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Genomics Biomarkers of Environmental Health
Newsletter ISSUE 1 • NOVEMBER 2010
www.envirogenomarkers.net • An FP7 molecular epidemiology project
What is EnviroGenomarkers?EnviroGenomarkers is a European FP7 research project aiming at the development and ap-plication of a new generation of biomarkers, based on -omics technologies, to study the envi-ronmental aetiology of human disease.
In the project, advanced technologies, including high-density -omics technologies in combina-tion with sophisticated bioinformatics, are being utilised with the following aims:
the discovery and validation of novel biomarkers predictive of increased risks of chronic • diseases in which the environment may play an important role (breast cancer, B-cell lym-phoma, childhood diseases including allergy, neurological and immune diseases),
the exploration of the association of such risk biomarkers with exposure to a number of • high-priority or emerging environmental pollutants with carcinogenic, immunotoxic or hor-mone-like properties, including polychlorinated biphenyls, polycyclic aromatic hydrocar-bons, cadmium, lead, phthalates, brominated flame retardants (polybrominated diphenyl ethers), as well as ambient air pollutants and water treatment byproducts,
the discovery and validation of biomarkers of exposure to the above pollutants.•
Project identityFP7 medium-scale collaborative project; priority area “Environment (including climatic change)”, sub-activity “Environment and health”; Grant Agreement 226756 • Co-ordinator: S.A. Kyrtopoulos, National Hellenic Research Foundation, Athens, Greece • Project dura-tion: March 2009 – February 2013 • Budget (EC contribution): 3.5 Meuro.
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Conceptual basis of the project
Data integration using bioinformatics and systems biology methodologies
exposure
chemicals: PCBs, PAHs, cadmium, phthalates, brominated flame retardants, ambient air pollutants, water disinfection byproducts
biomarkers of exposure intermediate -omicsbiomarkers of early effects
disease
Food frequency • questionnaires
Environmental • monitoring
GIS•
Concentrations • in urine, serum & erythrocytes
DNA adducts•
Metabonomics• Epigenomics• Proteomics• Transcriptomics•
Breast cancer•
Non-Hodgkin’s • lymphoma
Childhood allergy, • neurological and immune diseases
Biosamples and data from 3 prospective cohorts
Northern Sweden Health and Disease Study1. 2. EPIC Italy 3. Rhea mother-child cohort (Crete, Greece)
Risk-predictive intermediate biomarkersExposure – risk predictive intermediate biomarkers relationships
Exposure – disease risk relationships
Message from the coordinator The EnviroGenomarkers project is now well into its second year of operation and can look back at a successful establishment of its operational basis and the fulfillment of the basic prerequi-sites that enable it to progress towards its primary objectives.
EnviroGenomarkers is an ambitious project in a number of ways: First of all, it set out to apply highly demanding analytical techniques, including modern, high-density -omics methods, to a particularly large number of biosamples, being in fact one of the largest molecular epide-miology projects in the area of environmental health conducted in Europe. During the past 18 months or so, we were able to put together the project’s adult cohorts, consisting of 600 matched case-control pairs for breast cancer and 300 for B-cell lymphoma, coming from the consortium’s two adult cohorts and biobanks, the Northern Sweden Health and Disease Study and EPIC Italy. Altogether, more than 1800 sets of samples (buffy coats, erythrocytes, plasma) have been successfully distributed to the five laboratories conducting biomarker measure-ments, and approximately half of these samples have already been analysed. This progress confirms that the project has been completely successful in this respect.
Secondly, perhaps the most ambitious aim of the project is to use human biological samples, collected and placed in cold storage before the advent of -omics technologies and without the special precautions nowadays used for the preservation of samples destined for such analyses. The question whether such samples can be meaningfully analysed by these modern technologies, especially in relation to the transcriptomics, is one that many people were asking when the project was in it planning stages, and in fact a number of anecdotal reports suggest-ed that this is not possible. The EnviroGenomarkers team approached this question at an early stage in the project, by conducting a systematic pilot study which led it to identify criteria which samples stored without special precautions should satisfy and to develop handling procedures which permitted such samples to yield -omics data of quality comparable to that obtained with freshly collected samples. Does this mean that such samples have not suffered, during their storage or handling, any damage that might distort the -omics profiles they yield? It will be possible to begin answering this question only after these profiles are evaluated against the exposures and diseases of interest. If it turns out that these profiles correspond to biologically plausible pathways then one may be optimistic that the answer is affirmative. Should this prove to be so, it will mean a major advance in the prospects of exploiting the unique and enormous resource which existing biobanks represent (containing more than 1.5 million biosamples). This is an exciting prospect to look forward to as EnviroGenomarkers continues its work over the next couple of years.
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S.A. Kyrtopoulos Athens, November 2010
The EnviroGenomarkers cohorts
EnviroGenomarkers is a case-control study nested within 3 prospective cohorts:
The Northern Sweden Health and Disease • Study (NSHDS, Umea, Sweden)
EPIC Italy •
The Rhea mother-child birth cohort (Crete, Greece)•
These cohorts contain information (mainly on dietary habits) as well as blood fractions (white cells, plasma or serum, erythrocytes) stored in freezers.
i) The Northern Sweden Health and Disease Study biobank contains samples and data from more than 100,000 individuals, collected in the context of various studies. In the largest among these, the Västerbotten Intervention program, blood samples and lifestyle and environmental exposure information have been collected since 1985 from all individuals of the county aged 40, 50 and 60 years, and currently more than 100,000 samples from 80,000 unique individuals are available. Since 1995, a second blood sample (and questionnaire) is taken with a 10-year interval of the individuals within the cohort, an important asset which is exploited in the Envi-roGenomarkers project.
ii) EPIC-Italy is part of the continent-wide European Investigation into Cancer and Nutrition (EPIC) study in the context of which, since 1992, subjects without known disease, aged 35-70 years, were invited to provide blood samples and information on diet and lifestyle. EPIC-Italy contains samples and information from approximately 47,700 subjects in 5 different areas of the country.
iii) The Rhea mother-child birth cohort, set up in 2007, has enrolled approx. 1,700 pregnant women in the Heraklion region of Crete at the 3rd month of pregnancy and has followed them up until birth and after birth together with their children. Urine and blood samples were col-lected from the mothers at different times during pregnancy and at birth, along with cord blood, as well as information on dietary habits and lifestyle.
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NSHDS - the Northern Sweden Health & Disease Study
EPIC - ItalyRhea mother - child cohort
Crete
Diseases and environmental chemicals investigated
Breast cancer is a leading cause of death for women aged from their late 30s to their early • 50s, with more than 270,000 women being diagnosed with breast cancer each year in the EU. The large geographic variation in its frequency suggests that environmental factors (nutrition, lifestyle, environmental chemicals) may contribute to its causation. There is evi-dence that breast cancer risk may be increased by exposure to polychlorinated biphenyls (PCBs, pollutants with carcinogenic, immunotoxic and hormone-like activities, which accu-mulate in food and in human fatty tissues), polycyclic aromatic hydrocarbons (PAHs, car-cinogens found mainly in charcoaled foods and the atmospheric air polluted with exhaust gases) and cadmium (found in food as a result of its presence in fertilisers).
B-Cell lymphoma is one of the two major sub-types of non-Hodgkin lymphoma, the fifth • most common malignancy in the Western world, with more than 50,000 new cases being diagnosed in the European Union each year. Its frequency has risen dramatically in the lat-ter half of the 20th century, exceeding 4% annually between 1985 and 1992 in some of Eu-ropean countries. Little is known about its causes. Several recent studies have implicated PCBs in the causation of the disease.
Chronic neurological, immune and allergy effects with childhood origins. During recent • years, there has been growing awareness of an emerging risk related to the exposure dur-ing pregnancy or early life to environmental chemicals which give rise to health effects dur-ing childhood persisting into adulthood. Such effects include persistent effects on behav-iour and cognitive function (altered play behaviour pattern, overall cognitive performance, greater impulsivity, poorer concentration, and poorer verbal, pictorial, and auditory working memory), deficient immune function leading to long-term effects (into adulthood) including allergies and asthma. There is growing epidemiological and experimental evidence that en-vironmental chemicals like PCBs, phthalates (widely used as plasticisers) and brominated flame retardants can cause such effects owing to their hormone-like properties.
Diseases and environmental chemicals from the leaflet material
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Disease
Breast cancer •
Non-Hodgkins lymphoma •
Chronic neurological, • immune and allergy effects of childhood origin
Chemical
PCBs, PAHs, Cd•
PCBs•
PCBs, phthalates, • polybrominated diphenyl ethers
Biomarkers in EnviroGenomarkers
Biomarkers of exposure, as well as intermediate biomarkers based on -omics technologies, are being measured in biosamples coming from matched pairs of subjects with or without disease and collected prior to the appearance of the disease.
Biomarkers of exposure
plasma concentrations of PCBs and polybrominated diphenyl ethers•
erythrocyte cadmium concentrations •
PAH-DNA adducts in white blood cells•
urine concentrations of phthalate metabolites•
Intermediate, - omics biomarkers
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Why -omics?“-omics” technologies make possible the simultaneous examination and measurement of all, or a large fraction of, features of a given type in the cell (see below). This provides unprecedented opportunities for biomarker discovery since one can search for biomark-ers of interest among an enormous number of observations, without being limited by prior hypotheses.
omics technologies in • EnviroGenomarkers
Metabonomics: analysis, • using advanced liquid chromatography-mass spectrometry, of thou-sands of low-molecular weight substances present in serum
Epigenomics: • Measurement, using mi-croarray technology, of the levels of 5-methylcytosine at more than 450,000 of CpG dinucleotides in DNA scattered throughout the genome
Transcriptomics: • Measurement, using microarray technology, of the level of expression of all genes in the cell
Proteomics: Analysis of the • levels of 47 inflammation-related proteins in plasma
Examples of RNA yield and quality ob-• served with “real” biobank samples
Results pilot experiment 3
Comparison of microarray gene expression data obtained with fresh and biobank samples•
Transcriptomics: distribution of signal intensity (unnormalised data)
Transcriptomics: fraction of good probes
Epigenomics: distribution of beta values (fraction of methylation at CpG sites)
Fresh samplesFresh samples Biobank samplesFresh samples Biobank samplesBiobank samples
EnviroGenomarkers pilot study: Can human samples from existing biobanks be analysed by -omics methodologies?
One of the key questions addressed by the EnviroGenomarkers project relates to the possibil-ity of analyzing, using -omics technologies, biological samples which were collected before the advent of -omics technologies, without the special precautions which are taken nowadays. This question is particularly critical in relation to the conduct of genome-wide gene expression analysis (transcriptomics) using microarrays.
To address this question, the EnviroGenomarkers consortium conducted a pilot study. Ini-tially, blood samples were freshly collected from volunteers and handled in different ways that mimicked the range of conditions used by biobanks when their samples were collected, in relation to the use of anticoagulants, time between sample collection and processing, storage conditions etc. These samples were subsequently analysed by the various -omics techniques employed in the project, including microarray-based transcriptomics and epigenomics (CpG methylation), wide-target proteomics and full plasma metabonomics (UPLC-MS/MS), as well as target-specific methods like RT-PCR and bisulphite treatment/pyrosequencing. In addition, methodological adaptations to minimize RNA damage after removal of the samples from stor-age were developing and applied. In this way cut-off criteria were identified within which sat-isfactory -omics profiles could be obtained from such samples, which were confirmed by the analysis of “real” samples in long-term (10 years or even longer) storage in the project’s col-laborating biobanks.
Using the above approach, more than 400 samples, stored in the project’s biobanks for 2-15 years, have been analysed using the -omics technologies employed in the project, with more than 80% of them yielding -omics profiles of a quality comparable with that observed with freshly collected samples.
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Statistical analysis Statistical analysis of all omics and other data is carefully designed
Data warehouse
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omics data warehouse
Data analysis:
T-test / Chi-square•
Evolutionary Stochastic Search •
Adaptive empirical Bayesian thresh-holding•
Random Forrest / CART•
Partial Least Squares•
Matroids•
Neurological networks•
Ontology•
Mechanistic insights•
Pathway analyses• }
Project progress to date
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EnviroGenomarkers in Nature Magazine Soon after its launch, EnviroGenomarkers was featured in a Nature Magazine article on environ-mental cancer research. The article, published on 9 April 2009, presented the changing face of funding in this area, in the United States and in Europe, citing EnviroGenomarkers as an example of the European Union’s increasing interest and support.
According to the US National Institutes of Health (NIH), environmental factors such as pollution, cigarette smoke and diet contribute to 80–90% of all cancers. Despite these numbers, research in environmental oncology — which aims to track down environmental contributions to cancer — receives only a fraction of the amount spent on the hunt for cures. …This attitude may be changing with the recent emphasis on early disease diagnosis and personalized health care. “It is now being realized that an exclusive focus on genetic aspects of cancer is not providing solutions,” says So-terios Kyrtopoulos, director of the chemical carcinogenesis and genetic toxicology programme at the National Hellenic Research Foundation in Athens, Greece. “So the pendulum is swinging back again to studying the environment.”
“Prevention by numbers”. Nature, vol. 458 (2009), 792-3.
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Envirogenomarkes in international Conferences
The project and its activities have been presented, through oral and poster presentations, in a large number of international scientific meetings, including the following:
Genomics in cancer molecular epidemiology: The EnviroGenomarkers project (S.A. Kyr-• topoulos, oral presentation at the Workshop on Genomics in Cancer Risk Assessment, Venice, 27-28 August 2009; satellite meeting of the 10th ICEM-International Conference on Environmental Mutagens)
Genomics in cancer molecular epidemiology: The EnviroGenomarkers project (S.A. Kyr-• topoulos, oral presentation at the ECNIS Workshop on “Biomarkers and Cancer”, Porto, 21-23 September 2009
EnviroGenomarkers: A European molecular epidemiology project on genomics biomark-• ers of environmental health (S.A. Kyrtopoulos et al., poster presentation, AACR Workshop on “The Future of Molecular Epidemiology “, Miami, 6-9 June 2010)
Cancer and the environment: Experience from the EU-funded ENVIROGENOMARKERS, • NEWGENERIS and ECNIS projects (S.A. Kyrtopoulos, oral presentation at the EU Work-shop on Environmental Health Research, XII International Congress of Toxicology, 18-22 July 2010)
Exploring the potential of ‘omics’ technologies in population studies: The EnviroGenom-• arkers project (S.A. Kyrtopoulos, oral presentation, ICA-LRI & JRC Workshop on “Integrat-ing new approaches in exposure science and toxicity testing: Next steps”, Stresa, 16-17 July 2010
Applicability of -omics technologies to population studies for the discovery of biomarkers • of environmental health: results from the EnviroGenomarkers project (S.A. Kyrtopoulos, on behalf of the EnviroGenomarkers project consortium; poster presentation at the EEMS conference, Oslo, 15-19 September 2010).
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Project meetings
The kickoff meeting of EnviroGenomarkers was held at the National Hellenic Research • Foundation, Athens, on 9-10 March 2010.
Subsequent meetings of the project consortium have been held in Athens (29 September • 2009), Brussels (23 June2009) and Brussels (27 September 2010).
The 1st Annual General Meeting of the consortium was held on 3-4 March 2010 on San-• torini island, Greece, along with a Workshop on “Bioinformatics approaches to biomarker discovery”.
Project partners
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Partner Principal investigator
National Hellenic Research Foundation
Athens, Greece
S.A. Kyrtopoulos
(co-ordinator); [email protected]
University of Maastricht
Netherlands
J. Kleinjans
Imperial College London
United Kingdom
P. Vineis
Umeå University
Sweden
I. Bergdahl
Istituto per lo Studio
e la Prevenzione Oncologica, Florence, Italy
D. Palli
University of Crete
Greece
E. Stephanou
University of Utrecht
Netherlands
R. Vermeulen
Istituto Superiore di Sanita
Rome, Italy
P. Comba
National Institute for Health and Welfare Kuopio,
Finland
H. Kiviranta
University of Leeds
United Kingdom
M. Gilthorpe
Lund University
Sweden
B.G. Jönsson
ENVIRO NEWSLETTER details:
Project Officer:• Dr Tuomo Karjalinen EC, Research Directorate General Directorate I Environment Unit I.5, B-1049 Brussels Tel. +32-2-2984660, E-mail: [email protected] /Photos :• M. Botsivali Graphic Designer :• Dimitra PelecanouContact Point :• Dissemination officer M. Botsivali, National Hellenic Research Foundation, Tel. +210 7273740, E-mail: [email protected]
ISSEU 1 • NOVEMBER 2010
www.envirogenomarkers.net