Kina23347enc 002

KI-NA-23347-EN-C
Diagnostics
Diagnostics refers to the tools and technologies used in early diagnosis of disease, monitoring of disease progression and response to therapy. Use of diagnostic methods can greatly increase the effectiveness of therapy. Research into new diagnostics formed part of the EUs Sixth Framework Programme (2002-2006) and the purpose of this catalogue is to demonstrate the activities started over the duration of the programme and the initial results obtained. Diagnostics research involves many disciplines, including molecular biology, physiology, biomedical engineering and information technology, where new research is opening up exciting opportunities, and is a particularly active area for industry, especially SMEs.
Diagnostics
PROJECT SYNOPSES
Diagnostics
EUR 23347
EUR 23347
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Research*eu is our quarterly magazine keeping you in touch with main developments (results, programmes, events, etc.). It is available in English, French and German. A free sample copy or free subscription can be obtained from: European Commission Directorate-General for Research Information and Communication Unit B-1049 Brussels Fax (32-2) 29-58220 E-mail: research@ec.europa.eu Internet: http://europa.eu.int/comm/research/rtdinfo/index_en.html
European Commission Genomics and Biotechnologies for Health DIAGNOSTICS Luxembourg: Office for Official Publications of the European Communities 2008 168 pp. 17.6 x 25.0 cm ISBN 978-92-79-08527-7
This publication is also available online at: ftp://ftp.cordis.europa.eu/pub/fp7/docs/diagnostics_en.pdf
EUROPEAN COMMISSION Directorate F Health Unit F5 Health Biotechnology Contacts Philippe Jehenson Office CDMA 2/123 Tel (32-2) 29 86454 Fax (32-2) 29 94693 Directorate F Health Unit F5 Health Biotechnology Head of Unit Arnd HOEVELER (arnd.hoeveler@ec.europa.eu) Secretary: Andrea JAEGER (andrea.jaeger@ec.europa.eu) Diagnostics Jean-Luc SANNE (jean-luc.sanne@ec.europa.eu) Torbjoern INGEMANSSON (torbjoern.ingemansson@ec.europa.eu) Philippe JEHENSON (philippe.jehenson@ec.europa.eu) Jean-Luc Sanne Office CDMA 2/115 Tel (32-2) 29 92589 Fax (32-2) 29 94693
EUROPEAN COMMISSION
DIAGNOSTICS
EU-supported research in Genomics and Biotechnology for Health Sixth Framework Programme (2002-2006)
Edited by Charles Kessler
Directorate-General for Research 2008 Life Sciences, Genomics and Biotechnology for Health EUR 23347
NEW THERAPIES DIAGNOSTICS
TABLE OF CONTENTS
INTRODUCTION GENETIC TESTING AND BIOMARKERS
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11 GLYFDIS Glycans in body fluids potential for disease diagnostics 44 MolDiag-Paca Novel molecular diagnostic tools for the prevention and diagnosis of pancreatic cancer 47 COBRED Colon and breast cancer diagnostics
EuroGentest Genetic testing in Europe network for test development, harmonisation, validation and standardisation of services 13 EUROGENGUIDE Patient led education and development for genetic testing in research and medicine 19 SAFE Special non-invasive advances in foetal and neonatal evaluation 22 QuAGSIC Quantitative analysis of genes in single cells 28 IBDchip Usefulness of a new DNA array (IBDchip) to predict clinical course, development of complications and response to therapy in patients with inflammatory bowel disease (IBD) 30 AntePrion Development of a preclinical blood test for prion diseases 33 TSEUR An integrated immunological and cellular strategy for sensitive TSE diagnosis and strain discrimination 37 ADDNET Paradigm shift from kidney biopsies to advanced molecular diagnostics from patient urine 41
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EuroFlow Flow cytometry for fast and sensitive diagnosis and follow-up of haematological malignancies 54 DRoP-ToP Integration of DNA, RNA and protein markers in a tool for the prognosis and diagnosis of human disease 57 TB-trDNA Evaluation of transrenal-DNA detection to diagnose tuberculosis 62 GENEPARK Genomic biomarkers for Parkinsons disease 64 PREGENESYS Development of early non-invasive biomarkers and means for the diagnosis and progression monitoring of preeclampsia and tailoring putative therapies 67
Diagnostics - Table of Contents
TABLE OF CONTENTS
EDAR Beta amyloid oligomers in the early diagnosis of AD and as marker for treatment response 69 NeuroScreen Sensitive and differential blood and cerebrospinal fluid test for neurodegenerative dementia diagnostics 71
IMAGING, NANOPARTICLES AND BIOSENSORS
75 FLUOROMAG Multiparameter sensing for high sensitivity diagnostics using fluorescent and magnetic nanoparticles 103 BONSAI Bio-imaging with smart functional nanoparticles 106 NanoSense Moving sensitive immunoassays from slow and expensive to fast and affordable nanoparticlebased methods 110 DiaNa Predictive diagnostics for diabetic nephropathy novel nanotechnology based test platforms 112 NANOMYC Multiparametric detection of bio-molecule conjugated nanoparticles for the diagnostic investigation of mycobacterial infections of humans and animals 114 DETECTHIV Sensitive nanoparticle assay for the detection of HIV 117 NACARDIO Nanoparticle-based electronic biosensor for diagnostics of cardiovascular disease 120
DiMI Diagnostic Molecular Imaging (DiMI): a European network of excellence for the development of new molecular imaging strategies aiming to improve the diagnostic and therapy of human diseases 77 DASIM Diagnostic Applications of Synchroton Infrared Microspectroscopy 84 NeuroTAS Microfluidic total analysis system for the early diagnostic of neurodegenerative disorders 88 POC4life Multiparametric quantum dot bioassay for point of care diagnosis 91 DIAGNOSIS Development of new and cost-effective methods for non-invasive diagnosis of human pathogens 94 USDEP Capture and enrichment of emerging pathogens for multiple and ultra-sensitive diagnostic 97 NEMO Nano based capsule-endoscopy with molecular imaging and optical biopsy 100
DIAGNOSTICS
SLIC SLIC-Biosensors in molecular diagnostics: nanotechnology for the analysis of species specific microbial transcripts 124
eBIOSENSE Electrical biosensor arrays for analyses of harmful micro-organisms and microbial toxins 125
FP7 PROjECTS Biomedical imaging
129
CARS Explorer Innovative contrast imaging by non-linear optics (NLO) for the observation of biological tissues in vivo and in real time, at cellular and molecular levels 130 FLUODIAMON Ultra-high resolution and ultra-sensitive fluorescence methods for objective sub-cellular diagnosis of early disease and disease progression in breast and prostate cancer 132 FUN OCT Functional Optical Coherence Tomography 135 nEUROPT Non-invasive imaging of brain function and disease by pulsed near infrared light 137 FMT-XCT Hybrid Fluorescence Molecular Tomography X-ray Computed Tomography method and system 139 HYPERImage Hybrid PET-MR system for concurrent ultrasensitive imaging 141 MEGMRI Hybrid MEG-MRI Imaging System
SKINSPECTION Multimodal skin inspection with hybrid acoustic and optical spectroscopic imaging 145 EURIPIDES European Research initiative to develop Imaging Probes for early In vivo Diagnosis and Evaluation response to therapeutic substances 147 ENCITE European Network for Cell Imaging and Tracking Expertise 150
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FP7 PROjECTS Molecular testing

SPIDIA
153
Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics 154 EURO-GENE-SCAN European Genetic Disease Diagnostics
NMD-Chip Development of targeted DNA-Chips for High Throughput Diagnosis of NeuroMuscular Disorders 159 TECHGENE High throughput molecular diagnostics in individual patients for genetic diseases with heterogeneous clinical presentation 163
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INDEX OF PROjECTS INDEX OF COORDINATORS
166
167
DIAGNOSTICS
INTRODUCTION
iagnostics refers to techniques for determining the nature and cause of disease. As part of its Sixth Framework Programme for Research (2002-2006, FP6) , the EU supported diagnostics research notably under the heading Applications of knowledge and technologies in the field of genomics and biotechnology for health in the Life sciences, genomics and biotechnology for health thematic priority. Details of this research have been compiled in the present volume, whose objectives are to demonstrate the range of activities undertaken and initial results obtained. Diagnostics research is continued in the EUs Seventh Framework Programme (2007-2013, FP7), and brief details of research projects started in 2007 and 2008 are also included. The overall objective of the diagnostics research described here is to develop new tools and techniques for early diagnosis of disease, for monitoring disease progression and for guiding therapeutic interventions. A particular feature of the techniques is that they should be non- or minimally-invasive towards the patient. Since the projects are essentially technology-driven they have been ordered on the basis of similarity in approach and have been grouped into two chapters reflecting the major technologies employed. These are followed by summaries of work proposed in FP7 projects. GENETIC TESTING aNd BIOMaRKERS. Genetic testing refers to determining the susceptibility to inherited disease. It is an area that has advanced rapidly as a result of recent developments in genomics, a major thrust of the FP6. Genetic testing is also linked to the use of biochemical tests for the possible presence of genetic disease. More
generally, a biomarker refers to a substance whose detection indicates a particular disease state. The aim of projects described in this section is the development of genetic testing and biomarker technology in a general way. Some projects deal with specific disease applications chosen by scientists as a way of developing technology rather than necessarily targeting a specific disease objective. Since genetic testing may open up ethical questions, this aspect has also been included in projects. IMaGING, NaNOPaRTICLES aNd BIOSENSORS. Diagnostics tools are also expanding rapidly as a result of progress in non-medical disciplines and the tool development described in this section derives particularly from nanotechnologies, physics, electronics, molecular biology and computer science. Imaging work described here aims at the visualisation of biological processes at the cellular and molecular level. Bioimaging and molecular tests can also be used in combination and share the need for specific biomarkers and probes. Recent developments in nanotechnology have led to design of very powerful probes, allowing detection in vivo through bioimaging and in vitro through lab-on-a-chip designs. Biosensors combine a biological component, such as an antibody or nucleic acid, with a physico-chemical detector and an electronic processor to display the results. As in the previous section, projects aim at technology development in a general way but often with application to prototype stage in a particular disease situation. FP7 Biomedical imaging. Advances in imaging techniques have had a major impact in everyday medical practice. Following earlier work in FP6,
Diagnostics Introduction
INTRODUCTION
biomedical imaging research was a major thrust in the first calls for proposals in FP7. Bioimaging allows the visualisation and characterisation of both structure and function, and combining imaging technologies can lead to even more powerful tools to diagnose, track and treat a variety of diseases. The projects described here cover novel optical methodologies, hybrid imaging systems, the development of imaging probes to evaluate response to therapy, and tracking cells used in cell therapy. FP7 Molecular testing. In vitro diagnostics have allowed a great deal of progress in medicine but are limited by lack of guidelines in collection, handling, stabilisation and storage of biosamples. The first project described in this section aims to develop standard guidelines for molecular in vitro diagnostics and to develop pre-analytical tools to use with free biomolecules from samples. The second area of work concerns high-throughput molecular diagnostics for genetic diseases. The following table shows the number of projects supported in diagnostics research and the EC financial contribution to them. It shows that during FP6 34 projects were supported with an EC contribution of around EUR 105.7 million, Area FP6 Genetic testing and Biomarkers Imaging, nanoparticles and biosensors Total FP7* Biomedical imaging Molecular testing Total
* 2007 and 2008 only
and that in the first 2 years of operation of FP7, 14 projects are planned to be supported with an EC contribution of around EUR 73.7 million. In order to achieve their objectives, FP6 projects were built around five different funding schemes: Integrated Projects. These are the largest size projects and integrate a range of different activities, such as research, demonstration and training. They also permit projects to take a multidisciplinary approach, to link underlying biology and tool development and enable scientists, clinicians and other stakeholders to work together to achieve their deliverables. Networks of Excellence, whose objective is to reduce fragmentation in EU research, structure the way it is carried out and strengthen its excellence. Specific Targeted Research Projects. These are smaller projects which focus on specific research issues. They may have an applied focus but are less multi-disciplinary and wide-ranging than the Integrated Projects. EC financial contribution (million ) 61.7 44.0 105.7
Number of projects
18 16 34
10 4 14
55.9 17.8 73.7
Number of projects supported and EC financial contribution to diagnostics research
DIAGNOSTICS
SME-Specific Targeted Research Projects. Targeted Research Projects designed to encourage research and innovation efforts of small and medium-sized enterprises (SMEs) and where researchled SMEs play a leading role. Specific Support actions for training, conferences or prospective studies in support of the programme. FP7 projects described here are all categorized as Collaborative Projects and receive EU financial contributions of between around EUR 3 million and EUR 12 million. The smaller ones approximate to the format of the specific targeted research projects described above and the larger ones to the integrated projects. The number of projects supported by the different funding schemes and the EC financial contribution to them is shown in the table below. This was distributed in FP6 among over 400 research teams, around 80 of which are SMEs, including some as coordinators. Funding scheme FP6 Integrated project Network of excellence Specific Targeted Research Project SME-Specific Targeted Research Project Specific Support Action Total FP7* Collaborative project
* 2007 and 2008 only
FP7 has included in its first 2 years of operation around 150 teams, including about 25 SMEs. European research projects are encouraged to be open towards the general public and to engage with stakeholders and interest groups. This is particularly important in the field of genetic testing which is of considerable public interest. Accordingly many projects organise public meetings and dialogue and set up websites on the consortium, the research and results. Website addresses are given in the details of each project and provide more detailed and more up-to-date information than this publication. This compilation shows that diagnostics is an extremely active area of research for European scientists both from the public and private sectors. These efforts in the health programme are complemented by related research undertaken by the EUs nanosciences, nanotechnologies, materials & new production technologies programme. EU diagnostics research is an especially attractive area for health-related SMEs since there are many opportunities for placing of products on the market. EC financial contribution (million ) 8.5 32.7 36.4 27.3 0.8 105.7
Number of projects
1 3 16 12 2 34
14
73.7
Funding schemes used in diagnostics research and EC financial contribution
The EU will continue to support diagnostics research as FP7 develops. Further information can be found at: http://cordis.europa.eu/fp7/health/home_en.html
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Diagnostics
Genetic Testing and Biomarkers
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Diagnostics - Genetic Testing and Biomarkers
DIAGNOSTICS
EuroGentest
Contract No Project type EC contribution Starting date duration Website
Genetic testing in Europe network for test development, harmonisation, validation and standardisation of services
LSHB-CT-2004-512148 Network of Excellence e 10 000 000 1 January 2005 60 months www.eurogentest.org
Background and objectives: The EuroGentest NoE (Network of Excellence) is a five-year EU-funded programme aiming to develop the necessary infrastructure, tools, resources, guidelines and procedures that will lead to the establishment of harmonised, quality genetic testing services in Europe. This will be achieved by bringing together, in a real long-term partnership, experts and expert centres in Europe that are active in different aspects of testing. The Network includes researchers, small and medium-sized enterprises (SMEs), testing laboratories, quality management and public health experts, ethicists, lawyers, sociologists, educational authorities and consumers. EuroGentest is focusing on the following four major areas of activities: quality of the laboratories; quality of the clinical aspects of the s rvices; e translation of technologies into routine diagnostic practice; education aspects. For operational reasons, the NoE was structured into six different units, each with a specific focus on one of the activities of the Network, as well as with a unit board which coordinates the different Work Packages (WPs) of the unit and the interactions between them.
The management group, together with the Steering Committee that is composed of the unit leaders, is responsible for the coordination and financial management of the NoE, as well as for the smooth implementation of the horizontal activities, which include the provision of fellowships and communication about the NoE. In the figure (next page), the different units and the interacting networks and projects are indicated. In the centre, the management and the coordination are shown to interact with an advisory board composed of representatives of different international organisations. Expected outcome: Quality management Evaluation of the quality assurance information database. Training session on the use of LIMS and quality management of informatic systems. Final report with guidelines and requirements for quality management of genetic testing labs. Organisation and coordination of followup workshops and training for accredited labs and internal auditors. Final conclusion, and if necessary, ISO draft guidelines for genetic testing labs submitted to the ISO / TC212 committee. Model for sustainability of the quality management services.
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EuroGentest
Report on the impact of measures to coordinate, harmonise and make sustainable molecular genetics External Quality Assessments (EQAs) across Europe within a recognised governance and standards framework. Endpoint evaluation of the measures taken to expand molecular genetics EQA participation, and to meet the developing needs of diagnostic service laboratories across Europe. Report on the impact and future of best practice guidelines for molecular genetics in Europe. Proposal for the governance of cytogenetics EQA schemes and for the certification for cytogenetics EQA schemes has been approved. Cytogenetics EQA schemes flexibility and responsiveness to changing technologies has been successfully tested. A pan-European cytogenetics EQA available to all Europe cytogenetics laboratories, online registration and management of electronic cytogenetics EQA scheme has been established. Steps for certification of biochemical genetics EQA schemes in harmonisation with cytogenetics and molecular genetic schemes have been initiated. A European directory of biochemical genetic service providers linked to EQA participation has been established. Prepare sustained activities of the Network for Validation and Technology Testing. A number of technologies has been validated and generic SOPs are now available. New control materials have been developed and the field has been tested.
Information information on all testing activities in Belgium, Bulgaria, the Czech Republic,
ECA
EMQN
ORPHANET
Quality Issues
Unit 1
ERNDIM
Database
CANGENETEST EUROCARE-CF CAPABILITY PHGEN RELAGH
Clinic
OECD CDC ACMG WHO EFB ESHG EUROPABIO
Education
ESHG
Technology
Ethics, Legal
GeneBanC
INDUSTRY SAFE NoE
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DIAGNOSTICS
Denmark, Germany, Estonia, Ireland, Greece, Spain, France, Italy, Cyprus, Latvia, Lithuania, Hungary, the Netherlands, Austria, Poland, Portugal, Romania, Sweden, the UK and Norway. A unique European portal for information on genetic testing activities and diseases has been established and contains information on all testing activities in Europe. The data are updated once per year.
Clinical genetics and public health Major needs for professional education in the field have been identified. Endorsed recommendations for counselling will be published in the NoE website and elsewhere. Tools for assessing the quality of genetic counseling have been tested. Agreement on and establishment of a formal basis for widely accepted guidelines in relation to accreditation/certification of clinical genetic services. Ethical legal social issues development of European guidelines for genetic testing (in close collaboration with patient organisations and geneticists); evaluation of the convergences and divergences in the protection of rights of patients between the different EU members in the light of the common European framework offered by the European Convention. Research and emerging technologies description and analysis of the current European and International IP framework for diagnostic patents; technological platform for beta-site testing of new diagnostic technologies will be available. Education Training programmes in collaboration
with existing facilities will be available. A syllabus for each genetic course, with a minimal common basis among the partner countries for the education of the different categories of professionals, will be available. A collection with the existing regulations controlling the licence to practise as a designated genetic professional to facilitate harmonisation within the MS, will be available. New flyers with information for patients are available in several languages.
Main findings: In general, the different WPs and units achieved their milestones and deliverables in the first two years of the programme, and their achievements are already clearly visible, including the following. A European central database of laboratories with quality criteria is being developed in collaboration with Orphanet and will be online soon. Very successful training sessions about quality management and accreditation have been organised. EQAs for molecular, cytogenetic, biochemical tests (electronic EQA for cytogenetics) have been organised and have been extended. Control materials for different genetic tests are available and more are under development. Generic SOPs for DNA extraction MLPA are being finalised. Expert groups have been set up for quality aspects, counselling, clinical validation, ethical issues and educational aspects. Surveys of stakeholders and literature surveys were held on different aspects of counselling, quality issues, educational materials and ethical aspects. Newsletters were published, a website was developed, press releases were drafted and scientific publications were submitted.
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EuroGentest
EuroGentest participated in different expert meetings (EU, OECD, ISO, etc.). EuroGentest gave a whole series of presentations at international congresses. Plans for communication with the stakeholders, for sustainability and for gender issue analysis were drafted. Van Overwalle, G., van Zimmeren, E., Verbeure, B., Matthijs, G., Models for facilitating access to patents on genetic inventions, Nature Reviews Genetics, doi:10.1038/nrg1765 Cassiman, J.J., EuroGentest a European Network of Excellence aimed at harmonizing genetic testing services, European Journal of Human Genetics, 2005, 13, 11031105. doi:10.1038/sj.ejhg.5201484; published online 10 August 2005. Verbeure B., Matthijs, G., Van Overwalle, G., Analysing DNA patents in relation with diagnostic genetic testing, European Journal of Human Genetics, advance online publication 12 October 2005, doi:10.1038/sj.ejhg.5201503. Borry, P., Fryns, J.P., Schotsmans, P., Dierickx, K., Carrier testing in minors: a systematic review of guidelines and position papers, European Journal of Human Genetics advance online publication, 2005, Nov 2; doi:10.1038/sj.ejhg.5201509. Coordinator In conclusion, international awareness of the existing problems has been improved. The will to make the necessary improvements is present within the NoE, and the tools necessary to make these improvements are becoming available. Major publications: Chedraui, P., Landivar, X., Eurogentest, DS News, Vol. 13, No 2, 2006, pp. 27. Chedraui, P., CLP in Ecuador, DS News, Vol. 12, No 2, 2005. Chedraui, P., Standardization of molecular biology techniques for Downs syndrome diagnosis in amniotic fluid in Guayaquil-Ecuador: The intiation of a new era, DS News, 2004, Vol 11, No 2. Jean-Jacques Cassiman Center Human Genetics/ Centrum Menselijke Erfelijkheid Campus gasthuisberg Herestraat 49, Box 602 B 3000 Leuven E-mail: jean-jacques.cassiman@med.kuleuven.be Partners antoon Vyverman Ascent Consultancy Leuven, Belgium Michael Morris Geneva University Hospitals Geneva, Switzerland
Training activities In addition to the training sessions detailed in the WP reports, the NoE has provided six fellowships for training. These fellowships have allowed scientists either to be trained in a laboratory of one of the participants of the NoE, or to attend a training course relevant to the activities of the NoE. dissemination of knowledge In addition to a very active website, www. eurogentest.org, and the production of regular newsletters and flyers, the participants of the NoE are regularly solicited to participate in international expert meetings, and are invited to present the achievements of the network at international congresses and symposia (more than 40 in 2006).
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DIAGNOSTICS
Robert George Elles The University of Manchester Manchester, UK Clemens Reible-Mller Bayerische Julius-Maximilians Universitt Wrzburg Wuerzburg, Germany Gyrgy Fekete Semmelweis University Budapest, Hungary Michal Witt International Institute of Molecular and Cell Biology Warsaw, Poland Milan Macek Jr Charles University Prague Prague 5, Czech Republic david Barton National University of Ireland, Dublin Dublin, Ireland Philippe Corbisier European Commission, Joint Research Centre Geel, Belgium Rosalind Janice Hastings Oxford Radcliffe Hospital Oxford, UK Brian Fowler University Childrens Hospital Basel Basel, Switzerland Sgolne aym Institut National de la Sant et de la Recherche Mdicale Paris, France Bruno dallapiccola Istituto Casa Sollievo Della Sofferenza, San Giovanni Rotondo Rome, Italy
Helena Kriinen University of Turku Turku, Finland Ulf Kristoffersson Lunds Universitet Lund, Sweden Jrg Schmidtke Medizinische Hochschule Hannover Hannover, Germany Jorge Sequeiros Instituto de Biologia Molecular e Celular Porto, Portugal Bert Bakker Leiden University Medical Center Leiden, Netherlands Guillermo antiolo Hospitales Universitarios Virgen del Roco-Servicio Andaluz de Salud Sevilla, Spain Maj Hutten The University of Warwick Coventry, UK alastair Kent Genetic Interest Group London, UK Beverly Searle The Rare Chromosome Disorder Support Group Surrey, UK dada Radojkovic Institute of Molecular Genetics Belgrade, Serbia Peter Chedraui Universidad Cat de Santiago de Guayaquil Guayaquil, Ecuador
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EuroGentest
Jan Schouten MRC-Holland Amsterdam, Netherlands david Bishop Waypoint Systems Ltd Shewsbury, Shropshire, UK Peter Rosseel Management Consulting and Research Hevertee, Belgium Orfeu Flores Stab Vida investigao e servios em cincias biolgicas lda Oeiras, Portugal david atlan Phenosystems SA Brussels, Belgium Juergen Oster Chemagen Biopolymer Technologie AG Baesweiler, Germany Irmgard Nippert Westflische Wilhelms-Universitt Mnster, Universittsklinikum Mnster Muenster, Germany domenico Coviello Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena Milan, Italy
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DIAGNOSTICS
EUROGENGUIDE
Patient led education and development for genetic testing in research and medicine
LSHB-CT-2006-518156 Specific Support action e 499 476 1 January 2007 36 months www.gig.org.uk/teammember_ projectofficereuro1.htm
Background and objectives: Health professionals are often unaware of the ethical, legal and psychosocial implications genetic tests may have. What is more, these tests are often not in compliance with ethical and clinical guidelines especially regarding informed consent and indications for testing. Health professionals who are poorly informed and ill-prepared for engaging patients in an appropriate informed consent process can have a detrimental effect on the welfare of clients and their families. Shared practices in the informed consent process are urgently needed to help both professionals and the public. The key objectives of EuroGenGuide are to create tools, a manual for patients and consumers, and educational materials for health professionals, to: empower the European public to make informed choices about participation in genetic research and genetic testing , which will be understandable, culturally competent and widely available; educate researchers and health professionals to engage patients/clients in an appropriate informed consent process. The tools EuroGenGuide proposes to develop will reflect a clear, consumer/patient-focused perspective on issues common to all genetic human subject research and clinical testing situations, such as informed consent, storage
of genetic information, confidentiality, risks and benefits, communication of results, etc. EuroGenGuide will advance these objectives through a patient-led collaborative approach involving stakeholders from patient support groups representing a wide range of genetic conditions, and academic experts (as partners and advisors) in the field of genetics, biotechnology, science communication, social sciences, bioethics and law. Approach and methodology: EuroGenGuide will create a multidisciplinary European stakeholder task force that will develop the following: 1. The first European consensus based consumer manual for use by patients, family members of patients and the general public who are recruited, elected or coopted into genetic/genome research projects and clinical trials and/or will have to decide whether or not to undergo genetic testing. The consumer manual will help them to better understand the implications of informed consent in genetic research and testing; it will inform them about issues that may be important for them to consider before a decision is made and will provide sets of questions people may want to ask researchers/doctors before they decide
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EUROGENGUIDE
2. Educational materials to prepare researchers and health professionals about how best to enable potential research participants and patients to make informed decisions, and to raise professionals awareness for patients/consumer concerns and needs. These materials will help to structure, harmonise and improve the quality of professional training, and will fill the current gap in this field.
The process leading to the compilation of the consumer manual and the creation of corresponding training materials for professionals will be based upon an extensive dialogue between different stakeholders and an emphasis on collaborative partnership and open discussion. By developing the manual and training materials in this way, EuroGenGuide will embark upon a set of measurable and verifiable activities including: establishment of a working collaboration of different stakeholders including a network of European patient organisations and academic experts in the field of genetics, including bioethics, law and social sciences; identification of issues pertinent to informed decision-making in genetic research and testing for both the consumer manual and training materials by a multidisciplinary task force; the drafting of a culturally competent and easily understandable consumer manual, as well as training materials for professionals by providing a structured internal and external review process to ensure consensus and validity; effective EU-wide delivery and dissemination.
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DIAGNOSTICS
Coordinator alastair Kent Genetic Interest Group Ltd 436 Essex Road London, N1 3QP, UK E-mail: mail@gig.org.uk Partners domenico Coviello University Hospital Milan Milan, Italy david Bennett Cambridge Biomedical Consultants Ltd Delft, Netherlands Irmgard Nippert Frauengesundheits-forschung Muenster, Germany Cor Osterwijk Vereniging Samenwerkende Ouder- en Patintenorgani-saties Soestdijk, Netherlands Michael Livingston Heart Europe Hoofddorp, Netherlands Rodney Elgie Global Alliance for Mental Illness Advocacy Networks-Europe Brussels, Belgium Jean Georges Alzheimer Europe Luxemburg, Luxembourg avril daly Fighting Blindness Dublin, Ireland
Robert Gerard Rare Disorders Belgium Jambes-Namur, Belgium Ysbrand Poortman World Alliance of Neuromuscular Disorder Associations-Europe Baarn, Netherlands Selena Freisens Central and Eastern European Genetic Network Konstanz, Germany
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SAFE
Special non-invasive advances in foetal and neonatal evaluation
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2004-503243 Network of Excellence e 12 000 000 1 March 2004 60 months www.safenoe.org
Background and objectives: SAFE, a Network of Excellence (NoE), has drawn together European expertise in the area of noninvasive prenatal diagnosis (NIPD) to provide significant impetus to the objective of replacing the potentially risky procedures of amniocentesis and chorionic villus sampling. These techniques, which are currently the norm for prenatal diagnosis of common chromosomal disorders (e.g. aneuploidies), impart a small but significant risk of miscarriage during the procedure. SAFE has drawn on a rich background of research on the presence and isolation of foetal cells in the maternal circulation that has existed in Europe for decades, and a host of new research activity following the discovery of free foetal DNA in maternal plasma in 1997. SAFE has served to centralise and unify a common technical approach and programme of activities for a relatively new area of medical diagnostics, which has the potential to become a multibillion Euro market within the next decade. NIPD, if technically proven, would completely replace invasive prenatal diagnosis, as the NIPD procedures impart no risk to the foetus and are of negligible cost difference per assay; furthermore, they would also reduce clinicians time in performing a surgical procedure to procure foetal material for diagnosis. Some aspects of NIPD can be applied on a mass scale. For example, prenatal diagnosis of the
foetal RhD blood group can reduce the level of usage of human-derived blood products (prophylactic anti-D) administered during pregnancy, to prevent immunisation of the mother to a paternally-inherited blood group antigen on foetal red blood cells. This new mass-scale diagnosis requires careful cost-evaluation of the impact on the different health economies in Europe, and one of SAFEs areas of activity is the socioeconomic evaluation of new NIPD technology and implementation. Mass-scale adoption of NIPD will have significant psychological and ethical implications. Their impact on decision-making and policy decisions that need to be considered are also core areas of activity within the project. In particular, sex-selection was a specific focal point, the subject of detailed evaluation during the first half of the project. In common with all NoEs, SAFE hopes to create long-term partnerships of research groups in Europe that are performing and developing NIPD. Approach and methodology: The project is divided into eight Work Packages (WPs), five of which explore the new technology necessary to develop a diverse range of new tests that will be applied to a range of disorders of the foetus and pregnancy, including Aneuploidy (Downs Syndrome), foetal sex, blood group, cystic fibrosis, -thalassemia, preeclampsia and preterm labour. This involves the analysis of foe-
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DIAGNOSTICS
tal cells, free foetal DNA and foetally-derived proteins that are present in maternal blood and offer diagnostic potential. In some cases, such as foetal sex and RhD blood group, the technology is far advanced, and is currently in routine diagnostic use. SAFE laboratories were amongst the first to implement such tests, and are leading a series of workshops to spread this standard of good practice within and outside the consortium. Research projects have a priority to aid the development of a new generation of diagnostic tests for chromosomal abnormality, which may involve the analysis of foetal cells isolated from the maternal circulation, the analysis of foetally-derived biomarkers in maternal blood, or epigenetic markers present or absent on fetal DNA found in maternal plasma. The three socioeconomic WPs focus on the current and potential impact of NIPD on health economics, parental choice and ethics. NIPD will have a significant impact on all aspects of prenatal care, and it is critical that any controversial issues are considered and debated openly, within and outside the consortium. A particular area of focus has been the potential mass-scale application of foetal sexing, and the impact this may have in different cultures. Expected outcome: SAFE is currently driving the introduction of NIPD within the EC. SAFE workshops are attended by both SAFE and non-SAFE partners, with the sole objective of spreading the excellence generated within the project to the wider medical community. Whilst the application of NIPD to the most common request for prenatal diagnosis for foetal chromosomal disorders is technically challenging, SAFE hopes to generate internationally competitive researchers in this field. Realistically, NIPD for Downs syndrome and other chromosomal disorders may not be widely available by the end of SAFE EC funding in 2009. However,
SAFE has been a key driver in the assembly of a critical mass of European researchers that will play a key role in its eventual introduction in the not too distant future. The NoE will also provide the infrastructure for new diagnostic laboratories wishing to implement NIPD, to adopt a standardised approach. Main findings: Since its launch, SAFE has held three large standardisation workshops for foetal sexing and RhD blood group genotyping using maternal plasma as the source of foetal DNA. These workshops have focused on a standardised approach to DNA extraction and standard PCR protocols for foetal sexing and RhD blood group determination. SAFE has effectively delivered a set of protocols within the public domain which are the consortiums recommendations for the implementation of RhD and foetal sexing in diagnostic laboratories worldwide. A workshop has also been held, in which state-of-the-art approaches for the automated detection of rare foetal cells in maternal blood were presented. SAFE is also focusing on the implementation of maternal plasma-based typing for single gene disorders, in particular for -thalassemia, and has organised a series of workshops specifically intended to assess the implementation of NIPD for this disorder, which is widespread in southern Europe. Coupled with all maternal plasma research
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SAFE
programmes, is the production of a microfluidicbased device for enrichment and/or purification of foetal DNA from this rich source of foetal material. This technology exploits the known physical characteristic of foetal DNA in being significantly smaller than fragmented maternal DNA. SAFE has a large laboratory programme for the identification of protein biomarkers and chromosomal markers which are hyper or hypomethylated in foetuses. Novel protein biomarkers for Downs syndrome, trisomy 18, foetal sex, preeclampsia and preterm labour have all been identified and are currently undergoing validation using a large biobank of clinical samples that is being assembled by SAFE partners. This biobank will be of critical importance to prove the effectiveness of any candidate biomarkers identified. Overall, SAFE has implemented a framework of health technology evaluation which can be tested against any of the emerging technologies under investigation by the consortium. Psychosocial and ethical evaluation of parental decisions have revealed significant variation between Western and Eastern cultures, which warrant further investigation, especially as NIPD may bring diagnostic tools to the free market. Major publications: Avent, N.D., Chitty, L.S., Non-invasive diagnosis of fetal sex; utilisation of free fetal DNA in maternal plasma and ultrasound, Prenatal Diagnosis, 2006, 26(7):598-603. Hall, S., Chitty, L., Dormandy, E., et al., Undergoing prenatal screening for Downs syndrome: presentation of choice and information in Europe and Asia, European Journal of Human Genetics, 2007. Li, Y., Zimmermann, B., Rusterholz ,C., et al., Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms, Clincal Chemistry, 2004, 50(6):1002-11. Li, Y., Di Naro, E., Vitucci, A., et al., Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma, Journal of the American Medical Association, 2005, 293(7):843-9. Bianchi, D.W., Avent, N.D., Costa, J.M., van der Schoot, C.E., Noninvasive Prenatal Diagnosis of Fetal Rhesus D: Ready for Prime(r) Time, Obstet Gynecol, 2005, 106(4):841-4. Zhong, X.Y., Holzgreve, W., Hahn, S., Direct quantification of fetal cells in maternal blood by real-time PCR, Prenatal Diagnosis, 2006, 26: 850-854. Huang, D., Nelson, M.R., Zimmermann, B., Dudarewicz, L., Wenzel, F., Spiegel, R., Nagy, B., Holzgreve, W., Hahn, S., Reliable detection of trisomy 21 using MALDI_TOF mass spectrometry, Genetics in Medicine, 2006, 8: 728-735. Special edition of Prenatal Diagnosis, 2006, 26(7):587-647. Coordinator Kate Hughes University of Warwick Research Support Services University House CV4 7AL Coventry, UK E-mail: kate.hughes@warwick.ac.uk Scientific coordinator Sinuhe Hahn Universitt Basel Department of Research laboratory for Prenatal Medicine Spitalstrasse 21 4031 Basel, Switzerland E-mail: shahn@uhbs.ch
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DIAGNOSTICS
Partners Maj Hulten University of Warwick Department of Biological Sciences Coventry, UK ala Szczepura University of Warwick Warwick Medical School Coventry, UK Neil avent University of the West of England Faculty of Applied Sciences Bristol, UK William Clocksin Oxford Brookes University Department of Computing Oxford, UK Theresa Marteau Kings College London Psychology and Genetics Research Group London, UK Lucia Savadori Dipartimento di Scienze della Cognizione e della Formazione Universit degli Studi di Trento Rovereto, Italy Ciara OSullivan Department of Chemical Engineering Universitat Rovira i Virgili Tarragona, Spain Barbara Pertl Medizinische Universitaet Graz Department of Obstetrics and Gynaecology Graz, Austria
Peter Sedlmayr Medizinische Universitaet Graz Institute of Cell Biology, Histology and Embryology Graz, Austria Ellen van der Schoot Stichting Sanquin Bloedvoorziening Department of Experimental Immunohematology Amsterdam, Netherlands Ilona Hromadnikova Charles University 2nd Medical Faculty Prague, Czech Republic andreas Plesch Metasystems GmbH Altlussheim, Germany Franoise Soussaline IMSTAR SA Paris, France Jim Schrder University of Helsinki Department of Biosciences Helsinki, Finland andres Metspalu Estonian Biocentre Tartu, Estonia Stan Urbaniak University of Aberdeen Academic Transfusion Medicine Unit Aberdeen, UK Laura Cremonesi Fondazione Centro San Raffaele Del Monte Tabor Unit of Genomics for Diagnosis of Human Pathologies Milan, Italy
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SAFE
dorine Swinkels University Nijmegen Medical Centre Nijmegen, Netherlands Jean-Marc Costa American Hospital of Paris Paris, France ariadni Mavrou National and Kapodistrian University of Athens Department of Medical Genetics Athens, Greece Esther Guetta Chaim Sheba Medical Center Danek Gertner Institute of Human Genetics Tel-Hashomer, Israel Francisco alvarez Hospital Universitario Central de Asturias Laboratory of Biochemistry Oviedo, Spain Nicholas Fisk Imperial College Paediatrics, Obstetrics and Gynaecology London, UK Marina Kleanthous Cyprus Institute of Neurology and Genetics Thalassaemia Department Nicosia, Cyprus Philippos Patsalis Cyprus Institute of Neurology and Genetics Department of Cytogenetics Nicosia, Cyprus Peter Soothill University of Bristol Obstetrics and Gynaecology Bristol, UK andres Lpez Bernal University of Bristol Dorothy Hodgkin Building Bristol, UK Michael Siegrist Universitt Zrich Division of Psychology Zurich, Switzerland Gian Carlo di Renzo Universit Degli Studi di Perugia Department of Medical-Surgical Specialties and Public Health Perugia, Italy Tobias Legler Georg-August-Universitt Gttingen Transfusionsmedizin Gttingen, Germany Patrizia Paterlini-Brchot and Jean-Jacques Toulm Institut National de la Sant et de la Recherche Mdicale (INSERM) Paris and Bordeaux, France Mauro Buser Statistik Buser Riehen, Switzerland Maurizio dEsposito Institute of Genetics and Biophysics Naples, Italy alastair Kent Genetics Interest Group London, UK Janet Grant Open University Open University Centre for Education in Medicine Milton Keynes, UK
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DIAGNOSTICS
Yiming Wang Sun Yat-Sen University Department of Medical Genetics Guangxhou, Peoples Republic of China Madhulika Kabra All India Institute of Medical Sciences Pediatrics Genetics Subdivision New Delhi, India Geoff daniels National Blood Authority International Blood Group Reference Laboratory Bristol, UK Martin Olsson Lund University Hospital Lund, Sweden Charles Rodeck University College London Obstetrics and Gynaecology London, UK Lyn Chitty University College London Institute of Child Health London, UK Lars Larsen Kbenhavns Universitet Department of Medical Genetics Copenhagen, Denmark Tadeusz Tyszka Wysza Szkoa Przedsibiorczoci i Zarzdzania im. Leona Komiskigo (LKAEM) Warsaw, Poland Etienne Mullet Ecole Pratique des Hautes Etudes Toulouse, France
Renate Burgemeister P.A.L.M. Microlaser Technologies GmbH Bernreid, Germany Giovanni Romeo Universit degli Studi di Bologna Bologna, Italy Elisabeth Blennow and The-Hung Bui Karolinska Institutet Department of Molecular Medicine Stockholm, Sweden Jan Schouten Microbiology Research Centre-Holland Amsterdam, Netherlands Beverly Searle Rare Chromosome Disorder Support Group (Unique) Caterham, UK Michael Christiansen and Paal Skytt andersen Statens Serum Institut Copenhagen, Denmark Ray OConnor Genomed Ltd Paisley, UK Vincenzo Cirigliano General Lab Department of Molecular Genetics Barcelona, Spain Cees Oudejans VU Medical Centre Amsterdam, Netherlands Olivia Willcocks Waypoint Systems Ltd Shrewsbury, UK
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QuAGSIC
Quantitative analysis of genes in single cells
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037293 SME_Specific Targeted Research Project e 1 433 600 1 September 2006 24 months www.uni-ulm.de/quagsic/index.php
Background and objectives: The partners of the QuAGSIC project will develop methods and instruments to analyse the copy number of nucleic acid sequences down to the single cell / single molecule level, with the goal of developing an early diagnosis system for the childrens disease hemophagocytic lymphohistiocytosis (HLH). The underlying technique is amplification-based counting (ABC), enabling the quantification of the copy number of genetic sequences with a resolution of about 100 base pairs in single cells. The method provides a resolution, i.e. orders of magnitudes higher than for fluorescence in situ hybridisation (FISH), and works quantitatively with much lower sample amounts than quantitative polymerase chain reaction (PCR). To prove ABCs effectiveness in clinical applications, the partners will develop a single cell manipulation unit that picks cells from a solution and transfers them onto an integrated PCR and
Picture of a semi-automatic 3D cellector
hybridisation slide (AmpliGrid). The AmpliGrid contains dried-on PCR reagents, as well as hybridisation probes to detect the presence and specificity of the PCR products. The single cells on the AmpliGrid will then be processed automatically in an integrated PCR and hybridisation machine (AmpliHyb). Clinical samples will be investigated, in which copy number deviations are pathologic as in genetic diseases. As a model system, QuAGSiC chose HLH which is hard to diagnose and fatal without specific therapeutic measures, as well as trisomy 21 which is relevant in prenatal and postnatal diagnostics. Many basic questions in biology and medicine demand methods for the analysis of the basic unit of life: the single cell. The QuAGSIC partners will thus develop methods and instruments to analyse the genetic content of single cells with regard to the quantitative variation of sequences (copy number variations), as well as the qualitative variation of sequences (single nucleotide polymorphisms). Once the project is finalised, QuAGSIC will put on the market machines and consumables that allow gene measurements down to the single cell level with high precision, in a parallel format (an Ampligrid can accommodate 48 cells), with high amount of automation. Role of SMEs The three SMEs in QuAGSIC have central roles:
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DIAGNOSTICS
Imaging of an Ampligrid
MMI (Molecular Machines & Industries) is developing an automated cell picker from its semi-automatic, two dimensional cell selection tool, capillary-based cell handling system, to a fully automated threedimensional cell finding and sorting system (cellector 3D). Adavlytix is using its photolithographically structured microscope slide AmpliGridTM suitable for performing 48 different 1l PCR reactions on the same substrate. The special AmpliGridTM surface chemistry will be used to define a physical platform for the integrated PCR and hybridisation at the single cell level. Genewave is developing an integrated system which will allow the performance of both the PCR and optical detection of hybridisation in an unsupervised single machine.
Without doubt, the single cell is the basic entity of a living organism (or it is even the whole organism). Therefore, applying the methods mentioned above to single cells is not only a technological challenge but it is also the critical task in system biology for the coming years, especially for addressing medical diagnostics in the regime of single cells. Single cell analysis becomes more and more attractive because of limited cell population of interest, cell heterogeneity of samples or when cells are isolated automatically as early dissemination of tumour cells. Hopefully, the QuAGSIC effort will bring breakthrough analytic tools and systems for prevention, diagnosis, or monitoring of a broad range of diseases. Coordinator Claude Weisbuch Genewave SAS XTEC - Btiment 404 Ecole Polytechnique 91128 Palaiseau, France E-mail: claude.weisbuch@polytechnique.fr Partners Wolfgang Mann Advalytix AG Munich, Germany Stefan Niehren Molecular Machines & Industries AG (MMI) Glattbrugg, Switzerland andres Metspalu Estonian Biocentre Tartu, Estonia Marion Schneider Clinical Center University of Ulm Ulm, Germany
Expected outcome: By the end of the project, the partners will be in a position to quickly market systems that allow gene analysis at the single cell level at low cost, with high speed, reliability and throughput. Potential applications: The technologies developed in QuAGSIC demand a minimum amount of material to be analysed.
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Usefulness of a new dNa array (IBdchip) to predict clinical course, development of complications and response to therapy in patients with inflammatory bowel disease (IBd)
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-37319 SME-Specific Targeted Research Project e 2 467 314 1 december 2006 36 months http://ibdchipproject.eu
IBDchip
Background and objectives: The IBDchip project targets the development of an easy-to-use DNA array and accompanying innovative chip reading device. The IBDchip will be a non-invasive tool with the capacity to simultaneously analyse around 100 relevant mutations to predict the following: clinical evolution; risk of developing IBD-related complications; likelihood of responding to certain drugs for each IBD (inflammatory bowel disease) patient. IBD includes Crohns disease (CD) and ulcerative colitis (UC). Both are increasingly common chronic illnesses, and are currently impacting the lives of nearly 1 million patients in Europe. CD and UC affect patients early in life, seriously impairing their quality of life, and resulting in enormous personal, social and economic costs. There is evidence suggesting that genetic factors play a key role in IBD pathogenesis, pointing towards a polygenic mode of inheritance for CD and UC. To date, however, studies have only addressed the influence of single mutations on IBD, resulting in a poor prediction of clinical course or response to therapy in individual patients. The main aim of this project is to provide doctors, for the first time, with a non-invasive, predictive tool to optimise treatment in IBD patients, thus
resulting in better clinical outcomes and improved cost-effectiveness of treatment. Role of SMEs Progenika Biopharma SA, one of the two SMEs involved in the project, has been the main driver of IBDchip, having originated the idea, and identified and engaged the partners. It will perform tasks that use a major part of the EC contribution to the budget. Building on development work done for their existing products, Progenika will use this project to validate new technology and knowledge, as well as to create a prototype IBDchip that has been clinically tested, and is therefore close to final commercialisation. The innovative prototype chip will give Progenika a clear advantage over competitors, since they will be the first to bring to the market their new knowledge-intensive product. Innopsys, the second SME involved in the project, will develop a slide reader to be commercialised alongside the IBDchip. This slide reader will be smaller and much cheaper than existing machines and will take the company into a new market (the reading of DNA arrays) while reinforcing its scientific and technological capacity for future innovations. Expected outcome: IBDchip anticipates the following results: a fully validated innovative prototype IBD-
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DIAGNOSTICS
(i.e. in 2011). These illnesses are increasing, and the project team expects that the IBDchip and new reader will be embedded as a routine part of treatment over the coming decade. It is also probable that the IBDchip project R&D processes and the resulting technologies can be adapted to address problems in the prediction and treatment of other polygenic inflammatory conditions, such as rheumatoid arthritis. Over the duration of the project, the team will use the consortium and its work as the foundation for future projects, to explore other potential applications of the technology. Ccoordinator
Analysing the results
chip that will give doctors vastly improved capacities to make more accurate individualised predictions of clinical outcomes of IBD and to choose the optimum and most cost-effective therapy for each patient; a new DNA array reader that will be faster and one fifth of the price of existing machines, to optimise the reading of the IBDchip and help make it ubiquitous; a clear understanding of the pathways to clinical service, ethical and legal issues, and cost-effectiveness of the IBDchip, which will result in a maximum uptake of the IBDchip in routine clinical practice; results for the academic partners will be new knowledge derived from the research undertaken in the project and published in various leading academic publications.
Miquel Sans Hospital Clnic / IDIBAPS Department of Gastroenterology Barcelona, Spain E-mail: msans@clinic.ub.es Partners Marta artieda Progenika Biopharma SA Parque Tecnolgico de Zamudio Derio, Spain Stphane Le Brun Innopsys SA Carbonne, France derek Jewell University of Oxford Nuffield Department of Medicine and Oxford GKP Oxford, UK Severine Vermeire The University Hospital in Leuven Leuven, Belgium
Potential applications: The IBDchip will have very wide application across the EU and beyond. The team has the working objective of using the IDBchip for 15% of both UC and CD patients (a total of approximately 320 000 people) within 3 years of the end of the project
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IBDchip
Salvador Pea VU University Medical Center The Laboratory of Immunogenics Department of Pathology Amsterdam, Netherlands Stefan Schrei University Hospital Schleswig-Holstein The Institute for Clinical Molecular Biology (ICMB) Keil, Germany Milan Lukas The University Hospital in Prague Prague 2, Czech Republic Silvio danese Istituto Clinico Humanitas Milan, Italy
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DIAGNOSTICS
AntePrion
development of a preclinical blood test for prion diseases
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-019090 Specific Targeted Research Project e 2 450 000 1 June 2006 36 months www.antePrion.eu
Background and objectives: There are currently no tests for early prion diagnosis. Such tests would help to prevent transmission through blood, biologicals, the food chain and medical procedures, as well as to identify candidates for emerging anti-prion therapies and prophylactic procedures, especially among silent carriers of pathogenic PrP mutations. Current tests are based on the identification of PrPSc or of characteristic pathologies in brain samples collected post-mortem. Recent advances have shown that minute amounts of PrPSc exist in body fluids, such as blood. Although these PrPSc levels are too low to be detected reliably by existing methods, they point to the feasibility of using these fluids with improved methods. Backed by a powerful consortium that is comprised of partners with proven clinical, experimental and industrial achievements, AntePrion is seeking to develop methods for the preclinical detection of prions in body fluids, based on PrPSc and novel surrogate (non-PrP) markers of prion diseases. PrPSc detection in body fluids will be achieved by systematically improving every step in the process: sample fractionation: fluids will be separated to identify fractions enriched in PrPSc; PrPSc concentration; PrPSc amplification; PrPSc detection.
Surrogate markers and molecular signatures of prion diseases will be identified using novel sensitive proteomic and genomic approaches, such as SELDI, MALDI and DNA microarrays. Proprietary software will be developed to analyse these emerging signatures. Prions are infectious proteins. There is currently no effective therapy for prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), and the disease is lethal. There is no sensitive and reliable preclinical or even ante-mortem diagnostic test for detection of prions in blood or other body fluids. Post-mortem diagnosis relies on pathological findings in the CNS and especially on the detection of PrPSc. Due to the difficulty of detecting low levels of prion infectivity and PrPSc, little is known about prion infectivity and PrPSc in peripheral tissues. The sensitivity of existing PrPSc detection methods is insufficient for the identification of PrPSc in body fluids (blood, CSF, milk) or in non-neural tissues. The use of 14-3-3 and S-100 as surrogate markers to PrPSc has been proposed as the basis for diagnostic TSE tests, but such markers are not very specific. AntePrion is therefore striving to find novel TSEspecific markers using novel mass spectrometric and genomic approaches, as well as immunological methods. Thus, the search for non-PrP markers, in parallel with improved procedures for detecting both the protease-sensitive (s) and -resistant (r) PrPSc, may give rise to a reliable preclinical test.
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AntePrion
Approach and methodology: AntePrions goal is to use a combination of research and engineering approaches to attain the following goals: improve PrPSc detection in blood and fluids; discover novel proteomic, genomic, and immunologic surrogate markers of TSE; integrate these advances into a preclinical test for TSEs. To achieve these goals, experts in the field of prions, cell biology, antibody discovery and production, and diagnosis are present in the consortium, as are end-users, a national blood bank, a national biosafety regulatory agency and a centre for neurodegenerative diseases. Four types of activities can be discerned in AntePrion: 1. collection and preparation of human and animal samples; 2. analysis of samples (finding enriched fractions, discovering surrogate markers); 3. development of novel detection systems (e.g. cultured cells), reagents (such as antibodies) and novel software; 4. integration and assessment of the developing technologies. The principal aim is to devise a preclinical blood test to do the following: identify human TSE cases early enough so that emerging therapies can be administered in time (i.e. prior to extensive neurodegeneration); prevent transfusion of tainted human blood; remove TSE-tainted meat and animal products from the food chain. Expected outcome: With fluorescence-activated cell sorting (FACS), subpopulations of white blood cells (WBCs) from humans and animals will be isolated, and PrPSc in fractionated populations of cells will be measured using conformation-dependent immunoassay (CDI). Localisation of PrPSc will be studied by immunoelectron microscopy. The AntePrion partners expect to reveal the blood cell type(s) with a high PrPSc content. Enrichment gained by FACS may yield a blood test for PrPSc detection at a phase before any clinical symptoms appear. Mass spectrometry will be used to obtain profiles of molecules from body fluids, as well as brain and muscle from prion-infected animals which bind to different chromatographic surfaces. The aim is to identify prion-infection specific biomarkers, most likely proteins. Measurements of such a biomarker might provide a target for development of a non-PrP based ante-mortem test. Alternatively, specific patterns of protein expression or signatures detected by mass spectrometry might be used as the basis of such a test. European countries aim for high consumer protection standards and target high standards concerning the safety of blood and blood products. The presence of prion infections in cattle (sheep and goats) and the transmission of the disease to human beings seriously threaten public health. Low levels of PrPSc have been detected in WBCs in the blood circulation of prion-infected animals. Moreover, recent data suggest that vCJD can be transmitted from humans to humans via blood or blood products. AntePrions initiative will help to establish markers and methods for the early, preclinical diagnosis of prion infections in animals and human beings, and will lead to the development of a new and sensitive ante-mortem diagnostic test for prion diseases. Since several partners are involved in national and international research activities through networks and control/surveillance programmes on other infectious or transmissible diseases, AntePrion will have a large impact on public health.
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DIAGNOSTICS
Major publications: Dollinger, S., Lber, S., Klingenstein, R., Korth, C., Gmeiner, P., A Chimeric Ligand Approach Leading to Potent Anti-Prion Active Acridine Derivatives: Design, Synthesis and Biological Investigations, Journal of Medicinal Chemistry, 2006, 49:6591-659. Klingenstein, R., Melnyk, P., Leliveld, S.R., Rykebusch, A., Korth, C., Similar structure activity relationships in the antiprion and antimalarial activity of quinoline derivatives, Journal of Medicinal Chemistry, 2006, 49:5300-8. Collins, S.J., Sanchez-Juan, P., Masters, C.L., Klug, G.M., van Duijn, C.M., Poleggi, A., Pocchiari, M., Almoti, S., Cuadrado-Corrales, N., de Pedro Cuesta, J., Budka, H., Gelpi, E., Glatzel, M., Tolnay, M., Hewer, E., Zerr, I., Heinemann, U., Kretszchmar, H., Jansen, G.H., Olsen, E., Mitrova, E., Alperovitch, A., Brandel, J.P., Mackenzie, J., Murray, K., Will, R.G., Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease, Brain, 2006, 129: 2278-2287. Cepek, L., Brechlin, P., Steinacker, P., Mollenhauer, B., Klingebiel, E., Bibl, M., Kretzschmar, H.A., Wiltfang, J., Otto, M., Proteomic Analysis of the Cerebrospinal Fluid of Patients with CreutzfeldtJakob Disease, Dementia and geriatric cognitive disorders, 2007, 23:22-8 Campana, V., Caputo, A., Sarnataro, D., Paladino, S., Tivodar, S., Zurzolo, C., Characterization of the properties and trafficking of an anchorless form of the prion protein, J Biol Chem, 2007, 282:22747-56. Fasano, C., Campana, V., Schiavo, G, and Zurzolo, C,. Gene expression profiles of quinacrine-cured prion-infected mouse neuronal cells, J. Neurochemistry, 2008, epub Jan 8.
Coordinator Peter J. Peters Nederlands Kanker Instituut Department of Tumor Biology Plesmanlaan 121 H4 1066 CX Amsterdam, Netherlands E-mail: p.peters@nki.nl Partners albert Taraboulos Department of Molecular Biology Hebrew University Hadassah Medical School Jerusalem, Israel Krister Kristensson Karolinska Institutet Department of Neuroscience Stockholm, Sweden Jesus Requena Universidade de Santiago de Compostela Prion Research Unit Santiago, Spain R. G. Will University of Edinburgh National CJD Surveillance Unit Edinburgh, UK Markus Otto University of Ulm Medical Faculty Department of Neurology Ulm, Germany Eva Mitrova Slovenska Zdravotnicka Univerzita Department of Prion diseases and National Reference Centre for prion diseases Institute of Preventive and Clinical Medicine Bratislava, Slovakia
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AntePrion
Chiara Zurzolo Institute Pasteur Department of Cell Biology and Infection/ Unit Trafic Membranaire et Pathogense Paris, France Carsten Korth Heinrich Heine Universitt Dsseldorf Department of Neuropathology Dsseldorf, Germany Michael Baier and Georg Pauli Robert-Koch-Institut Berlin, Germany Lothar Stiz Friedrich-Loeffler-Institut Federal Institute for Animal Health Greifswald - Insel Riems, Germany Martin Wiesenfeldt Matrix Advanced Solutions Germany GmbH Gttingen, Germany H. Schuitemaker Stichting Sanquin Bloedvoorziening Amsterdam, Netherlands
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DIAGNOSTICS
TSEUR
an integrated immunological and cellular strategy for sensitive TSE diagnosis and strain discrimination
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-018805 Specific Targeted Research Project e 1 750 000 1 June 2006 36 months www.pathol.uzh.ch/tseur
Background and objectives: Prion infections, or Transmissible Spongiform Encephalopathies (TSEs), result in progressive, fatal neurodegeneration. No effective therapies are available, and medical interventions, possibly including blood transfusions, have resulted in human-to-human transmission of prions. However, no biomarkers are available for the preclinical diagnosis of prion infection in body fluids. All approved methods of diagnosis rely exclusively on the detection of pathological prion protein (PrPSc), which may not be present in all TSEs. The TSEUR consortium proposed to develop, validate, and exploit innovative reagents and technologies that will address the above challenges, within the following three areas: enhanced detection of PrPSc; direct measurement of prion infectivity; validation of new TSE biomarkers in body fluids. Each partner brings to the TSEUR project a significant body of existing knowledge and data; this includes a potent new panel of picomolar-affinity anti-PrP monoclonal antibodies for a variety of PrP domains. Their work will enable the highly sensitive detection of PrPSc and discrimination of prion strains (epitope coding). The project partners will field-test the validity of their recently identified secreted surrogate markers whose levels are significantly higher in preclinical prion infections for identifying potentially contaminated body fluids. Immuno-PCR technology
will be explored as a means to enhance the sensitivity threshold of each assay. Finally, the partners will extend the use of scrapie cell assays to the rapid and sensitive detection of actual prion infectivity under different sets of conditions. Ongoing work indicates that all Work Packages are highly feasible. TSEUR will develop innovative diagnostic technologies to address the current gaps in prion detection. The projects goals are to enhance the safety of the blood supply, to provide minimally invasive techniques for diagnosing human and animal TSEs, and to develop highly sensitive tools for identifying prion strains. The current need for sensitive tests to detect prions or the surrogate markers of prion diseases in affected individuals before the clinical onset of disease, has intensified the search for new technologies and tools. Currently, diagnosis relies exclusively on the detection of PrPSc which may not be present in all TSEs. Significantly, even by using detection methods specifically for PrPSc, no sensitive tests are available to detect prions in the blood or urine of affected individuals, such as human CJD patients or scrapie-infected sheep. Therefore, not only are new tools required for detecting PrPSc in various body fluids or organs at different stages of the disease, but also new surrogate markers are needed that can sense the presence of prions. Therefore, in order to detect affected individuals at the subclinical stage, it is crucial to find sur-
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TSEUR
rogate markers other than PrPSc. Moreover, the appearance of new prion strains that do not differ in their primary amino acid sequence but are believed to vary in their three-dimensional structure, shows that there is a big demand for tools that enable scientists to differentiate among prion strains. Prions strains possess different organ tropisms, can be replicated in different hosts with different efficiency, and can induce diseases with varying incubation times, lesion profiles, etc. Therefore, it will be of great importance to generate tools that can differentiate between a range of prion strains. The TSEUR consortium proposes to develop, validate, and exploit innovative reagents and technologies that will address the current challenges faced by prion diagnostics in three areas: developing innovative diagnostic technologies addressing the current gaps in prion detection; providing minimally invasive diagnostic techniques for human and animal TSEs (e.g. detection of prions in blood and urine of affected individuals; surrogate markers found in urine or blood that can be used to identify subclinically infected individuals); producing highly sensitive tools for identifying prion strains. Approach and methodology: Work Package (WP) 1 investigates whether other inflammatory conditions will give rise to ectopic prion replication (e.g. granulomas). For this reason, the partners have established a granuloma mouse model, in which granulomas are induced subcutaneously, and prions are peripherally administered to mice with granulomatous inflammation. They also set up in vitro-based cell systems (e.g. stable transformation of various neuronal and non-neuronal cell lines with or without PrP expression) to investigate prion replication competence of various cell lines (e.g. for sheep scrapie). This is expected to lead to in vitro assays for monitoring infectivity in various organs, infectious properties of various prion strains (e.g. ovine prions) and other species, including elk and deer with Chronic Wasting Disease or sheep scrapie). In WP2, the partners are attempting to map regions that could be of importantance to prion conversion and replication. To this end, they have generated various PrP mutants with additional disulphide bridges which induce folding and different three-dimensional structures at the particular sites investigated. The disulphide mutants will be expressed in vitro, purified and analysed by mass spectrometry and by NMR. After transfection into cell lines, prion replication competence of the respective mutants will be analysed in a scrapie cell assay-like set-up. WP3 and WP4 are producing highly sensitive monoclonal antibodies for human Cystatin F, a surrogate marker of transmissible spongiform encephalopathy found in body fluids of infected individuals, including cerebral spinal fluid and urine. The antibody will be used to establish a diagnostic test. In addition, the partners will deliver a technological platform in which low levels of the antigen are amplified by a PCR technology (quantitative immuno-PCR). Tools will be produced for the benefit of the whole consortium (POM antibodies). The extraneuronal prion distribution in scrapie infected sheep is being investigated in WP5. Several different methods are being followed that include the analysis of organs and body fluids from both healthy sheep and those infected by scrapie by natural means. The partners are also analysing prion distribution and body fluids including milk, blood and urine in sheep artificially infected with scrapie. PrPSc deposition is monitored by NaPTA precipitation followed by conventional Western blot analysis and PMCA.
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DIAGNOSTICS
Furthermore, inoculation studies with urinary proteins or other tissue homogenates (e.g. kidney, mammary gland) are being performed. In order to investigate whether inflammation induces prion deposition in previously prion-free organs, mammary glands derived from scrapie-infected sheep with mastitis were isolated. Homogenates of these organs are transmitted into bank voles. Similarly, inflamed lungs are monitored, analysed and transmitted into susceptible ruminants to examine infectivity. In addition, milk proteins are transmitted into New Zealand lambs, believed to be devoid of scrapie. Moreover, urine will be carefully analysed for prion infectivity and PrPSc. By using TaqMAN analysis, a sensitive and qualitative assay will be established in WP6, that can differentiate among the various Maedi Visna viruses responsible for inflammatory disorders, in sheep, for example. Furthermore nanotechnology will be specifically used as a detection system for prions. In addition, WP6 provides various tissues and body fluids of naturally scrapie-infected and healthy sheep used for the investigation of peripheral prion pathogenesis, as well as for establishing various systems (immuno-PCR) to detect prions within organs or body fluids of scrapie-infected ruminants. Main findings: WP 1 has established a mouse model with longlasting, subcutaneous granulomas. The partners can show that after peripheral prion administration these granulomas have gained prion replication competence. Furthermore, they can demonstrate that the stromal compartment in the granuloma is responsible for expressing the cellular prion protein. Additionally, histoblot analysis has localised PrPSc to the granulomatous nodules which are composed of epitheloid macrophages, fibroblasts and dendritic cells. In WP2, cells expressing various forms of the prion protein were established and analysed:
1.
2.
Generation of retroviral vectors/supernatants for: Myc tagged mouse PrP (92, 95, 97, 190, 195, 202, 230); Flash tagged mouse PrP (97, 202, 230); ovine PrP (VRQ, ARQ, ARR); 3F4 antibody epitope tagged ovine PrP (VRQ, ARQ, ARR). Generation of cell lines stably expressing: tagged mouse PrP (HpL3-4 MoPrP-Myc and -Flash); ovine PrP (HpL3-4 and RK13 ovPrP-VRQ, -ARQ, -ARR); tagged ovine PrP (HpL3-4 ov3F4-VRQ, -ARQ, -ARR).
Confirmation of cell surface expression of all constructs, as depicted above, except 202PrPFlash, as well as subcloning of RK13 cells expressing ovPrP was performed. In WP3 and WP4, multiple human Cystatin F-specific antibodies were isolated and the strongest binders have been identified by ELISA. Sequence analysis of the antibodies shows that most are related to one another. The 3 strongest binders are being expressed in 293T cells and have been purified as single chain FC fusion proteins while 12 high binders from the last sort experiment are being further investigated. These antibodies or single chain fusion proteins are currently being evaluated in an ELISA-like set-up. The first batches of POM antibodies were produced as a basis for providing Abs to the whole TSEUR consortium (POM1, POM2, POM3). In addition, the Abs are used for the establishment of immuno PCR and other techniques for detecting PrPSc or PrPC . As part of WP5, PrPSc was identified in the lungs, kidneys and urine of sheep naturally infected with scrapie; a paper is now being prepared. Further transmission experiments were begun, using milk from sheep infected with scrapie and
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TSEUR
suffering from mastitis. Milk proteins were orally transmitted into lambs from New Zealand sheep. Urinary proteins from sheep with PrPSc deposits in kidney as well as all control samples were also transmitted into bank voles, and into transgenic mice expressing the bank vole protein. In addition to test prion infectivity in renal homogenates, transmission experiments in bank voles were also performed. So far, prion infectivity has been detected in the kidneys of scrapie-infected sheep, as well as PrPSc by PMCA in the urine of sheep infected with scrapie, but not in controls. In WP6, various tissues and body fluids from healthy sheep and sheep naturally infected with scrapie were provided to the whole consortium. In addition a multicolour TaqMAN analysis was established as a sensitive and qualitative assay for different Maedi visna viruses. Coordinator adriano aguzzi Institute of Neuropathology University Hospital Zurich Schmelzbergstrasse 12 8091 Zurich, Switzerland E-mail: adriano.aguzzi@usz.ch Partners Ina Vorberg Institute of Virology Technical University of Munich Munich, Germany Roman Jerala National Institute of Chemistry Ljubljana, Slovenia Ciriaco Ligios Istituto Zooprofilattico Sperimentale della Sardegna Sassari, Italy Martin Bachmann Cytos Biotechnology AG Schlieren, Switzerland Ingolf Lachmann AJ Roboscreen GmbH Leipzig, Germany Max Basagni Prion Diagnostica Srl Rho (MI), Italy
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DIAGNOSTICS
ADDNET
Paradigm shift from kidney biopsies to advanced molecular diagnostics from patient urine
Contract No Project type EC contribution Starting date duration LSHB-CT-2003-503364 Specific Targeted Research Project e 2 000 000 1 January 2004 36 months
Background and objectives: Proteinuria is a common medical symptom often found in association with infectious, inflammatory or immunological diseases. However, the most important cause of proteinuria is progressive kidney damage due to diabetes. The level of proteinuria correlates with the severity of glomerular damage, and persistent proteinuria leads to scarring and end-stage renal disease (ESRD) requiring dialysis treatment or ultimately renal transplantation. Both treatments are severely and chronically debilitating for the patient and increase the risk for cardiovascular complications. In Europe, the need for these treatments is projected to clearly exceed their availability during the next few years. At present, the diagnostics of proteinuric kidney diseases consists of selected serum markers and urine analysis. However, mainly due to their limited sensitivity and specificity, the diagnostic method of choice is still direct kidney biopsy. Although inherently accurate, this is invasive and carries a notable risk for severe complications. There is therefore a recognised need for a more accurate, predictive, non-invasive and modern diagnostics reflecting the precise molecular pathogenesis of proteinuric diseases. The main objective of the ADDNET project was to identify and evaluate kidney damage associated molecules in the urine to develop, establish
and validate new diagnostics directly from urine, an easily accessible source. Furthermore, the project aimed at gaining information about the structure and interactions of the podocyte proteins, as well as identifying novel pathways in the development of proteinuria. Approach and methodology: The consortium produced the first knowledge of new and previously unknown molecules involved in determining the kidney glomerular filtration barrier in health and disease. For this purpose, identification of candidate genes/proteins for proteinuric diseases as derived from various activities in Work Packages (WP) 1-5 was performed. The first level of verification came from using in vitro methods, further extending to established experimental models of disease and, finally, to the use of unique database collections of human sample material and associated clinical information. The project combined state-of-the-art tools to understand the mechanisms of proteinuric kidney disease. An expanding set of crucial podocyte molecules and their role in proteinuric damage was achieved. Specific objectives included: exploitation of data from gene expression analysis of the human single gene disease CNF as well as its transgenic animal model (nephrin deficient TRAP) and, particularly, clustering of the Affymetrix derived expression data to achieve
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ADDNET
a comparative genomics approach; sophisticated bioinformatics tools, such as gene promoter analyses, were used to identify new molecules involved in proteinuric damage as based on identification of transcription factor patterns of differentially expressed genes in specific tissues; equipment including the SELDI-tof (Ciphergen) to search for molecular differences between urines of healthy individuals and diabetic patients with nephropathy to identify potential molecules involved in diabetic nephropathy was used; establishment of the role of novel key molecules, their regulation and protein partners in the pathogenesis of early kidney disease was initiated using appropriate experimental models; information generated to establish simple and non-invasive test methods from urine for diagnostics, prognostics and follow-up of kidney patients was gathered. For this, conventional immunologic methods including Western blot, and ELISA techniques were used. The collection of urine and kidney samples from two experimental models of nephropathy was completed, and the urine samples were analysed by SELDI-tof technology. The expression of novel target hits were shown within distinct structures of the normal and diseased kidney and regulation also by RT-PCR and Western blot verified in kidney injury. Immunohistochemistry was used to show semiquantitatively the regulation of novel target antigens within the tubular and glomerular compartments. Type 1 diabetic patients and healthy control subjects were phenotyped for multiple parameters, which were correlated with findings in the urine biomarker profiling by SELDI-technology. Several potentially interesting markers were identified. A wide range of antibodies against candidate molecules were acquired from commercial as well as collaborative sources, and then tested and used with human and animal kidney samples to gain biological information and validation with respect to diseases on these molecules. Work on these potential markers is continuing. Major publications: Main findings: Based on microarray analyses of nephrin-deficient mouse embryonal kidneys, a ranking list of proteinuria associated genes was generated and analysed further. The expression of selected candidate genes as teased out from the bioinformatics platforms was replicated using classical biochemical and immunochemical methods in an independent set of samples of wildtype, as well as gene-deficient kidneys using quantitative RTPCR to confirm the results of microarray analysis. Methods to analyse nephrin-binding proteins in silico were used with novel candidate interacting proteins identified. Dual immunofluorescence staining was used to verify the appropriate colocalisation within the kidney. Ihalmo, P., Schmid, H., Rastaldi, M.P., Mattinzoli, D., Langham, R.G., Luimula, P., Kilpikari, R., Lassila, M., Gilbert, R.E., Kerjaschki, D., Kretzler, M., Holthofer, H,. Expression of filtrin in human glomerular diseases, Nephrol Dial Transplant, 2007, Jul;22(7):1903-9. Heikkila, E., Ristola, M., Endlich, K., Lehtonen, S., Lassila, M., Havana, M., Endlich, N., Holthofer, H. (on behalf of the Addnet consortium), Densin and beta-catenin form a complex and co-localize in cultured podocyte cell junctions, Mol Cell Biochem, 2007. Nov;305(1-2):9-18. Epub 2007 Jun 21.
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DIAGNOSTICS
Coordinator Harry Holthfer University of Helsinki POB 21 (Haartmaninkatu 3) 00014 Helsinki, Finland E-mail: harry.holthofer@helsinki.fi Partners Klaus-Robert Mller Fraunhofer Institute Berlin, Germany Per-Henrik Groop Samfundet Folkhlsan i Svenska Finland r.f. Helsinki, Finland Jesus Egido Fundacin Jimenez Diaz Autnoma University Madrid, Spain Kimmo Kaski Helsinki University of Technology Espoo, Finland Peter N. Iversen Ciphergen Biosystems A/S Copenhagen, Denmark
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GLYFDIS
Glycans in body fluids potential for disease diagnostics
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037661 SME-Specific Targeted Research Project e 2 793 722 1 November 2006 36 months www.glyfdis.org
Background and objectives: Developing effective tools to screen for cancer is an important endeavor, where there is much research taking place. The GLYFDIS projects objective is to develop methods for earlier diagnostic and effective disease screening of prostate and pancreatic cancer that will lead to better treatment outcomes. Early diagnosis of cancer is of far greater prognostic importance than any attempts to treat the disease in its late stages. Even in cases where the eventual outcome cannot be changed, treatment is simpler and quality of life improved for those cases where early diagnosis is achieved. For this purpose, GLYFDIS proposed a method of a simple noninvasive blood testing. Accurate monitoring of a cancerous state following diagnosis can contribute significantly to prognosis determination and online evaluation of therapeutic regimens. The most widespread and diverse post-translational modification is glycosylation. The location and variation of glycans place them in a position to mediate cellular and intracellular signalling events, as well as participate in different biological processes including pathology states such as cancer. Therefore, the project proposes to use analyses of glycans for identifying novel biomarkers that can be used for the diagnostics and monitoring of cancer. Cancer is a significant burden on individuals, families and society. The economic impact of
cancer is substantial. In 2002, the overall cost of cancer, as published by the National Cancer Institute, was USD 172 billion. This does not account for the psychological toll that it takes on individuals and families. Early detection and diagnosis of cancer is based on the observation that treatment is more effective when the disease is detected earlier in its natural history, prior to the development of symptoms, than in an advanced stage. Diagnosis of cancer in the early stages of the disease influences many aspects of life. It can significantly decrease cancer-associated morbidity and mortality and to relieve the burden on patients, their families and society. Accurate monitoring of a cancerous state following diagnosis can significantly contribute to prognosis determination
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DIAGNOSTICS
and online evaluation of therapeutic regimens. Developing effective tools to screen for cancer is an important endeavour and there is much research taking place to develop these tools. The goal is to detect the cancer when it is localised to the organ of origin without invasion of surrounding tissues or distant organs. Approach and methodology: The GLYFDIS project will make use of glycans. Their diversity, compared to genome or proteome, makes the glycans ideal for diagnosis and monitoring of cancer. Cancer-associated changes in the glycome of the tumoural tissue are very frequent. Currently, one of the main obstacles is the lack of sufficient technology. Glycome-analysis technologies today fall behind the rapidly developing genome- and proteome-analysing technologies. The consortium hopes to identify biomarkers that can be used to develop a non-invasive method for the early diagnosis of prostate and pancreatic cancer based on glycan analysis. GLYFDIS main objectives are: optimising high-throughput methods of glycan analysis for the diagnosis of prostate and pancreatic cancer by the analysis of glycans in blood; identifying cancer associated glyco-markers in serum samples of prostate and pancreatic cancer patients; developing and validating protocols for lectin-based microarrays intended for large scale screening of cancer associated glyco-markers in serum samples. Role of SMEs RNTech Diagnostics RNTech Diagnostics is an SME specialising in early stage diagnosis of digestive tract cancers with special focus on colorectal and pancreatic can-
cer. The company operates a dedicated biobank of biological samples selected from patients that have undergone surgery for the removal of digestive tract solid tumour. RNTechs role in GLYFDIS has three facets: 1. To provide biological samples collected from pancreatic and prostate cancer patients and healthy control subjects for the discovery and validation phases of the project. To contribute to the project database by providing genomics data on pancreatic cancer patients, as well as clinical data on all selected patients and healthy subjects and by participating in the bio-statistical and bio-informatics treatment of such data and research results. To contribute to the validation of identified potential biomarkers with its network of clinical oncologists and cancer biology specialists.
2.
3.
Procognia Procognia has developed a lectin-array based technology for rapid analysis of glycosylation profiles of intact glycoproteins. The array contains 2530 well-characterised lectins (carbohydrate binding proteins) with overlapping specificities. The binding of a glycoprotein to the array results in a characteristic fingerprint that is highly sensitive to changes in the proteins glycan composition. The large number of lectins, each with its specific recognition pattern, ensures high sensitivity to changes in the glycosylation pattern. Automatic algorithms were constructed for deconvoluting these signals into a glycan profile output. The major advantages of this technology, in comparison to traditional methods of glycoanalysis, are its short analysis times, the relatively low protein amount needed for analysis, the possibility to analyse multiple samples in parallel, and the relatively low costs compared to the classical analysis methods (HPLC/MS).
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GLYFDIS
A highly sophisticated, automated platform (GlycoScope) was first developed for use in the biopharmaceutical industry for the analysis of recombinant glycoprotein drugs. By tailoring algorithms for various protein families, this product provides accurate, quantitative glycoanalysis for single proteins. In addition, Procognia is developing a line of products for the life science and academic research market. The products are a line of off-theshelf kits for glycoanalysis, distributed by QIAGEN. The first product, QproteomeTM GlycoArray, launched in 2006, provides a rapid and simple tool for glycoanalysis of glycoproteins. This kit can be used without sophisticated equipment, and generic interpretation algorithms provide semi-quantitative glycoanalysis for purified glycoproteins. The second product was launched in 2007 for analysis of global glycosylation patterns of membrane protein extracts. The kit is intended for analysing global changes in glycosylation patterns in extracts of cell membrane proteins of cultured mammalian cells, with the aim of enabling characterisation of glycosylation-related biological effects. As a partner in GLYFDIS, Procognia will optimise the existing technology for analysis of global glycosylation patterns to allow the characterisation of complex protein mixtures in serum from healthy donors and donors with pancreatic or prostate cancer. Expected outcome: The project partners expect: the identification of biomarkers using glycomic and proteomic methods together with computer-based algorithms; the development of a non-invasive, modest, diagnostic kit that will identify specific markers for cancer in the blood; the construction of a website and a gly come bio-bank integrating GLYFDIS results and serving as a basis for a continuously growing public glycome databank; the dissemination of the information to the scientific community and community at large.
Potential applications: The project will generate knowledge relevant for non-invasive diagnosis of cancerous states with the effort in developing a standard protocol for diagnosis of serum samples.
Coordinator angel Porgador Ben-Gurion University of the Negev Beer-Sheva, Israel E-mail: angel@bgu.ac.il Partners Rakefet Rosenfeld Procognia Ltd Ashdod, Israel Pauline Rudd The National Institute for Bioprocessing Research and Training (NIBRT) Dublin, Ireland Jasna Peter-Katalinic Muenster University Muenster, Germany Julien Taieb RNTech Diagnostics Charleroi-Gosselies, Belgium
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DIAGNOSTICS
MolDiag-Paca
Novel molecular diagnostic tools for the prevention and diagnosis of pancreatic cancer
LSHB-CT-2006-018771 Integrated Project e 8 495 490 1 august 2006 36 months www.moldiagpaca.eu
Background and objectives: Malignant tumours of the pancreas, known as pancreatic carcinomas, remain among the most serious challenges in modern medicine. Although they are not among the most common tumours, they are among the most frequent causes of cancer-related deaths, with approximately 28 000 deaths per year in the USA and 40 000 per year in Europe. There are currently no means for reliable diagnosis of early stages and for curative treatment of late stages of the tumour. The overall aim of the MolDiag-Paca project is to make use of genetic profiles of pancreatic cancer and precursor lesions to improve the outcome of pancreatic cancer patients, by providing novel and highly efficient molecular diagnostic tools. In order to achieve this ambitious aim, an integrated multidisciplinary research approach is required, as it calls for a strong interaction between technology, biology and medicine to translate genome data into practical, clinical applications. Approach and methodology: The basic structure of the Work Programme comprises 3 levels and 7 Work Packages (WPs): Level 1 comprises WP1 and WP2. They provide the patient resources required to develop and validate molecular diagnostic tools. They also generate and collect data on genome, transcriptome and proteome profiles of preneoplastic lesions, early
and advanced tumours, as well as prognostic and therapeutic tumour subgroups. At this level, essential resources and datasets are created for the successful completion of all other WPs. Level 2 comprises WPs3 through WP6. They generate novel molecular diagnostic tools based on the profiles of genetic alterations defined in Level 1. Diagnostic tools on the level of the proteome and transcriptome are also being produced, and they make use of epigenetic changes in tumours, so as to develop diagnostic tools. The WPs specifically focus on developing molecular imaging approaches to be tested initially in animal models of preneoplastic lesions and pancreatic cancer, and later intended as in vivo diagnostic imaging tools. First, the ideal targets or signatures are selected for the proposed molecular diagnostic tools.
Examples of comet assays (left) and micronuclei assays (right) to uncover DNA repair defects.
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MolDiag-Paca
The WPs initially design preclinical prototypes, and then validate them using animal models and patient samples archived in Level 1. Then, together with the participating small and medium-sized enterprises (SMEs) and the pharmaceutical industry, the prototype molecular diagnostic tools will be prepared and standardised for use in clinical trials. Molecular diagnostic tools developed in Level 2 aim at improving the diagnosis and prognosis for pancreatic cancer patients or patients at risk, in the following major areas: diagnosis of preneoplastic lesions in patients at risk for pancreatic cancer; diagnosis of early stage tumours in patients at risk and sporadic pancreatic cancer patients; diagnosis of different types and origins of pancreas tumours; prognostic and therapeutic risk stratification of pancreatic cancer patients. Level 3 contains WP7, which represents the final stage of the development of novel molecular diagnostic tools. Experienced clinical oncologists focused on clinical trials of pancreatic cancer patients and populations at risk for pancreatic cancer, work together to jointly organise prospective clinical trials for the newly developed tools. The first clinical trials will involve studies on the following: pancreatic juice of populations at risk for pancreatic cancer to detect preneoplastic lesions or early tumours; resected pancreatic tumours to predict the responsiveness to an adjuvant therapy; fine needle aspirates of primary pancreatic tumours or liver metastases to differentiate distinct pancreatic tumours; fine needle aspirates of primary tumours or liver metastases of pancreatic adenocarcinomas to obtain prognostic and therapeutic signatures. Expected outcome: The MolDiag-Paca partners expect that global differential genetic analyses on the genome and transcriptome and proteome level of PanINs, as well as on early and advanced tumours, may help to identify complex genetic patterns associated with the individual progression steps. Combined with innovative diagnostic approaches, such as molecular imaging technology or detection of minimal amounts of marker genes in secretions, these analyses will create the necessary tools to devise early diagnostic strategies for patients at risk. Equally important is that the collection of aberrantly expressed genes and proteins generated by such analyses may prove instrumental in the identification of targets for innovative chemoprevention strategies, which may in future replace or augment the only existing curative treatment option (radical surgery) with a pharmacological approach. Main results: During the first reporting period (months 1-12), the consortium laid the fundamental prerequisites for the final project goals (development of molecular tools for early diagnosis of pancreatic cancer). In summary, that means that material (i.e. tissue, serum, plasma, DNA, RNA, tissue arrays) was collected, databases and gene lists were generated, and protocols optimised. At the same time a number of assays were established.
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DIAGNOSTICS
Coordinator Thomas M. Gress Universittsklinikum Gieen und Marburg GmbH Standort Marburg Klinik fr Innere Medizin SP Gastroenterologie und Endokrinologie Baldingerstrasse 35043 Marburg, Germany E-mail: gress@med.uni-marburg.de Scientific coordinator Bjrg V. Pauling BMFZ-Building Hans-Meerwein-Str. 2 35043 Marburg, Germany E-mail: pauling@staff.uni-marburg.de Partners Sven N. Reske University of Ulm Nuclear Medicine Clinic Ulm, Germany John Neoptolemos, Bill Greenhalf, Eithne Costello University of Liverpool School of Cancer Studies Division of Surgery and Oncology Liverpool, UK Nick Lemoine and Tatjana Crnogorac-Jurcevic Institute of Cancer Molecular Oncology Unit London, UK Jrg Hoheisel and andrea Bauer German Cancer Research Centre (DKFZ) Division of Functional Genome Analysis Heidelberg, Germany Francisco Real (Paco) and Nria Malats Institut Municipal dInvestigacio Medica, IMIM Barcelona, Spain
aldo Scarpa, Patrick Moore, Claudio Sorio University of Verona Department of Pathology Verona, Italy Claudio Basso University of Verona Department of Surgery Verona, Italy Stephan Hahn, Irmgard Schwarte-Waldhoff University of Bochum Molecular GI-Oncology Department of Internal Medicine Bochum, Germany Hans-dieter Pohlenz and annette Sommer Bayer Schering Pharma AG Berlin, Germany Helmut Friess and Jrg Kleeff University of Heidelberg Department of General Surgery Heidelberg, Germany Ian Fleming and Simon Green Cyclacel Ltd Dundee, UK Gnther Klppel and Bence Sipos University of Schleswig-Holstein Department of Pathology Kiel, Germany Martin Highett Tepnel Life Sciences Manchester, UK Christoph Bremer University of Mnster Department of Clinical Radiology Muenster, Germany
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MolDiag-Paca
Matthias Wassermeier and Zeno von Guttenberg Advalytix AG Brunnthal, Germany Katrin Sak Asper Biotech Tartu, Estonia Johan Permert and Magnus Nilsson Karolinska Institute Department of Surgical Gastroenterology Stockholm, Sweden Jutta Ltges Karolinska Institute Department of Pathology Stockholm, Sweden Jrg Rademann and Jens Peter von Kries Leipniz Institute for Molecular Pharmacology (FMP) Berlin, Germany
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DIAGNOSTICS
COBRED
Colon and breast cancer diagnostics
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2007-037730 SME-Specific Targeted Research Project e 2 985 102 1 March 2007 36 months www.cobred.eu
Background and objectives: COBRED seeks to discover colon cancer or colorectal cancer (CRC) and breast cancer (BC) biomarkers for patient follow-up (monitoring markers) by exploiting the capacity of three state-of-theart high-throughput technologies in an integrated systems biology approach. The specific RTD objectives are: the design of a clinical protocol for prospective clinical CRC and BC collections that fit the needs of the three high-throughput -omics technologies used (i.e. transcriptomics, proteomics, metabolomics); the identification of biomarker candidates (metabolites, proteins, peripheral blood Leucocytes (PBL)-derived mRNAs) capable of detecting and assessing the status of minimal residual disease, metastases and recurrence after surgery and chemotherapy; the development of a centralised database to integrate the data generated by the three technology platforms with the clinical and pathological information of the collections; the discovery of biomarkers with better specificity and sensitivity using cross-platform advanced data mining techniques on the combined data from the consolidated database; the validation of the biological relevance and diagnostic potential of the identified
biomarkers by testing their specificity on tissue arrays and in relevant preclinical models. COBRED gathers the expertise and RTD resources of three biotech small and medium-sized enterprises (SMEs), leading academic partners and two leading cancer treatment centres renowned for their expertise in BC and CRC treatment. An apparent paradox of current cancer epidemiology is that while new therapies and diagnostics improve survival rates in common cancers, e.g. colon and breast cancer, the incidence rates are also increasing, and thus the net effect is negative. CRC is the third commonest cancer type worldwide; in the year 2000 the global incidence was about 1 million, close to 10% of all cancers, and it resulted in about 0.5 million deaths, equalling some 8% of all cancer mortality (Midgley R. et al, Nat Clin Pract Oncol. 2005 Jul; 2(7):364-9). Lifetime risk of colorectal adenocarcinoma is one of the highest of all cancers, approximately 6%, and of colorectal adenoma the benign but precancerous lesion it is approximately 50%. The risk of CRC rises with age, especially in patients over the age of 60. BC is the most common cancer in women from western countries. In these patients, it is not the primary tumour, but its distant metastases that are the main cause of mortality. The yearly incidence rate is over 0.5 million (630 000 new breast cancer cases) that results in about 0.2
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COBRED
million deaths. Recently, the rates of metastasis and mortality in BC patients have decreased as a result of early diagnosis by mammographic screening and the implementation of systemic adjuvant therapy similarly to CRC. However, as the population ages, the incidence of breast cancer increases (Parkin D.M. et. al., Int J Cancer 1999; 80: 827-841. (1999), Elmore J.G. et al., JAMA; 293:1245-1256 (2005)). Continuous improvements in the treatment of another major life terminating illness, namely cardiovascular disease, has lead to an increase in the overall life expectancy. This contributes to the increase of the incidence rate in CRC and BC, among others, due to population ageing. The speed of population ageing is higher than the decreased mortality, due to the accumulated treatment benefits from new cancer diagnostics (Shen Y. J. et al., Natl Cancer Inst. 17;97(16):1195-203 (2005)] and therapies (Nygren P. et al., Acta Oncol. 44(3):203-17. (2005)) thus leading, paradoxically, to a negative net effect. There is ample, but only circumstantial evidence derived from survival data of patients with early stages of cancer, suggesting that earlier diagnosis would allow a 10% to 20% survival rate improvement. In fact, the potential benefits of early CRC and BC diagnosis are so high that an extensive range of community and governmental efforts have been implemented for population-wide screening. Biomarkers are substances found in the blood, other body fluids (e.g. urine) or tissues that alone or in combination may signal the presence of cancer or the risk for cancer. Diagnostics based on biomarkers have the potential to significantly improve current cancer diagnostic means, thus providing a higher sensitivity (i.e. much smaller tumours can be detected), easier, faster and at a much lower cost (Baker M., Nat Biotechnol. 23(3):297-304. (2005)). Biomarker discovery and validation, similarly to drug discovery and validation, is a long process with a high rate (60-80%) of attrition of candidate biomarkers along the major steps of qualification, that ultimately ends in the approval by the US Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products (EMEA) in the EU. Often, seemingly good candidates that have been identified and found valuable in one study do not show the expected predictive values in another study. In fact, the number of new diagnostics approved per year is decreasing in sharp contrast to the intensifying biomarker discovery efforts. Thus, despite having the highest potential value in numbers, COBRED chose not to pursue the discovery of screening markers because of the economic and logistic impracticalities of a large-scale screening-marker validation in BC and CRC. Instead, the partners will focus on the second largest clinical need the improvement of patient follow-up by the discovery of monitoring markers, which are expected to report relapse, metastasis and minimal residual disease at earlier stages. They are more amenable to surgical and chemotherapy treatment, and are more likely to improve cancer patient survival. Role of SMEs The three SMEs involved (BioSystems International, Biocrates Life sciences and Ipsogen) are actually research-intensive SMEs and they play leading roles, since their expertise in -omics (genomics, proteomics and metabolomics) technologies is at the heart of the COBRED project and forms the technology basis for the achievement of the project objectives. The targeted project results are clearly of interest and potential benefit to SMEs, since those create business opportunities for them in the field of diagnostic tools and methods (and related intellectual property rights (IPR)). The SME ARTTIC is responsible for the project management.
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DIAGNOSTICS
Expected outcome: COBRED will deliver candidate protein, metabolite and mRNA biomarkers tested in preclinical studies, ready for large-scale clinical validation and further development for commercialisation by the respective SME partners: Ipsogen for mRNA derived markers, BioSystems International for protein markers and Biocrates Life sciences for metabolomics markers. Specific project results will include: sets of biomarkers (gene signatures, proteins, metabolites or a combination of these) that will be considered clinically relevant for early diagnosis of primary BC and CRC and relapses; a central repository system hosting the results from the technological platforms and the relevant clinical data; prospective clinical collection of BC and CRC; clinical validation for the diagnostic potential of subsets of the identified biomarkers in comparison to existing biomarkers and for currently available imaging techniques; preclinical models for the biomarker evaluation and biological studies. Potential applications: Diagnostic kits for BC and CRC patient follow-up.
Coordinator Laszlo Takacs BioSystems International 4 rue Pierre Fontaine 91058 Evry cedex, France E-mail: laszlo.takacs@biosys-intl.com Partners armin Graber Biocrates Life sciences Innsbruck, Austria Fabienne Hermitte Ipsogen Luminy Biotech Entreprises, Marseille, France Xavier Sastre-Garau Institut Curie Paris Cedex 05, France david Malka Institut Gustave Roussy Villejuif, France Lszl Fss University of Debrecen Debrecen, Hungary andras Guttman Universitt Innsbruck Innsbruck, Austria Jaak Vilo University of Tartu Tartu, Estonia Bruno Cucinelli ARTTIC Paris, France
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Flow cytometry for fast and sensitive diagnosis and follow-up of haematological malignancies
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-018708 Specific Targeted Research Project e 2 182 340 1 april 2006 36 months www.EuroFlow.org
EuroFlow
Background and objectives: Laboratory diagnostics of haematological malignancies has three major applications: establishment of the diagnosis, prognostic classification, and evaluation of treatment effectiveness. Over the past decade, several molecular techniques have brought new insights into the classification and monitoring of treatment effectiveness. However, they have several major disadvantages: they are time-consuming, not applicable in all categories of patients, and cannot focus on cellular subpopulations without purification steps. These limitations can be overcome by innovations in flow cytometry. Flow cytometric immunophenotyping is the sole technique that fulfils the requirements of highspeed, broad applicability at diagnosis and follow-up, and accurately focuses on the malignant cell population using membrane-bound and intracellular proteins as targets. The required, innovative steps concern the development of novel antibodies, immunobead technology, flow cytometry software, and eight-color immunostaining protocols. These are all key objectives of the EuroFlow consortium. A multidisciplinary translational research approach is needed, using cutting-edge technologies and biological data arising from genomic research, which can be addressed most successfully via a close collaboration between industry and academia. Consequently, the EuroFlow con-
sortium is comprised of two SMEs and eight diagnostic research groups, regarded as experts in the fields of flow cytometric and molecular diagnostics. Together, the 10 participants have sufficient complementarity and congruence to cover all aspects of the development, standardisation, and validation of highly sensitive tests for diagnosis and follow-up. The successful implementation of the proposal and the dissemination of the results to European haematological laboratories should result in improved health care, through more precise diagnosis and individualised treatment, and stronger biotechnology enterprises across Europe. Approach and methodology: The EuroFlow project is focusing on the following: design of new antibodies, particularly for detection of intracellular proteins; development of an immunobead system for the detection of leukaemia-derived fusion proteins and other aberrant oncoproteins; development of new software for fast and easy handling of large data sets and for the integration of eight-color stainings into a single multicolor data file; development of software for automated pattern recognition of normal, reactive, and aberrant (malignant) leukocyte populations in blood and bone marrow.
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DIAGNOSTICS
Expected outcome: The consortium anticipates the following results by the end of the project: multiplex immunobead assay for detection of fusion proteins and oncoproteins per disease category (particularly ALL and AML); standardised 8-color immunostaining protocols for fast and easy flow cytometric diagnosis and classification of haematological malignancies; standardised 8-color immunostaining protocols for highly sensitive monitoring of leukaemia and lymphoma patients for evaluation of treatment effectiveness; large database with hundreds of welldefined normal, reactive and malignant cell samples, which can be utilised as a ready-to-use template for a fully automated comparison with newly analysed patient samples. Main findings: In 2006/2007, the partners successfully designed the first immunobead assays for the detection of all three types of BCR-ABL fusion proteins (p190, p210, and p230) and for E2A-PBX1 fusion proteins. The first version of novel software for fast and easy integration of eight-color staining results was completed (INFINICYT) in 2007 as well. EuroFlow is selecting the fluorochromes for the eight-color immunostainings from 14 available fluorochromes, and new antibodies against 12 new intracellular proteins/domains are being generated. Major publications: Orfao, A., Lopez, A., Flores, J., Almeida, J., Vidrialez, B., Perez, J., Kneba, M., Macintyre, E., Parreira, A., Richards, S., Szczepanski, T., Trka, T., Van der Velden, V.H.J., Van Dongen, J.J.M., Diagnosis of hematological malignancies: new applications for flow cytometry, Hematology, 2006, 2:6-13.
Pedreira, C.E., Costa, E.S., Barrena, S., Lecrevisse, Q., Almeida, J., Van Dongen, J.J.M., Orfao, A., A new automated flow cytometry data merging and estimation approach for the generation of n-multi-dimensional spaces: comparison between originally measured and estimated immunophenotypic data on a series of B-cell chronic lymphoproliferative disorders, Submitted. Coordinator Jacques J.M. van dongen Erasmus MC Department of Immunology Dr Molewaterplein 50 3015 GE Rotterdam, Netherlands E-mail: b.vanbodegom@erasmusmc.nl Partners alberto Orfao University of Salamanca Salamanca, Spain Frank J.T. Staal DYNOMICS Rotterdam, Netherlands Marta Martin-ayuso CYTOGNOS Salamanca, Spain antnio Parreira Instituto Portugus de Oncologia Lisbon, Portugal Michel Kneba University of Schleswig-Holstein Kiel, Germany Elisabeth Macintyre Hpital Necker-Enfants Malades Paris, France
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EuroFlow
Stephen Richards University of Leeds Leeds, UK Jan Trka Charles University Prague, Czech Republic Tomasz Szczepanski Silesian Academy of Medicine Zabrze, Poland
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DIAGNOSTICS
DRoP-ToP
Integration of dNa, RNa and protein markers in a tool for the prognosis and diagnosis of human disease
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037739 SME-Specific Targeted Research Project e 1 834 331 1 January 2007 36 months www.drop-top.net
Background and objectives: The management of patients with superficial bladder cancer is difficult. No reliable means exists to determine whether a tumour will progress towards an infiltrative form, which requires radical surgery (cystectomy), or whether it will remain superficial, which requires only conservative surgery (resection). In addition, no dependable marker exists to predict whether a primary tumour will reappear or not during the years following surgical resection, forcing patients to undergo constant revisions that reduce their quality of life and overburden healthcare systems. Numerous markers of various types (genes, transcripts, proteins) have been analysed in bladder cancer studies. Some of them have been found to harbour potential for the prognosis (progression and recurrence) of superficial tumours. However, analyses have often been limited to a single type of marker or even to a single marker. To the best of the project partners knowledge, no study has attempted to integrate different types of markers for an increased predictive power. The main scientific goal of DRoP-ToP is to identify a set of markers with high predictive power for tumour progression and recurrence. To this end, the project proposes to collect tumour and urine samples from bladder cancer patients with a detailed clinical record, to measure in them markers of different types, to find statisti-
cally significant correlations between measurements and clinical records, and to select a predictor set. In addition, DRoP-ToP pursues an ambitious technological challenge: the development of a prognosis microarray for the detection of the above-mentioned predictor set. In order to achieve this, the project proposes to measure all three types of marker biomolecules by means of a single type of probe: oligonucleotides. Specifically, it proposes to use short, long and aptamer oligonucleotides for the detection of gene, transcript and protein markers respectively. Cancer is the second major cause of death in the western world and its incidence is increasing due to the overall ageing of the population. Although advances in our understanding of the mechanisms of tumour onset and progression have been enormous, major impact on survival has been restricted to haematopoietic malignancies, some paediatric tumours, and very few solid tumours. Improvement in survival can be attributed not only to advances in standard chemo- and radio-therapy and to the recent implementation of targeted-drug therapy, but also to advances in the diagnosis and identification of high-risk groups, which allow for earlier and better treatment selection. In contrast, the overall prognosis of the most common cancers, such as lung, colon, prostate, breast and bladder, remains poor, particularly when the
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DRoP-ToP
tumour cannot be cured by surgery. One limitation is that the pathologists interpretation of the tumours histological features remains the gold standard. Recent advances in microarray technology, together with information derived from the sequencing of the human genome, have raised hopes that this situation will change dramatically in the coming years. For these hopes to be realised, it is essential to make appropriate use of information, technology and clinical resources. The project believes that resources are often wasted because of inappropriate approaches and inadequate collaboration between clinical, academic and industrial partners. Transcriptome analysis by DNA microarrays has been successfully used for the identification of biomarkers of tumour progression. However, and due to the use of different microarray platforms and patient selection strategies, among others, the biomarkers identified for a given clinical condition vary from study to study. Therefore, their application to common clinical practice has not yet taken place, and requires prospective studies. The DRoP-ToP proposal intends to overcome the above limitations with a two-fold approach: 1) Prospective validation of the information acquired through retrospective studies. For this, a collaborative multicentre effort is essential. 2) Integration of biomarkers from genome, transcriptome and proteome analysis in a single predictor set. Even though each of these three analyses by itself will likely contribute, it is expected that the combination of biomarker types will result in an enhanced predictive power. This type of strategy has rarely been used due to: the high cost of microarray technology; the need to have access to different platforms for the detection of different biomolecules; the fragmentary nature of most of the published work (multiple DNA, mRNA and protein markers studied, but only individually and in most cases weakly associated to disease phenotype); the lack of bioinformatics and biostatistics tools to handle heterogeneous data; the limited amount of clinical and followup information usually available. In addition, most of these studies are performed
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DIAGNOSTICS
without taking into consideration potential bias in the patient population under study (i.e. large tumour cases are more likely to be studied than small tumour cases for sample availability reasons). Approach and methodology: DRoP-ToP has two major objectives: one technological and one scientific. Regarding the former, the project proposes to develop a tool for multiparametric analyses (mRNA levels, large genetic rearrangements, genetic mutations, genetic polymorphisms, protein levels, post-translational modifications) of biological samples, to better predict tumour progression and recurrence. The evaluation of such heterogeneous parameters will be performed on a single microarray: the triple microarray, an oligonucleotide microarray for simultaneous DNA, RNA and protein assessment). The triple microarray constitutes the test surface of a workstation that integrates technology for the hybridisation, scanning and detection of biomarkers. Its simplicity should facilitate a wide implementation of this tool in the clinic. As a scientific objective, the project proposes to identify a set of biomarkers with of the ability to predict the clinical behaviour of bladder cancer. The selection of such a set of biomarkers will be the end-point of a five-phase endeavour: 1) identification of candidate biomarkers for bladder cancer progression and recurrence from the scientific literature and from existing data generated by two DRoP-ToP partners specialised in bladder cancer; 2) pre-selection of biomarkers on the basis of the strength of their association to tumour behaviour and on the scientific and technical quality of the study; 3) measurement and validation of said candidate biomarkers in a set of samples from patients with a detailed clinical record and follow-up; 4) application of bioinformatics and biostatistics tools for the identification of a set of biomarkers with a strong association to tumour behaviour.
The DroP-ToP strategy should be applicable to the study of any tumour type, and more generally to any disease with a genetic or gene-expression component. However, and as a proof of concept, the project proposes to apply it to bladder carcinoma because it represents a paradigm of the need for useful biomarkers in the clinical setting: 1) it is one of the best models of tumour progression; 2) its incidence ranks fifth among all cancer types (the fourth most common in males and the ninth in females); 3) despite widely variable outcomes, the diagnosis and prognosis tools used in the clinic are few and the same, and they are invasive even for asymptomatic patients (cystoscopy); 4) it is the most expensive cancer type, as it can recur many times after treatment; 5) its evolution is very difficult to predict, whereas the therapeutic approach for its two forms is completely different: when invasive at the time of diagnosis, it has a poor prognosis and requires aggressive surgery (cystectomy); when non-invasive, prognosis is favourable and it only requires conservative surgery (resection); 6) its recurrence is also difficult to predict, which leads to unnecessary visits and cystoscopies for about 50% of patients, whose tumours will never recur; and 7) a number of highly promising biomarker candidates have already been identified and reported in the literature. Role of SMEs The DRoP-ToP consortium is made of eight multidisciplinary partners coming from three European Member States (Germany, Spain, France), two Associated States (Israel and Switzerland), and one INCO country (the Former Yugoslav Republic of Macedonia). Among the partners, the participation of three European high-tech SMEs (Progenika Biopharma, Genewave and NuAce Technologies) must be noted. Progenika Biopharma is the coordinator of the DRoP-ToP consortium.
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DRoP-ToP
Expected outcome: Cancer is the second leading cause of death worldwide. In the year 2002 there were 10 million new cases of cancer in the world, 6 million deaths and approximately 22 million people living with cancer. It is estimated that by year 2020 there will be 15 million new cases per year, and 10 millions deaths. Bladder cancer is a highly common neoplasia, mainly among men, and its incidence is rising in several countries in Europe. Approximately 125 000 new cases with bladder cancer are diagnosed each year in the EU. Despite continued interest in the development of novel tests to better predict bladder cancer prognosis, there has been very limited progress. This is in part due to the fact that all tests developed until present are based on the detection of only one type of biomolecule (i.e. RNA, DNA or protein). The projects approach is radically different: from a systematic review of current knowledge on biomarkers of bladder cancer and existing research results of the participating academic partners, the partners propose to develop a microarray that can detect the three major types of molecules in human biological samples. This should provide a much more solid basis to identify molecular predictors of the disease. The DRoP-ToP proposed technology will bring about three main improvements: the number of invasive tests will be strongly reduced, leading to a reduction in costs by decreasing hospital admissions and the number of working hours lost; reduction in the number of invasive tests will also diminish morbidity and improve the quality of life of patients; a better prognosis will allow more adequate choice of treatment i.e. avoiding therapy to patients who do not need it and applying more aggressive therapy to patients at risk. While most patients develop bladder tumours with a relatively good prognosis in terms of survival, their management is very expensive because of the multiple recurrences that most patients suffer, the need for invasive follow-up procedures, and the frequent hospitalisations. Overall, bladder cancer patients generate the highest cost per patient and lifetime among patients with cancer. In conclusion, bladder cancer generates very high costs to society. At the present time, there is no test recommended or approved to help establish the prognosis of patients with bladder cancer. Over the past 10 years several products have been approved by the FDA for use in the early detection of bladder cancer recurrence (i.e. nmp22, BTA, Diagnocure Immunocyt, Vysis). However, none of these tests is used routinely in the clinical setting yet because they do not provide a substantial benefit. Therefore, there is a tremendous need for better tests. Patients with bladder cancer develop multiple (up to 30) tumour recurrences, thus requiring continued follow-up after the initial diagnosis. For this reason, most patients undergo at least two medical visits over the first few years after diagnosis. Subsequently, the frequency of medical examinations varies according to the evolution of the disease. Therefore, a test that would allow the early detection of recurrences and an improved establishment of prognosis would be applied very frequently to the patients. The DRoP-ToP proposed test could even be used more commonly than cystoscopies are performed today, given that it would not be invasive. Its availability would allow demonstration of the concept that early detection of tumour recurrence is associated with improved overall outcome. Potential applications: Diagnosis and prognosis for bladder cancer and other diseases.
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DIAGNOSTICS
Coordinator Gorka Ochoa Progenika Biopharma, SA Parque Tecnolgico de Bizkaia Edificio 801 B 48160 Derio-Vizcaya, Spain E-mail: gochoa@progenika.com Partners Francois Radvanyi Institut Curie-CNRS Paris, France Nuria Malats Universitat Pompeu-Fabra Institut Municipal dInvestigacio Medica Barcelona, Spain Gordana Cerovic Genewave Ecole Polytechnique Palaiseau Cedex, France Melanie Hilanio University of Geneva Geneva, Switzerland Zivko Popov University Cyril and Methodius-Faculty of Medicine Skopje, Former Yugoslav Republic of Macedonia Hader Kless NuAce Tecnologies, Ltd Rehovot, Israel Joerg Hoheisel Deutsches Krebsforschungszentrum Heidelberg, Germany
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TB-trDNA
Evaluation of transrenal-dNa detection to diagnose tuberculosis
Contract No Project type EC contribution Starting date duration LSHP-CT-2006-037785 SME-Specific Targeted Research Project e 2 000 000 1 January 2007 36 months
Background and objectives: Tuberculosis (TB) continues to be a global threat to public health. It is also of significant social and financial concern to the expanding European Union and a cause of enormous morbidity and mortality in much of the developing world. Timely and accurate diagnosis is a critical obstacle to TB control, and the currently available diagnostic methods are marked by being insensitive, slow, and/or cumbersome to use. Nucleic acid amplification is the only rapid detection method with proven sensitivity and specificity, but it is difficult to implement in its current format. A method that avoided complex sputum processing and cell lysis steps that was applicable across multiple amplification formats (e.g. in addition to PCR) would be a tremendous advantage. There is growing evidence that short DNA fragments, arising from human or bacterial cells dying throughout the body, pass through the renal barrier and appear in urine as transrenal DNA (TrDNA). In a preliminary study conducted at the National Institute of Infectious Diseases in Rome, it has been shown that Tr-DNA from M. tuberculosis was detectable in the urine by polymerase chain reaction (PCR) in 100% of patients with pulmonary tuberculosis, and that these DNA fragments disappeared following anti-TB drug therapy. The TB tr-DNA project aims to validate the diagnostic potential of Tr-DNA detection for TB, to optimise and simplify the sample preparation methods, and to explore the feasibility of us-
ing a diagnostic approach based on this method in a developing world setting. Tuberculosis remains among the most prevalent causes of death from an infectious disease in the world. While global targets for rates of cure have been reached in many areas, case detection remains a significant bottleneck to effective disease control. Microscopy, the only widely available laboratory diagnostic test for tuberculosis, is both difficult to implement and insensitive. Consequently, the availability of new diagnostic tools that are more accurate and accessible may greatly benefit individual patients and significantly contribute to the control of the disease. TB tr-DNA aims to develop a new and highly innovative platform for the detection of povertyrelated diseases (TB followed by HIV, malaria). This platform is based on the principle that dying cells release cell-free DNA into the blood stream that then passes through the renal barrier and can subsequently be detected in urine. Role of SMEs The small and medium-sized enterprise (SME) Foundation for Innovative New Diagnostics (FIND) is an independent, not-for-profit, foundation wholly dedicated to the development, evaluation and demonstration of diagnostics for infectious diseases relevant for the developing world. FIND has a minor role in most of the Work Packages of the project, but will coordinate
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DIAGNOSTICS
the interface between test development and product evaluation, and conduct a project workshop in 2007 and project public health advisory meeting in 2009. FIND will provide documentation and technical expertise related to the customer requirements and design of the product version(s) to be tested. It will ensure that clinical protocols will yield data that will have the greatest utility to determining the future of the technology for the public health sector. Expected outcome: TB-trDNA is designed to develop a rapid diagnostic procedure for utilising transrenal DNA as a target sample for the identification of Tuberculosis patients. The findings of TB-trDNA will also contribute to policy development through knowledge and awareness of the importance of TB diagnosis, with close association with the respective ministries of health and international organisations, such as the World Health Organization. Potential applications: Given the significant challenges of Mtb detection and monitoring in developing countries, the application of the Tr-DNA test could provide a very useful new diagnostic tool. By simplifying the sampling procedure and combining this with improved molecular detection methods (which could eventually lead to simple dip-stick methods), the findings of TB tr-DNA could ensure that simple, cheap, efficacious TB diagnosis is made available to the developing world to ensure targeted use of the available therapy.
Coordinator Jim Huggett Centre for Infectious Diseases and International Health Windeyer Institute, University College London 46 Cleveland St. London, W1T 4JF, UK E-mail : j.huggett@ucl.ac.uk Partners david Tomei Spaxen Italia, c/o National Institute of Infectious Diseases in Rome Lazzaro Spallanzani IRCCS Rome, Italy Peter Mwaba University Teaching Hospital D Block Department of Medicine Lusaka, Zambia Michael Hoelscher University of Munich Department of Infectious Diseases & Tropical Medicine Munich, Germany Leonard Maboko Mbeya Medical Research Programme Mbeya, Tanzania Enrico Girardi Istituto Nazionale per le Malattie Infettive L. Spallanzani IRCCS Dipartimento di Epidemiologia Rome, Italy Giorgio Roscigno Foundation for Innovative New Diagnostics (FIND) Cointrin, Switzerland
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GENEPARK
Genomic biomarkers for Parkinsons disease
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037544 Specific Targeted Research Project e 2 987 966 1 January 2007 36 months www.genepark.org
Background and objectives: Parkinsons disease (PD) is the second most prevalent neurodegenerative disease after Alzheimers disease. There is currently no specific clinical or laboratory diagnostic test available for PD. The diagnosis of PD, which relies on expert opinion, is only about 75% accurate when compared with brain autopsy, which is regarded as the gold standard.. The most important differential diagnosis of idiopathic PD remains atypical Parkinsonism, such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and diffuse Lewy body disease (DLBD), These atypical forms of Parkinsonism may initially be clinically indistinguishable from idiopathic PD, and a definite diagnosis can currently only be established upon autopsy. In early stages of the disease and in patients with atypical presentations, the functional and structural neuroimaging can sometimes improve the diagnostic accuracy, as it provides objective markers for the patterns of dopaminergic neurodegeneration in the basal ganglia (SPECT, PET) and of regional atrophy (structural MRI, VBM). Despite these advances in neuroimaging, the need for better diagnostic accuracy in PD and related disorders remains a great challenge for researchers and clinicians in the field of neurology and movement disorders. In addition to idiopathic PD, more than 10 autosomal dominant and recessive genes or gene loci have been linked to PD. Mutations in the
three most common genetic forms of PD (Parkin-, PINK1- and LRRK2-associated PD) can lead to a phenotype indistinguishable from that of idiopathic PD. The similarities between genetic PD and idiopathic PD extend beyond the clinical picture. For example, PET findings in individuals with mutations in the Parkin gene resemble those obtained in idiopathic PD. Since genetic PD can be diagnosed prior to the onset of clinical symptoms, it offers a unique opportunity to study PD in asymptomatic individuals. Such studies could prove useful for the understanding of disease pathogenesis, as well as for providing a therapeutic window for possible preventive treatments. In order to develop and monitor the effects of such preventive treatments, it would be important to develop surrogate markers of PD in asymptomatic individuals. Preliminary data suggest that such markers can be detected in blood by microarray technology. The microarrays for gene expression profiling are rapidly becoming an important research tool for identifying potential biomarkers and for novel classifications of the disease. The microarray-based biomarkers have been described for numerous diseases, such as ovarian and prostate cancer, but the drawback of such studies still remains the accessibility of tissues in live patients. On the other hand, analysis of blood-derived mRNA by microarrays may represent an important advancement for the development of biomarkers in neurological and other diseases.
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DIAGNOSTICS
In its preliminary work, the GENEPARK team used genome-wide expression profiling of human blood to identify biomarkers for Huntingtons disease (HD), a neurodegenerative disease that also affects basal ganglia. There are no reported studies on gene expression profiling from blood in PD patients; however, there is evidence for specific gene expression signatures in the brain. Preliminary studies have demonstrated that significant changes in gene expression could be detected in blood from PD patients when compared with expression in samples from healthy individuals (Fig. 1). Moreover, there is evidence that possible pathogenetic mechanisms in PD, such as inflammation, apoptosis and oxidative stress, can be at least in part detected in lymphocytes of PD patients. In this STREP (Specific Targeted Research Project), the team is proposing to employ microarrays to identify the diagnostic and prognostic tool for the diagnosis of PD. In addition, they are planning to develop new sophisticated analysis methods and user-friendly bioinformatic tools that are necessary to make accurate functional interpretation of these large-scale data sets. The genes were selected from 6 PD patients and 6 healthy control subjects according to P value, fold change (>1.8 or <0.6) and expression maximum greater than 100. Each column represents a sample and each row a gene. Colorgram depicts high (red) and low (green) relative levels of gene expression. PD denotes patients; PC denotes controls. Approach and methodology: Key methodologies and techniques include clinical workup, neuroimaging, microarray analysis, RT-PCR validation and bioinformatic analysis and development (Fig. 2). The researchers are aiming to include a total of 150 carriers of mutations in known PD genes and 400 idiopathic PD patients. In relation to disease controls, 50 MSA, 50 PSP, 50
DLBD, 100 HD and 50 doparesponsive dystonia (DRD) patients are involved, along with the implementation of 300 healthy controls. In relation to the structural and functional MRI studies, 75 PD mutation carriers, 200 patients with idiopathic PD, 50 patients with MSA, 25 patients with PSP and 25 patients with DLBD are involved. RNA will be isolated from peripheral blood and prepared, as needed, for microarray analysis. Using microarray data, a set of established computational tools will be applied. Novel methods and new bioinformatic software tools for the selection of genomic biomarkers will also be developed. Selected biomarkers will be validated with the real-time PCR method. Selected differentially expressed genes will be used for the design and production of a dedicated spotted microarray. Expected outcome: Three outcomes have been identified as a result of GENEPARK. 1. The improvement of European competitiveness in the field of genomic approaches to the development of new diagnostic methods for nervous system disorders. To this end, a unique collection of patients (not available to this extent in countries outside of the EU), as well as expertise in microarray profiling, bioinformatic analysis and diagnostics will be pooled across research groups in five European countries and Israel. 2. The development of a new diagnostic test for PD based on the haemogenomic biomarkers. To date, there has been no such test for neurodegenerative disorders reported or in use. It has the advantage of being non-invasive and has the potential to provide early diagnosis of disease, estimation of disease progression and evaluation of treatment efficacy of the existing or newly-developed drugs. In addition, development of new bioinformatic software
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GENEPARK
3. tools for microarray analysis is planned, which is likely to improve genomic marker selection through disease modelling and existing knowledge integration. Diagnosis and treatment of the disease. As PD is one of the most common chronic neurodegenerative diseases in Europe, improving public health is crucial. Due to the high costs associated with physicians, drugs, and hospitalisation rates, the decreased quality of life in PD patients extends to a decreased quality of life for the families of sufferers. Thus, PD causes considerable strain on both patients and society, as a whole. It is therefore of utmost importance to develop new diagnostic and prognostic tests for early diagnosis of PD, as well as for the monitoring of the neuroprotective efficiency of existing and new candidate drugs for PD. Coordinator Borut Peterlin University Medical Centre Division of Medical Genetics Slajmerjeva 3 1000 Ljubljana, Slovenia E-mail: borut.peterlin@guest.arnes.si Partners alexis Brice Institut National de la Sant et de la Recherche Mdicale (INSERM) Paris, France Christine Klein University of Lebeck and Neuroimage Nord Department of Neurology Lebeck, Germany dimitri Krainc Mediterranean Institute for Life Sciences Split, Croatia Gorka Ochoa Progenika Biopharma SA Vizcaya, Spain Olaf Riess Eberhard-Karls-University Tebingen Tebingen, Germany Ron Shamir Tel Aviv University Tel Aviv, Israel Mojca Zajc RR & CO. Business Consulting, d.o.o. Ljubljana, Slovenia
Major publications: Binkofski, F., Reetz, K., Gaser, C., Hilker, R., Hagenah, J., Hedrich, K., v Eimeren, T., Thiel, A., Bchel, C., Pramstaller, P.P., Siebner, H.R., Klein, C., Morphometric fingerprint of asymptomatic Parkin and PINK1 mutation carriers in the basal ganglia, Neurology, 2007;69:842-850.
Cluster analysis of the 36 most differentially expressed genes on Affymetrix microarrays.
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DIAGNOSTICS
PREGENESYS
development of early non-invasive biomarkers and means for the diagnosis and progression monitoring of preeclampsia and tailoring putative therapies
LHSB-CT-2005-037244 SME-Specific Targeted Research Project e 2 326 567 1 december 2006 36 months http://pregenesys.net/main
Background and objectives: Preeclampsia, a multi-system disorder, complicates 5-7% of all pregnancies, and is responsible for 18% of maternal deaths during pregnancy and for a third of prematurity. Major motor and cognitive newborn disabilities, and blindness and life-long complications place a heavy toll on obstetrics and paediatric expenditures. The complex disorder, expressed as a newly onset hypertension developed in previously normotensive women after 20 weeks of gestation along with protein loss in the urine, is coupled to complications of the kidney, liver, blood system and brain. While clinically diagnosed after the second half of pregnancy, the underlying patho-physiology is associated with deleterious alterations of implantation and placentation already starting in the first trimester. In the PREGENESYS project, the partners propose to apply an inter-disciplinary approach, combining cell and molecular biology, tissue culture, microscopy and biochemistry in order to rigorously assess the effectiveness of first trimester non-invasive markers, integrated with the power of sonography, and leverage on their prediction power for drug tailoring thus optimising therapies for preventing preeclampsia or reducing its severity. The consortiums working hypothesis is: first trimester markers are the key for diagnosis and tailor putative medications for preventing preeclampsia. The group has an unprecedented rich
repertoire of diversified candidates; each contributes to preeclampsia prediction. The partners will apply multi-facet pathways for systematically identifying and characterising additional ones. Moreover, they have direct access to stored large specimen banks and to new patient enrolment. Their marker battery will be utilised for monitoring disease progression. Putative medications will be tailored based on preassessment models thereby increasing their medical effectiveness. Approach and methodology: 1. A scientific core will further characterise markers and bring new ones based on genomics and proteomics and will develop in vitro and in vivo systems for detecting markers in human body specimens. A clinical core will design and conduct nested case-control (Phase I) and a multi-centre (phase II) clinical trial for collecting medical records and human specimens along with randomised patients to putative medications according to good clinical practice, correct definitions, ethics and logistics. A quantitative core will apply rigorous statistics to determine the effectiveness of markers and medications. A biotechnology core that develops kits and assays will turn the research results into viable commercial products and utilise its selling power to generate economic growth ensuing from the project, implementing principles of good manu-
2.
3. 4.
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PREGENESYS
5. facturing and laboratory practice. Tailoring drug treatment for women at risk will progress individual medicine suitable for such a multi-system disorder and the vision of deploying medicine beyond 2010. Coordinator Hamutal Meiri Diagnostic Technologies Ltd 2 HaCarmel St., Building B 4th Floor 20692 Yokneam, Israel E-mail: Hamutal.meiri@pregenesys.com Partners Kevin Spencer Barking, Havering & Redbridge Hospitals NHS Trust Romford, UK Berthold Huppertz Medical University Graz Graz, Austria Nandor Gabor Than Semmelweis University Hospital Budapest, Hungary Pedro Seada ImunoSTAR Porto, Portugal Yvonne Parker Wallac Turku, Finland Kypros H. Nicolaides Foetal Medicine Foundation London, UK Sinuhe Hahn Basel University Basel, Switzerland Irene Cetin University of Milan Milan, Italy Michael T. Jones Innomedica Oy Ltd Turku, Finland
Members of the PREGENESYS group have already led the development of first trimester prenatal screening for detecting chromosomal aberrations, and will lead the consortium in moving from the research and clinical study bench into education and training programmes, and into successful implementation of prenatal screening of preeclampsia and drug tailoring to prevent it.
Immunohistochemistry for PP13 in placenta. A normal term placenta (left) and two cases of late-onset preeclampsia cases (right) stained for PP13. Note the more intense staining for PP13 in the preeclamptic cases especially at the apical membrane of the syncytiotrophoblast. In the lower right image the frame in the middle right image has been enlarged to highlight the disturbed surface of the syncytiotrophoblast showing evidence for necrotic release of subcellular fragments only in preeclampsia (arrows). Confocal microscopy of normal term placental villi. Placental villi from a normal term placenta stained for PP13 (red) and actin (green) are shown. Nuclei were counter stained with DAPI. Shown is an overlay of a stack of 20 single images.
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DIAGNOSTICS
EDAR
Beta amyloid oligomers in the early diagnosis of ad and as marker for treatment response
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2005-037670 Specific Targeted Research Project e 621 002 1 January 2007 36 months www.edarstudy.eu/
Background and objectives: Alzheimers disease (AD) is one of the most common neurodegenerative disorders, yet there are no accurate biomarkers for the early stage of the disease. The goal of the EDAR project is to develop new diagnostic markers of AD that can be used for the early diagnosis and for the monitoring of treatment response in drug trials. This STREP (Specific Targeted Research Project) focuses on beta amyloid oligomers and how these are affected by genes involved in beta amyloid processing. Oligomers have only recently been recognised as a key pathogen in AD. Due to their low concentration, they could not be measured with regular techniques. In the present study, ultra-sensitive assays are being used in order to measure oligomers in vivo. Thus, the project will transfer recent scientific discoveries in the early pathophysiology of AD to clinical application, using innovative technological methods. The project has the following objectives. Develop an assay for the detection of beta amyloid oligomers in cerebrospinal fluid (CSF) and plasma using ultra-sensitive multiplex immuno-polymerase chain reaction (IPCR) with nano-structured DNAprotein conjugates; this objective has five sub-objectives: develop and characterise different oligomer isoforms; develop antibodies against different oli
gomer isoforms and an ELISA based on these antibodies; validate the pathological relevance of oligomers in AD; experimentally validate the relevance of oligomers as early biomarkers in body fluids and brain homogenates in a mouse model; develop an IPCR detection method using relevant antibodies. Measure oligomers using the IPCR and immunoprecipitation assay in subjects with AD, other types of dementia, mild cognitive impairment, and control subjects. Investigate whether genes involved in beta amyloid processing actually modify the levels of beta amyloid oligomers. Investigate the diagnostic value of beta amyloid oligomers in CSF, serum, and plasma in subjects with AD across the disease spectrum from MCI (mild cognitive impairment) to mild dementia. Perform a cost-effectiveness analysis of the oligomer assays for the diagnosis of AD. Investigate the change over time of beta amyloid oligomers in CSF and plasma.
Approach and methodology: The project consists of six Work Packages (WPs). WP1 aims to measure the oligomers in CSF and plasma in patients with AD, other types of dementia, MCI (a prodromal state of AD), and control subjects. In WP2, a number of genes involved
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EDAR
in beta amyloid processing will be genotyped. In WP3, clinical data, and CSF and blood samples of the patients and controls in the study will be collected. In WP4, the data generated in WP1 to WP3 will be analysed, and the diagnostic accuracy of the oligomer assay and the utility of the assay as a marker of treatment response will be investigated. The results from the study will be disseminated and exploited in WP5, and WP6 deals with project management. Expected outcomes: 5 to 10 antibodies against oligomers; 1 IPCR antibody-DNA probe and 2 to 3 multiplex IPCR probes for beta amyloid oligomer measurements; data on oligomers levels and other biomarkers in patients and controls; guidelines for the use of oligomer assays in the diagnosis of AD and as a marker of treatment response. Coordinator Pieter Jelle Visser VU University Department of Neurology Outpatient Clinic PO Box 5075 1007 MB Amsterdam, Netherlands E-mail: pj.visser@np.unimaas.nl Partners Michael adler Chimera Biotech Dortmund, Germany Wim Buurman Hycult Biotechnology Uden, Netherlands Frans Verhey University of Maastricht Maastricht, Netherlands Heinz Hillen Abbott GmbH & Co KG Ludwigshafen, Germany Wiep Scheper University of Amsterdam Academic Medical Centre Amsterdam, Netherlands Lars-Olof Wahlund Karolinska Institutet Stockholm, Sweden Magda Tsolaki Aristotle University of Thessaloniki Thessaloniki, Greece Rik Vandenberghe Katholieke Universiteit Leuven Leuven, Belgium Gunhild Waldemar Rigshospitalet Copenhagen, Denmark Harald Hampel Ludwig Maximilian University of Munich Munich, Germany
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DIAGNOSTICS
NeuroScreen
Contract No Project type EC contribution Starting date duration
Sensitive and differential blood and cerebrospinal fluid test for neurodegenerative dementia diagnostics
LSHB-CT-2006-037719 Specific Targeted Research Project e 2 797 521 1 January 2007 36 months
Background and objectives: The NeuroScreen research project aims to develop an integrated system allowing differential diagnosis of neurodegenerative diseases based on several patented, unique, sensitive and robust technologies. This system will be based on the detection of specific direct and indirect amyloid-related markers in the cerebrospinal fluid, and in blood with new derivative products of nano- and/or micro-biosciences. Among those neurodegenerative diseases with amyloid deposits, NeuroScreen will focus on Alzheimers disease and Prion diseases, for which some ambiguities still exist with respect to their differential diagnosis. At present, and in most cases, the diagnosis of these diseases must be confirmed by postmortem cerebral analysis. Furthermore, the consortium will determine the effectiveness of the technology developed within the project for the diagnosis of other neurodegenerative diseases, particularly those associated with the accumulation of -synuclein (e.g. Parkinsons disease, Lewy body dementia and multiple system atrophy). Sensitivity is needed for early diagnosis, as this will permit more cost-effective therapeutic intervention, before the disease concerned has progressed to a stage where considerable damage to the brain has already occurred. In the case of prion diseases, and especially in the instances where there is a risk of transmission of prions, like blood transfusions,
there are concerns not only for patient care, but for the wider community as well. Specificity is needed, firstly to discriminate the non-degenerative causes of dementia (alcoholic, psychiatric, metabolic, etc.) from degenerative dementia, and secondly to discriminate the different molecular aetiologies in order to choose an appropriate therapeutic treatment. For example, anticholinesterase drugs show certain efficacy in Alzheimers disease, whereas frontal temporal dementia seems to be resistant to this kind of drug. In a similar manner, some compassionate treatments can be proposed for patients with probable Creutzfeldt-Jakob disease (CJD); the latest treatment proposed in the UK and France is highly invasive with an intraventricular delivery of the drug. A mistake in the diagnosis of CJD would be extremely unfortunate. Approach and methodology: The work is being carried out by a consortium comprised of 12 partners from 6 European Member States. A high-quality multidisciplinary partnership with complementary expertise, the NeuroScreen network includes academics, research centres and a technology transfer centre, two hospitals, two industrial SMEs (small and medium-sized enterprises) and an SME expert in regulatory aspects. The work involves developing an integrated assay system based on two steps: the first concerns
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the concentration of the disease markers and the second involves the molecular detection of these markers. The application of the work for biological diagnosis of Alzheimers disease, CJD and possibly other neurodegenerative diseases too, presents incontestable advantages in comparison with current technologies, and also in guaranteeing increased protection of public health. The tests will meet the requirements of the end users, namely the medical biology laboratories involved in carrying out the tests following the clinicians prescriptions. The experimental work is divided into 7 Work Packages (WP): WP1: design, optimisation and production of the early test. neuroaptamer prototypes, neuro-strips, neuro-magnetic beads, validation of packaging and sterilisation process, and stabilised and validated supports. WP2: Alzheimer Diagnostic: tau-181 and -231 markers. choice of the marker candidate and the prototype to be tested in a multiplex trial. WP3: Dementia diagnostic and Lewy bodies: -synuclein marker. development and validation of a suitable assay for -synuclein oligomers. WP4: Creutzfeldt-Jakob: PRION. validation of aptamers, neurostrips and iPCR. WP5: Therapeutic follow-up. biological markers for future therapeutic follow-up validation WP6: Integrative section and fundamental knowledge derived from the other WPs. validation of the multiplex assays systems. WP7: Accompaniment and regulatory follow-up. constant growth. The medical objectives are as follows: 1) to diagnose the kind of disease (especially CJD, Alzheimers disease, Lewy Body Dementia) with specific markers, e.g. pathological Prion Protein (PrPsc), abnormally phosphorylated tau proteins (tau-181 and tau 231) and pathological -synuclein proteins with an ultra-sensitive detection level; 2) to determine if any of the diagnostic markers studied in this project would be suitable as a potential marker for therapeutic follow-up, notably with regards to patients affected by Alzheimers disease. Scientific objectives 1) to develop scientific knowledge (i.e. adhesion /non-adhesion) of surfaces and interfaces in the field of life sciences; 2) to develop ultra-sensitive quantitative iPCR dosing tests, already established for prion proteins, in order to adapt them for other disease markers; 3) to develop a biochemical procedure for the concentration and purification of prion proteins from blood and CSF samples; 4) to develop new binding molecules (aptamers) for the purification and detection of direct and indirect markers of neurodegenerative disorders; 5) to develop biofunctional supports and magnetic beads (or neuro supports) whose specific chemical and physicochemical features will enhance via integrated systems the different markers of interest capture and detection; 6) to investigate the usefulness of -synuclein as a biochemical diagnostic marker for synucleinopathies; 7) to develop new biochemical preparation protocols for samples containing markers of interest to be analysed by (q)-iPCR.
aim:
aim: aim: aim: aim: aim:
Expected outcome: Medical objectives The project concerns public health for neurodegenerative diseases, which are experiencing
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Technological objectives: 1) To adapt engineering technologies (notably, surface technology adapted to manufactured storage and analysis supports), generating higher signal/background, such as q-iPCR tubes and the magnetic beads. These latter will be in encapsulated magnetite, surfactant-free, with a high binding capacity, a high surface area, and an excellent lot-to-lot reproducibility, as well as with mechanical and chemical stability. 2) To allow the partnership to acquire industrial property rights in a sector which may become very competitive in the near future, and thus give the European market the opportunity to be at the forefront of this technology, 3) To make small pilot production of prototypes validated for the neurodegenerative disease area, with a view to rapid industrial development. Coordinator Willy Zorzi Universit de Lige Centre de Recherche sur les Protines Prion Institut de Pharmacie B36, n1 avenue de lHpital 4000 Lige, Belgium E-mail: willy.zorzi@ulg.ac.be Partners Gilbert Legeay and arnaud Coudreuse Association pour les Transferts de Technologies du Mans Le Mans, France Thierry daviet EUDICA Annecy-le-Vieux, France
Jean-Francois delforge, Benjamin Klein, Isabelle Zitte, Julien Lacauste ALCIS Besanon, France awad Osman, daniela Liche, Heinrich Ina, Ingolf Lachmann AJ ROBOSCREEN Leipzig, Germany Fabienne Poncin-Epaillard Universit du Maine Le Mans, France armand Perret-Liaudet, Isabelle Quadrio, Jrmie Seguin CHU-Lyon Hpital Neurologique Laboratoire Diagnostic Maladies prions Bron, France Miran Mozetic, anton Zalar, Janez Kovac, alenka Vesel, Tatjana Filipic, Uros Cvelbar, Janez Trtnik Institut Jozef Stefan Slovenia, Ljubljana david allsop Lancaster University Lancaster, UK Gabor Kovacs, Zsuzsanna Fldvri, Katalin Majtnyi Orszagos Pszichiatriai es Neurologiai Intezet Hungary, Budapest andreas Kage and Kathleen Grttner Charit Universittsmedizin Berlin Institut fr Laboratoriumsmedizin und Pathobiochemie Berlin, Germany
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Imaging, Nanoparticles and Biosensors
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Diagnostics - Imaging, Nanoparticles and Biosensors
DIAGNOSTICS
DiMI
diagnostic Molecular Imaging (diMI): a European network of excellence for the development of new molecular imaging strategies aiming to improve the diagnostic and therapy of human diseases
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2005-512146 Network of Excellence e 10 700 000 1 april 2005 60 months www.dimi.eu
Background and objectives:
Cardiovascular Early detection of atherosclerosis and cardiac dysfunction and imaging disease progression Neuroscience Phenotyping of animal models/patients for early diagnosis and imaging disease progression
Animal Models Animal Imaging Library for validation of molecular markers in vitro and in vivo Diagnostic Molecular Imaging Probes Development of improved smart diagnostic imaging agents Diagnostic Molecular Imaging Technology Integration multimodal imaging technology (MRI, PET, SPECT, OI)
In ammation & Regeneration In vivo detection of transcriptional regulation and migration of in ammatory and stem cells
Integrating Activities Sharing facilities, equipment Exchange of personnel Integration of SMEs
Dissemination Activities Training/Education Communication Exploitation
Innovations, Exploitations, Publications, Training, Meetings, Common Knowledge
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DiMI
The main objective of the DiMI project is the creation of a Network of Excellence: to integrate multidisciplinary research aiming towards the development of new probes and novel multimodal non-invasive imaging technology for early diagnosis, assessment of disease progression and treatment evaluation of diseases of the central nervous, cardiovascular and immune system; to achieve efficient training of young researchers, dissemination of new common knowledge and integration of SMEs and industry; to reach the European leadership role in topics related to molecular imaging for diagnostic purpose, especially with respect to the creation of common data platforms, standards and guidelines. Approach and methodology: The Network consists of research groups from 13 European countries with expertise covering nearly the entire field of molecular imaging. The consortium is performing research tasks within the fields of probe and technology development, generation and evaluation of animal models, as well as preclinical and clinical evaluation and diagnostics in cardiology, neuroscience, inflammation and regeneration. Furthermore, the project features 12 training platforms with a wide offer in imaging modalities and topics. Expected outcome: The Joint Production Agreement of DiMI will join and reinforce researchers and scientists from all specialties in the six main topics of the field
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of molecular imaging for diagnostic purposes. These main topics are inter-related on horizontal tasks for developing technological aspects of molecular imaging, like integration of multimodal radiotracer, magnetic resonance and optical imaging methods, as well as creating new diagnostic and smart imaging probes and validating animal models. Results obtained within these horizontal activities will serve as the basis for the vertical tasks that target applications in major disease models and patients (CNS, heart, inflammation). Besides these scientific results and their uses, the main focus within the DiMI Network lies in strengthening the Diagnostic Molecular Imaging research activities at the European level, the formation of a European Society of Molecular Imaging (ESMI), and the education of scientists, clinicians and society about the potential impact of molecular imaging for diagnostic purposes. Main findings: New ultra-high resolution SPECT with reconstruction software; The SimSET+GEANT4 (SimG4) simulation programme has been extended to model the microPET P4 detector and end shield geometry; Various new radiotracers and MR-based and optical imaging probes were developed and tested with regards to toxicity and imaging; A number of animal models useful for imaging studies of neurological and cardiovascular diseases were characterised by imaging to test new tracers, to validate the biological targets, to investigate the fate of transplanted cells, and to test the effect of therapeutic agents; Over 200 cases and controls have been screened for suitability for identification of novel neuroimaging targets in neurodegenerative disease; Clinical protocols were prepared and harmonised to enable imaging studies
Post-contrast (0.5 mmol/kg GdDTPA) T1W image of a rat acquired at 4.7T following infection with Streptococcuspneumoniae. Note the significant meningeal
for early diagnosis of neurodegenerative disease; Various experimental and clinical imaging studies have been performed by multiple partners for imaging neuroinflammation; Protocols to allow multimodal imaging of arteriosclerotic lesions, as well as stem cells in the CNS and heart have been developed.
Major publications: Kopka, K., et al., 5-Pyrrolidinylsulfonyl Isatins as a Potential Tool for the Molecular Imaging of Caspases in Apoptosis, J Med Chem, 2006, Nov 16; 49(23):6704-6715. Ottobrini, L., et al., Molecular imaging: A new way to study molecular processes in vivo, Mol Cell Endocrinol, 2006, 246, 69-75. Himmelreich, U., et al., A responsive MRI contrast agent to monitor functional cell status, NeuroImage, 2006, 32:1142-1149. Chalon, S., et al., Pharmacological Characterization of (E)-N-(4-Fluorobut-2-enyl)-2carbomethoxy-3-(4-tolyl) nortropane (LBT-999)
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as a Highly Promising Fluorinated Ligand for the Dopamine Transporter, JPET, 2006, 317, 147-152. Lucas, A.J., et al., Development of a Combined microPET-MR System. Technol Cancer Res Treat, 2006, Aug; 5(4):337-341. Vercammen, L., et al., Parkin Protects against Neurotoxicity in the 6-Hydroxydopamine Rat Model for Parkinsons Disease, Mol Ther, 2006, Nov;14(5):716-723. Fulton, D.A., et al., Glycoconjugates of gadolinium complexes for MRI applications, ChemComm, 2006, 1064-1066. Soraya Benderbous University of Tours Laboratoire Biophysique Mdicale et parmaceutique Tours, France ann Planas Institut dInvestigacions Biomdiques August Pi i Sunyer (IDIBAPS) Barcelona (Spain) adriana Maggi University of Milan Centre of Excellence on Neurodegenerative Diseases Milan, Italy Gitte Moos Knudsen Copenhagen University Hospital Rigshospitalet Neurobiology Research Unit Copenhagen, Denmark annemie van der Linden University of Antwerp Vision-Lab, Bio-Imaging Lab Laboratory of Cell Biology and Histology, and Digital Cell Imaging Labs Antwerp, Belgium Chrit Moonen Universit Victor Segalen Bordeaux Molecular and Functional Imaging: From Physiology to Therapy Technological Research Team Bordeaux, France Pascal Laugier Universit Paris VI Targeted and Functional Ultrasound Laboratoire dImagerie Paramtrique UMR 7623 CNRS Paris, France Roland Matrippolito University of Paris Sud Institute of Nuclear Physics UMR 8608 CNRS Paris, France
Coordinator andreas H. Jacobs University of Cologne Laboratory for Gene Therapy and Molecular Imaging Gleuelerstr. 50 50931 Cologne, Germany E-mail: dimi.admin@nf.mpg.de Partners John Clark University of Cambridge Wolfson Brain Imaging Centre Cambridge, UK Silvio aime University of Torino Department of Chemistry IFM and Centre for Molecular Imaging Turin, Italy denis Guilloteau University Hospital Tours Unit INSERM 316 Tours, France
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Markus Schwaiger Technical University of Munich Department of Nuclear Medicine Munich, Germany Harald Carlsen University of Oslo Faculty of Medicine Institute for Nutrition Research and Institute of Immunology Oslo, Norway Bertrand Tavitian CEA - Experimental molecular medicine laboratory Orsay, France Philippe Hantrave CEA The ImaGene Program Fontenay-aux-Roses / Orsay, France Philippe Rizo CEA LETI: Dpartement Systmes pour lInformation et la Sant Grenoble, France Elena Ceccarelli and Corinne Carreaux CEA National Institute for Nuclear Science and Technology Saclay, France Bruno Brisson Biospace Lab Paris, France Veerle Baekelandt University of Leuven Division of Molecular Medicine Leuven, Belgium Luc Mortelmans University of Leuven Division of Nuclear Medicine, Medical Imaging Leuven, Belgium
deniz Kirik Lund University Department of Physiological Sciences Lund, Sweden Rikard Holmdahl Lund University Medical Faculty Medical Inflammation Research (MIR) at the Biomedical Center Lund, Sweden david Brooks Imperial College Neurology Group MRC Clinical Sciences Centre London, UK Hazel ann Jones Imperial College College of Science Technology and Medicine London, UK Ignasi Carri Hospital Sant Pau and CETIR Foundation Autonomous University of Barcelona Department of Nuclear Medicine Barcelona, Spain Klaus P. Ebmeier University of Edinburgh SHEFC Brain Imaging Centre for Scotland Edinburgh, UK Bernd Fleischmann University of Bonn Institute of Physiology I Bonn, Germany Christer Halldin Karolinska Institute Department of Clinical Neuroscience Psychiatry Section Stockholm, Sweden
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agneta Nordberg Karolinska Institute Department Neurotec Division of Molecular Neuropharmacology Uppsala, Sweden andreas Bauer Research Centre Juelich Institute of Medicine Juelich, Germany Matthias Hoehn MPI for Neurological Research In vivo NMR Laboratory Cologne, Germany Chris Reutelingsperger Cardiovascular Research Institute Maastricht Maastricht, Netherlands Michael Horn University of Gteborg Center for Bio-Imaging Gothenburg, Sweden andreas Jacobs and Wolf-deiter Heiss University Medical Center Groningen Department of Neurology and Neuroimaging Center Groningen, Netherlands Philip Elsinga Groningen PET-Center Groningen, Netherlands Christopher Morris University of Newcastle Institute for Ageing and Health Wolfson Research Centre Newcastle, UK Klaas Nicolay Eindhoven University of Technology Biomedical NMR Department of Biomedical Engineering Eindhoven, Netherlands Sabina Pappata University of Naples Federico II CNR Naples and Department of Clinical and Experimental Medicine Naples, Italy alberto auricchio University of Naples Telethon Institute of Genetics and Medicine Biostructure and Bioimaging Institute of the National Research Center Naples, Italy david Parker University of Durham Department of Chemistry Durham, UK Ferrucio Fazio Vita-Salute San Raffaele University Scientific Institute Hospital San Raffaele Institute of Bioimaging and Molecular Physiology CNR Milan, Italy adriana Gittenberger-de Groot Leiden University Medical Centre Department of Anatomy and Embryology Leiden, Netherlands Hans Romijn Leiden University Medical Centre Endocrinology Research Laboratory Department of Endocrinology Leiden, Netherlands Eric Salmon University of Liege Cyclotron Research Centre Liege, Belgium
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Michael Schfers University of Muenster Interdisciplinary Molecular Imaging Network for Cardiovascular Diseases Muenster, Germany Walter Heindel University of Muenster Department of Clinical Radiology Muenster, Germany denis Vivien University of Caen Cerebral Imaging Centre for Research on Neuroscience Caen, France Harald Carlson and Jan O. Moskaug Cgene AS (former MiceTech) Oslo, Norway Jose Masdeu University of Navarre Neuroscience Centre Pamplona, Spain Jean Bernard deloye Cyclopharma Laboratories Saint Beauzire, France Stefan Wecker Medical Research GmbH Cologne, Germany Ivan Lukes Charles University Department of Inorganic Chemistry and Department of Organic Chemistry Prague, Czech Republic Renata Mikolajczak POLATOM Research and Development Department of Radioisotope Centre Otwock, Poland
Eva Sykova Charles University Institute of Experimental Medicine ASCR Department of Neuroscience and Center for Cell Therapy and Tissue Repair Prague, Czech Republic Ulrich Bogdahn University of Regensburg Department of Neurology Regensburg, Germany Karl Herholz University of Manchester Wolfson Molecular Imaging Centre and Centre for Clinical Neuroscience Manchester, UK andrian Lammertsma VU University Medical Centre Department of Nuclear Medicine and PET Research Amsterdam, Netherlands Bodo Levkau University of Duisburg-Essen Institute of Pathophysiology University Hospital Essen Essen, Germany
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DASIM
diagnostic applications of Synchroton Infrared Microspectroscopy
Contract No Project type EC contribution Starting date duration Website LSSB-CT-2005-005326 Specific Support action e 280 000 1 July 2005 36 months www.dasim.eu
Background and objectives: Diagnosis of disease is the basis for all clinical medicine. The primary requirement is reliability, in order to ensure that therapies are appropriate and successful. However, the modern requirements of clinicians from diagnostic services go beyond a simple yes or no to a particular disease. Successful therapy requires information on disease subtype classification, assessment of the disease stage and extent such as the grading of tumours, as well as the monitoring of disease progress and therapeutic success. Rapid pathological analysis can also be a requirement, to ensure timely clinical intervention and maximise the chances of therapeutic success. New technologies are thus constantly sought to assist pathologists in this demanding and important clinical area. Infrared spectroscopy can deliver a very quick biochemical fingerprint of cells and tissues, and it has been demonstrated convincingly in many studies that infrared spectroscopy can be used to classify tissues as normal or pathological, as well as for classifying and grading pathological samples. This is the motivation for more than 70 scientists and clinicians from 9 European countries involved in the DASIM project, who have joined forces to investigate whether a practical tool for pathologists can be developed on this basis. The common factor in this diverse group is their interest in using synchrotrons as high-performance light sources for infrared spectroscopy of cells and tissues.
Synchrotrons are particle accelerators dedicated to the production of light for scientific research. Within a synchrotron, electrons are accelerated almost to the speed of light around an approximately circular path several hundreds of meters in length. As the electrons fly around the curves, they emit extremely broadband radiation covering the entire spectral range from hard x-rays to millimeter waves. The primary advantage of a synchrotron light source for infrared spectroscopy is its high brilliance - that is, a high intensity, low divergence beam from an almost point source. When a sample area of only a few microns is to be investigated, a synchrotron light source can put up to a 1000 times more infrared light onto the sample than a conventional instrument. At each of Europes synchrotrons offering infrared spectroscopy ANKA and BESSY in Germany, DAFNE and ELETTRA in Italy, SRS and DIAMOND in the UK, SOLEIL and ESRF in France, MAX in Sweden and SLS in Switzerland local collaborations exist or are being established to explore biomedical applications. The DASIM project is networking this research effort on a European scale, providing a forum for international cross-fertilisation of ideas and multidisciplinary expertise, facilitating access to synchrotrons for researchers from EU countries without their own facility, and promoting methodological validation by adding a multi-centric quality control perspective. In this way, DASIM is accelerating the transfer of this emerging technology into clinical practice.
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Main findings: In this framework, several workshops and a summer school on synchrotron infrared microspectroscopy have been organised. The first DASIM meeting was organised by the University of Leeds (19-20 July 2005) and hosted at the SRS synchrotron radiation facility in Daresbury. The meeting, chaired by Dr Mark Tobin, triggered the DASIM activity presenting the capabilities of the available European synchrotrons and, in order to use IR microspectroscopy, the needs of the clinical environment covering screening, diagnosis, surgery, post operative investigation and therapeutics. Teams already working on clinical problems through the use of spectroscopic tools presented contributions about diagnostic applications and the international collaborations in the scientific areas already operative. At the meeting, working parties began identifying the key issues of the research areas involved in the project. Cooperation between teams of biologists, clinicians and synchrotron scientists started and continuous progress was achieved as witnessed by the programme of the second DASIM meeting. The event was chaired by Dr Augusto MarInfrared microspectroscopy image of protein, lipid and nucleic acid distributions in a single human prostate cancer cell (picture courtesy of DASIM consortium member Dr Peter Gardner, University of Manchester, UK)
celli and hosted at the Laboratori Nazionali di Frascati of the INFN (21-23 June 2006) where one of the IR synchrotron radiation facilities is now running. During the meeting specific issues such as the full understanding of the advantages of synchrotron-based instruments, the coordination of the ongoing research, the attempt to define standard methodologies and the support to new research teams joining the initial participating teams were discussed. The admission of new teams has been a unique opportunity to discuss the status and the perspectives of the European IR synchrotron facilities working in this area, including those under construction, like DIAMOND, and to extend the international collaborations to overseas countries such as Australia, China and the USA. The most recent DASIM workshop was organised by the University of Reims and held at the French synchrotron facility SOLEIL in SaintAubin (10-11 September 2007), chaired by Dr Pascale Roy, Dr Paul Dumas and Dr Ganesh Sockalingum. A greater and interdisciplinary community of scientists working in both academic and medical environments attended the third workshop. The event was arranged with for parallel sessions dedicated to the DASIM workgroups: Clinical trials, Single Cell Spectroscopy, Unified Merit Parameters and Complementary Spectroscopy Approaches. A central feature of the DASIM project is indeed the formation of working parties to address specific aspects of the project at the annual meetings and at their own special meetings, such as the Joint Single Cell Spectroscopy/Raman Working Group Meeting held in Krakow, Poland on 7 and 8 February 2008. Detailed information is available on the DASIM website (http://www.dasim.eu/) the active virtual forum of the initiative open to the whole EU community where the entire DASIM databank is available.
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Finally, DASIM members are working to complete an original textbook that will be published by the Royal Society of Chemistry and will represent a guide for future generations of scientists involved in the scientific areas covered by this SSA. The interdisciplinary book written for nonexperts with an accessible language has been organised in several chapters presenting the various aspects of the field plus a limited number of case studies discussed in great details. Major publications: Conti, C., Ferraris, P., Giorgini, E., Rubini, C., Sabbatini, S., Tosi, G., Anastassopoulou, J., Arapantoni, P., Boukaki, E., Konstadoudakis, S., Theophanides, T., Valavanis, C., FT-IR microimaging spectroscopy: discrimination between healthy and neoplastic human colon tissues, Journal of Molecular Structure, 2008, in press.
Poster advertising the second DASIM meeting in Frascati, 21-23 June 2006
In addition to the results of the cooperation among teams, their scientific exchange and the publications, workgroup reports will represent the main feedback of this SSA to the European community. Their summaries are the main achievements foreseen for the fourth and final workshop that will be held in Dublin (12-13 June 2008), hosted by the Dublin Institute of Technology. Among the DASIM initiatives to be mentioned is the International Summer School on synchrotron infrared microspectroscopy and biomedical applications, the first in the world that will be held at Karlsruhe (23-27 June 2008), hosted by the ANKA laboratory. The school is open to a wide range of students both undergraduate and postgraduate, and scientists belonging to the different disciplines involved in DASIM. The summer school will be organised in small groups attending parallel modules that will cover all aspects of the field, both theoretically and with practical training of the IR instrumentations.
Gazi, E., Dwyer, J., Lockyer, N.P, Gardner, P., Shanks, J.H, Roulson, J-A., Hart, C.A., Clarke, N.W., Brown, M.D., Biomolecular profiling of metastatic prostate cancer cells in bone marrow tissue using FTIR microspectroscopy: a pilot study, Analytical and Bioanalytical Chemistry, 2007, 387, 16211631. Gazi, E., Gardner, P., Lockyer, N.P., Hart, C.A, Clarke, N.W., Brown, M.D., Probing Lipid Translocation Between Adipocytes and Prostate Cancer Cells with Imaging FTIR Microspectroscopy, Journal of Lipid Research, 2007, 48, 1846-1856. Generosi, J., Piccinini, M., Marcelli, A., Belardinelli, S., Pozzi, D., Congiu Castellano, A., Characterization of solid supported lipoplexes by FTIR microspectroscopy, Infrared Physics Technology, 2007, 50, 14-20. Kwiatek, W.M., Banas, A., Banas, K., Cinque, G., Dyduch, G., Falkenberg, G., Kisiel, A., Marcelli, A., Podgrczyk, M., Micro and bulk analysis of prostate tissues classified as hyperplasia, Spectrochimica Acta Part B: Atomic Spectroscopy, 2007, 62, 707-710.
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Lyng, F., Faolin, E., Conroy, J., Meade, A., Knief, P., Duffy, B., Hunter, M., Byrne, J., Kelehan, P., Byrne, H.J., Vibrational spectroscopy for pathology, from biochemical analysis to diagnostic tool, Experimental and Molecular Pathology, 2007, 82, 121-129. Paluszkiewicz, C., Kwiatek, W.M., Banas, A., Kisiel, A., Marcelli, A., Piccinini, M., SR-FTIR spectroscopic preliminary findings of non-cancerous, cancerous, and hyperplastic human prostate tissues, Vibrational Spectroscopy, 2007, 43, 237.
Coordinator david Moss Synchrotron Light Source ANKA Forschungszentrum Karlsruhe P.O. Box 3640 76021 Karlsruhe, Germany E-mail: david.moss@anka.fzk.de Partners: augusto Marcelli INFN-LNF Frascati, Italy Ganesh Sockalingum University of Reims Reims, France Marco Colombatti University of Verona, Italy Fiona Lyng DIT Dublin, Ireland Sheila Fisher University of Leeds Leeds, UK
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Microfluidic total analysis system for the early diagnostic of neurodegenerative disorders
Contract No Project type EC contribution Starting date duration Website LHSB-CT-2006-037953 Specific Targeted Research Project e 2 499 999 1 January 2007 36 months www.neurotas.eu
NeuroTAS
Background and objectives: The NeuroTAS project aims to develop a prototype of a miniaturised system for diagnostics in the early stage of Alzheimers disease and other neurodegenerative diseases, or as a point-of-care instrument for patient follow-up. The system to be developed belongs to the emerging field of labon-chip systems. It incorporates several innovative enabling technologies, including microfluidic flow control, highly sophisticated nanobiodevices with integrated detection, and novel magnetic nanoparticles. These approaches will lead to unprecedented integration and automation, and allow routine implementation of tests that can, at the moment, only be performed in a small number of specialised research laboratories. The system will use biomarkers present in blood, such as differently cleaved amyloid peptides and post-translational modifications of the tau protein. The miniaturisation and integration of innovative detection technologies are intended to extend the sensitivity of biomarker detection, and thus improve the precocity of diagnosis. This is of paramount importance for the treatment of neurodegenerative diseases, since therapeutic approaches able to retard the evolution of the diseases are progressing and appear promising, but little hope exists for the repair of existing brain damage. The method also aims at allowing the simultaneous study of a wide range of markers, improving
the early discrimination between different neurodegenerative diseases, and thus the choice of treatment. Indeed, the NeuroTAS system will have a modular and evolving structure, and will be able to progressively test and integrate new biomarkers into its diagnostic scheme that may be discovered during development of the prototype. The consortium is a combination of four academic, methodology-oriented laboratories (with complementary competences in biochemistry, analytical chemistry, biophysics and microfabrication), two small and medium-sized enterprises in the field of microfluidics, and two end-users directly involved in patient diagnosis and treatment. Approach and methodology: The envisioned device will present the following advantages: full automation (from plasma to result) for low cost, reproducibility and usability in routine diagnosis or patient follow-up; fast analysis (the ultimate objective is one hour, and the criterion for success will be set at three hours total time, to be compared with present protocols that last several days); high sensitivity; multi-parameter, multiplexed analysis to allow for differentiation between different types of neurological diseases that cannot presently be distinguished at an early stage, and to permit a detailed in-
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vestigation of the evolution of biomarkers in patients. It is hoped that this will help researchers to better understand the disease, and also be useful in the patients follow-up routine. The prototype to be developed in the project is an automated system capable of performing sequential capture, analysis and detection of protein and peptide biomarkers. For both A peptides and tau proteins, two strategies will be considered. 1. Direct track: this strategy consists of multiplexed immunocapture followed by direct detection by miniaturised ELISA. This strategy will be relevant if antibodies specific enough for a complete medically relevant profiling are available. 2. Electrophoretic profiling track: this strategy, more powerful but also more demanding on the instrumental side, involves immunocapture followed by electrokinetic separation and post-separation detection. The lab-on-chip instruments will be designed in a modular format incorporating the following principal modules. The multiplexed immunocapture module (MI) is common to both tracks. The protein capture will be performed by circulating plasma in a sequential series of immunoaffinity microcolumns dedicated to the capture of specific peptides (one A species) or one specific tau form per chamber. The main strategy will rely on an innovative approach based on magnetic nanoparticles. This system uses self-organisation of the nanoparticles to create a compact microcolumn with auto-calibrated micronsized pores. It combines a high-loading capacity and fast affinity reaction, thanks to a high surface-to-volume ratio and extremely thin diffusion layer. Electrochemical detection module (ECD). In the direct track option, an electrochemical
detection will be performed directly after immunocapture. The system to be developed will have the capability of detecting in parallel biomarkers from several capture chambers, using a secondary antibody labelled with an enzyme that is able to transform a substrate into an electrochemically active compound. For A peptides, specific antibodies against several amyloid species exist, so that a direct capture and electrochemical detection strategy of the ELISA type can be contemplated. Microchannel electrophoresis-laser induced fluorescence profiling module (ME-LIF). For some other biomarkers, a more elaborate profiling strategy (electrophoretic profiling track) should be used. In this approach, the preconcentrated proteins/peptides will be transferred to an electrokinetic profiling module, in which they will be profiled by high-resolution capillary electrophoresis (CE) on chips. Two different detection strategies will be developed; the first one will be based on fluorescent labelling and direct on-chip detection using an innovative technology centred on the incorporation of planar waveguides integrated into the chip during fabrication. It dramatically simplifies the detection scheme, avoiding complicated and expensive optics, and is thus particularly well-suited to the development of an integrated lab-on-chip. As part of the project, this approach will be implemented in an all-polymer scheme to retain full compatibility with the other modules of the project. Hyphenated microchannel electrophoresis-immunodetection module (MicroWestern). As an alternative to the ME-LIF module outlined above, a second strategy will combine CE separation followed by translation into a secondary multiplexed immuno-immobilisation element. This new approach, which will be a major pre-
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scient and innovative aspect of the project, will combine the resolution of CE with the sensitivity and specificity of ELISA-type immunoassays. Essentially, this development will introduce the power of Western blotting to the lab-on-chip world. This option will be selected if either the online fluorescent labelling strategy (ME-LIF) does not provide sufficient resolution (this may be the case in particular for tau proteins, since differences in phosphorylation levels only lead to small differences in mobilities that may be hidden by the labelling step), or to increase the sensitivity and the specificity of the detection. Sensitivity enhancement will be made possible, particularly because this approach allows for an ELISA-type amplification step. The final system will comprise two sequential blocks, one dedicated to A peptides, and the other to tau proteins. Depending on the options chosen for each family of biomarkers, the corresponding block will itself comprise several modules, selected among the ones described above, depending on the option chosen for profiling. The direct track involves an immunoaffinity module and an electrochemical detection module, whereas electrokinetic profiling will involve an immunoaffinity module, followed by an electrokinetic profiling module, involving either the MELIF strategy or the MicroWestern strategy. The modular strategy will allow for parallel development and optimisation, but a requirement of compatibility between the different modules will be included from the start to allow the integration of all elements into a single microfluidic system. Coordinator Jrg P Kutter Technical University of Denmark Department of Micro and Nanotechnology Building 344 2800 Lyngby, Denmark E-mail: jku@mic.dtu.dk Scientific coordinator Jean Louis Viovy UMR 168 CNRS/Curie Institute 26 Rue dUlm 75005 Paris, France E-mail: jean-louis.viovy@curie.fr Partners Myriam Taverna University of Paris-Sud Paris, France Zusana Bilkova University of Pardubice Pardubice, Czech Republic Frdric Reymond Diagnoswiss Lausanne, Switzerland Markus Otto University of Ulm Ulm, Germany Jens Wiltfang University of Erlangen-Nrnberg Erlangen, Germany
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POC4life
Multiparametric quantum dot bioassay for point of care diagnosis
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037933 SME-Specific Targeted Research Project e 2 497 820 1 January 2007 36 months www.poc4life.eu
Background and objectives: This project aims to improve the healthcare of patients by elaborating a unique Point of Care (POC) diagnosis platform that will help specialists to deliver an earlier diagnosis and to decide on appropriate treatment. The goal is to provide the clinicians with multiparametric measurement of the main 4/5 essential markers and to support decisions with a software tool. This will be a costeffective breakthrough in the diagnosis market. Each year, 377 000 new European citizens develop lung cancer and 340 000 die from it. Studies show that early diagnosis and accurate cancer typing could save a number of lives. Laboratories nowadays have a wide panel of reproducible diagnostic tests at their disposal: these are mostly routine tests realised in centralised laboratories. The needs for early diagnosis, multiparametric analysis of results and quick monitoring of disease progression or therapeutic sensitivity were progressively left aside, whereas they could be fulfilled with using decentralised diagnostic tools, close to the patient. Among the possible applications of this new concept, the partners have chosen to work on the primary diagnosis of the histological types of lung cancer to help give an improved initial diagnosis and to eliminate the 1520% problematic or late diagnosed cases. The study will pay special attention to women (lung cancer death rates for women have been increasing
in Europe since the 1990s and marker patterns may be different). It will then have a huge impact on health by contributing to the fight against cancer and the development of gender dimension in research. For this purpose, the multidisciplinary project will involve academic researchers (from Germany, Spain and France) and small and medium-sized enterprises (SMEs) (from Sweden and the UK) gathering around the initiators of the project (the SME Cezanne, the University of Strasbourg in France and the University of Potsdam in Germany) which have the skills to build a multiparametric device, to develop immunoassays and to design an interpretation software. The projects objectives are: to elaborate an innovative medical device: a unique point of care diagnosis platform which will help specialists to make earlier diagnosis and provide more appropriate treatments; to provide the best possible integration of parameters by means of a consortium composed of public and private partners such as research intensive SMEs and academic entities in Europe.; to elaborate the new device for a specific application: the primary diagnosis of the different types of lung cancer. The project will then have a huge impact on health by contributing to the fight against cancer and the development of gender dimension in research.
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Approach and methodology: The objective of the project is to generalise this approach to combinations of immuno-assay measurements to deliver a clear diagnosis of the disease or monitoring information. The generalisation means first that the diagnosis should be accessible to various types of medical practices (medical doctor to hospitals) and thus a low cost, easy-to-use POC device should be developed. However, this should not be done to the detriment of quality and precision. A homogeneous technology known for high-level precision can therefore be a judicious choice. Generalisation also means that various pathologies could be addressed: typically from two to four or five immuno-assays. The project therefore aims to allow the simultaneous measurement of four to five immuno-assays on the POC device with one draw of patient sample (one droplet). Finally, generalisation means universality of the measurement technique and of the data reduction process. In this way, fluorescent measurement based on FRET (Fluorescence Resonance Energy Transfer) seems an excellent choice, since it may be extended in the future to other diagnostics such as DNA analysis, coagulation, microbiology, etc. Role of SMEs The SMEs will bring to the consortium the complementary and comprehensive expertise required by the other public researchers (CEZA brings competence in two complementary scientific and technical fields: immunoassay development and scientific instrumentation, which over the years CEZA managed efficiently to cross-fertilise, FDAB brings competence related to antibody development and characterisation (hybridoma technology, phage display, gene expression analysis, c-DNA immunisation, DNA-shuffling, affinity maturation) and competence in assay development, and EI brings experience in flash lamps, laser diodes, fluorescence spectrometry and a prototype nanosecond plate reader. Expected outcome: The project should attain the following challenging objectives: The development of a functional prototype of POC multi-parametric measurement for immunoassays, based on Homogeneous Time Resolved Fluorescence (HTRF), for which main characteristics are: it is equivalent to an A4 sheet of paper, costs less than EUR 2 000, works with a sample droplet deposited on a disposable reagent vessel containing dried reagents; it defines the panel of assays and how to combine them into a decision making software (to be developed). Potential applications: Multiparametric diagnostics in the following fields: cancer, prenatal diagnostics, sepsis, cardiac
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Scientific coordinator Emmanuel Bois Cezanne SAS 280 alle Graham Bell Parc Scientifique Georges Besse 30035 Nmes cedex 1, France E-mail: contact@cezanne.fr Partners Olle Nilsson Fujirebio Diagnostics (FDAB) Gothenburg, Sweden S. desmond Smith Edinburgh Instruments Livingston, Scotland, UK Philippe Pieri Centre National de la Recherche Scientifique (CNRS) Paris, France Hans-Gerd Lhmannsrben University of Potsdam Potsdam Golm, Germany Yves Caristan Commissariat a lEnergie Atomique (CEA) Grenoble, France Klaus-dieter Weltmann Institute of Low Temperature Plasma Physics Greifswald, Germany Rafael Molina Hospital Clinic Barcelona Barcelona, Spain Petra Stieber Institute for Clinical Chemistry University Hospital Munich-Grosshadern Munich, Germany
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development of new and cost-effective methods for non-invasive diagnosis of human pathogens
Contract No Project type EC contribution Starting date duration LSHB-CT-2006-037212 SME-Specific Targeted Research Project e 2 499 999 1 January 2007 36 months
DIAGNOSIS
Background and objectives: Infectious diseases continue to be a serious burden around the world, in both developing and industrialised countries. The main objective of DIAGNOSIS is to deal with the issue by developing a novel, easy to use, low-cost and mostly non-invasive biotechnological platform for infectious diseases detection. The challenging aim is to reach a short and efficient sample treatment, which can be implemented into existing and newly developed portable PCR laboratory for multiplex fluorescent pathogen detection. The concept will be proven on human critical pathogens but will be applicable also for animals, and plants pathogens. The concept will be applicable for samples taken from affected organisms, as well as from food/feed and environments (water, air, soil, etc.). The scientific and technological objectives will include the development of: 1) new principles of nucleic acids sorption/desorption; 2) innovative concentrating methods and materials for the enrichment of viruses, micro-organisms and nucleic acids; 3) the adaptation of the phosphoramidite chemistry to alternative fluorescence dyes in order to broaden the labelling assortment for multiplex PCR analyses a portable non-dependent on external power supply PCR laboratory; and 4) the exemplary demonstration of the whole technological chain on several groups of human pathogens, including the Mycobacterium complex, the periodontal pathogens, the causal
agents of population shifts within the intestinal microflora associated with inflammatory bowel diseases and fungal skin infections, completed by the diagnosis of main representatives of foodborne pathogens. Infectious diseases are a global concern. Of the annual 12 million deaths attributable to infectious diseases on the planet, 95% occur in the developing world, particularly in the most impoverished areas where individual and general hygiene standards remain very low and prevention policies are non-existent, poorly adapted or insufficiently funded. Conversely, economic and industrial development also accounts for the emergence of infections, such as food-borne pathogens that take advantage of the increasing industrialisation of the food chain; nosocomial infections in the increasingly complex hospital environment; and travel-related infections. In addition, the demographic trend towards an ageing population in Europe enhances the risk of increasing infection as elderly people are prone to invasive medical procedures and are, in general, more susceptible to infectious diseases. European science must play a major role in this fight to curb infectious diseases, particularly through the establishment of a stronger and more coherent surveillance and control system, and through a substantially increased investment in research to underpin this endeavour.
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Only with this investment will Europe be able to manage infectious diseases within its own boundaries. Furthermore, Europe will have the capacity to help prevent the emergence and spread of infections prevailing elsewhere in the world and to pursue its historical tradition of providing assistance to the poorest countries. The challenging aim is to reach a short and efficient sample preparation followed by highly sensitive, accurate, cost-efficient multiplex diagnosis implemented i.a. into a portable PCR fluorescent laboratory. The concept will be proven on human critical pathogens but will be applicable also for animals, plants and environmental, and food samples. Role of SMEs DIAGNOSIS attempts to enhance the competitiveness of Europes biotechnology industry by the development of fast and reliable nucleic acids separation and purification, as well as the creation of new methods for separation from inhibitor enriched samples and new instruments and kits for fast and cost-effective detection of critical human pathogens. Most of the partners are industrial partners, thus taking the outcome of the project directly to the European biotechnology industry: The consortium consists of 73% SMEs, all of which are involved in innovative technologies and research. Of the overall budget, 67% is allocated to SMEs. The main idea is to promote their technologies and develop new products by enhancing their business plans. The project management is fully performed by two SMEs: one an expert in technological work and the other in administrative management and coordination of EU-funded projects. DIAGNOSIS is allocating one Work Package to exploitation and dissemination by its SMEs partners, including internal
workshops and staff exchange between the relevant entities. A detailed plan of staff exchange will be implemented during the project. The consortium includes three partners from INCO countries that are highly recommended by the EU; one of them is an SME (DNAT) and the other two are research institutes which will provide essential know-how for the project.
Expected outcome: DIAGNOSIS will realise new technologies and products as listed below: Novel technologies New principles for NA separation and purification New sorbent methods and materials Protocols for sample preparation Protocols for samples target concentration New products Kits for ultra-rapid separation and purification of DNA and RNA Kits for separation and purification of NA from difficult (inhibitor rich) samples Kits for separation of different classes of NA Kits for non-cultural enrichment of micro-organisms
Kits for diagnostic Chemistry for oligoassays of critical nucleotides synthesis pathogens Multiplex PCR Protocols for diagnostic assays Prototype instrument for fluorescent detection Portable PCR laboratory
Protocols for POC Kits for sample (point of care) sample preparation outside preparation the laboratory
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Potential applications: The application Work Package will focus, but not limit, its activities on the following groups of pathogens and critical sample sources for multiplex PCR systems with fluorescence detection, thus approving and demonstrating the broad and beneficial universal applicability of the technological development in the technological Work Packages: mycobacterium complex: in sputum; difficult culturable fastidious periodontal pathogens in gingival crevicular fluid; predominant food-borne human pathogens; gut flora pathogens in stool samples for analyses of intestinal microbiota associated with inflammatory bowel disease; fungal pathogens in hair, skin and nail samples. Scientific coordinator Robert-Matthias Leiser Agrobiogen GmbH Thalmannsdorf 25 Larezhausen 86567 Hilgertshausen, Germany E-mail: matthias.leiser@agrobiogen.de Partners denis Rebrikov DNA-Technology Moscow, Russia Claus-detlev Bauermeister Labor Dr. Bauermeister & Co Moers, Germany Vendula Pachmanova Generi Biotech s.r.o. Hradec Kralove, Czech Republic Camilla Giammarini DIATHEVA Srl Fano, Marche, Italy Martin Gehri PreentTec AG Fribourg, Switzerland Ronald arthur Bosch Hilbrands Laboratorium BV Wijster, Netherlands Sergey Zavriev M.M. Shemyakin-Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of RAS Moscow, Russia Hamlet Balayan Institute of Fine Organic Chemistry of NAS Yerevan, Armenia Ulf Goebel Charit Universittsmedizin Berlin Institut fr Mikrobiologie und Hygiene Berlin, Germany Pnina dan OSM-DAN Ltd Rehovot, Israel
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USDEP
Capture and enrichment of emerging pathogens for multiple and ultra-sensitive diagnostic
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037560 SME-Specific Targeted Research Project e 2 004 952 1 November 2006 36 months www.usdep.eu
In the 1970s, the World Health Organization (WHO) proclaimed that eradication of smallpox should be attempted. This goal was successfully achieved in 1979. Nonetheless, presently there is a general consensus that the list of newly emerging or re-emerging pathogens is continuously growing. Indeed, during the last decades, patho-
gens such as Marburg, Ebola, Hepatitis-C, Hantavirus, HIV and more recently, SARS coronavirus, have emerged. Furthermore, the apparent risk of a new influenza pandemic again highlights the global threat of infectious diseases. In addition, the possibility of bioterrorist attacks using highly pathogenic viruses and bacteria cannot be ig-
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nored. As a consequence, the current requirements for novel, highly sensitive and specific diagnostics technologies have increased. A major obstacle for the detection of pathogens in clinical or environmental samples are false negative results, e.g. for HCV occult infections. This is mainly due to the lack of a rapid and reliable pathogen concentration methodology, and the inability of most of the currently used technologies to eliminate or neutralise interfering molecules natural inhibitors present in most complex samples. The aim of this research programme is to exploit the non-self recognition and binding properties of human apolipoprotein H (ApoH) for the development of novel tools to isolate pathogens from complex biological mixtures. ApoH binds pathogens enabling their capture and concentration from different biological samples. Magnetic beads coated with ApoH protein can be efficiently used as a pre-treatment step to greatly improve the detection threshold, thereby increasing the sensitivity for diagnosis of emerging pathogens, regardless of the molecular or immunological techniques used in the final diagnostic step. The project will focus on increasing the sensitivity and specificity of the currently available detection methods used for pathogenic bacteria and viruses, and on the development of novel techniques for pathogen detection from clinical and environmental samples including those that could be used in bioterrorism.
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While the publicly funded partners as well as the Robert Koch Institut (RKI), the Institut de Recherches pour le Dveloppement (IRD), and the privately funded Pontifica Universidad Catolica de Chile working in virology and public health will incorporate ApoH technology into their panel of regular techniques for pathogen detection, the SME will develop and standardise novel technologies for rapid, multiple and ultra-sensitive pathogen diagnosis such as mini-array systems. ApoH-Technologies develop ApoH-coated supports for diagnostic purposes, GenExpress specialises in the development and optimisation of molecular biology assays, the Institut fr Siliziumtechnologie (ISIT) specialises in the development and production of microelectronic components and will supply the electronic biochips, eBiochip Systems is focused on manufacturing of technology for electronic biochip applications, SKULD-TECH is developing a mini array system for virus detection and IMMUNOCLIN provides strategic direction as well as management and laboratory services for clinical development and preclinical contract research.
Scientific coordinator Heinz Ellerbrok Robert Koch-Institut Centre for Biological Safety Nordufer 20 133353 Berlin, Germany E-mail: EllerbrokH@rki.de Partners dorothy Bray ImmunoClin Ltd London, UK Elias Stefas ApoH-Technologies Montpellier, France Francisco Veas University of Montpellier 1 Faculty of Pharmacy Montpellier Cedex 5, France Marcelo Lopez Lastra Pontificia Universidad Catlica de Chile Santiago, Chile Roland Lauster GenExpress Gesellschaft fr Proteindesign mbH Berlin, Germany Rainer Hintsche Fraunhofer Institut fr Silizium Technologie Itzehoe, Germany Ralf Wrl eBiochip Systems GmbH Itzehoe, Germany didier Ritter and Stamatis Varsamos Skuld-Tech Montpellier, France
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NEMO
Nano based capsule-endoscopy with molecular imaging and optical biopsy
Contract No Project type EC contribution Starting date duration Website LHSB-CT-2006-037362 Specific Targeted Research Project e 2 832 020 1 November 2006 36 months www.nemo-strep.org
Background and objectives: Gastrointestinal (GI) malignancy, especially colorectal cancer, makes a significant impact on the quality of life and well-being of European citizens, and has significant economic consequences. The World Health Organization reported that in 2000, 940 000 of the 10 million cancer deaths were colorectal in origin, 870 000 were derived from the stomach, and 410 000 were of oesophageal origin. The ageing of the European population means that the death rate will continue to rise. GI cancer in general, and more specifically colorectal cancer, develops slowly and usually takes years before expressing symptoms. It is most curable during its early stage. Consequently, earlier detection of cancer using screening methods is likely to be the most practical way of addressing the epidemic of gastrointestinal cancers. Today, gastrointestinal cancers are detected mainly by gastroscopy or colonoscopy, followed by biopsy. There is good evidence that such screening can locate early cancers and that lives can be saved if they are discovered and removed. Many patients, however, are reluctant to have screening gastroscopies and colonoscopies because of the discomfort of the procedures. Women are especially reluctant to undergo screening colonoscopy. The acceptability of capsule endoscopy, which is pain-free, requires no sedation and does not
necessarily entail a hospital visit, is attractive for patients. It is likely that they would find such a screening method much more acceptable than screening colonoscopy, for example. It would be very helpful if capsule endoscopic video imaging could be combined with more sensitive and specific methods for the detection of early cancer, to avoid the need for biopsy. The aim of the NEMO project is to develop a new and advanced cancer screening method, which is user-friendly enough to significantly increase compliance, simplify the diagnosis procedure and enhance the sensitivity and specificity of early detection. The focus is on advanced non-invasive method for cancer screening of the GI tract, that on the one hand, will be user-friendly enough to increase the compliance of the population, and on the other hand, will raise the sensitivity and capability of early detection of colorectal cancer. Approach and methodology: The concept of the proposed medical device is to combine capsule endoscopy with molecular recognition that highlights cancerous and precancerous lesions in the GI tract, in a way that revolutionises the accuracy and ease of screening for this major cause of death. The system unites the verity of technological platforms: immunoassay technology is used for targeting the disordered tissue or body fluid; nanotechnology is used for marking and optical tagging; advanced in vivo
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DIAGNOSTICS
capsule endoscopy, based on spectral technology, is used to detect the marked disorder; and manoeuvring and navigation is based on magnetic fields and forces technologies. Two types of miniaturisation are required to enable the assembly of the new imaging system, coils, wireless communication and batteries into a capsule that will remain a reasonable size: mechanical (increasing space) and electrical (reducing power requirement). Technologies like MEMs switches and improved electronic packaging, based on array of chips using flip-chip bonding, are essential. The ability of light to penetrate the tissue depends on its wavelength. Blue light has limited penetration capacity, while NIR can reach deep tissue layers. By comparing the light scattered in a short wavelength (providing information from the surface) to light scattered in a long wavelength (providing deep tissue information), optical biopsy can be achieved. A new medical diagnostic tool may be set up by fusing visual imaging and analytical data based on molecular recognition technology. The NEMO partners will explore the use of specific optical filters used in conjunction with light
emitting diodes (LEDs) in order to make a miniature narrow band imaging device which can be contained within a capsule endoscope. Narrow band imaging reduces the surface reflection of broad band illumination, and can allow small or flat precancerous lesions to be seen clearly, in comparison with conventional illumination. The combination of conventional and narrow band imaging may provide an overview of the gastrointestinal tract, indicating one of many sites of interest. It could be alternated with conventional imaging. Expected outcome: The project partners expect that the new capsule endoscopy screening method will offer improved clinical solutions for cancer screening: wireless capsule endoscopy is painless; does not need to be administered in hospital; does not require a team of nurses to clean the devices since it is disposable; and does not require great skill to perform (although skill in interpretation of the images is needed). It is much preferred by patients to gastroscopy or colonoscopy. The new device will be free to manoeuvre in stomach. By combining this capability with molecular recognition, which highlights cancerous and precancerous lesions in the GI tract, would revolutionise the accuracy and ease of screening for gastric cancer. Moreover, by combining the capability to analyse secretions with localisation, the solution offers pancreatic and liver cancer detection as a by-product of stomach screening. The partners hope to develop a combination of miniature spectral and Magnetic Resonance nanotechnology so as to create virtual biopsy methods, and incorporate these within the capsule endoscope. They also anticipate that the new autonomous NEMO capsule will be able to move backwards and forwards in the gastrointestinal tract, for example, in order to re-examine a suspicious lesion.
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Coordinator Elisha Rabinovitz Given Imaging Ltd 2 Hacarmel Street 20692 Yoqneam, Israel E-mail: elisha.rabinovitz@givenimaging.com Partners Mikael Svensson Zarlink Semiconductor AB Jarfalla, Sweden avraham Rubinstein The Hebrew University of Jerusalem Jerusalem, Israel Meike Reimann-Zawadzki Fraunhofer-Gesellschaft zur Foerderung der Angewandten Forschung e.V. (FhG) Institute for Biomedical Engineering (IBMT) Sulzbach, Germany alberto Lui Fondazione Bruno Kessler (Istituto Trentino di Cultura) Trento, Italy Jutta Keller Israelitisches Krankenhaus Hamburg, Germany Paul Swain Imperial College of Science, Technology and Medicine (Imperial College London) London, UK Heinz Jochim List Indivumed GmbH Hamburg, Germany Emil Katz Novamed Ltd Jerusalem, Israel Elie Berdugo Ernst & Young (Israel) Ltd Tel Aviv, Israel
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FLUOROMAG
Multiparameter sensing for high sensitivity diagnostics using fluorescent and magnetic nanoparticles
LHSB-CT-2006-037465 Specific Targeted Research Project e 2 552 300 1 November 2006 36 months www.mpibpc.gwdg.de/groups/jovin/ index.php/Main/Fluoromag
Background and objectives: The objective of the FLUOROMAG project is to produce noble metal nanoclusters or nanodots (NDs), and core-shell (CS) nanoparticles by a variety of methods that will ensure uniform size distributions and the transfer of this technology to partner NANOGAP-sub-nm-powders SA. NANOGAP will then scale up the synthesis of these nanoparticles for commercial production and supply the consortium with nanoparticles (NPs) for the characterisation of their extinction, fluorescent and magnetic properties, and the further development of diagnostic tests. FLUOROMAG, as a STREP (Specific Targeted Research Project), will devise conjugation strategies to couple biomolecules to noble metal NDs and commercially available quantum dots (QDs) to produce probes that can specifically target macromolecules, such as proteins and DNA/ RNA in vitro, and in cells and tissues. The consortium will take advantage of ND electrochemical synthesis to introduce specific molecules in the shells that permit efficient derivatisation and coupling to biomolecules. The consortium is also targeting the development of multiparametric diagnostic assays, using combinations of bioconjugated QDs and noble metal NDs as novel, fluorescent and extinction probes. The goal is to achieve high sensitivity (down to single virus/biomolecule detection) in molecular and cellular recognition. New assays
are being proposed that will monitor several antigens in multiplexed kinetic and end-point determinations. Another objective is development of a commercial, low-cost programmable array microscope (PAM) module for wide-field microscopes, that utilises a spatial light modulator to achieve highspeed sectioning and simultaneous measurement of multiple fluorescence modalities as a detection system for single and multiplexed diagnostic assays, using nanoparticles developed within the project. The research consortium will test and improve the capabilities of this instrument in collaboration with the partner, Cairn Research Ltd, so as to bring it to market for both research and clinical laboratories, by the end of the project. Approach and methodology: Controlled electrochemical as well as microemulsion techniques for NP synthesis are being pursued. New methods for separation and characterisation of the nanoparticles are also under development. The NP technology is being transferred to NANOGAP, which is increasing the syntheses of these nanoparticles for commercial production proportionally, as well as supplying the consortium with NPs. AFM, STM, X-RAY, SQUID, Raman, fluorescence and mass spectrometry measurement tech-
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niques are being used for the characterisation of NPs. Prospective candidate material is being directed into bioconjugation for diagnosis and fluorescence microscopic studies. An alpha version of PAM is undergoing rigorous testing and development at Max Planck Institute (MPI) for Biophysical Chemistry, Germany, in collaboration with Cairn Research Ltd. Two beta units are under construction at Cairn Research Ltd for the Biophysical Engineering Department at University of Twente, Netherlands, and for the Analytical Chemistry Department of Nottingham Trent University, UK. Expected outcome: The consortium will have identified a range of well-characterised NDs and CSs with interesting properties and potential that can be produced commercially for the research and diagnostic market. New assays with high sensitivity for diagnosis using unique nanoparticle material will be developed; some of these will use the new PAM platform. PAM units will enter the market for both the research and medical communities at the later stages of the FLUOROMAG project. This powerful, new high-speed fluorescence sectioning microscope will have capabilities not yet combined in other microscope systems.
(B) X-ray diffraction of large clusters.
Main findings: A market analysis for the nanoparticles produced by NANOGAP is to be finalised in mid-2007. The alpha version of the PAM at the MPIBPC has been improved and two beta units for Twente and Nottingham Trent Universities are under development at Cairn Research Ltd, and will be delivered to the partners in the very near future. Characterisations of nanoparticle syntheses made by the University of Santiago de Compostela and NANOGAP are ongoing at the MPIBPC and Twente. Scientists from all 3 universities met in February 2007 to conduct tests jointly and discuss further experiments. Scientists from Nottingham Trent University have visited the University of Santiago to set up toxicity studies on the nanoparticles.
Major publications: Hagen, G., Lidke, K., Rieger, ., Caarls, W., ArndtJovin, D., Jovin, T., Dynamics of membrane receptors: single molecule tracking of quantum
(A) Transmission electron micrograph image of large Au clusters. The size of clusters is 2.1 0.5 nm.
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dot liganded epidermal growth factor, In Single Molecule Dynamics, 2007, in press, Y. Ishii and T. Yanagida, editors. Hagen, G., Caarls, W., Thomas, M., Hill, A., Lidke, K., Rieger, B., Fritsch, C., van Geest, B., Jovin, T., Arndt-Jovin, D., Biological applications of an LCosbased Programmable Array Microscope (PAM), Proc. SPIE, 2007, 6441:64410S1-12.
Coordinator: donna J. arndt-Jovin Max Planck Institute for Biophysical Chemistry Department of Molecular Biology 37070 Gttingen, Germany E-mail: djovin@gwdg.de Partners Vinod Subramaniam University of Twente Department of Biophysical Engineering Enschede, Netherlands M. arturo Lpez-Quintela University of Santiago de Compostela Faculty of Chemistry Department of Physical chemistry Santiago de Compostela, Spain Quentin S. Hanley Nottingham Trent University School of Biomedical and Natural Sciences Nottingham, UK Martin Thomas Cairn Research Ltd Faversham, UK Tatiana Lpez de Rio NANOGAP-subnm-powders SA Santiago de Compostela, Spain
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BONSAI
Bio-imaging with smart functional nanoparticles
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037639 Specific Targeted Research Project e 2 909 005 1 November 2006 36 months www.bonsai-project.eu
Background and objectives: The overall objective of the BONSAI project is the development of ultrasensitive bio-imaging techniques based on novel multifunctional nanoparticles (NPs) with tailored optical and magnetic properties for visualising complex cellular structures (tissues and organs), receptors, tumour cells and masses. True innovation rests on the capability to combine the preparation of ad-hoc nanoparticles, having different properties and functions, with the development of advanced bio-imaging techniques, in the same project. The expected improvements of labelling cells and cellular structures with tailored nanoparticles are sensitivity, speed and specificity in the visualisation of biological systems. The improved resolution, sensitivity and versatility of diagnostics based on the NPs developed by BONSAI is likely to have a strong impact on biomedical research, by allowing the unravelling of some aspects of the cellular response to pathological perturbation and tissue repair, including signal transduction and cell-cell interaction. The success in reaching the project objectives would contribute both to a better understanding of the molecular mechanisms of cellular processes, and to achieving higher sensitivity in early detection of tumour cells and lesions.
Approach and methodology: Currently, cells are visualised through fluorophores, such as organic dyes. These have several drawbacks, including their tendency to blink (that limits their efficiency), photo-oxidation (that limits the detection time) and the necessity of different wavelengths to activate each dye (that slows data acquisition). Semiconductor nanocrystals (quantum dots) were recently introduced for biolabelling, since one wavelength can make them glow in a rainbow of colours depending on their size. The most widely used quantum dots, however, are cytotoxic. BONSAIs goal is the preparation by laser pyrolysis (Fig. 1) of sizeable quantities of light-emitting Si (Fig. 2) and Si-based NPs that have a broad-band pumpability, size-dependent optical emission (Fig. 3), a reduced tendency to photo-bleach, and are not cytotoxic as compared with largely investigated, toxic CdSe quantum dots. Si-NPs, with different colours of fluorescence, can be excited with one single wavelength, which can be selected to minimise the cell autofluorescence that usually masks signals from labelled molecules. This is an added advantage in high-resolution bio-imaging. The applications in biology and medical diagnosis (Fig. 4) require that the NPs do not interfere with biological processes or poison the cells. Thus, the second task of BONSAI is the development of stable colloidal solutions containing non-cytotoxic, colloidally stable Si-based NPs, which are properly
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Fig. 1: Schematic of laser pyrolysis set-up (courtesy of partner CEA).
Fig. 2: Si nanoparticles prepared at ENEA by laser pyrolysis (HREM-TEM image: care of partner UNIPD)
Fig. 3: Photoluminescence (PL) emission from size-selected Si nanoparticles (NPs) under illumination with UV lamp. The arrow indicates NP size variation from 8 nm to 2.5 nm. (Courtesy of partner MPI for Astronomy).
functionalised on the surface in order to improve and tune their optical properties and increase their selectivity in specific biological targets. Imaging can also be generated by exposure of cells or tissues to ultrasound and by measuring the resulting signal with the help of contrast agents, as in MRI (Magnetic Resonance Imaging). MRI shows excellent spatial resolution, but as its sensitivity is relatively low, MRI requires amplification mechanisms for its use in molecular imaging. To this purpose, the help of suitable contrast agents is required. The objective of BONSAI is the development of stable colloids of nontoxic, non-immunogenic NPs with: high magnetisation, so that NP can be moved in the blood and accumulated in the target organ; particle size that is small enough to remain in circulation after injection and to pass
through the capillary system of organs and tissues with little vessel embolism; narrow size distribution for differential uptake of various tissues (Fig. 5,6).
Nanoparticle coating and hydrodynamic size are key features for driving the pharmacokinetics of NPs and their access to deep compartments of the body. A very ambitious and risky objective of the project is to combine optical (for optical detection and imaging) and magnetic (for manipulation and/or MRI) properties in the same NP that will acquire the capability to be moved, followed and eventually energised when introduced in specific biological tissues. Prior to using nanoparticles in biological and medical diagnostics, the partners will verify that they have no adverse effect on cells health or development. Non-toxic, luminescent nanoparticles will then be used to develop optical bioimaging techniques that aim for the following:
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better understanding of how the genome instructs and orchestrates the function of cell, organs and organisms; whole-cell labelling for cell or pathogen detection, cell tracking and cell lineage studies; optical imaging of in vitro and in vivo tumour cells for early cancer diagnostics.
Expected outcome:
Fig. 4: Bio-applications of Diffuse Reflectance and Fluorescence Spectroscopy of nanoparticles (courtesy of GPI-Natural Sciences Center).
Fig. 5: HREM-TEM image of Fe2O3 magnetic nanoparticles prepared by laser pyrolysis at CSIC. Fig. 6: HREM-TEM image of siloxane polymer-iron/ iron oxide shell-core nanoparticles prepared at the National Institute for Lasers, Plasma and Radiation Physics Institute in Bucharest, Romania.
The primary aim of the BONSAI project is to achieve a real industrial breakthrough, based on scientific and technological excellence through interdisciplinary work. In this respect, BONSAI has joined together 11 participants from 4 EU Member States (Germany, Spain France and Italy) and a Russian partner with complementary, well-documented and high-profile expertise in the following: material science (synthesis of new nanoparticles through bottom-up strategies, like laser synthesis from gas-phase precursors and laser ablation in liquids); nanotechnology (exploration and exploitation of novel optical and magnetic properties that appear only at nanoscale and are truly size dependent); colloidal chemistry (bio-compatibilisation of the colloidal nanoparticles by building up a passive interface between the artificial material and the physiological fluids); biotechnology (advanced imaging technologies in biological samples, development of magnetic NPs as contrast agents in Magnetic Resonance); biomedicine (toxicity studies of NPs, uptake of NPs by specific tumour models, NPs distribution to specific organs in relation to administration routes). Specific magnetic Fe-based nanoparticles will be prepared for the project at the National Institute for Lasers, Plasma and Radiation Physics Institute in Bucharest, Romania. Moreover, the European dimension of the project will contribute to the
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strengthening of the European Research Area. The expected breakthrough is related mainly to the development of advanced bio-imaging diagnostics made possible by the availability of adhoc nanoparticles, produced within the project and having superior properties with respect to commercially available nanoparticles. After a careful testing process, which is needed for pharmaceutical products, these materials will make up a source of long-term innovation of contrast agents for medical imaging, especially in MRI. Alternative long-term implementation and exploitation approaches of the projects diagnostics in early tumoural cell and tissues detection will be considered in the course of the project, with the cooperation of hospitals and oncological centres. Coordinator Elisabetta Borsella ENEA (Ente Nazionale per le Nuove Tecnologie, lEnergia e lAmbiente) Via Enrico Fermi 45 Frascati (Roma), Italy E-mail: borsella@frascati.enea.it Partners Nathalie Herlin-Boime CEA Commissariat lEnergie Atomique-Direction des Sciences de la Matire Gif-sur-Yvette, France Sabino Veintemilas Verdaguer Consejo Superior de Investigaciones Cientificas Instituto de Ciencia de Materiales Madrid, Spain Miguel angel Garcia Universidad Complutense Madrid Instituto de Magnetismo Aplicado Madrid, Spain
Giovanni Mattei Universit di Padova Dipartimento di Fisica Padova, Italy dayang Wang Max Planck Gesellschaft Max Planck Institute of Colloids and Interfaces Potsdam, Germany Friedrich Huisken Max Planck Institute for Astronomy Heidelberg, Germany Giuseppe Miserocchi Universit di Milano-Bicocca Dipartimento di Medicina Sperimentale, Ambientale e Biotecnologie Mediche Milan, Italy Jean Marc Idee GUERBET Aulnay-sous-Bois, France Vladimir Pustovoy General Physics Institute Natural Sciences Centre Moscow, Russia Klaus Palme University of Freiburg Center for Applied Biosciences Inst. Biology II Freiburg, Germany Maria Rosa GaSCO NANOVEC-NANOVECTOR srl Turin, Italy Rainer UHL TILL-TILL Photonics GmbH Graefelfing, Germany
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Moving sensitive immunoassays from slow and expensive to fast and affordable nanoparticle-based methods
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037245 SME-Specific Targeted Research Project e 1 433 600 1 January 2007 36 months www.nanosense.eu
NanoSense
Background and objectives: There is a need for high sensitivity non-separation immunoassay technology for general clinical chemistry instrument platforms, in particular for large protein disease markers of low concentration, such as NT-proBNP and PSA, and a long list of other plasma proteins, protein hormones and specific antibodies. Such new technology will significantly change the diagnostic industry and healthcare providers, in a step towards greater efficacy. Traditionally, immunoassays have been separation based, meaning that the analyte of interest goes through several steps of antibody binding, washing and separation before final detection. This type of assay requires high use of consumables, which is expensive as well as time-consuming due to all the steps involved. While no separation steps are involved and the use of consumables is limited in a non-separation assay, making the assay less expensive and with a much shorter assay time is key. A non-separation assay will typically be run on a clinical chemistry platform intended for high-throughput of analytes, making homogeneous non-separation immunoassays a high potential market-growth opportunity. The aim of the NanoSense project is to move immunoassays from slow and expensive methods to fast, high-throughput super-sensitive nanoparticle-based methods, to demonstrate that it is working within the specifications, and to generate intellectual property for such technology.
Role of SMEs There are five participants in this project; three of them are small and medium-sized enterprises (SMEs). Coordinator of the project is Dalen Diagnostics AS, a Norway-based SME. In addition to the coordination, the company has the necessary expertise for improved signal generation in homogeneous nanoparticle assays. The other two SMEs are 77 Elektronika Kft in Budapest, Hungary (i.e. a specialist in the field of medical electronics and manufacturer of blood glucose meters, urine analysers and rapid test readers) and Getica AB (i.e. a specialised Swedish small biotech company concentrating on bioorganic coupling chemistries and bioprocessing of protein conjugates and nanoparticles). The consortium also includes a large industrial company, Merck Chimie SAS, which is Europes largest producer of nanoparticles, and an academic participant, the Groningen University Medical Center, providing a reference laboratory for measurements in large epidemiological studies. Expected outcome: To achieve the goal of this project, the consortium will use specific methods and techniques so as to optimise each component in the assay. When technology, as described above, was developed for small molecule markers 15 years ago, a big change in the diagnostic industry was seen and two SMEs grew into big industrial corporations. The NanoSense partners foresee that similar ef-
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fects will emerge when such technology is developed for large molecule markers as well. Potential applications: New assay high sensitivity, high-speed assay products on automated clinical chemistry platforms for use in laboratories throughout the healthcare system, for higher throughput and improved cost-effectiveness.
Coordinator Guri Skjeltorp Dalen Diagnostics AS Kolsroedveien 120 1599 Moss, Norway E-mail: email@dalendiagnostics.no Partners Erling Sundrehagen and Ingrid Hulthn Getica AB Tsse, Sweden Richard Vidal and Ccile Genies Merck Chimie SAS Estapor FontenaySousBois, France Uzonka Farkas and Pter Jakus 77 Elektronika Muszeripari Kft Budapest, Hungary dick de Zeeuw and Stephan J. L. Bakker Groningen University Medical Center Department of Clinical Pharmacology Groningen, Netherlands
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Predictive diagnostics for diabetic nephropathy novel nanotechnology based test platforms
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037681 SME-Specific Targeted Research Project e 2 977 400 1 december 2006 36 months www.diana-eu.fi
DiaNa
Background and objectives: The DiaNa project uses the latest knowledge of the pathophysiology of diabetic nephropathy and newly identified urinary markers to develop predictive diagnostic tests to follow disease progression. Through metabolomic approaches, DiaNa aims to find additional markers from diabetic urine. In parallel, two separate approaches will be used to develop diagnostic tests: one will be based on nanobead technology and the other on a multiplexing platform allowing a combination of several parameters in a single test. This will translate into early identification of patients at high risk of rapid loss of kidney function. After validation with clinical material, subsequent steps will include a transfer of the test into patient use by a small and medium-sized enterprise (SME). This approach directly aimed at developing a clinical urinary test will be supported by extensive basic research on the mechanisms/ biomarkers of diabetic nephropathy. DiaNa combines the expertise accumulated from the previous FP5 EU project, Mechanisms of Proteinuria (QLG1-2000-00691) and the FP6 project, ADDNET (LSHB-CT-2003-503364). Diabetic nephropathy is the main cause of endstage renal failure in the western world. The World Health Organization (WHO) estimates that presently there are over 200 million people with diabetes worldwide. The prevalence is constantly rising and is estimated to reach over 300 million by the year 2025. Overall, up to 40%
of diabetic patients will develop debilitating kidney complications. Diabetic nephropathy is becoming not only a severe health problem for individual patients but a major economic burden of all societies as well. With DiaNa, the partners aim to achieve a better understanding of the pathophysiologic mechanisms underlying the development of diabetic nephropathy a major complication shared by both type I and type II diabetes. Role of SMEs The SMEs involved will (1) utilise their expertise to pull out antibodies specific for the molecules involved in the pathogenetic sequelae of diabetic nephropathy and used for diagnostics (Philogen S.P.A, Siena, Italy); (2) combine the antibodies produced to novel test platforms, including the appropriate packaging into existing and to-bedeveloped concepts (Future Diagnostics B.V., Wijchen, The Netherlands); (3) develop and screen for appropriateness of the platforms developed for daily routines with incoming patient samples (United Laboratories Ltd, Helsinki, Finland); and (4) develop novel magnetosensors for detecting low amounts of target molecules in patients urine (Philips Electronics Netherlands BV, The Netherlands). In addition, market analysis and surveys, IPR strategies, competitor analysis will be provided by an additional party as subcontractor of Partner University of Helsinki.
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Expected outcome: The ultimate goal of DiaNa is to develop novel diagnostic tests based on the best molecular understanding reflections in the urine to reveal the early diabetic damage during the premicroalbuminuric stage of the disease. Potential applications: The applications will include novel magnetosensor-based sensors for detection of diabetes-diabetic kidney damage.
Coordinator Harry Holthofer Haartman Institute Haartmaninkatu 3 FI-00014 University of Helsinki, Finland. E-mail: harry.holthofer@helsinki.fi and National Centre for Sensor Research/ BioAnalytical Sciences Dublin City University Dublin 9, Ireland E-mail: harry.holthofer@dcu.ie Partners Per-Henrik Groop Samfundet Folkhlsan Helsinki, Finland Rob van der Heijden Universiteit Leiden Leiden, Netherlands dario Neri Swiss Federal Institute of Technology Zurich, Switzerland Reinerio Gonzales Philogen SpA Siena, Italy Menno Prins Philips Electronics Nederland BV Eindhoven, Netherlands Mike Martens Future Diagnostics BV Wijchen, Netherlands Jussi Vilpo United Laboratories Ltd Helsinki, Finland
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Multiparametric detection of bio-molecule conjugated nanoparticles for the diagnostic investigation of mycobacterial infections of humans and animals
Contract No Project type EC contribution Starting date duration LSHB-CT-2006-036812 Specific Targeted Research Project e 2 194 020 1 January 2007 36 months
NANOMYC
Background and objectives: The WHO reports that tuberculosis causes millions of deaths or disabilities each year, especially in poorer areas of the planet. The problem is also exacerbated by the AIDS epidemic that increases disease incidence in developed countries. However, in addition to tuberculosis, exposure to mycobacteria has also been linked to the pathogenesis of sarcoidosis and Crohns disease that affect millions of people in Europe alone. Diagnostic investigation of mycobacterial infections is hampered by the difficulty to detect in a specific manner low populations of mycobacteria or the immunology markers associated with the infections they cause. The NANOMYC project aims to develop a highly sensitive and specific quantifiable detection system for molecular and immunology diagnostic markers associated with infection caused by M. tuberculosis complex (human and animal tuberculosis, implicated in sarcoidosis) and M. paratuberculosis (animal paratuberculosis, implicated in Crohns disease). To meet this goal, the consortium will combine nanotechnology and molecular biology incorporating the recent advances in the sequencing of mycobacterial genomes to routine diagnostics. NANOMYC will develop a set of functionalised quantum dots (QDs) with different emission characteristics that will allow a sensitive, differential and quantifiable detection of mycobacte-
rial-specific oligonucleotides and polypeptides in solid and liquid clinical samples. The consortium will focus on Mycobacterium tuberculosis (MTB) complex (the cause of human and animal tuberculosis, and possibly a causative agent of sarcoidosis) and Mycobacterium avium subsp. paratuberculosis (MAP), (associated with Crohns disease of man and causative agent of paratuberculosis of ruminants). The project partners will also develop the assay for: in-field diagnostics using portable devices for evaluation of liquid samples; manual and automated evaluation of solid samples using fluorescence resonance energy transfer (FRET). Approach and methodology: Currently, in vitro diagnostic investigation of mycobacterial infection relies on two kinds of tests: those that aim to detect the pathogen and those that target specific antibody. Of the former, culture and microscopic examination of specimens by auramine-rhodamine or Ziehl-Neelsen staining are among those more traditionally used, but have been largely substituted by molecular techniques that can allow detection and differentiation of mycobacterial pathogens in a clinical sample, and in a rapid (culture may require up to 8 months in the case of MAP), highly sensitive and specific manner. Numerous assays of the polymerase chain reaction (PCR) have been
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proposed for this. More recently, Real Time PCR (ReT-PCR) has further increased sensitivity (lower minimum detection limit) and has allowed a quantifiable assessment of the result. However, the latter method has not yet been incorporated in practice; not only does it require very detailed calibration but it also requires costly equipment. All the techniques mentioned above share the same disadvantage of low negative predictive value (NPV), i.e. although positive results mean definite presence of mycobacteria in the sample, negative results require further confirmation. The problem of low NPV of these tests is solved by assays that focus on the detection of substances produced as a consequence of infection. However, tests in this category (e.g. ELISA, CFT, AGIT, g-inteferone, tuberculin testing, etc) often fail to produce specific results due to cross reactions with other mycobacterial species. Furthermore, immuno-compromised patients, especially those infected with HIV representing a very considerable risk-population for tuberculosis, often produce false negative results due to the impaired ability of their immune system to respond to infections. It becomes evident that a method that will combine the advantages of the diagnostic tests mentioned above will greatly improve diagnostic investigation of mycobacterial infections. NANOMYC, however, will go a step further since it is being developed to allow, in addition to the above, the assessment of mycobacterial viability through detection of proteins produced exclusively by viable cells (this assessment currently relies on reverse transcriptase PCR or NASBA, both of which have failed to be used routinely mainly because of their low reliability, i.e. positive results do not always signify microbial death and negative results are often false due to RNA degradation). The consortium will develop QDs with different oproelectrochemical characteristics. To this purpose, the approach will involve construction of QDs (CdSe, ZnS, Au) that have already proven
their efficiency for conjugation with biomolecules and detection of specific target regions, and new ones that can revolutionise the diagnosis of mycobacterial infections (InN, and GaN) since they can be combined with other semiconductor-based QDs for direct and indirect immobilisation onto biomolecules. These substrates are unique, in that they have enhanced stability, selectivity, but most of all surface characteristics that are dependent on the surrounding chemical environment. Conjugation of these biomolecules with the QDs will be performed. Know-how for the completion of this stage is already well established and thus the only foreseeable risk is a delay in timely completion. This is associated with the fact that QDs are expected to show a shift in their optical characteristics after their conjugation with the biomolecules. Therefore, the final products of this stage will have to be described in detail with reference to their electrical-physico-chemical characteristics. The QD-conjugates that will be constructed will be used for the development of NANOMYC for application with liquid samples (in-field diagnosis) and with solid samples (manually and automated). For the former, QDs will become soluble by coating with siloxane shells and specific capping macromolecules; for the latter, GaN and InN will be used as matrixes for biomolecule and polymer immobilisation for the production of reversible sensor arrays through Fluorescent Resonance Energy Transfer combined with Field Effect Transistors. Expected outcome: The consortium expects to improve quality of life through better and more effective diagnosis, prevention and treatment of mycobacterial infections with reference too to children who are by definition more susceptible. Considering indications that early exposure to mycobacteria may lead not only to tuberculosis but also to immune-
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dysregulation that has been associated with various manifestations, including soarcoidosis (the primary cause of eye-loss in children), Crohns disease and asthma, the NANOMYC project will provide considerable support to health maintenance and disease prevention. This is also in agreement with the efforts of the EU to combat poverty-related diseases one of which is certainly tuberculosis. The partners anticipate improving competitiveness of the European biotechnology sector by providing a World Innovative diagnostic tool that addresses a market of several million euros per year and has strong commercialisation potential. The project will also allow more effective detection of mycobacterial pathogens among animals and animal products which will decrease the loss of capital and revenue associated with the relevant diseases. Moreover, it will gradually decrease the level of the required alertness of the European Food Industry. Coordinator John Ikonomopoulos Agricultural University of Athens Faculty of Animal Science Department of Anatomy and Physiology of Farm Animals Iera Odos 75 11855 Athens, Greece E-mail: ikonomop@aua.gr Scientific coordinator Maria Gazouli University of Athens School of Medicine Department of Biology E-mail: mgazouli@med.uoa.gr Palmiro Poltronieri Biotecgen Srl Lecce, Italy Joers Titt Quattromed Ltd Molecular Diagnostic Laboratory Tartu, Estonia Birgit Glaubitz Medizone GmbH Munich, Germany Panos Malamis Malamis & Malamis Co Athens, Greece Partners Nikos Chaniotakis University of Crete Laboratory of Analytical Chemistry Heraklion, Crete, Greece Leonardo Sechi University of Sassari Sezione di Microbiologia Dipartimento di Scienze Biomediche Sassari, Italy Mirjana omor Vinca Institute for Nuclear Sciences Laboratory for Radiation Chemistry and Physics Belgrade, Serbia Nikos Goutas Eugenidion Infirmary Agia Trias Athens, Greece Spanos Stamatios Biosure Cell & Co Athens, Greece
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DETECTHIV
Sensitive nanoparticle assay for the detection of HIV
Contract No Project type EC contribution Starting date duration LSHP-CT-2006-037118 Specific Targeted Research Project e 2 026 260 1 January 2007 36 months
Background and objectives: The DETECTHIV project aims to develop a new platform and assay for the detection of the HIV p24 antigen in serum or blood. The p24 test has two advantages: it can detect HIV at an early stage of the infection, before antibodies develop, and it is quantitative. This STREP will develop an extremely simple viral load test with only one reactant (grafted colloids). In the first phase of the project, the DETECTHIV partners will set up a magnetic nanoparticles assay to be used in a microtiter plate with the goal of detecting concentrations as low as 1 ng/ml. In the second phase, the use of nanoparticles on a microfluidic chip will allow the detection of p24, to levels as low as 0.1 picog/ml one to two orders of magnitude more sensitive than classical Enzyme Linked Immuno-Sorbent Assay (ELISA) p24 tests. The partners will use both recombinant p24 and patient samples to validate the platform. Approach and methodology: The test primarily involves optically detecting the formation of a colloidal gel of magnetic nanoparticles (agglutination test). The gel forms in a magnetic field under the presence of antigens that are capable of irreversibly linking two colloidal particles. Therefore, the latter are grafted with antibodies that are specific to the p24 antigen. The detection is achieved through simple optical
absorbance measurements, owing to the strong optical scattering modification when passing from nanometric colloids to the gelled state. In the microfluidic chip test, a sample solution of serum or blood is transported through suspended magnetic nanoparticles that are magnetically retained within a microfluidic channel. When brought into a magnetic field, the particles will be able to approach each other, form chains and will be irreversibly linked if the p24 antigen is present. Subsequently, on-chip light scattering techniques will be used to quantify the concentration of permanent chains or clustered beads, which are proportional to the p24 antigen concentration. Given that immunoassays are the most widely used assays in clinical diagnostics, the impact of lowering their cost while enhancing their efficiency would provide a much-needed boost towards lowering healthcare costs. The chosen assay, i.e. the p24 antigen-based viral test, would be of great benefit in the global battle against HIV infection and AIDS, especially in the developing world that constitutes over 70% of the affected world population. An arsenal of laboratory methods is available to screen blood, diagnose infection and monitor disease progression in individuals infected by HIV. ELISA is the most commonly used test to screen for HIV infection. One advantage of using magnetic colloids arises from the fact that the particle surface has a very
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strong colloidal stability in any type of buffer, serum or plasma, and has functional groups or receptors so that the particles can be further grafted with the biomolecules of interest. After trapping molecules (present in small amounts in solution) on the nanoparticles, the latter can be handled with applied magnetic fields. There is a net advantage due to faster reaction rates, trapping of the analyte and separation without losses. With this innovative and sensitive p24 assay, DETECTHIV seeks to approximate the sensitivity of the current viral load assays with the added advantage of being more robust (because of the superior physical stability of the p24 antigen). Although measurement of HIV-1 RNA (viral load) is the acknowledged gold standard marker for monitoring disease activity in patients receiving highly active antiretroviral therapy, it remains a very expensive test in resource-constrained settings. As this STREP promises to establish a more sensitive, faster and simpler test than a conventional ELISA p24 test, as well as a more affordable test than an HIV-1 RNA viral load test, it has the full potential to set a new standard in the diagnosis and follow-up of HIV-infected and AIDS patients during the complete duration of the infection. Expected outcome: The final goal of this project is the development of an agglutination test for the HIV p24 antigen, with a detection sensitivity that is one to two orders of magnitude better (0.1 pg/ml) and up to one order of magnitude faster than standard ELISA p24 tests. A single platform that handles magnetic actuation, microfluidics, optical detection and signal processing on a disposable microfluidic chip will be provided. Magnetic nanoparticles with appropriate surface chemistry for selective recognition of the p24 antigen will be the first to be developed. Clinical validation of the assay for the detection and quantification of p24 antigens is an important milestone. For ultimate sensitivity, magnetic retention and actuation will be used to separate nanoparticle clusters from nonreactive nanoparticles in a microfluidic chipbased system. By means of an optical detection system, with integrated polymerbased waveguides, it will be possible to exploit the scattered light to retrieve information from the nanoparticles. Potential applications: With the recent availability of low-cost/free antiHIV drugs, a remaining major hurdle is the availability of an affordable viral load test. Viral load tests help determine the correct regimen of anti-HIV drugs. The current gold standard for viral load testing (the Polymerase Chain Reactionbased test) is unfortunately out of reach for the budgets of developing nations. Consequently, most of these countries commence treatment without viral load monitoring. The new p24 assay platform developed within the DETECTHIV project will be of considerable benefit to the development of a low-cost viral load test for HIV. The aim is to use South Africa as the beta test site. The total number of people with HIV infections in South Africa is currently estimated at around 4.7 million. South Africa is the ideal pilot site because it has conditions approximating both the developed and developing world, as well as an established medical infrastructure.
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Coordinator Martin Gijs Ecole Polytechnique Fdrale de Lausanne Swiss Federal Institute of Technology Lausanne Institute of Microelectronics and Microsystems 1015 Lausanne, Switzerland E-mail: Martin.Gijs@epfl.ch Partners Marc Van Ranst Katholieke Universiteit Leuven Clinical and Epidemiological Virology Aids Refrence Lab Leuven, Belgium Solly Makholiso Ayanda Biosystems SA Lausanne, Switzerland Bruno Vallayer Bertin Technologies Biotech Systems Department Montigny-le-Bretonneux, France Jrme Bibette Ecole Suprieure de Physique et de Chimie Industrielles de la ville de Paris Laboratoire Collodes et Matriaux Diviss Paris Cedex 05, France Jrg P. Kutter Technical University of Denmark MIC Department of Micro and Nanotechnology Lyngby, Denmark Bernard Plichon ADEMTECH SA Pessac, France
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Nanoparticle-based electronic biosensor for diagnostics of cardiovascular disease

Contract No Project type EC contribution Starting date duration Website LSHB-CT-2006-037672 Specific Targeted Research Project e 2 221 185 1 October 2006 36 months www.nacardio.com
NACARDIO
Background and objectives: NACARDIO is a multidisciplinary project aiming to develop and commercialise a nanobiosensor technology, capable of analysing extremely small amounts of protein in small sample volumes. The technology can be used to quantify proteins involved in lipid storage to investigate whether any of these proteins are potential biomarkers for the development of insulin resistance and cardiovascular disease. The sensor technology is based on single electron tunnelling (SET), a phenomenon well explored for low temperature applications. Stateof-the-art nanofabrication utilising metallic nanoparticles now make this technology platform available for operation at room temperature. SET-technology provides unique possibilities for biosensing. To achieve the goals, the NACARDIO network will perform extensive multidisciplinary work to address questions at the interface between nanotechnology, physics, electrical engineering, surface chemistry, biotechnology and medical sciences. Frontline experimental approaches encompassing peptide-stabilised gold nanoparticles, electron-beam lithography, nano-imprint, molecular self-assembly, engineered antibody-fragments, protein expression and fluidic simulations will be used to fabricate the sensor and ensure biological functionality and usability.
Approach and methodology:
Figure shows how the different Work Packages of NACARDIO are assembled into an instrument prototype
The individual scientific, technical and innovation objects of NACARDIO are as follows. 1. To express proteins, which have been identified as potential biomarkers for insulin resistance and cardiovascular disease through investigation of the mechanism for the storage of lipids in the cell. The proteins will be used to obtain a few specific antibody fragments directed against each of the respective biomarkers. A unique route for the production of small, highly stable single chain antibody fragments will be used, giving antigen-binding fragments that are perfectly suited for the SET-biosensor. The antibody fragments will have a tailored affinity ligand that meets the immobilisation requirements for the biosensing by SET
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2.
3.
4.
5.
To evaluate different methods for the preparation of gold nanoparticles to be used for the island and active site of the biosensing SET. The gold nanoparticles will be fabricated with different sizes (between 3 nm and 15 nm) and narrow size distribution. The surface of the nanoparticles will be provided by appropriate chemistry that stabilises the particles and allows chemical coupling to sensor electrodes and antibody fragments. Frontline approaches, including peptide stabilised gold nanoparticles, will be used in order to create the nanoparticle-antibody fragment conjugates that are needed in the sensor. To use advanced lithography for the fabrication of metallic electrodes on silicon separated by 5 to 20 nm, and to evaluate the reproducibility of this fabrication. Reproducible fabrication on this scale is a giant leap forward for the state of the art in lithographic methods. The extremely small nanostructures are, together with nanoparticles, making up the SET, which is the foundation of the sensor. To make the nanogaps, three innovative lithography methods will be used: electron-beam, nanoimprint and focused ion beam. To couple tailored antibody fragments to the nanoparticle and to provide the surfaces surrounding the active site of the SET with chemical functionality, modified in order to reduce unspecific proteins interaction using self-assembly techniques. Innovative instrumental approaches utilising surface plasmon resonance, ellipsometry and quartz crystal microbalance with dissipation makes it possible to distinguish unspecific protein binding from specific antigen-antibody binding. To fabricate fluidic microchips in elastic polymer material to be easily positioned on top of the biochip. The fluidic microchip should enable optimal sample transfer to the active sites of the biochip with
6.
minimised unspecific binding, allowing for extremely sensitive detection. The inventive design of the fluidic chip will also seal the biochip so as to avoid leakage during sample screening. Simulations will be performed in order to optimise microfluidics regarding parameters, such as channel design, temperature, density, concentration, electric fields, flow speed, etc. To assemble electrode structures and gold nanoparticles to achieve functional SETdevices for operation at room temperature. To perform biosensing experiments and develop a model for SET-biosensing. An electrical measurement set-up with an associated fluidic system is available. The purpose of these experiments is also to optimise the sensor chips and provide important information concerning fabrication of the SET-biosensor. The knowledge gained will then be used to refine the measurement set-up into a first prototype.
Expected outcome: The project partners anticipate the following results: small, highly stable single chain antibody fragments with great specificity towards identified potential biomarkers within cardiovascular disease; successful functionalisation of gold nanoparticles with these fragments; routes to avoid unspecific binding to the device; a method to fabricate device electrodes in an industrial way; a method to assemble a device in an industrial way; a sensing mechanism model; market analysis; a SET biosensing prototype.
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Coordinator Main findings: Whilst NACARDIO has only been running since autumn 2006, the project partners already have some progress to report. The first potential biomarker has been identified and is now ready for production of small, highly stable single chain antibody fragments via the immunisation of camels. As regards the investigation of the design and synthesis of biologically functional metallic nanoparticles, the preparation of robust biocompatible and functional gold nanoparticles has begun and the initial results look very promising. Low temperature measurements of SET device confirm room temperature measurements and show that the partners can fabricate the devices. Olle Isaksson Gteborgs Universitet Department of Medicine Grna strket 8 413 45 Gteborg, Sweden E-mail: Olle.Isaksson@medic.gu.se Partners Linda Olofsson Midorion AB Gteborg, Sweden albert van den Berg University of Twente Twente, Netherlands Serge Muyldermans Vlaams Interuniversitair Instituut Biotechnologie Brussels, Belgium Sergey Kubatkin Chalmers University of Technology Gteborg, Sweden d Mathias Brust The University of Liverpool Liverpool, UK
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SLIC
SLIC-Biosensors in molecular diagnostics: nanotechnology for the analysis of species specific microbial transcripts
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2005-513771 Specific Targeted Research Project e 1 999 980 1 January 2005 36 months www.fp6-slic.eu
Background and objectives: Molecular diagnostics of microbial pathogens is an integral part of modern medicine. The growing need for direct genotyping and/or the screening of the transcriptome calls for the development of alternative technologies. The consortium of SLIC, a Specific Targeted Research Project, planned to develop a cost-effective platform for the identification of bacterial species based on the SLICNanobiosystem. Using tmRNA transcripts of the bacterial ssrA gene, the partners were able to detect, quantify and identify bacterial species in a single homogenous assay format. The SLICNanobiosystem consists of a self-assembled lipid bilayer membrane that integrates a synthetic ligand-gated ion channel (SLIC). The SLIC comprises a capture molecule that can specifically bind a given analyte, a process that is monitored via electrical impedance spectroscopy. With this system, the effect from even a few channels can be resolved, thus providing an ultra-sensitive, highly stable and versatile biosensor platform. The consortium utilised transcripts (tmRNA) of the ssrA gene in order to identify bacterial species present in clinical samples. These transcripts occur in high abundance and contain a core sequence that is species specific, a feature that was exploited to identify infectious disease pathogens. Identification of the different bacterial tmRNA transcripts was accomplished by displaying a library of nucleic acid capture probes on the SLIC. This enabled species identification
and discrimination between one or more species present in the sample if there was mixed species infection. Since the detection equipment was based on electronics, miniaturised/compact and cost-effective instrumentation was possible. The consortiums approach aimed to lay the foundation for a new generation of multiparametric molecular testing systems, which would open novel opportunities within the area of point-of-care applications in the clinical diagnostics market. Approach and methodology: In the first half of the project, one particular pathogen, S. pneumoniae, out of the three (S. pneumoniae, H. influenza and M. tuberculosis) was selected as the initial focus. During this phase, the consortium focused on developing and assembling the building blocks of the biosensor for the assay to detect the S. pneumoniae pathogen. The RTD activities involved in this period can be broadly classified into the following categories: development of a panel of capture probes that can bind target RNA, as well as methodologies and strategies to optimise capture probe performance; development of simplified methods for producing the SLIC biosensor constituent elements, such as the SLIC peptide units and ligation of capture of capture probe on biosensor platform; development of a microfluidics-based sample pre-treatment platform in order to carry out on-chip target RNA extrac-
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tion, purification and transportation to SLIC analysis site; assessment of SLIC biosensor robustness when in contact with media of complex mixtures (e.g. blood) as would be the case with clinical samples. Patent ALU: Vulto, P., Urban, G., A microfluidic chip for RNA purification and hybridisation detection, German patent, filed end of 2006. Coordinator Solomzi a. Makohliso Ayanda Biosystems SA PSE Parc Scientifique Swiss Federal Institute of Technology CH-1015 Lausanne, Switzerland E-mail: s.makohliso@ayanda-biosys.com Partners Horst Vogel Swiss Federal Institute of Technology Institute of Chemical Sciences & Engineering Lausanne, Switzerland Gerald a. Urban IMTEK Albert Ludwigs Universitat Freiburg Freiburg, Germany Majella Maher DNA Diagnostics Laboratory The National Diagnostics Centre National University of Ireland Galway, Ireland ants Kurg University of Tartu Estonian Biocentre, Laboratory of Gene Technology Tartu, Estonia andrea degen Iseli eurelations AG Zurich, Switzerland
Expected outcome: The following outcomes are expected: novel automated sample preparation platform for both cell lysis extraction and separation of RNA; methodology on a bioinformatics platform for optimising oligo probe design for RNA target.
Major publications: Vulto, P., Medoro, G., Igel, G., Kieninger, J., Urban, G., Tartagni, M., Guerrieri, R., Manaresi, N., Phaseguide structures for pipette actuated laminar flow-based selective sample recovery, Proc. of MicroTAS, 2005, pp. 1093-1095. Vulto, P., Medoro, G., Altomare, L., Urban, G.A., Tartagni, M., Guerrieri, R., Manaresi, N., Selective sample recovery of DEP-separated cells and particles by phaseguide-controlled laminar flow, J. Micromech. Microeng., 16, 2006, pp. 1847-1853 Glynn, B., Lacey, K., Reilly, J., Barry, T., Smith, T., Maher, M., Quantification of Bacterial tmRNA using in vitro Transcribed RNA Standards and Two-Step qRT-PCR, Research Journal of Biological Sciences, 2, 2007: 564-570. Vulto, P., Klaunick, C., Igel, G., Urban, G., tmRNA purification by electrophoretic filtration for genomic identification of bacteria, Submitted to MicroTAS, 2006. Healthtech Institutes Nucleic-Acid Based Technologies conference, 2006.
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eBIOSENSE
Electrical biosensor arrays for analyses of harmful micro-organisms and microbial toxins
Contract No Project type EC contribution Starting date duration Website LSHB-CT-2004-512009 Specific Targeted Research Project e 2 370 900 1 January 2005 36 months www.ebiosense.org
Background and objectives: The eBIOSENSE project is developing a platform for the analysis of harmful micro-organisms and their toxic products. The platform is based on silicon chips, which are manufactured with standard silicon technology. These chips are then activated for specific analyses via the immobilisation of different sample binding molecules on an array of microelectrodes on the chip surface. The electric biochip arrays enable parallel and simultaneous identification and quantification of specific nucleic acids (DNA or RNA), microbial proteins and toxic microbial products. Figure 1 shows a silicon chip with electrodes for the analysis of 16 different molecules in a sample.
The use of DNA- and antibody-based analyses for identification of micro-organisms, rather than traditional classification in species, offers a great advantage in the assessment of health hazards, since in many cases only certain strains of a species are pathogenic and many toxins are produced by several species. Thus, eBIOSENSE is seeking to assess the pathogenicity factors, rather than the species name. Approach and methodology: Two versions of arrays are being developed: nucleic acid-based chips and antibody-based chips.
Fig. 1. An electric biochip. Left: The 9x10 mm electric chip array with 16 positions. Right: The biochip reader with chip-holding cassette, and sample-handling equipment for the assay.
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The function of an electric DNA-chip is illustrated in Figure 2. When the chip surface is exposed to a sample, the target DNA molecules simultaneously hybridise with the capturing DNA-probe on the electrode and with a soluble detection probe. The detection probe is subsequently labelled with an enzyme. The signal is then generated by the addition of a chemical substance (pAPP). pAPP reacts with the enzyme and generates p-aminophenol (pAP), which is oxidised to quinoneimine (QI) at the positive electrode. The two electroactive components pAP and QI are then redox recycled between the anode and cathode, generating a current from the chip, as illustrated in Fig. 2. In an antibody-based biochip, the DNA capture probe is replaced by a specific antibody which recognises and binds the target molecule where an enzyme-antibody conjugate is then used to label the bound-target molecules. Enzyme reaction and signal generation are the same as for the DNA chip. The different steps in the analysis are automatically controlled by an instrument
Fig. 2. Left: Principle of signal generation with an electric DNA chip. Right: responses from 16 electrodes of a chip. The left arrow shows time of pAPP application. The second arrow shows the subsequent stop-flow. The initial slope (nA/sec) generated during the stop-flow is used as signal.
(Fig. 1), so the operator only prepares and adds the sample to a container in the instrument and then collects the signal from the different electrode positions. Expected outcome: Time chips will be developed for pathogenic strains of the Bacillus cereus group, shiga-toxin producing E. coli strains (usually called EHEC), pathogenic Staphylococcus, Salmonella enterica and the mycotoxins ochratoxin A and fumonisin. However, the electric biochips are generic and can be applied to varied analyses by altering the DNA probes or the antibodies. Many applications are found in clinical and food analyses, and in the future, environmental analyses may also be based on electric biochips.
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Main findings: Silicon chip arrays with 16 positions have been designed and manufactured for the analysis of several pathogens and toxins: a DNA chip has been applied for the simultaneous detection of all genes involved in synthesis of all known toxins produced by many strains of the common food pathogens of the Bacillus cereus group. In this way, the toxin-producing capacity of Bacillus strains, causing different types of food poisoning, could be assessed in one assay. A DNA chip for the analysis of the pathogenic E. coli strains called EHEC has also been developed. This chip gives information on whether the suspected E. coli strain can produce the feared shiga-like toxin or not. These chips are intended for rapid confirmative analysis of suspected bacterial colonies from the primary enrichment culture, which is usually applied in food analysis. Such an assay takes about 30-40 minutes with the DNA chips, in comparison with standard ISO methods that require at least one extra day, due to the demand for repeated cultivations. Similar chips for analysis of Salmonella and fumonisinproducing fungi will also be delivered. The performance of the Salmonella chips is compared with commercial methods and the ISO method. An antibody-based chip has been developed for analysis of several staphylococcal toxins and its performance is compared with standard methods. The chip for quantitative analysis of staphylococcal enterotoxin B currently detects 15 ng toxin in a 250 L sample. Major publications: Liu, Y., Elsholz, B., Enfors, S.O., Gabig-Ciminska, M., Confirmative electric DNA array-based test for food poisoning Bacillus cereus, Journal of Microbiological Methods (in press), 2007. Gabig-Ciminska, M., Liu, Y., Enfors, S.O., Gene-
based identification of bacterial colonies with an electric chip, Analytical Biochemistry, 2005, Vol. 345, pp. 270-276. Los, M., Los, J., Blohm, L., Spillner, E., Grunwald, T., Albers, J., Hintsche, R., Wegrzyn, G., Rapid detection of viruses using electrical biochips and anti-virion sera, Letters in Applied Microbiology, 2005, Vol. 40, pp. 479-485. Coordinator Sven-Olof Enfors School of Biotechnology Royal Institute of Technology (KTH) Roslagstullsbacken 21 10691 Stockholm, Sweden E-mail: enfors@biotech.kth.se Partners Rainer Hintsche The Fraunhofer Institute for Silicon Technology Itzehoe, Germany Thomas Schweder Ernst-Moritz-Arndt Universitt Greifswald, Germany Peter Neubauer University of Oulu Oulu, Finland Grzegorz Wegrzyn University of Gdansk Gdansk, Poland Lars Blohm eBiochip Systems GmbH Itzehoe, Germany antje Breitenstein Scanbec Oy Oulu, Finland
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Cees Waalwijk Plant Research International BV Wageningen, Netherlands Julien Bourjault Centre de Recherche et dtude pour lAlimentation Paris, France Mika Tuomola Raisio Benecol Ltd Raisio, Finland
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FP7 PROJECTS
Biomedical imaging
Innovative contrast imaging by non-linear optics (NLO) for the observation of biological tissues in vivo and in real time, at cellular and molecular levels
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2008-200820 Collaborative Project e 3 091 129 1 March 2008 36 months http://www.carsexplorer.eu
CARS Explorer
Background and objectives: The CARS Explorer project seeks to demonstrate the concept of innovative light-based contrasting technologies for functional in situ imaging in life science and biomedical research. The partners ultimate goal is to develop an endoscope based on non-linear optics (NLO) and laser pulse phase shaping. Non-linear laser pulse interactions with living tissues provide unique possibilities, such as an absence of sample preparation, direct multiparametric visualisation with molecular specificity and cellular resolution, and deep sample penetration. Nevertheless, the effective transfer of NLO to biomedical applications faces major technological challenges that are related to the delivery of ultra-short laser pulses, the weakness of the signal produced in biological samples and the difficulty in interpreting generated contrasts.
The CARS Explorer interdisciplinary consortium includes partners with expertise ranging from optical physics to the clinic. The work plan is split into five research and technology development (RTD) Work Packages (WPs): three are intended to overcome specific technological problems and the other two will determine the assets and constraints in NLO imaging through appropriate experimental biological models. Finally, to bring the concept to the diagnostic level, the consortium will explore the molecular and morphological NLO signatures associated with tumour development in skin cancer. In addition to the challenge of developing pulse shaped NLO-based endoscope technology, this project will have a strategic and economic impact by providing a non-invasive functional exploration method. Approach and methodology: The work plan is broken down into WPs that are partially or totally interdependent and will be developed in parallel. As the structure of the WPs reflects the complementarity of the approaches and expertise of the individual partners, each partner contributes to several WPs. The WPs that will overcome the specific technological problems and validate the CARS Explorer project are the following: WP2, NLO contrast propagation using pulse shaping;
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DIAGNOSTICS FP7
WP3, photonic crystal fibres for NLO imaging; WP4, signal recovery and imaging processing.
Partners Herv Rigneault Centre National de la Recherche Scientifique (CNRS) Institut Fresnel Marseille, France Benot Van den Eynde De Duve Institute Brussels, Belgium andreas Volkmer Universitt Stuttgart Stuttgart, Germany Jonathan Knight University of Bath Bath, UK
WP1 and WP5, which are more biologically related, will delineate through appropriate experimental models the assets and constraints in NLO in a microscopy mode and in an endoscope configuration, respectively: WP1, assets and constraints in imaging tissues by NLO; WP5, molecular & morphological NLO signatures associated with tumour development.
Coordinator didier Marguet Institut National de la Sant et de la Recherche Mdicale (INSERM) 18 Avenue Mozart 13276/09 Marseille, France E-mail: marguet@ciml.univ-mrs.fr Franois Lacombe Mauna Kea Technologies Paris, France Christiane dascher-Nadel Inserm Transfert SA Marseille, France
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Ultra-high resolution and ultra-sensitive fluorescence methods for objective sub-cellular diagnosis of early disease and disease progression in breast and prostate cancer
Grant agreement No Project type EC contribution Starting date duration HEaLTH-F5-2008-201837 Collaborative Project e 4 197 774 1 July 2008 42 months
FLUODIAMON
Background and objectives: FLUODIAMON seeks to develop and validate a quantitative, minimally invasive diagnostic tool for early and conclusive detection, diagnosis and monitoring of breast and prostate cancer. By making use of a combination of what are probably the most exciting recent advances in the field of light microscopy, the partners will develop a methodology for fluorescence-based optical imaging of individual sample cells. The methodology will include advances that will take the spatial resolution far beyond the fundamental limits of optical resolution and the sensitivity down to an ultimate single-molecule level. Multi-parameter detection schemes will significantly increase the fluorescence information by which these cellular images can be analysed. Apart from detecting and identifying tumour markers in the samples, FLUODIAMON will exploit tumour-specific spatio-temporal molecular distributions within the intact sample cells. To date, this has been an almost unexploited dimension of diagnostic information. By combining and supporting these novel optical methods with state-of-the-art affinity molecule biotechnology, fluorophore chemistry, proteomicsbased biomarker identification, and bioinformatic validation tools, all possible means will be exploited to extract a maximum amount of information out of very small amounts of sample material. The partners expect that an improved, early and reliable diagnosis of breast and prostate cancer will be possible from amounts of sample material which are small enough for a minimally invasive procedure such as Fine-needle aspiration (FNA) to be used. In addition, with the minimally invasive FNA-based sampling, serious sampling related side effects, such as seeding and spread of cancer cells, can be completely avoided. Given the high incidence of breast and prostate cancer, and the utmost importance of an early and conclusive diagnosis for the prognosis of these diseases, the relevance of this project cannot be overestimated. Approach and methodology: Recent biophysical research has made remarkable progress in the field of fluorescence-based optical imaging. With an attainable spatial resolution of a few tens of nanometres, a detection sensitivity down to the single-molecule level and multiparameter fluorescence detection schemes, the amount of information that is now obtainable has reached a quantitatively new level. However, despite shortcomings in the current analytical methods, clinical diagnostics have so far had little benefit from these developments. The FNA technique is a prominent example where these developments can lead to considerable improvements. Being minimally invasive and without major side effects, like tumour cell spreading, FNA is a superior sample extraction method for breast
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and prostate cancer sampling, for example. However, providing only small amounts of (often lowquality) sample, the investigation of the material is often impossible with the currently available analytical methods. To address this particular problem, the FLUODIAMON consortium seeks to bring together experts from both clinical and biophysical research, as well as from the instrument development side to devise new diagnostic methods to analyse FNA samples reliably and without the requirement of expert knowledge. The partners are distinguished researchers in their respective fields and cover the following fields of expertise: super-resolution fluorescence nanoscopy; transient-state microscopy; multidimensional fluorescence detection; low-level light detection and data acquisition; fine-needle aspiration, FNA; cancer proteomics; affinity molecules and fluorescent labels; bioinformatics. Potential risks can be identified. However, the partners do not consider them as being significant, but more localised to the detailed activities along the whole chain of activities. Being a field-tested method, the consortium does not expect major risks related to FNA-based sample extraction. Regarding the identification of specific biomarkers, some libraries may not be readily available. Difficulties may also occur in the peptide synthesis, genetic immunisation of camels, and development of variants of libraries. However, due to the numerous biomarkers already known and the variants identified within the project, the partners anticipate that they can focus on a moderate number of markers compatible with their antibody/affibody and optical methods. The use of clinical, FNA-based biopsy smears instead of cell lines may pose additional challenges
for the microscopy methods. Clustered cells, tissue fat or cell detritus will cause additional light scattering. This could be addressed by established wash- and filter-clean up of the aspirates and/or by shifting the employed wavelengths into the near-infrared region and using time-gated detection. Possible failures in the labelling or delivery of labelled antibodies/affibodies into cells or nuclei of clinical tissue can be circumvented by using multiple alternative penetration techniques. In summary, as far as risks for the project are concerned, alternative approaches both with respect to affinity molecules, the arsenal of complementary imaging techniques and tumour markers will make it possible to define combinations by which one can overcome the practical problems that may arise.
Coordinato Jerker Widengren Albanova University Center 10691 Stockholm, Sweden E-mail: jerker@biomolphysics.kth.se Partners Hans Wiksell VibraTech AB Stockholm, Sweden Gert auer Karolinska Institutet Stockholm, Sweden Stefan Hell Max-Planck-Gesellschaft zur Frderung der Wissenschaften e.V. Institute for Biophysical Chemistry Goettingen, Germany
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Claus Seidel Heinrich-Heine University Duesseldorf, Germany John Murphy SensL Inc Cork, Ireland Wolgang Becker Becker&Hickl GmbH Berlin, Germany Jens Habermann University of Luebeck Luebeck, Germany Karl drexhage University of Siegen Siegen, Germany Pekka Hnninen University of Turku Turku, Finland Sampsa Hautaniemi University of Helsinki Helsinki, Finland Silvere van der Maarel Academisch Ziekenhuis Leiden (Leiden University Medical Center) Leiden, Netherlands
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DIAGNOSTICS
FUN OCT
Functional Optical Coherence Tomography
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2008-201880 Collaborative Project e 5 411 720 1 april 2008 48 months www.funoct.eu
Background and objectives: The aim of FUN OCT is to expand the noninvasive optical biopsy capability of optical coherence tomography (OCT) and combine it with multiphoton tomography (MT) to develop novel functional capabilities enabling morphofunctionalperformance, i.e. the fusion of anatomic and functional imaging at the cellular resolution level. These methodologies will enable unprecedented non-invasive detection of depth-resolved physiological, metabolic and molecular specific tissue information. In other words, a novel, powerful medical imaging platform is envisioned. This novel platform fills an important gap in todays medical imaging technology. The hypothesis is that the combination of cellular resolution, real time imaging of morphology and depthresolved tissue function could enable a major step forward in early cancer diagnosis and in the early detection of retinal pathologies that are worldwide leading causes of blindness. This will be accomplished due to a synergistic effect from joining complementary international expertise in the fields of laser sources, OCT, MT and beam delivery system technology. The consortium comprises seven research groups and two small and medium-sized enterprises (SMEs). It will make use of its existing relations to clinical collaborators in order to achieve proof-of-principle validation of the imaging modalities.
The outcome will contribute directly to improving and maintaining the quality of life and living conditions of the European ageing population through early diagnosis of cancer and retinal pathologies, as well as more efficient therapy monitoring. Moreover, the envisaged imaging modality may in the long term act as a screening device to investigate the prevalence of cancer as a function of geographic (regional) or gender-related parameters. Finally, the diagnosis of other age-related diseases in a variety of medical fields, such as cardiology, neurology, gynaecology, and gastroenterology, will benefit from this novel diagnostic platform provided by FUN OCT. Approach and methodology: The functional extensions of OCT comprise polarisation-sensitive OCT, Doppler OCT, spectroscopic OCT and OCT for optophysiology. Furthermore, OCT is combined with MT, thus providing additional functional imaging and diagnostic capabilities. It should be emphasised that the choice of applications illustrate the general clinical impact and applicability of the developed novel imaging platform. In order to facilitate such development, the consortium has identified two key enabling device and system technologies, namely novel light source technology and probe delivery and applicator systems. Accordingly, in order to implement the main project objectives, the work plan is divided into nine Work Packages (WPs), two of which deal with project management:
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FUN OCT
WP1: project management; WP2: development of novel light sources for multi-modal, functional and ultrahigh speed biomedical imaging; WP3: application systems for functional OCT and multiphoton tomography; WP4: design and implementation of polarisation sensitive OCT; WP5: design and implementation of Doppler OCT for depth resolved and quantitative blood flow imaging; WP6: design and implementation of spectroscopic optical coherence tomography for contrast enhancement and depth resolved metabolic tissue information; WP7: design and implementation of optophysiology for depth resolved physiologic tissue information; WP8: combination of multiphoton tomography and optical coherence microscopy (OCM); WP9: dissemination and exploitation. Coordinator Peter andersen Technical University of Denmark 1 Anker Engelunds Vej 2800 Kgs. Lyngby, Denmark E-mail: peter.andersen@risoe.dk Partners Gereon Httmann Universitt zu Lbeck Luebeck, Germany Wolfgang drexler Cardiff University Cardiff, UK Robert Huber Ludwig-Maximilians-Universitt Mnchen Munich, Germany Christoph Hitzenberger and Rainer Leitgeb Medizinische Universitt Wien Vienna, Austria andreas Stingl and Tuan Le Femtolasers Produktions GmbH Vienna, Austria Karsten Koenig JenLab GmbH Jena, Germany
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DIAGNOSTICS
nEUROPT
Non-invasive imaging of brain function and disease by pulsed near infrared light
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2008-201076 Collaborative Project e 5 738 653 1 april 2008 48 months www.neuropt.eu
Background and objectives: The nEUROPT project targets the development and clinical validation of advanced non-invasive optical methodologies for in vivo diagnosis, monitoring, and prognosis of major neurological diseases (e.g. stroke, epilepsy, ischemia), based on diffuse optical imaging by pulsed near infrared light. Established diagnostic imaging modalities (e.g. X-ray Computed Tomography, Magnetic Resonance Imaging, Positron Emission Tomography) provide 3D anatomical, functional or pathological information with spatial resolution in the millimetre range. However, these methods cannot be applied continuously or at the bedside. Diffuse optical imaging is expected to provide a valuable complementing tool to assess perfusion and blood oxygenation in brain tissue and their time evolution in a continuous or quasicontinuous manner. Not only will the devices be portable and comparably inexpensive, they can be applied in both adults and children. Timedomain techniques are acknowledged as offering superior information content and sensitivity compared to other optical methods, allowing for separation between contributions of surface tissues (skin and skull) and brain tissue. Timedomain imaging can also differentiate between scatter and absorption effects. The nEUROPT consortium plans major developments in technology and data analysis that will enhance time-domain diffuse optical imaging
with respect to spatial resolution, sensitivity and robustness of quantification, as well as performance of related instruments in clinical diagnosis and monitoring. The diagnostic value of timedomain diffuse optical imaging will be assessed by clinical pilot studies addressing specific neurological disorders, in comparison with established neurophysiological and neuroimaging techniques. Perspectives regarding clinical application of time-domain diffuse optical brain imaging will be estimated and a reliable basis for a potential commercialisation of this novel technique by European system manufacturers will be created. Approach and methodology: According to this general framework, the timing of the activities will be divided into two main parts scheduled for months 1-24 and months 25-48. Within each part, two parallel research lines will be conducted: Applied Research, involving upgrade, adaptation to clinical environment, standardisation, and use in clinical tests of existing/novel time-domain instrumentation. Basic Research, focussing on exploration of novel approaches, identification of potentially successful methodologies, and development of novel instrumentation. A continuous feedback from the applied research to the basic research lines will provide valuable input for the optimisation of novel methodologies.
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nEUROPT
Coordinator Rinaldo Cubeddu Politecnico di Milano Physics Department 32 Piazza L. da Vinci 20133 Milan, Italy E-mail : rinaldo.cubeddu@fisi.polimi.it Partners Sergio Cerutti Politecnico di Milano Department of Biomedical Engineering Milan, Italy Heidrun Wabnitz Physikalisch-Technische Bundesanstalt Department of Biomedical Optics Braunschweig, Germany Simon arridge Department of Computer Science Jeremy Hebden Department of Medical Physics and Bioengineering Judith Meet Institute of Woman Health University of College London London, UK adam Liebert Institute of Biocybernetics and Biomedical Engineering Warsaw, Poland Silvana Fanceschetti Fondazione Istituto Nazionale Neurologico Carlo Besta Milan, Italy Helmuth Obrig Charit - Universittsmedizin Berlin Department of Neurology Berlin, Germany Ewa Mayzner-Zawadzka Medical University of Warsaw Warsaw, Poland alvin Kienle Institut fr Lasertechnologien in der Medizin und Metechnik UlmUniversitt Ulm, Germany Fabrizio Martelli Universita degli Studi di Firenze Dipartimento di Fisica Florence, Italy William Wadsworth University of Bath Centre for Photonics and Photonic Materials Bath, UK John Clowes Fianium Ltd Southampton, UK Franco Zappa Micro Photon Devices S.r.l. Bolzano, Italy Wolgang Becker Becker & Hickl GmbH Berlin, Germany Carla Finocchiaro CF Consulting S.r.l. Milan, Italy
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DIAGNOSTICS
FMT-XCT
Hybrid Fluorescence Molecular Tomography X-ray Computed Tomography method and system
Contract No Project type EC contribution Starting date duration HEaLTH-F5-2008-201792 Collaborative Project e 4 512 303 To be confirmed 48 months
Background and objectives: The FMT-XCT project aims to combine X-ray computed tomography (XCT) and fluorescence molecular tomography (FMT) into a hybrid, quantitative system. The project builds on stateof-the-art knowledge that has only recently become available in Europe, and which is brought to the project by the partners herein. In return, it should deliver the first such hybrid system worldwide. The system will operate by: (1) co-registering XCT images with highly performing FMT images for merging anatomical, functional and molecular contrast; and (2) combining XCT information into the FMT inversion to provide a system with superior imaging performance. XCT-based correction can improve FMT performance in a more radical way than corresponding correction methods used for improving PET or SPECT images. In this way, FMT-XCT can reach the imaging accuracy of radio-nuclei-based tomography hybrid systems. By using fluorescence, FMT-XCT can then enable high flexibility in targeting physiology and molecular function, especially in multi-spectral mode, and high dissemination potential because virtually any biomedical laboratory has access to fluorescence reporting compared to radio-nuclei based research that requires access to radiochemistry and cyclotron facilities.
FMT-XCT aims to advance small animal imaging and drug discovery with a view to the clinical application of non-invasive breast cancer imaging. For this reason, the focus is on imaging breast cancer and response to therapy. Overall, the technology is ideally suited for commercial translation and has the potential to become the method of choice for in vivo imaging in most biomedical laboratories and in select clinical applications. While it appreciates the value of nuclear imaging methods, this project will hopefully raise the funding necessary to establish in Europe a potent new paradigm of in vivo imaging with high dissemination and application potential, and have a large social and health-care impact. Approach and methodology: The work is split into nine Work Packages (WPs). WP1 focuses on the management and coordination of the project while WP9 considers dissemination and training activities. WP2-WP4 build unique XCT and FMT technology and know-how that is then integrated into one system prototype in WP5. WP6 and WP7 research appropriate imaging strategies for in vivo imaging and perform preclinical imaging. WP8 compares the FMT-XCT system with a previously developed PET-XCT system to test the hypotheses in this proposal.
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FMT-XCT
Coordinator Vassilis Ntziachristos Helmholtz Center Munich 1 Ingolstaeder Landstrasse 85764 Neuherberg, Germany E-mail: vasilisn@comcast.net Partners Bertrand Tavitian Laboratory for Experimental Molecular Imaging Paris, France Philippe Rizo LETI, Commissariat lEnergie Atomique Grenoble, France Jorge Ripoll Foundation for Research and Technology Hellas Heraklion, Crete, Greece Simon arridge University College London London, UK Manuel desco Fudacion para la Investigacion Biomedica del Hospital Gregorio Maraon Madrid, Spain Markus Rudin Universitt Zrich Zurich, Switzerland Willi Kalender Verfahren und Apparate der Medizinischen Physik GmbH Erlangen, Germany
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DIAGNOSTICS
HYPERImage
Hybrid PET-MR system for concurrent ultra-sensitive imaging
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2008-201651 Collaborative Project e 4 940 000 1 april 2008 36 months www.hybrid-pet-mr.eu/
Background and Objectives: The HYPERImage project will develop a novel hybrid system for simultaneous whole-body PET-MR (Positron Emission Tomography-Magnetic Resonance) imaging for humans. It will advance the required Time-of-Flight (ToF) PET technology, and the software for MR-mediated compensation of motion artefacts. The hybrid system will be validated in preclinical and initial clinical studies, for application in cardiovascular disease and in breast cancer, as one of the most relevant applications in oncology. For the latter application, the concept will be extended from pure imaging towards image-guided therapy. PET is the most sensitive molecular imaging modality. Hybrid PET/CT systems using Computer Tomography (CT) in order to provide the anatomical reference for lesion localisation have evolved to become the best choice for a number of applications in cardiology and oncology. However, PET/CT has drawbacks and limitations: CT is associated with a radiation dose and lacks soft tissue contrast, and PET suffers from restrictions in small lesion detectability, both due to motion artefacts during the scan, and detector limitations.
A hybrid combination of ToF-PET with a 3T MR has the potential to overcome these shortcomings by fully exploiting the superior soft-tissue contrast of MR in combination with a new MRcompatible solid-state PET detector technology. Sophisticated motion compensation enabled by concurrent acquisition of both MR and timestamped PET data will also be used. In addition, the versatility of MR allows imaging of supplementary functional parameters like temperature, elasticity, and diffusion, enabling this new hybrid imaging concept to open up new fields of applications in therapy guidance and therapy response monitoring. The consortium consists of one large company, four academic partners and three research institutes from six different EU Member States. It combines leadership in technology with pioneer experience in using biomedical imaging for diagnosis and therapy monitoring. Approach and methodology: The project is divided into three major research topics: development of hybrid PET/MR test systems for concurrent image acquisition; development of multimodality methods to advance quantitative technologies; preclinical and clinical tests with focus on oncology and cardiology applications.
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HYPERImage
Coordinator Volkmar Schulz Philips Technologie GmbH Forschungslaboratorien 2 Weibhausstrabe 52066 Aachen, Germany E-mail: volkmar.schulz@philips.com Partners Peter Fischer University of Heidelberg Mannheim, Germany Claudio Piemonte Fondazione Bruno Kessler-IRST Trieste, Italy Tobias Schaeffter Kings College London London, UK Julia Redondo Fundacin Centro Nacional de Investigaciones Cardiovasculares, Carlo III Madrid, Spain Gijsberta Krenn Netherlands Cancer Institute Amsterdam, Netherlands Gerhard adam Universittsklinikum Hamburg-Eppendorf Hamburg, Germany Stefaan Vandenberghe Interdisciplinair Instituut voor Breedb and Technologie Ghent, Belgium
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DIAGNOSTICS
MEGMRI
Hybrid MEG-MRI Imaging System
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2008-200859 Collaborative Project e 4 865 656 1 May 2008 48 months http://megmri.tkk.fi
Background and objectives: The MEGMRI project will develop and validate hybrid magnetoencephalography (MEG) and magnetic resonance imaging (MRI) technology that will allow simultaneous structural (MRI) and functional (MEG) imaging of the human brain. MEG is a non-invasive 3D functional imaging technology with a high temporal resolution compared to other functional imaging but often suffers from a precise structural localisation that will be improved by the dual modality approach of the MEGMRI hybrid scanner. In parallel, low-field MRI, a new very promising alternative to conventional high-field MRI, will provide enhanced image contrast in certain applications, improved geometric accuracy, improved safety (for patients with pacemakers and other implants, pregnant women and infants), and reduced costs. These new opportunities are based on recent advances in ultra-sensitive magnetic sensors. Two US teams recently used superconducting magnetometers based on quantum interference devices (SQUIDs) to provide 2D-MRI images at very low fields. In parallel, the Paris group of the MEGMRI consortium has developed a new type of magnetometer based on their Nobel-prize winning (Albert Fert, 2007) GMR technology, called mixed sensor, and is used for low-field NMR. The first part of the project will focus on sensor optimisation and low-field MRI development. This
covers the development of field-tolerant SQUIDs and optimised mixed sensors, as well as 3D-MRI low-field hardware and software development. The second part of the project will be devoted to a prototype building with the best kind of sensor and preclinical validation, covering major brain disorders for adults and children. The consortium has the necessary skills to perform all the tasks: sensor developers, MRI experts, MEG developers and clinical validators. It comprises 13 partners from 5 countries, including 3 small and mediumsized enterprises (SMEs) and 1 large medical device manufacturer. Approach and methodology: The partners will develop an MEG-MRI scanner for brain studies. The MRI mode will function at low field with an initial resolution of 333 mm3. Partners will compare and integrate lowfield MRI and high-field MRI on volunteer subjects and patients. This device will be the first whole-head low-field 3D MRI system where MEG source localisation maps and MR images are obtained in the same session. To realise the goal in an optimal way, they will develop and test both optimised field-tolerant SQUIDs and mixed sensors. The partners will select their optimal combination for the prototype. The project has three main tasks corresponding to the steps described above. To succeed in these, the work plan is divided in Work Packages (WPs), each with a clear end point and a designated, responsible WP leader.
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MEGMRI
Coordinator Risto Ilmoniemi Helsinki University of Technology Box 1000 02015 Espoo, Finland E-mail: risto.ilmoniemi@tkk.fi Partners Jari Sakari Penttil Aivon Oy Espoo, Finland Patrick Meneroud CEDRAT Technologies SA Meylan, France dag Winkler Chalmers University of Technology Gothenburg, Sweden Gian Luca Romani Universita degli studi Gabriele DAnnunzio Department of Clinical Sciences and Bioimaging Chieti, Italy Claude Fermon Commissariat lEnergie Atomique Paris, France antti ahonen Elekta AB Stockholm, Sweden Paolo Maria Rossini Assiociazione Fatebenefratelli per la Ricerca Biomedica e Sanitaria Rome, Italy Jyrki Mkel Hospital District of Helsinki and Uusimaa Helsinki, Finland antonello Sotgiu Imaging Technology Abruzzo LAquila, Italy Lutz Trahms Physikalisch-Technische Bundesanstalt Braunschweig, Germany Giacomo Rizzolatti University of Parma Parma, Italy Panu Helist VTT Technical Research Centre of Finland Espoo, Finland
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DIAGNOSTICS
SKINSPECTION
Grant agreement No Project type EC contribution Starting date duration Website
Multimodal skin inspection with hybrid acoustic and optical spectroscopic imaging
HEaLTH-F5-2008-201577 Collaborative Project e 4 097 585 1 april 2008 48 months http://www.skinspection-fp7.eu
Background and objectives: The incidence of skin cancer in Europe, the US and Australia is rising rapidly. One in five people will develop some form of skin cancer during their lifetime. A person has a 1 in 33 chance of developing melanoma, the most aggressive skin cancer. Melanoma is the second most common cancer in women aged 20-29, and the sixth most common cancer in men and women. In 2007, more than 1 million new cases were expected to be diagnosed in the US alone. About 90% of skin cancers are caused by ultraviolet (UV) sunlight. The World Health Organization estimates that 60 000 people will die this year from too much sun: 48 000 from melanoma and 12 000 from other skin cancers. A significant improvement of the current diagnostic tools of dermatologists is required in order to identify dermal disorders at a very early stage, as well as to monitor directly the effects of treatment. The SKINSPECTION partners aim in this project to develop a non-invasive multimodal hybrid imaging system with the capacity to perform non-invasive high resolution threedimensional clinical (1) two-photon imaging with timecorrelated single photon detection; (2) autofluorescence lifetime imaging; (3) highfrequency acoustical imaging with novel miniaturised multiple detector arrays; and (4) optoacoustical imaging using ultrashort near infrared (NIR) laser pulses.
This novel multimodal approach will provide a wide-field acoustic/optoacoustic view with quantitative depth information of the dermatological lesion as well as a close optical look into particular intratissue compartments with quantitative hyperspectral information and subcellular resolution. It is intended to provide a novel unique tool for early diagnosis and treatment control of skin cancer and skin disease, which will significantly contribute to the improvement of the European healthcare system. Approach and methodology: The project implementation is divided into three logical phases: specification, development & in vitro / ex vivo testing and clinical evaluation. The work in all phases is structured into 10 Work Packages, 7 of which are directed towards research and development with others covering management aspects, regulatory affairs for clinical testing, and training and dissemination.
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Coordinator Volkmar Schulz Robert Lemor Fraunhofer IBMT 48 Ensheimer Strasse 66386 Sankt Ingbert, Germany E-mail : robert.lemor@ibmt.fraunhofer.de Partners Karsten Koenig JenLab GmbH Jena, Germany Chris dunsby Imperial College of Science, Technology and Medicine London, UK alberto Giannetti Universita degli studi di Modena e Reggio Emilia Modena, Italy Hanno Wittig tp21 GmbH Saarbruecken, Germany Christian Weiss kibero GmbH Saarbruecken, Germany
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DIAGNOSTICS
EURIPIDES
European Research initiative to develop Imaging Probes for early In vivo diagnosis and Evaluation response to therapeutic substances
Grant agreement No Project type EC contribution Starting date duration Website HEaLTH-F5-2007-201380 Integrated Project e 6 994 850 1 February 2008 48 months www.euripides-europe.com/
Background and objectives: The EURIPIDES project partners aim to develop an in vivo imaging biomarker of multi-drug transporter function as a generic tool for the prediction, diagnosis, monitoring and prognosis of major central nervous system (CNS) diseases, as well as to provide support and guidance for therapeutic interventions. Multi-drug transporters actively transport substrates (including multiple CNS drugs) against concentration gradients across the blood-brain barrier (BBB). Over-activity of these transporters results in inadequate access of CNS drugs to their targets and hampers the build-up of adequate tissue levels of these drugs in the brain, This greatly limits their therapeutic efficacy. As such, this transporter hypothesis of drug resistance is applicable to a broad range of CNS drugs and patients with a variety of CNS diseases who critically depend on these drugs. Efflux transporters may also influence brain elimination of A, the hallmark of Alzheimers disease (AD). Impaired multi-drug transporter function with reduced clearance of A could lead to accumulation within the extracellular space, contributing to the pathogenesis of AD. The partners will determine the contribution of multi-drug transporters to impaired brain uptake of drugs for the prediction of therapeutic responses, or the contribution of impaired transporter function to reduced clearance of
toxic substances for the early in vivo diagnosis of AD. Circumvention of pharmacoresistance, or increasing clearance, may involve inhibitors of multi-drug transporters or sophisticated alternative therapies, but demonstration of overexpression or underactivity of transporter function is an essential and necessary first step. An in vivo imaging biomarker of multi-drug transporter function is essential for identifying altered transporter activity in individual patients. If a relation between overexpression and therapy resistance, or underactivity and AD, can be demonstrated, such a biomarker will provide the means for predicting treatment response, or early diagnosis, in individual patients. Approach and methodology: EURIPIDES is a four-year research programme based on a multidisciplinary approach integrating radiochemistry, molecular and cellular biology, physics, physiology, pharmacology, pathology and clinical research topics related to the most important neurological and neurodegenerative issues. The overall goal is to provide a non-invasive molecular imaging tool for the prediction, diagnosis, monitoring and prognosis of major CNS diseases, and to provide support and guidance for therapeutic interventions. It will be determined whether multi-drug transporters contribute to drug therapy resistance in diseases of the CNS. If transporters are shown to be important in mediating resistance, the development of strate-
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EURIPIDES
gies to modulate their function will be important, and are feasible with the development of highly specific, potent and non-toxic transporter inhibitors. The work programmes selected are complementary, symbiotic and cross-fertilising, and address the underlying methodological issues: (A) RTD activity area (organised according to the flow of developing radioactive molecular probes): radiotracer development; in vitro molecular characterisation of compounds; biological evaluation in animal models; proof-of-concept studies in animal models; evaluation in healthy human subjects; proof-of-concept studies in human CNS disease. In parallel with activities aimed at newly-developed P-glycoprotein tracers, the role of existing PET tracers for two major neurotransmitter systems will be explored: serotonin ([18F]-MPPF) and GABAA ([11C]-FMZ). Use will be made of the unique possibility in certain CNS disorders to validate and inform in vivo imaging findings by comparing with autoradiography and immunohistochemistry the sacrificed animals and brain specimens resected by surgery. (B) Management activity area, which consists of the following main activities: Technical coordination of EURIPIDES components. Harmonisation and standardisation procedures will be implemented and shared within the EURIPIDES consortium and applied to: a) animal models; b) acquisition and analysis of PET data; c) structural imaging (e.g. MRI) with standard protocols for the detection of structural abnormalities; d) collection of human biological samples (brain tissue, blood, cells, DNA, surgical and post-mortem specimens). Quality assurance and monitoring, especially financial status and scientific relevance of EURIPIDES. Knowledge management. The major activities are: a) internal dissemination including internal communication, workshops and training courses; b) external dissemination of research progress through peer-reviewed manuscripts, through reports in lay language to patient organisations, the general public and through its own website; c) reporting.
WP 9: Trainind and dissemination
WP 1: Synthesis of new P-g tracer
WP 2: Biological evaluation of new P-gp tracer animal PET studies
WP 3: In-tro inhibition and induction of P-gp
WP 7: P-gp in AD C-VPM C-PIB
WP 6: P-gp in epilepsy C-VPM New PgP tracer
WP 5: P-gp in healthy controls C-VPM New PgP tracer
WP 4: C-FMZ F-MPPF human PET studies
WP 4: C-FMZ F-MPPF animal PET studies
WP 8: Ex-vivo studies
WP 10: Project Management
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DIAGNOSTICS
Coordinator Matthias Johannes Koepp University College London 33 Queen Square London, WC1N 3BG, UK Email: mkoepp@ion.ucl.ac.uk Partners adriaan Lammertsma VU medisch centrum Amsterdam, Netherlands Ekaterina Patraia Medizinische Universitaet Wien Vienna, Austria Oliver Langer Austrian Research Centres GmbH Seibersdorf, Austria Wolfgang Loescher Stiftung Tierrztliche Hochschule Hannover Hannover, Germany Heidrun Potschka Ludwig-Maximillians-Universitaet Munich, Germany Philippe Ryvlin Hospices Civils de Lyon Lyon, France Karl Herholz University of Manchester Manchester, UK Munir Pirmohamed University of Liverpool Liverpool, UK Elizabeth de Lange Universiteit Leiden Leiden, Netherlands
Rob Voskuyl Stichting Epilepsie Instellingen Nederland Heemstede, Netherlands alexandra Varvarigou National Center for Scientific Research Demokritos Aghia Paraskevi, Athens, Greece Gitte Moos Knudsen Rigshospitalet Copenhagen, Denmark Birgit Fuchs GABO.mi (Gesellschaft fr Ablauforganisation: milliarium mbH & Co. KG) Munich, Germany
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ENCITE
European Network for Cell Imaging and Tracking Expertise
Grant agreement No Project type EC contribution Starting date duration HEaLTH-F5-2008-201842 Collaborative Project e 11 997 946 1 June 2008 48 months
Background and objectives: Cell therapy can be defined as the transplantation of living cells for the treatment of medical disorders. Three different principles underlie the increasing interest in cell therapy: 1. Transplanted cells used as an active drug. 2. Transplanted cells used to replace damaged and degenerated tissue. 3. Cells used as a drug delivery vehicle. Promising results have been obtained in preclinical and clinical studies. However, success rates have been variable and clinical benefits have been limited. A major issue is the fact that the mechanisms by which cell therapy works in the different disease areas are still poorly understood. The ability to non-invasively monitor the fate and modes of action of transplanted cells over time is mandatory. The development of relevant imaging tools will lead to a better understanding of how cell therapy works, increase the possibility of response monitoring in patients, and offer sufficient safety of the treatment. The ENCITE project will provide tools to allow this by developing: new imaging methods to improve the spatio-temporal tracking of labelled cells; dual and multimodality imaging procedures to cross-validate each individual approach; new contrast agents and procedures that will improve the sensitivity and specificity of cellular labelling;
a combination of molecular biology for the generation of molecular and cellular imaging reporters with multimodal imaging techniques; novel cell and animal reporter systems detecting the location and function of individual cells and small cell subsets within the target organ; cellular labelling that does not interfere with cellular functions and therapeutic efficacy; methods for quantitative assessment to generate reliable biomarkers of the cell fate and therapeutic effects; cell homing for therapeutic delivery to target organs.
The tools and methodologies developed will be validated in five key disease areas: neurological, cardiovascular, musculoskeletal, diabetes and cancer. Approach and methodology: The key work of the project will be carried out within Subprojects (SPs), which represent the relevant expertise of the participating groups. While SPs will concentrate on the integration of new methods, technologies and approaches, they will be broken down into individual Work Packages (WPs) which will concentrate on the scientific and technology activities, as well as provide well defined, specific objectives and milestones.
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DIAGNOSTICS
Matched images of iron-labeled cells. (A) Light microscopy image showing the contours of the cells. (B) Fluorescent microscopy image showing the fluorescent labeled nuclei of the cells. (C) MR image showing the susceptibility artifacts caused by the intra-cellular iron particles. (D) Overlay of the images shown in A. B. and C.
The SPs and WPs will be coordinated at the project level and the success of the project will be assured by well-defined interaction and communication channels coordinated at the project level between the coordinator, and the SP and WP leaders. Coordinator Gabriel Krestin European Institute for Biomedical Imaging Research (EIBIR) Neutorgasse 9/2A 1010 Vienna, Austria E-mail: g.p.krestin@erasmusmc.nl Partners Monique Bernsen Erasmus Medical Center Department of Radiology Rotterdam, Netherlands Mike Modo Kings College London Centre for the Cellular Basis of Behaviour & MRC Centre for Neurodegeneration Research London, UK Michal Neeman Weizmann Institute of Science Rehovot, Israel Silvio aime Universit di Torino Turin, Italy Milan Hjek Institute for Clinical and Experimental Medicine Department of Diagnostic and Interventional Radiology Prague, Czech Republic Juergen Henning University of Freiburg Freiburg, Germany
Mathias Hoehn Max Planck Gesellschaft zur Foerderung der Wissenschaften E.V. Colgone, Germany Gil Navon Tel Aviv University School of Chemistry Tel Aviv, Israel
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ENCITE
Robert Muller University of Mons-Hainaut General, Organic and Biomedical Chemistry Mons, Belgium Olivir Clment University Paris Descartes Laboratoire de Recherche en Imagerie EA 4062 Paris, France Gerold Schuler Friedrich-Alexander University ErlangenNurenberg Department of Dermatology Nuernberg, Germany Clemens Lowik Leiden University Medical Center Endocrinology, Leiden University Medical Center, postal zone C4-R Leiden, Netherlands Francesca Granucci The University of Milano Bicocca Department of Biotechnology and Bioscience Milan, Italy Carl Figdor Radboud University Nijmegen Medical Centre Department of Tumor Immunology Nijmegen, Netherlands Ignacio Melero Foundation for Applied Medical Research Gene Therapy and Hepatology- CIMA Pamplona, Spain diego amigorena Institut Curie Dept. U653, Immunit et cancer Paris, France Pierre-alix dancer BioSpace Paris, France Stefan Wecker Medres-Medical research GmbH Cologne, Germany Giovanni B. Giovenzana Cage Chemicals SRL Turin, Italy Ignacio Melero University of Navarra Gene Therapy and Hepatology Pamplona, Spain
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FP7 PROJECTS
molecular testing
Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics
Grant agreement No Project type EC contribution Starting date duration HEaLTH-F5-2008-222916 Collaborative Project e 9 000 000 (proposed) To be confirmed 48 months
SPIDIA
Background and objectives: In vitro diagnostics have allowed a great deal of progress in medicine but are limited by two factors: (1) the lack of guidelines in collection, handling, stabilisation and storage of biosamples which limits the reproducibility of subsequent diagnoses, and (2) its scale is limited to the cellular level. To address this first point, the Integrated Project SPIDIA, comprising clinicians, academics, tool developers and assay developers, aims to develop quality guidelines for molecular in vitro diagnostics and to standardise the pre-analytical workflow in related procedures. Regarding the second point, SPIDIA aims to develop modern pre-analytical tools for diagnostics, improving the stabilisation, handling and study of free biomolecules within blood, plasma, serum, tissues and tumours. Recent discoveries have revealed that RNA, DNA or proteins released from pathological sites such as tumour cells or Alzheimers disease (AD) brain lesions into the blood or as a secondary blood based response to the disease can serve as biomarkers for early and reliable molecular diagnosis of such debilitating diseases. Further discoveries have shown that the cellular profiles of these molecules and structures in clinical samples can change during transport and storage, thus making clinical assay results and pharmaceutical research unreliable or even impossible. It will therefore be a decisive prerequisite for future and current diagnostic assays to develop
standards and new technologies, tools and devices that eliminate the human error in the preanalytical steps of in vitro diagnostics. At this crucial moment in the development of molecular diagnostics, SPIDIA proposes a project that reunites eight private research companies (including four small and medium-sized enterprises (SMEs)), six public research organisations (including universities, hospitals and biobanks) and an official European Standards Organisation. This strong consortium is balanced and empowered to maximise the impacts of in vitro diagnostics on human health. Approach and methodology: SPIDIA is organised into three activities, each consisting of multiple Work Packages (WPs): 1. Evidence-based, international guidelines and quality-assurance schemes: to evaluate solutions developed by the consortium by using ISO-certified analysis platforms and ring-trials in order to gather evidence needed to improve critical steps in the pre-analytical workflow and gather the data required for standardisation activities; to submit these data and quality-assurance schemes to the CEN and its partner organisations, including ISO, and the IFCC and WHO (members of the club of interest) for conversion into official technical guidelines;
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Diagnostics - FP7 Projects - Molecular Testing
DIAGNOSTICS
to discover one or more sets of biomarkers that serve as quality assurance indicators for artificial, post collection changes of biological samples.
2. Research leading to pre-analytical tools for molecular in vitro diagnostics and classical pathology: to discover chemical compositions that enable the simultaneous stabilisation of multiple cellular and biomolecular targets in blood, tissue and non-invasive samples (i.e. respiratory and cervical swab samples); to create prototypes of integrated kits based on new, innovative collection and stabilisation; to develop technologies for tissue and blood samples that can stabilise RNA, DNA, proteins and metabolites while leaving the morphology and antigenicity of the cells and tissues intact (these technologies would have a unique market position worldwide, as no such technology is currently available); to create a fully automated system for use with stabilised blood samples which combines the pre-analytical tool described above with the first critical steps of the analytical assay setup, thereby eliminating potential for human error in the workflow; to provide a proof-of-principle for noninvasive sample collection techniques improved with respect to the stabilisation and recovery of biomolecules and simultaneous inactivation of pathogens; to evaluate and provide feedback encouraging improvement and innovation of guidelines and tools used for the discovery and validatation of biomarkers.
3. Management, ethics and spreading of excellence: to perform training to diffuse information about guidelines and discoveries to concerned authorities in the scientific, clinical and biobanking communities; to disseminate non-proprietary results to the community; to encourage ethical sensitivity and compliance; to efficiently manage SPIDIA; to optimise implementation and maximise impact. Coordinator Uwe Oelmller QIAGEN GmbH 1 Qiagen Strasse 40724 Hilden, Germany E-mail : uwe.oelmueller@qiagen.com Partners Kurt Zatloukal Medical University of Graz Graz, Austria Ivano Bertini Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche Sesto Fiorentino, Italy Robert Sjback TATAA BIOCENTER AB Gothenburg, Sweden Christian Lenz PreAnalytiX GmbH Hombrechtikon, Switzerland
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SPIDIA
anders Lnneborg DIAGENIC ASA Oslo, Norway Mogens Kruhoffer AROS Applied Biotechnology AS Aarhus, Denmark Nanna Christensen Dako Denmark AS Glostrup, Denmark Bndicte Charrin ACIES Lyon, France Mikael Kubista Institute of Molecular Genetics Czech Academy of Sciences Prague, Czech Republic anna von Groote European Committee for Standardization (CEN) Brussels, Belgium Sbastien Weisbuch ImmunID Technologies Grenoble, France Pierre Hainaut International Agency for Research on Cancer Lyon, France Mario Pazzagli University of Florence Florence, Italy Pieter Riegman Erasmus Medical Center Rotterdam Rotterdam, Netherlands Heinz Hfler Institute of Pathology of the Technical University Munich Munich, Germany
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DIAGNOSTICS
EURO-GENE-SCAN
European Genetic disease diagnostics
Background and objectives: Molecular techniques have become more efficient, increasingly precise and much cheaper, resulting in an unprecedented discovery rate of inherited disease genes. In areas such as primary immunodeficiencies (PID), muscle disorders, growth deficiencies, hearing/vision impairments and metabolic diseases, very large numbers of different genes have been found to carry mutations in diseases with heterogeneous clinical presentation. For example, mutations in almost 150 genes have been found to cause PID. This means that even for well-defined subgroups of PID, mutations in different genes result in identical, or overlapping, phenotypes. Current mutation analysis is very complex, and often involves many different European laboratories. Thus, individual laboratories carrying out mutation detection normally only cover a few percent of all disease genes. Obtaining a correct diagnosis is both difficult and time-consuming. If multiple genes need to be analysed, the cost rises proportionately. New sequencing approaches have been used for the analysis of whole genomes. The EURO-GENE-SCAN project partners will adapt these technologies, based on massive, parallel sequencing, to specific disease fields. This will involve the development of an innovative multiplexing technology. The proposed prototype area is PID, where significant collaboration in Europe has been continuing over
the last two decades. By using high-throughput sequencing, the partners estimate that the cost for analysing all known 150 PID genes in a single run will be in the same range as the current cost for mutation detection in single disease genes. They will also develop chips to identify single nucleotide polymorphisms (SNPs) for the study of modifier genes. In addition, they will develop reverse-phase protein arrays for proteomics approaches in the diagnostics of PID patients during infancy. They will also disseminate information and transfer the developed technologies to other disease areas. Approach and methodology: The overall strategy is to develop high-throughput technologies, which will become available to all Europeans. The aim is that parallel sequencing of PID genes will become operational by the end of the project. This is also true for the SNP chip and the reverse-phase protein (RPP) arrays technology. For RPP the aim is not to include all, but selected disease proteins, representing both secreted and intracellular targets. In order to develop the massive sequencing effort in a complementary way, partners will run in parallel the Roche 454 and the Solexa (Illumina 1G) technologies for high-throughput sequencing. Solexa may be especially suitable for this purpose.
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EURO-GENE-SCAN
Coordinator Edvard Smith Karolinska Institutet 5 Nobels vg 17177 Stockholm, Sweden Email: edvard.smith@ki.se Partners Bodo Grimbacher University College London London, UK Johannes Regenbogen GATC Biotech AG Konstanz, Germany Ewa Bernatowska Childrens Memorial Health Institute Warsaw, Poland Mats Nilsson Uppsala University Uppsala, Sweden
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DIAGNOSTICS
NMD-Chip
development of targeted dNa-Chips for High Throughput diagnosis of NeuroMuscular disorders

Grant agreement No Project type EC contribution Starting date duration HEaLTH-F5-2008-223026 Collaborative Project e 2 907 735 To be confirmed 36 months
Background and objectives: Inherited Neuromuscular Disorders (NMDs) form a large and very heterogeneous group of genetic diseases that cause progressive degeneration of the muscles and/or motor nerves that control movements. Most NMD types result in chronic long-term disability posing a significant burden to the patients, their families and public healthcare. Life is always shortened by multiple and cumulative defects that occur during disease progression. Premature death may result from cardiac and respiratory muscle involvement. These pathologies are present in all populations, affecting children as well as adults. The overall prevalence of NMDs is very difficult to evaluate, but one can estimate that, given the incidence of every different types, around 1 among 1 000 people may have a disabling inherited neuromuscular disease. The precise diagnostics of NMDs require a conjunction of extensive clinical examination and targeted complementary tests: biological analyses, electromyography, imaging, and histological analysis of biopsies. Since many gene mutations responsible for these diseases are known, molecular genetics analyses are performed, both to confirm the clinical diagnosis and to make precise the genotype configuration in each patient. However, one cannot avoid the difficulties in making a molecular diagnosis in those diseases due to frequent overlaps of clinical phenotypes,
a large number of genes, large genes that lack hot spots of mutation etc. Most of the molecular approaches currently used for genetic diagnosis correspond to gene by gene explorations, starting by the most pertinent gene. Thus, a differential molecular genotyping is required, which is highly complex and time-consuming (two weeks to one year) according to presently available technologies. As a consequence, many patients remain devoid of genetic confirmation of their disease: to date this proportion amounts to 30 to 40% of NMD. Importantly, new cutting-edge therapies, such as exon skipping, cannot be envisaged when no precise genetic diagnosis is available. To overcome this situation, novel genomicsbased technologies could present an efficient alternative to develop new molecular diagnosis tools, enabling quick, reliable and cost-effective sequencing of numerous NMD genes in parallel. In particular, the potential of DNA-chip arrays, designed to examine all possible mutations in relevant gene(s) in only one step process, could certainly help to increase the ratio of precisely diagnosed patients, ameliorate genetics counselling and patients management, establish phenotype-genotype correlations, construct dedicated databases and include patients in current or future clinical trials in relation to the TREAT-NMD network of excellence. Such an approach will also dramatically reduce the costs associated to the diagnosis in the medium term. From a long-
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NMD-Chip
term perspective, this will probably contribute to decreasing the global NMD prevalence by developing an appropriate diagnostic counselling. Aim: The aim of NMD-Chip is to design, develop and validate new sensitive high-throughput DNA arrays to efficiently diagnose patients affected by NMDs, namely Duchenne/Becker muscular dystrophies (DMD/BMD), limb girdle muscular dystrophies (LGMD), congenital muscular dystrophies (CMD), and hereditary moto-sensory neuropathies or Charcot-Marie-Tooth neuropathies (CMT). The new sensitive and reliable tools (reliability from 95 to >99%) originating from this project will allow assessing all known genes implied in a group of disease at one time (2 100 000 probes), and analysing efficiently chip data through optimised read-out bioinformatic tools, within 72 hours to 1 week and thus be cheaper than any gene by gene approach. Developing these NMD-Chips could allow decreasing molecular dignostics cost by a factor of 10. Approach and methodology: The present challenge lies in increasing the detection rate, as well as abbreviating the timeto-diagnosis (down to 72 hours to 1 week) for patients and families via characterising all mutations types and reducing analyses costs by
Today Biochemical characterisation Molecular diagnostic
Clinical consulation NMD patient
Clinical diagnostic
Biopsy Blood
Test the most pertinent gene 1 NO Clinical consulation
Diagnosed patient
Participation to clinical trials
Therapy
Up to 10 genes tested Test most pertinent gene X
Mutation in untested gene Undetected mutation Unknown mutation 2 weeks to 1 year
Undiagnosed patient (40% of NMD patients)
Tomorrow with NMD Chips NMD CHIP (Test 2,000,000 in one shot)
Clinical consulation NMD patient
Clinical diagnostic
Blood
Diagnosed patient
Participation to clinical trials
Therapy
Unknown gene mutated 72 hours to 1 week
Undiagnosed patient (YY% NMD patients)
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DIAGNOSTICS
using platforms with high diagnostic capacities. These two goals will also allow the consortium to characterise the genotype in rare and atypical phenotypes, in genetically ambiguous sporadic cases and in NMDs whose pathophysiology is multiallelic or multigenic. One must keep in mind that patients with a well-characterised pathology, both on clinical and genetic sides, are the only ones eligible for clinical trials or protocols, which become more and more numerous. DNA-Chips really correspond to a one-shot technology that may considerably reduce both the times and the costs of the whole diagnostic process. To achieve NMD-Chip aims, the partners will: design specific Sequence Capture DNA arrays containing all the genes already known to be involved in LGMD, CMD and CMT; design a whole gene CGH array containing all the genes already known to be involved in LGMD, CMD and CMT. Only on CGH-chip will be designed for all these pathologies; develop bioinformatic tools to accurately and quickly analyse DNA-Chip data; assess the quality of these chips. Several hybridisation tests will be performed to assess a good reproducibility and a strong efficiency. An important part of the work will be to adapt existing algorithms to the partners specific goals (e.g. lowering of the inclusion threshold); validate these DNA-arrays on pre-diagnosed patient samples and test their robustness on undiagnosed samples; design distinct candidate genes SC- and CGH-chips for LGMD, CMT and CMD; test patients with unidentified gene mutations with candidate genes chips. This last step will provide the consortium with information on the reliability of the tools developed.
known genes implied in a given group of NMD. This, coupled with a high-throughput sequencing technology (pyrosequencing), will bring a quick molecular diagnosis to patients. The project will also comprise the design and validation of one CGH-array chip to scan genes for large rearrangements, deletions or insertions. Then, if no deleterious mutation is found with the first run on Known Genes-chips, a second series of chips dedicated to candidate genes will be hybridised with the patients DNA. That means that every gene implied in a given NMD group will be checked at a glance, whereas until now, diagnostic laboratories have to sequence one gene after another until the mutation is found. If deleterious mutations are identified in known genes, the delay to diagnosis will be reduced to less than a week.
The developed chips will consist first in a series of chips dedicated to sequence capturing of all
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NMD-Chip
Coordinator Nicolas Levy Institut National de la Sant et de la Recherche Mdicale (INSERM) 101 Rue de Tolbiac 75654/13 Paris, France E-mail : nicolas.levy@medecine.univ-mrs.fr Partners Pascal Soularue PARTNERCHIP Evry, France Thomas Sejersen Karolinska Institutet Stockholm, Sweden david atlan PhenoSystems SA Lillois Witterzee, Belgium Clemens Mueller-Reible University Wuerzburg Wuerzburg, Germany Volker Straub University of Newcastle upon Tyne Newcastle, UK Veronika Karcagi National Institute of Environmental Health Budapest, Hungary Isabelle Richard GENETHON Evry, France Thomas Voit Association Institut de Myologie Paris, France alessandra Ferlini University of Ferrara Ferrara, Italy Johan den dunnen Leiden University Leiden, Netherlands Francesco Muntoni University College London London, UK angela Huebner University Dresden Dresden, Germany
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DIAGNOSTICS
TECHGENE
High throughput molecular diagnostics in individual patients for genetic diseases with heterogeneous clinical presentation
Background and objectives: Since completion of the sequencing of the human genome, the demand for genetic analysis in the human healthcare system is increasing dramatically, and the extension of molecular genetic diagnostics is urgently needed. However, the majority of genetic diseases are molecularly and clinically highly heterogeneous, and until recently the available techniques lacked the required capacity to analyse several genes in parallel. The recently introduced high-throughput whole genome sequencing (WGS) technology now offers the unique opportunity to extend molecular genetic analysis by introducing these techniques, and developing tailor-made medical resequencing approaches for molecular genetic diagnosis of heterogeneous disorders. The TECHGENE project aims to deliver crucial innovations leading to these approaches, and to deliver a proof-
of-principle for its implementation in selected model disorders. The model disorders have been selected with increasing genetic complexity and represent the majority of non-multifactorial genetic disorders. The current momentum to perform these innovations by a European consortium of clinical genetic diagnostic laboratories and research laboratories, and industrial stakeholders will lead to a front-running position of European laboratories and small and medium-sized enterprises (SMEs) in this field. The consortium consists of leading scientists and established laboratories providing cutting-edge knowledge with respect to quality management aspects, ethical and societal issues, and cost-effectiveness issues. This is the only approach that will warrant the development of diagnostic tools designed to restrict genetic testing to relevant medical factors. For European SMEs, this project offers the opportu-
WP8 Management of TECHGENE WP1 Hemoglobinopathies Breast Cancer Few Genes with Multiple Mutations Long PCR Enrichment WP2 Sensory D isorders 1 Major Gene & Multiple Minor Genes Validation WP3 Ataxia & Paraplegia Multiple Equally Important Genes Array Enrichment WP4 Mental Retardation Rare Mutations in Rare Genes Read-out & Data Anal
WP5 Ethical Issues
WP6 Economic Issues
WP7 Dissemination & Training
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TECHGENE
nity to identify niches in the steadily increasing molecular genetic market. A specially designed training programme will take care of rapid dissemination of the acquired knowledge and tools across Europe. Approach and methodology: The overall objective of TECHGENE is to improve molecular analysis for genetic disorders, which can be caused by multiple possible underlying mutations (allelic heterogeneity) in multiple possible underlying genes (locus heterogeneity). This general objective can be reached by the interdisciplinary genetic approach of clinical scientists, researchers, clinicians, and commercial companies involved in the project. Obviously, for all molecular techniques a sliding window in time will be present, after which the particular techniques may be surpassed in performance by others. However, the partners are convinced that they have chosen the most promising new technology and available platforms that are currently emerging for further development of novel clinical applications, and they extend the application of genetic analysis towards clinical entities with extensive genetic and/or clinical heterogeneity. Milan Macek Charles University 2nd School of Medicine Prague, Czech Republic Xavier Estivill Center for Genomic Regulation Barcelona, Spain Paolo Gasparini University of Trieste Trieste, Italy Olaf Riess Eberhard-Karls-Universitaet Tuebingen Tuebingen, Germany Bert Bakker and Johan den dunnen Leiden University Medical Center The Netherlands Bakker Leiden, Netherlands Sandro Banfi Telethon Foundation Naples, Italy Vincenzo Nigro Second University of Naples Naples, Italy Coordinator Hans Scheffer Radboud University Nijmegen Medical Centre 9 Geert Grooteplein Noord 6525 EZ Nijmegen, Netherlands E-mail : h.scheffer@antrg.umcn.nl Partners Gert Matthijs Katholieke Universiteit (KU) Leuven Leuven, Belgium Katrin Sak Asper Biotech Ltd Tartu, Estonia anne Cambon Thomsen University Paul Sabatier INSERM U558 Toulouse, France Katherine Payne University of Manchester Manchester, UK
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INDEX OF PROjECTS
addNET antePrion BONSaI CaRS Explorer COBREd daSIM dETECTHIV dIaGNOSIS diaNa diMI dRoP-ToP eBIOSENSE EdaR ENCITE EURIPIdES EuroFlow EURO-GENE-SCaN EUROGENGUIdE EuroGentest FLUOdIaMON FLUOROMaG FMT-XCT FUN OCT GENEPaRK 41 33 106 130 51 84 117 94 112 77 57 125 69 150 147 54 157 19 13 132 103 139 135 64 GLYFdIS HYPERImage IBdchip MEGMRI Moldiag-Paca NaCaRdIO NaNOMYC NanoSense NEMO nEUROPT NeuroScreen NeuroTaS NMd-Chip POC4life PREGENESYS QuaGSIC SaFE SKINSPECTION SLIC SPIdIa TB-trdNa TECHGENE TSEUR USdEP 44 141 30 143 47 120 114 110 100 137 71 88 159 91 67 28 22 145 123 154 62 163 37 97
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Diagnostics Index of Projects
DIAGNOSTICS
INDEX OF COORDINATORS
NETWORK OF EXCELLENCE Cassiman, Jean-Jacques (EuroGentest) Hughes, Kate (SAFE) Jacobs, Andreas H. (DiMI) INTEGRATED PROjECT Gress, Thomas M. (MolDiag-Paca) SPECIFIC TARGETED RESEARCH PROjECT Aguzzi, Adriano (TSEUR) Arndt-Jovin, Donna J. (FLUOROMAG) Borsella, Elisabetta (BONSAI) Enfors, Sven-Olof (eBIOSENSE) Gijs, Martin (DETECTHIV) Holthfer, Harry (ADDNET) Ikonomopoulos, John (NANOMYC) Isaksson, Olle (NACARDIO) Kutter, Jrg P. (NeuroTAS) Makohliso, Solomzi A. (SLIC) Peterlin, Borut (GENEPARK) Peters, Peter J. (AntePrion) Rabinovitz, Elisha (NEMO) Van Dongen, Jacques J.M. (EuroFlow) Visser, Pieter Jelle (EDAR) Zorzi, Willy (NeuroScreen) 40 105 109 127 119 43 116 122 90 124 66 35 102 55 70 73 49 16 24 80 SME-SPECIFIC TARGETED RESEARCH PROjECT Bois, Emmanuel (POC4life) Ellerbrok, Heinz (USDEP) Holthofer, Harry (DiaNA) Huggett, Jim (TB tr-DNA) Leiser, Robert-Matthias (DIAGNOSIS) Meiri, Hamutal (PREGENESYS) Ochoa, Gorka (DRoP-ToP) Porgador, Angel (GLYFDIS) Sans, Miquel (IBDchip) Skjeltorp, Guri (NanoSense) Takacs, Laszlo (COBRED) Weisbuch, Claude (QuAGSIC) SPECIFIC SUPPORT ACTION Kent, Alastair (EUROGENGUIDE) Moss, David (DASIM) COLLABORATIVE PROjECTS (FP7) Marguet, Didier (CARS Explorer) Widengren, Jerker (FLUODIAMON) Andersen, Peter (FUN OCT) Cubeddu, Rinaldo (nEUROPT) Ntziachristos, Vassilis (FMT-XCT) Schulz, Volkmar (HYPERImage) Ilmoniemi, Risto (MEGMRI) Schulz, Volkmar (SKINSPECTION) Koepp, Matthias Johannes (EURIPIDES) Krestin, Gabriel (ENCITE) Oelmller, Uwe (SPIDIA) Smith, Edvard (EURO-GENE-SCAN) Levy, Nicolas (NMD-Chip) Scheffer, Hans (TECHGENE) 131 133 136 138 140 142 144 146 149 151 155 158 162 164 21 87 93 99 113 63 96 68 61 46 31 111 53 29
Diagnostics Index of Projects
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Edited by Charles Kessler

NEW THERAPIES DIAGNOSTICS

Diagnostics - Table of Contents

IMAGING, NANOPARTICLES AND BIOSENSORS

Diagnostics - Table of Contents

FP7 PROjECTS Biomedical imaging

Diagnostics - Table of Contents

FP7 PROjECTS Molecular testing

INDEX OF PROjECTS INDEX OF COORDINATORS

55.9 17.8 73.7

Number of projects supported and EC financial contribution to diagnostics research

Funding schemes used in diagnostics research and EC financial contribution

Genetic Testing and Biomarkers

Diagnostics - Genetic Testing and Biomarkers

Diagnostics - Genetic Testing and Biomarkers

CANGENETEST EUROCARE-CF CAPABILITY PHGEN RELAGH

OECD CDC ACMG WHO EFB ESHG EUROPABIO

INDUSTRY SAFE NoE

Diagnostics - Genetic Testing and Biomarkers

Diagnostics - Genetic Testing and Biomarkers

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Diagnostics - Genetic Testing and Biomarkers

Diagnostics - Genetic Testing and Biomarkers

Diagnostics - Genetic Testing and Biomarkers