zones • 479 children (age 9 months - 5 years) • Aflatoxin-albumin in blood • Anthropometry Gong et al., Brit. Med J. 2002

38. Exposure to aflatoxin associated with impaired growth 80 Z >0 Z 0 to-2 Z -2 to -3 Z <=-3 60 AF-alb (pg/mg) 40 20 0 Height for Age Weight for Age Growth Status (Z score)

39. Longitudinal study of aflatoxin exposure and child growth in Benin Subjects: 200 children, aged 16-37 months from four villages, two high, two low aflatoxin exposure Time: February May/June October Survey: 1 2 3 Serum AF-alb: X X X Anthropometry: X X X Questionnaire: X X X

40. Longitudinal Study of Aflatoxin Exposure and Child Growth in Benin Gong et al., Environ. Health Perspec. (2004) 112, 1334-1338 200 children, aged 16-37 months followed over 8 months aAdjusted for age, height, weaning status, mothers SES and village. cData labelled * are significantly different to **.

41. Are there critical windows of exposure during which exposure to environmental risk factors is most relevant? The public health relevance of an exposure should be considered in relation to all its adverse health effects

42. Biomarkers and Biological Plausibility • Demonstration of exposure • Evidence for a plausible mechanism

43. Demonstration of exposure –environmental tobacco smoke Nicotine/Cotinine Urinary TSNA 4-ABP-Hb Urinary mutagenicity Demonstration of exposure and plausibility of association with disease Anderson et al., JNCI, 93: 378-381, 2001

44. Plausible mechanism – ETS and bladder cancer in never smokers * Control, subjects only Adapted from Jiang et al., Cancer Res., 67: 7540-7545, 2007

45. Biomarkers and intervention studies • Proof of concept(e.g. anti-oxidants, induction of detoxification enzymes, avoidance of exposure) • Surrogate (earlier) outcome

46. Protocol for primary prevention study to reduce aflatoxin exposure in Guinea 20 Villages (10 intervention, 10 control), 30 subjects per village Sept/Oct Dec/Jan Feb/Mar Intermediate Survey 1 Intermediate Survey 2 Survey 1 Survey 2 Survey 3 Blood sample collection Groundnut sample collection

47. Mean levels of AF-alb are reduced in individuals following intervention Turner et al.,(2005) The Lancet, 365, 1950-1956

48. Intervention increases the number of individuals with non-detectable blood AF-alb Turner et al.,(2005) The Lancet, 365, 1950-1956

49. Future perspectives – summary 1 • Investment in exposure biomarkers to complement genetic analysis is required if large/expensive prospective cohort studies are to fulfill their promise • New methodologies (e.g. metabonomics) and knowledge of mechanisms (e.g. epigenetics) need to be applied to population-based investigations of environment and cancer

50. Future perspectives – summary 2 • The contribution of biological plausibility to establishing aetiology should be given higher priority, particularly in cases of modest risk elevation • Early life exposures merit consideration in the context of mother:child cohorts and related biobanks • Priorities for prevention need to be considered across the disease spectrum where appropriate