Diagnostics - CORDIS

Transcription

PROJECT SYNOPSES
Diagnostics
EUR 23347
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EUROPEAN COMMISSION
Directorate F – Health
Unit F5 – Health Biotechnology
Contacts
Philippe Jehenson
Jean-Luc Sanne
Office CDMA 2/123
Office CDMA 2/115
Tel (32-2) 29 86454
Tel (32-2) 29 92589
Fax (32-2) 29 94693
Fax (32-2) 29 94693
Directorate F – Health
Unit F5 – Health Biotechnology
Head of Unit
Arnd HOEVELER ([email protected])
Secretary: Andrea JAEGER ([email protected])
Diagnostics
Jean-Luc SANNE ([email protected])
Torbjoern INGEMANSSON ([email protected])
Philippe JEHENSON ([email protected])
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
DIAGNOSTICS
THERAPIES
TABLE OF CONTENTS
inTRODUCTION
Genetic Testing and
Biomarkers
7
11
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
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
51
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 Parkinson’s 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
5
TABLE OF CONTENTS
EDAR
Beta amyloid oligomers in the early diagnosis of
AD and as marker for treatment response
69
Imaging,
Nanoparticles and
Biosensors
75
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
6
NeuroScreen
Sensitive and differential blood and cerebrospinal
fluid test for neurodegenerative dementia
diagnostics
71
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
DIAGNOSTICS
SLIC
SLIC-Biosensors in molecular diagnostics:
nanotechnology for the analysis of species
specific microbial transcripts
124
FP7 Projects Biomedical imaging
eBIOSENSE
Electrical biosensor arrays for analyses of harmful
micro-organisms and microbial toxins
125
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
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
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
143
7
FP7 Projects Molecular testing
153
SPIDIA
Standardisation and improvement of generic
pre-analytical tools and procedures for in vitro
diagnostics
154
8
EURO-GENE-SCAN
European Genetic Disease Diagnostics
157
INDEX OF PROJECTS
166
INDEX OF
COORDINATORS
167
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
DIAGNOSTICS
Introduction
D
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
EU’s 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
9
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
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.
Number of projects
EC financial
contribution (million €)
FP6
Genetic testing and Biomarkers
18
61.7
Imaging, nanoparticles and biosensors
16
44.0
Total
34
105.7
10
55.9
4
17.8
14
73.7
FP7*
Biomedical imaging
Molecular testing
Total
* 2007 and 2008 only
10
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
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
EU’s 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.
Number of projects
EC financial contribution
(million €)
FP6
Integrated project
1
8.5
Network of excellence
3
32.7
Specific Targeted Research Project
16
36.4
SME-Specific Targeted Research Project
12
27.3
2
0.8
34
105.7
14
73.7
Specific Support Action
Total
FP7*
Collaborative project
* 2007 and 2008 only
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
11
12
Diagnostics
Genetic Testing and Biomarkers
14
Diagnostics - Genetic Testing and Biomarkers
DIAGNOSTICS
EuroGentest
Genetic testing in Europe – network for test development, harmonisation,
validation and standardisation of services
Contract No
Project type
EC contribution
Starting date Duration
Website
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
services;
•
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.
LSHB-CT-2004-512148
Network of Excellence
e 10 000 000
1 January 2005
60 months
www.eurogentest.org
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.
15
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
ORPHANET
EMQN
Unit 1
2
OECD
CDC
ACMG
WHO
EFB
ESHG
EUROPABIO
Database
CANGENETEST
EUROCARE-CF
CAPABILITY
PHGEN
RELAGH
ERNDIM
Quality Issues
3
6
Education
5
Technology
Clinic
4
INDUSTRY
SAFE NoE
Ethics, Legal
GeneBanC
16
ESHG
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.
17
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.
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).
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, 1103–1105.
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 Down’s syndrome diagnosis in
amniotic fluid in Guayaquil-Ecuador: The intiation
of a new era’, DS News, 2004, Vol 11, No 2.
18
Jean-Jacques Cassiman
Center Human Genetics/
Centrum Menselijke Erfelijkheid
Campus gasthuisberg
Herestraat 49, Box 602
B 3000 Leuven
Partners
Antoon Vyverman
Ascent Consultancy
Leuven, Belgium
Michael Morris
Geneva University Hospitals
Geneva, Switzerland
DIAGNOSTICS
Robert George Elles
The University of Manchester
Manchester, UK
Helena Kääriäinen
University of Turku
Turku, Finland
Clemens Reible-Müller
Bayerische Julius-Maximilians Universität Würzburg
Wuerzburg, Germany
Ulf Kristoffersson
Lunds Universitet
Lund, Sweden
György Fekete
Semmelweis University
Budapest, Hungary
Jörg Schmidtke
Medizinische Hochschule Hannover
Hannover, Germany
Michal Witt
International Institute of Molecular and Cell Biology
Warsaw, Poland
Jorge Sequeiros
Instituto de Biologia Molecular e Celular
Porto, Portugal
Milan Macek Jr
Charles University Prague
Prague 5, Czech Republic
Bert Bakker
Leiden University Medical Center
Leiden, Netherlands
David Barton
National University of Ireland, Dublin
Dublin, Ireland
Guillermo Antiñolo
Hospitales Universitarios Virgen del Rocîo-Servicio
Andaluz de Salud
Sevilla, Spain
Philippe Corbisier
European Commission, Joint Research Centre
Geel, Belgium
Rosalind Janice Hastings
Oxford Radcliffe Hospital
Oxford, UK
Brian Fowler
University Children’s Hospital Basel
Basel, Switzerland
Ségolène Aymé
Institut National de la Santé et de la Recherche Médicale
Paris, France
Bruno Dallapiccola
Istituto Casa Sollievo Della Sofferenza,
San Giovanni Rotondo
Rome, Italy
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 Cató de Santiago de Guayaquil
Guayaquil, Ecuador
19
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 investigação e serviços
em ciências biológicas lda
Oeiras, Portugal
David Atlan
Phenosystems SA
Brussels, Belgium
Juergen Oster
Chemagen Biopolymer – Technologie AG
Baesweiler, Germany
Irmgard Nippert
Westfälische Wilhelms-Universität Münster,
Universitätsklinikum Münster
Muenster, Germany
Domenico Coviello
Ospedale Maggiore Policlinico,
Mangiagalli e Regina Elena
Milan, Italy
20
DIAGNOSTICS
EUROGENGUIDE
Patient led education and development for genetic testing in research
and medicine
Contract No
Project type
EC contribution
Website
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
LSHB-CT-2006-518156
e 499 476
1 January 2007
36 months
www.gig.org.uk/teammember_
projectofficereuro1.htm
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
21
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.
22
DIAGNOSTICS
Coordinator
Alastair Kent
Genetic Interest Group Ltd
436 Essex Road
London, N1 3QP, UK
Robert Gerard
Rare Disorders Belgium
Jambes-Namur, Belgium
Ysbrand Poortman
World Alliance of Neuromuscular
Disorder Associations-Europe
Baarn, Netherlands
Partners
Domenico Coviello
University Hospital Milan
Milan, Italy
Selena Freisens
Central and Eastern European Genetic Network
Konstanz, Germany
David Bennett
Cambridge Biomedical Consultants Ltd
Delft, Netherlands
Irmgard Nippert
Frauengesundheits-forschung
Muenster, Germany
Cor Osterwijk
Vereniging Samenwerkende
Ouder- en Patiëntenorgani-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
23
SAFE
Special non-invasive advances in foetal and neonatal evaluation
Contract No
Project type
EC contribution
Website
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
24
LSHB-CT-2004-503243
e 12 000 000
1 March 2004
60 months
www.safenoe.org
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 SAFE’s 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.
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 (Down’s Syndrome), foetal sex, blood group,
cystic fibrosis, β-thalassemia, preeclampsia and
preterm labour. This involves the analysis of foe-
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 Down’s 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
consortium’s 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 β-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
25
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
Down’s 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 Down’s 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,
26
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
Scientific coordinator
Sinuhe Hahn
Universität Basel
Department of Research laboratory
for Prenatal Medicine
Spitalstrasse 21
4031 Basel, Switzerland
DIAGNOSTICS
Partners
Maj Hulten
Department of Biological Sciences
Coventry, UK
Ala Szczepura
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
King’s College London
Psychology and Genetics Research Group
London, UK
Lucia Savadori
Dipartimento di Scienze della Cognizione
e della Formazione
Università degli Studi di Trento
Rovereto, Italy
Ciara O’Sullivan
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
Ilona Hromadnikova
Charles University
2nd Medical Faculty
Prague, Czech Republic
Andreas Plesch
Metasystems GmbH
Altlussheim, Germany
Françoise Soussaline
IMSTAR SA
Paris, France
Jim Schröder
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
27
SAFE
Andres López Bernal
University of Bristol
Dorothy Hodgkin Building
Bristol, UK
Dorine Swinkels
University Nijmegen Medical Centre
Nijmegen, Netherlands
Jean-Marc Costa
American Hospital of Paris
Paris, France
Michael Siegrist
Universität Zürich
Division of Psychology
Zurich, Switzerland
Ariadni Mavrou
National and Kapodistrian University of Athens
Department of Medical Genetics
Athens, Greece
Gian Carlo Di Renzo
Universitá Degli Studi di Perugia
Department of Medical-Surgical Specialties and Public
Health
Perugia, Italy
Esther Guetta
Chaim Sheba Medical Center
Danek Gertner Institute of Human Genetics
Tel-Hashomer, Israel
Tobias Legler
Georg-August-Universität Göttingen
Transfusionsmedizin
Göttingen, Germany
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
28
Patrizia Paterlini-Bréchot and
Jean-Jacques Toulmé
Institut National de la Santé et
de la Recherche Médicale (INSERM)
Paris and Bordeaux, France
Mauro Buser
Statistik Buser
Riehen, Switzerland
Maurizio D’Esposito
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
DIAGNOSTICS
Yiming Wang
Sun Yat-Sen University
Guangxhou, People’s 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
Renate Burgemeister
P.A.L.M. Microlaser Technologies GmbH
Bernreid, Germany
Giovanni Romeo
Universitá 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
Beverly Searle
Rare Chromosome Disorder Support Group (Unique)
Caterham, UK
Michael Christiansen and Paal Skytt Andersen
Statens Serum Institut
Copenhagen, Denmark
Lyn Chitty
Institute of Child Health
London, UK
Ray O’Connor
Genomed Ltd
Paisley, UK
Lars Larsen
Københavns Universitet
Copenhagen, Denmark
Vincenzo Cirigliano
General Lab
Department of Molecular Genetics
Barcelona, Spain
Tadeusz Tyszka
Wyższa Szkoła Przedsiębiorczości i Zarządzania im.
Leona Koźmińskigo (LKAEM)
Warsaw, Poland
Cees Oudejans
VU Medical Centre
Etienne Mullet
Ecole Pratique des Hautes Etudes
Toulouse, France
Olivia Willcocks
Waypoint Systems Ltd
Shrewsbury, UK
29
QuAGSIC
Quantitative analysis of genes in single cells
Contract No
Project type
EC contribution
Website
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
children’s 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 ABC’s 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
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
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).
Picture of a semi-automatic 3D cellector
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:
30
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 1µl
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.
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:
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 - Bâtiment 404
Ecole Polytechnique
91128 Palaiseau, France
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
The technologies developed in QuAGSIC demand
a minimum amount of material to be analysed.
31
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)
Contract No
Project type
EC contribution
Website
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 Crohn’s 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.
LSHB-CT-2006-37319
SME-Specific Targeted
Research Project
e 2 467 314
1 December 2006
36 months
http://ibdchipproject.eu
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:
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
32
IBDchip anticipates the following results:
•
a fully validated innovative prototype IBD-
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.
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
Miquel Sans
Hospital Clínic / IDIBAPS
Department of Gastroenterology
Barcelona, Spain
Partners
Marta Artieda
Progenika Biopharma SA
Parque Tecnológico de Zamudio
Derio, Spain
Stéphane 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
33
IBDchip
Salvador Peña
VU University Medical Center
The Laboratory of Immunogenics
Department of Pathology
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
34
DIAGNOSTICS
AntePrion
Development of a preclinical blood test for prion diseases
Contract No
Project type
EC contribution
Website
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.
LSHB-CT-2006-019090
Specific Targeted
Research Project
e 2 450 000
1 June 2006
36 months
www.AntePrion.eu
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.
35
AntePrion
AntePrion’s 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
36
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.
AntePrion’s 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.
DIAGNOSTICS
Major publications:
Coordinator
Dollinger, S., Löber, 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.
Peter J. Peters
Nederlands Kanker Instituut
Department of Tumor Biology
Plesmanlaan 121 – H4
1066 CX Amsterdam, Netherlands
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.
Partners
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.
Albert Taraboulos
Department of Molecular Biology
Hebrew University Hadassah Medical School
Jerusalem, Israel
Krister Kristensson
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
37
AntePrion
Chiara Zurzolo
Institute Pasteur
Department of Cell Biology and Infection/
Unité Trafic Membranaire et Pathogenèse
Paris, France
Carsten Korth
Heinrich Heine Universität Düsseldorf
Department of Neuropathology
Düsseldorf, 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
Göttingen, Germany
H. Schuitemaker
Stichting Sanquin Bloedvoorziening
38
DIAGNOSTICS
TSEUR
An integrated immunological and cellular strategy for sensitive
TSE diagnosis and strain discrimination
Contract No
Project type
EC contribution
Website
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
LSHB-CT-2006-018805
Specific Targeted
Research Project
e 1 750 000
1 June 2006
36 months
www.pathol.uzh.ch/tseur
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 project’s 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-
39
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.
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.
40
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.
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
41
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
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
42
DIAGNOSTICS
ADDNET
Paradigm shift from kidney biopsies to advanced molecular diagnostics
from patient urine
Contract No
Project type
EC contribution
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
LSHB-CT-2003-503364
Specific Targeted
Research Project
e 2 000 000
1 January 2004
36 months
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.
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
43
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.
44
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.
DIAGNOSTICS
Coordinator
Harry Holthöfer
POB 21 (Haartmaninkatu 3)
00014 Helsinki, Finland
Partners
Klaus-Robert Müller
Fraunhofer Institute
Berlin, Germany
Per-Henrik Groop
Samfundet Folkhälsan i Svenska Finland r.f.
Helsinki, Finland
Jesus Egido
Fundación Jimenez Diaz
Autónoma University
Madrid, Spain
Kimmo Kaski
Helsinki University of Technology
Espoo, Finland
Peter N. Iversen
Ciphergen Biosystems A/S
Copenhagen, Denmark
45
GLYFDIS
Glycans in body fluids – potential for disease diagnostics
Contract No
Project type
EC contribution
Website
LSHB-CT-2006-037661
Research Project
e 2 793 722
1 November 2006
36 months
www.glyfdis.org
Developing effective tools to screen for cancer is
an important endeavor, where there is much research taking place. The GLYFDIS project’s 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 impor¬tance 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
46
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
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.
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.
RNTech’s role in GLYFDIS has three facets:
1.
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-
2.
3.
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.
Procognia
Procognia has developed a lectin-array based
technology for rapid analysis of glycosylation
profiles of intact glycoproteins. The array contains 25–30 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 protein’s 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).
47
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.
•
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.
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:
•
•
•
48
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-
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
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
DIAGNOSTICS
MolDiag-Paca
Novel molecular diagnostic tools for the prevention and diagnosis
of pancreatic cancer
Contract No
Project type
EC contribution
Website
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.
LSHB-CT-2006-018771
Integrated Project
e 8 495 490
1 August 2006
36 months
www.moldiagpaca.eu
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.
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
49
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.
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:
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.
50
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.
DIAGNOSTICS
Coordinator
Thomas M. Gress
Universitätsklinikum Gießen und Marburg GmbH
Standort Marburg
Klinik für Innere Medizin
SP Gastroenterologie und Endokrinologie
Baldingerstrasse
35043 Marburg, Germany
Björg V. Pauling
BMFZ-Building
Hans-Meerwein-Str. 2
35043 Marburg, Germany
Aldo Scarpa, Patrick Moore, Claudio Sorio
University of Verona
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
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
Jörg Hoheisel and Andrea Bauer
German Cancer Research Centre (DKFZ)
Division of Functional Genome Analysis
Heidelberg, Germany
Helmut Friess and Jörg Kleeff
University of Heidelberg
Department of General Surgery
Heidelberg, Germany
Ian Fleming and Simon Green
Cyclacel Ltd
Dundee, UK
Günther Klöppel and Bence Sipos
University of Schleswig-Holstein
Kiel, Germany
Martin Highett
Tepnel Life Sciences
Manchester, UK
Christoph Bremer
University of Münster
Department of Clinical Radiology
Muenster, Germany
Francisco Real (Paco) and Núria Malats
Institut Municipal d’Investigacio Medica, IMIM
Barcelona, Spain
51
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 Lütges
Stockholm, Sweden
Jörg Rademann and Jens Peter von Kries
Leipniz Institute for Molecular Pharmacology (FMP)
Berlin, Germany
52
DIAGNOSTICS
COBRED
Colon and breast cancer diagnostics
Contract No
Project type
EC contribution
Website
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
LSHB-CT-2007-037730
Research Project
e 2 985 102
1 March 2007
36 months
www.cobred.eu
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
53
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)).
54
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.
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.
Diagnostic kits for BC and CRC patient follow-up.
Coordinator
Laszlo Takacs
BioSystems International
4 rue Pierre Fontaine
91058 Evry cedex, France
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
László Fésüs
University of Debrecen
Debrecen, Hungary
Andras Guttman
Universität Innsbruck
Innsbruck, Austria
Jaak Vilo
University of Tartu
Tartu, Estonia
Bruno Cucinelli
ARTTIC
Paris, France
55
EuroFlow
Flow cytometry for fast and sensitive diagnosis and follow-up
of haematological malignancies
Contract No
Project type
EC contribution
Website
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-
56
LSHB-CT-2006-018708
Specific Targeted
Research Project
e 2 182 340
1 April 2006
36 months
www.EuroFlow.org
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.
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.
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
Partners
Alberto Orfao
University of Salamanca
Salamanca, Spain
Frank J.T. Staal
DYNOMICS
Rotterdam, Netherlands
Marta Martin-Ayuso
CYTOGNOS
Salamanca, Spain
António Parreira
Instituto Portugués de Oncologia
Lisbon, Portugal
Michel Kneba
University of Schleswig-Holstein
Kiel, Germany
Elisabeth Macintyre
Hôpital Necker-Enfants Malades
Paris, France
57
EuroFlow
Stephen Richards
University of Leeds
Leeds, UK
Jan Trka
Charles University
Tomasz Szczepanski
Silesian Academy of Medicine
Zabrze, Poland
58
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
Website
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-
LSHB-CT-2006-037739
Research Project
e 1 834 331
1 January 2007
36 months
www.drop-top.net
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
59
DRoP-ToP
tumour cannot be cured by surgery. One limitation is that the pathologist’s interpretation of the
tumour’s 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:
60
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
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).
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 project’s 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.
62
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.
Diagnosis and prognosis for bladder cancer and
other diseases.
DIAGNOSTICS
Coordinator
Gorka Ochoa
Progenika Biopharma, SA
Parque Tecnológico de Bizkaia
Edificio 801 B
48160 Derio-Vizcaya, Spain
Partners
Francois Radvanyi
Institut Curie-CNRS
Paris, France
Nuria Malats
Universitat Pompeu-Fabra
Institut Municipal d’Investigacio 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
63
TB-trDNA
Evaluation of transrenal-DNA detection to diagnose tuberculosis
Contract No
Project type
EC contribution
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-
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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
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.
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 : [email protected]
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
65
GENEPARK
Genomic biomarkers for Parkinson’s disease
Contract No
Project type
EC contribution
Website
Parkinson’s disease (PD) is the second most
prevalent neurodegenerative disease after
Alzheimer’s 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
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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.
DIAGNOSTICS
In its preliminary work, the GENEPARK team used
genome-wide expression profiling of human
blood to identify biomarkers for Huntington’s 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.
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.
Major publications:
Binkofski, F., Reetz, K., Gaser, C., Hilker, R.,
Hagenah, J., Hedrich, K., v Eimeren, T., Thiel, A.,
Büchel, 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.
Coordinator
Borut Peterlin
University Medical Centre
Division of Medical Genetics
Slajmerjeva 3
1000 Ljubljana, Slovenia
Partners
Alexis Brice
Institut National de la Santé et
de la Recherche Médicale (INSERM)
Paris, France
Christine Klein
University of Lüebeck and Neuroimage Nord
Lüebeck, Germany
Dimitri Krainc
Mediterranean Institute for Life Sciences
Split, Croatia
Gorka Ochoa
Progenika Biopharma SA
Vizcaya, Spain
Olaf Riess
Eberhard-Karls-University Tüebingen
Tüebingen, Germany
Ron Shamir
Tel Aviv University
Tel Aviv, Israel
Mojca Zajc
RR & CO. Business Consulting, d.o.o.
Ljubljana, Slovenia
68
DIAGNOSTICS
PREGENESYS
Development of early non-invasive biomarkers and means for the diagnosis
and progression monitoring of preeclampsia and tailoring putative therapies
Contract No
Project type
EC contribution
Website
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 consortium’s working hypothesis is: first
trimester markers are the key for diagnosis and
tailor putative medications for preventing preeclampsia. The group has an unprecedented rich
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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.
1.
2.
3.
4.
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-
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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.
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.
Coordinator
Hamutal Meiri
Diagnostic Technologies Ltd
2 Ha’Carmel St., Building B 4th Floor
20692 Yokneam, Israel
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
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.
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
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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
Website
LSHB-CT-2005-037670
Specific Targeted
Research Project
e 621 002
1 January 2007
36 months
www.edarstudy.eu/
Alzheimer’s 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.
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.
Wiep Scheper
University of Amsterdam
Academic Medical Centre
Lars-Olof Wahlund
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
Coordinator
Pieter Jelle Visser
VU University
Outpatient Clinic
PO Box 5075
1007 MB Amsterdam, Netherlands
Partners
Michael Adler
Chimera Biotech
Dortmund, Germany
Wim Buurman
Hycult Biotechnology
Uden, Netherlands
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Frans Verhey
University of Maastricht
Maastricht, Netherlands
Heinz Hillen
Abbott GmbH & Co KG
Ludwigshafen, Germany
DIAGNOSTICS
NeuroScreen
Sensitive and differential blood and cerebrospinal fluid test
for neurodegenerative dementia diagnostics
Contract No
Project type
EC contribution
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 Alzheimer’s
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 α-synuclein (e.g. Parkinson’s 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,
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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 Alzheimer’s 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.
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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NeuroScreen
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 Alzheimer’s 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.
•
Aim:
•
Aim:
•
Aim:
•
Aim:
•
Aim:
•
Aim:
•
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: α-synuclein marker.
development and validation of a suitable
assay for α-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.
Expected outcome:
Medical objectives
The project concerns public health for neurodegenerative diseases, which are experiencing
74
constant growth. The medical objectives are as
follows:
1)
to diagnose the kind of disease (especially
CJD, Alzheimer’s disease, Lewy Body Dementia) with specific markers, e.g. pathological Prion Protein (PrPsc), abnormally
phosphorylated tau proteins (tau-181 and
tau 231) and pathological α-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 Alzheimer’s 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 α-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.
DIAGNOSTICS
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
Université de Liège
Centre de Recherche sur les Protéines Prion
Institut de Pharmacie
B36, n°1 avenue de l’Hôpital
4000 Liège, Belgium
Partners
Gilbert Legeay and Arnaud Coudreuse
Association pour les Transferts de Technologies du Mans
Le Mans, France
Jean-Francois Delforge, Benjamin Klein,
Isabelle Zitte, Julien Lacauste
ALCIS
Besançon, France
Awad Osman, Daniela Liche,
Heinrich Ina, Ingolf Lachmann
AJ ROBOSCREEN
Leipzig, Germany
Fabienne Poncin-Epaillard
Université du Maine
Le Mans, France
Armand Perret-Liaudet,
Isabelle Quadrio, Jérémie Seguin
CHU-Lyon
Hôpital Neurologique
Laboratoire Diagnostic Maladies à 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 Földvári, Katalin Majtényi
Orszagos Pszichiatriai es Neurologiai Intezet
Hungary, Budapest
Andreas Kage and Kathleen Grüttner
Charité – Universitätsmedizin Berlin
Institut für Laboratoriumsmedizin und Pathobiochemie
Berlin, Germany
Thierry Daviet
EUDICA
Annecy-le-Vieux, France
75
76
Diagnostics
Imaging, Nanoparticles and Biosensors
78
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
Website
LSHB-CT-2005-512146
e 10 700 000
1 April 2005
60 months
www.dimi.eu
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
Inflammation
& Regeneration
In vivo detection of
transcriptional regulation
and migration of
inflammatory
and stem cells
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)
Integrating Activities
Sharing facilities, equipment
Exchange of personnel
Integration of SMEs
Dissemination Activities
Training/Education
Communication
Exploitation
Innovations, Exploitations, Publications,
Training, Meetings, Common Knowledge
79
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.
80
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
DIAGNOSTICS
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.
Post-contrast (0.5 mmol/kg GdDTPA) T1W image of a rat
acquired at 4.7T following infection with Streptococcuspneumoniae. Note the significant meningeal
•
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
•
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)-2βcarbomethoxy-3β-(4’-tolyl) nortropane (LBT-999)
81
DiMI
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 Parkinson’s 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.
Ann Planas
Institut d’Investigacions Biomèdiques August Pi i
Sunyer (IDIBAPS)
Barcelona (Spain)
Adriana Maggi
University of Milan
Centre of Excellence on Neurodegenerative Diseases
Milan, Italy
Coordinator
Gitte Moos Knudsen
Copenhagen University Hospital Rigshospitalet
Neurobiology Research Unit
Copenhagen, Denmark
Andreas H. Jacobs
University of Cologne
Laboratory for Gene Therapy and Molecular Imaging
Gleuelerstr. 50
50931 Cologne, Germany
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
Partners
Chrit Moonen
Université Victor Segalen Bordeaux
Molecular and Functional Imaging: From Physiology
to Therapy Technological Research Team
Bordeaux, France
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
82
Soraya Benderbous
University of Tours
Laboratoire Biophysique Médicale et parmaceutique
Tours, France
Pascal Laugier
Université Paris VI
Targeted and Functional Ultrasound
Laboratoire d’Imagerie Paramétrique
UMR 7623 CNRS
Paris, France
Roland Matrippolito
University of Paris Sud
Institute of Nuclear Physics
UMR 8608 CNRS
Paris, France
DIAGNOSTICS
Markus Schwaiger
Technical University of Munich
Department of Nuclear Medicine
Munich, Germany
Deniz Kirik
Lund University
Department of Physiological Sciences
Lund, Sweden
Harald Carlsen
University of Oslo
Faculty of Medicine
Institute for Nutrition Research and
Institute of Immunology
Oslo, Norway
Rikard Holmdahl
Lund University Medical Faculty
Medical Inflammation Research (MIR) at
the Biomedical Center
Lund, Sweden
Bertrand Tavitian
CEA - Experimental molecular
medicine laboratory
Orsay, France
Philippe Hantrave
CEA – The ImaGene Program
Fontenay-aux-Roses / Orsay, France
Philippe Rizo
CEA – LETI: Département Systèmes
pour l’Information et la Santé
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
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 Carrió
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
Department of Clinical Neuroscience
Psychiatry Section
Stockholm, Sweden
83
DiMI
Agneta Nordberg
Department Neurotec
Division of Molecular Neuropharmacology
Uppsala, Sweden
Sabina Pappata
University of Naples ‘Federico II’
CNR Naples and Department of Clinical
and Experimental Medicine
Naples, Italy
Andreas Bauer
Research Centre Juelich
Institute of Medicine
Juelich, Germany
Alberto Auricchio
University of Naples
Telethon Institute of Genetics and Medicine
Biostructure and Bioimaging Institute
of the National Research Center
Naples, Italy
Matthias Hoehn
MPI for Neurological Research
In vivo NMR Laboratory
Cologne, Germany
Chris Reutelingsperger
Cardiovascular Research Institute Maastricht
Maastricht, Netherlands
Michael Horn
University of Göteborg
Center for Bio-Imaging
Gothenburg, Sweden
Ferrucio Fazio
Vita-Salute San Raffaele University
Scientific Institute Hospital San Raffaele
Institute of Bioimaging and Molecular Physiology
CNR
Milan, Italy
Andreas Jacobs and Wolf-Deiter Heiss
University Medical Center Groningen
Department of Neurology and Neuroimaging Center
Groningen, Netherlands
Adriana Gittenberger-de Groot
Leiden University Medical Centre
Department of Anatomy and Embryology
Leiden, Netherlands
Philip Elsinga
Groningen PET-Center
Hans Romijn
Leiden University Medical Centre
Endocrinology Research Laboratory
Department of Endocrinology
Leiden, 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
84
David Parker
University of Durham
Department of Chemistry
Durham, UK
Eric Salmon
University of Liege
Cyclotron Research Centre
Liege, Belgium
DIAGNOSTICS
Michael Schäfers
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
Eva Sykova
Charles University
Institute of Experimental Medicine ASCR
Department of Neuroscience and Center for Cell Therapy
and Tissue Repair
Ulrich Bogdahn
University of Regensburg
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
Bodo Levkau
University of Duisburg-Essen
Institute of Pathophysiology
University Hospital Essen
Essen, Germany
Stefan Wecker
Medical Research GmbH
Cologne, Germany
Ivan Lukes
Charles University
Department of Inorganic Chemistry and
Department of Organic Chemistry
Renata Mikolajczak
POLATOM
Research and Development Department
of Radioisotope Centre
Otwock, Poland
85
DASIM
Diagnostic Applications of Synchroton Infrared Microspectroscopy
Contract No
Project type
EC contribution
Website
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.
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.
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.
At each of Europe’s 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.
86
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e 280 000
1 July 2005
36 months
www.dasim.eu
DIAGNOSTICS
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.
87
DASIM
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.
88
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, 1621–1631.
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., Podgórczyk, M., ‘Micro and bulk analysis of
prostate tissues classified as hyperplasia’, Spectrochimica Acta Part B: Atomic Spectroscopy, 2007,
62, 707-710.
DIAGNOSTICS
Lyng, F., Ó Faoláin, 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
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
89
NeuroTAS
Microfluidic total analysis system for the early diagnostic
of neurodegenerative disorders
Contract No
Project type
EC contribution
Website
The NeuroTAS project aims to develop a prototype of a miniaturised system for diagnostics in
the early stage of Alzheimer’s 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 β 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
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LHSB-CT-2006-037953
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Research Project
e 2 499 999
1 January 2007
36 months
www.neurotas.eu
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.
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-
DIAGNOSTICS
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 Aβ
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 Aβ 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 Aβ peptides, specific
antibodies against several β 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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NeuroTAS
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 Aβ 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.
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Coordinator
Jörg P Kutter
Technical University of Denmark
Department of Micro and Nanotechnology
Building 344
2800 Lyngby, Denmark
Jean Louis Viovy
UMR 168 CNRS/Curie Institute
26 Rue d’Ulm
75005 Paris, France
Partners
Myriam Taverna
University of Paris-Sud
Paris, France
Zusana Bilkova
University of Pardubice
Pardubice, Czech Republic
Frédéric Reymond
Diagnoswiss
Lausanne, Switzerland
Markus Otto
University of Ulm
Ulm, Germany
Jens Wiltfang
University of Erlangen-Nürnberg
Erlangen, Germany
DIAGNOSTICS
POC4life
Multiparametric quantum dot bioassay for point of care diagnosis
Contract No
Project type
EC contribution
Website
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 15–20% problematic or late diagnosed cases. The study will
pay special attention to women (lung cancer
death rates for women have been increasing
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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 project’s 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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POC4life
Expected outcome:
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.
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).
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.
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Multiparametric diagnostics in the following fields:
cancer, prenatal diagnostics, sepsis, cardiac
DIAGNOSTICS
Emmanuel Bois
Cezanne SAS
280 allée Graham Bell
Parc Scientifique Georges Besse
30035 Nîmes cedex 1, France
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 Löhmannsröben
University of Potsdam
Potsdam – Golm, Germany
Yves Caristan
Commissariat a l’Energie 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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DIAGNOSIS
Development of new and cost-effective methods for non-invasive diagnosis
of human pathogens
Contract No
Project type
EC contribution
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
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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.
DIAGNOSTICS
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 Europe’s 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 products
New principles for
NA separation
and purification
Kits for ultra-rapid
separation and
purification of DNA
and RNA
New sorbent
methods and
materials
Kits for separation
and purification of NA
from difficult (inhibitor rich) samples
Protocols for sample
preparation
Kits for separation of
different classes of NA
Protocols for samples
target concentration
Kits for non-cultural
enrichment of
micro-organisms
Kits for diagnostic
Chemistry for oligoassays of critical
nucleotides synthesis
pathogens
Multiplex PCR
Prototype instrument
for fluorescent
detection
Protocols for
diagnostic assays
Portable PCR
laboratory
Protocols for POC
Kits for sample
(point of care) sample preparation outside
preparation
the laboratory
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DIAGNOSIS
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.
Robert-Matthias Leiser
Agrobiogen GmbH
Thalmannsdorf 25
Larezhausen
86567 Hilgertshausen, Germany
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
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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
Charité – Universitätsmedizin Berlin
Institut für Mikrobiologie und Hygiene
Berlin, Germany
Pnina Dan
OSM-DAN Ltd
Rehovot, Israel
DIAGNOSTICS
USDEP
Capture and enrichment of emerging pathogens for multiple
and ultra-sensitive diagnostic
Contract No
Project type
EC contribution
Website
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-
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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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USDEP
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
100
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.
DIAGNOSTICS
While the publicly funded partners — as well as
the Robert Koch Institut (RKI), the Institut de Recherches pour le Développement (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 für 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.
Heinz Ellerbrok
Robert Koch-Institut
Centre for Biological Safety
Nordufer 20
133353 Berlin, Germany
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 Católica de Chile
Santiago, Chile
Roland Lauster
GenExpress Gesellschaft für Proteindesign mbH
Berlin, Germany
Rainer Hintsche
Fraunhofer Institut für Silizium Technologie
Itzehoe, Germany
Ralf Wörl
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
Website
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
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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.
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
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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NEMO
Coordinator
Elisha Rabinovitz
Given Imaging Ltd
2 Hacarmel Street
20692 Yoqneam, Israel
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
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Elie Berdugo
Ernst & Young (Israel) Ltd
Tel Aviv, Israel
DIAGNOSTICS
FLUOROMAG
Multiparameter sensing for high sensitivity diagnostics using fluorescent
and magnetic nanoparticles
Contract No
Project type
EC contribution
Website
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
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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.
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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FLUOROMAG
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.
Main findings:
•
•
•
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.
106
•
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, Β., 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.
DIAGNOSTICS
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 Göttingen, Germany
Partners
Vinod Subramaniam
University of Twente
Department of Biophysical Engineering
Enschede, Netherlands
M. Arturo López-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 López de Rio
NANOGAP-subnm-powders SA
Santiago de Compostela, Spain
107
BONSAI
Bio-imaging with smart functional nanoparticles
Contract No
Project type
EC contribution
Website
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.
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.
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.
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www.bonsai-project.eu
BONSAI’s 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
DIAGNOSTICS
Fig. 1: Schematic of laser pyrolysis set-up (courtesy of
partner CEA).
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).
Fig. 2: Si nanoparticles
prepared at ENEA by laser
pyrolysis (HREM-TEM
image: care of partner
UNIPD)
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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BONSAI
•
•
•
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.
110
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
DIAGNOSTICS
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 project’s 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.
Giovanni Mattei
Università 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
Università di Milano-Bicocca
Dipartimento di Medicina Sperimentale, Ambientale e
Biotecnologie Mediche
Milan, Italy
Coordinator
Elisabetta Borsella
ENEA (Ente Nazionale per le Nuove Tecnologie, l’Energia
e l’Ambiente)
Via Enrico Fermi 45
Frascati (Roma), Italy
Jean Marc Idee
GUERBET
Aulnay-sous-Bois, France
Vladimir Pustovoy
General Physics Institute
Natural Sciences Centre
Moscow, Russia
Partners
Nathalie Herlin-Boime
CEA
Commissariat à l’Energie Atomique-Direction des
Sciences de la Matière
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
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
111
NanoSense
Moving sensitive immunoassays from slow and expensive to fast
and affordable nanoparticle-based methods
Contract No
Project type
EC contribution
Website
Role of SMEs
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.
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
chem¬istries and bioprocessing of protein conjugates and nanoparticles). The consortium
also includes a large industrial company, Merck
Chimie SAS, which is Europe’s largest producer of
nanoparticles, and an academic participant, the
Groningen University Medical Center, providing
a reference laboratory for measurements in large
epidemiological studies.
Traditionally, immunoassays have been separation based, meaning that the analyte of in¬terest
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.
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www.nanosense.eu
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 corpora¬tions.
The NanoSense partners foresee that similar ef-
DIAGNOSTICS
fects will emerge when such technology is developed for large molecule markers as well.
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
Partners
Erling Sundrehagen and Ingrid Hulthén
Getica AB
Tösse, Sweden
Richard Vidal and Cécile Genies
Merck Chimie SAS Estapor
Fontenay–Sous–Bois, France
Uzonka Farkas and Péter Jakus
77 Elektronika Muszeripari Kft
Budapest, Hungary
Dick de Zeeuw and Stephan J. L. Bakker
Groningen University Medical Center
Department of Clinical Pharmacology
113
DiaNa
Predictive diagnostics for diabetic nephropathy — novel nanotechnology
based test platforms
Contract No
Project type
EC contribution
Website
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%
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www.diana-eu.fi
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.
DIAGNOSTICS
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.
Coordinator
Harry Holthofer
Haartman Institute
Haartmaninkatu 3
FI-00014 University of Helsinki, Finland.
and
The applications will include novel magnetosensor-based sensors for detection of diabetes-diabetic kidney damage.
National Centre for Sensor Research/
BioAnalytical Sciences
Dublin City University
Dublin 9, Ireland
Partners
Per-Henrik Groop
Samfundet Folkhälsan
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
115
NANOMYC
Multiparametric detection of bio-molecule conjugated nanoparticles for the
diagnostic investigation of mycobacterial infections of humans and animals
Contract No
Project type
EC contribution
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 Crohn’s 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 Crohn’s 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-
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Research Project
e 2 194 020
1 January 2007
36 months
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 Crohn’s
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).
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
DIAGNOSTICS
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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NANOMYC
dysregulation that has been associated with various manifestations, including soarcoidosis (the
primary cause of eye-loss in children), Crohn’s
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.
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 Čomor
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
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
118
Palmiro Poltronieri
Biotecgen Srl
Lecce, Italy
Joers Titt
Quattromed Ltd Molecular
Diagnostic Laboratory
Tartu, Estonia
Birgit Glaubitz
Medizone GmbH
Munich, Germany
Maria Gazouli
University of Athens
School of Medicine
Department of Biology
Panos Malamis
Malamis & Malamis Co
Athens, Greece
DIAGNOSTICS
DETECTHIV
Sensitive nanoparticle assay for the detection of HIV
Contract No
Project type
EC contribution
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.
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
LSHP-CT-2006-037118
Specific Targeted
Research Project
e 2 026 260
1 January 2007
36 months
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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DETECTHIV
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.
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.
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.
120
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.
DIAGNOSTICS
Coordinator
Martin Gijs
Ecole Polytechnique Fédérale de Lausanne
Swiss Federal Institute of Technology Lausanne
Institute of Microelectronics and Microsystems
1015 Lausanne, Switzerland
Partners
Marc Van Ranst
Katholieke Universiteit Leuven
Clinical and Epidemiological Virology
Aids Refrence Lab
Leuven, Belgium
Solly Makholiso
Ayanda Biosystems SA
Bruno Vallayer
Bertin Technologies
Biotech Systems Department
Montigny-le-Bretonneux, France
Jérôme Bibette
Ecole Supérieure de Physique et
de Chimie Industrielles de la ville de Paris
Laboratoire Colloïdes et Matériaux Divisés
Paris Cedex 05, France
Jörg P. Kutter
MIC – Department of Micro and Nanotechnology
Lyngby, Denmark
Bernard Plichon
ADEMTECH SA
Pessac, France
121
NACARDIO
Nanoparticle-based electronic biosensor for diagnostics
of cardiovascular disease
Contract No
Project type
EC contribution
Website
LSHB-CT-2006-037672
Specific Targeted
Research Project
e 2 221 185
1 October 2006
36 months
www.nacardio.com
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. State–of-the-art
nanofabrication utilising metallic nanoparticles
now make this technology platform available for
operation at room temperature. SET-technology
provides unique possibilities for biosensing.
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 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.
122
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
DIAGNOSTICS
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.
123
NACARDIO
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
Göteborgs Universitet
Department of Medicine
Gröna stråket 8
413 45 Göteborg, Sweden
Partners
Linda Olofsson
Midorion AB
Göteborg, Sweden
Albert van den Berg
University of Twente
Twente, Netherlands
Serge Muyldermans
Vlaams Interuniversitair Instituut Biotechnologie
Brussels, Belgium
Sergey Kubatkin
Chalmers University of Technology
Göteborg, Sweden
D Mathias Brust
The University of Liverpool
Liverpool, UK
124
DIAGNOSTICS
SLIC
SLIC-Biosensors in molecular diagnostics: nanotechnology for the analysis
of species specific microbial transcripts
Contract No
Project type
EC contribution
Website
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
LSHB-CT-2005-513771
Specific Targeted
Research Project
e 1 999 980
1 January 2005
36 months
www.fp6-slic.eu
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
consortium’s 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.
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-
125
SLIC
•
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.
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 Institute’s ‘Nucleic-Acid Based Technologies’ conference, 2006.
126
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
CH-1015 Lausanne, Switzerland
Partners
Horst Vogel
Institute of Chemical Sciences & Engineering
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
DIAGNOSTICS
eBIOSENSE
Electrical biosensor arrays for analyses of harmful micro-organisms
and microbial toxins
Contract No
Project type
EC contribution
Website
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.
LSHB-CT-2004-512009
Specific Targeted
Research Project
e 2 370 900
1 January 2005
36 months
www.ebiosense.org
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.
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.
127
eBIOSENSE
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
128
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.
DIAGNOSTICS
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 µL sample.
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
Partners
Rainer Hintsche
The Fraunhofer Institute for Silicon Technology
Itzehoe, Germany
Thomas Schweder
Ernst-Moritz-Arndt Universität
Greifswald, Germany
Peter Neubauer
University of Oulu
Oulu, Finland
Grzegorz Wegrzyn
University of Gdansk
Gdansk, Poland
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-
Lars Blohm
eBiochip Systems GmbH
Itzehoe, Germany
Antje Breitenstein
Scanbec Oy
Oulu, Finland
129
eBIOSENSE
Cees Waalwijk
Plant Research International BV
Wageningen, Netherlands
Julien Bourjault
Centre de Recherche et d’Étude
pour l’Alimentation
Paris, France
Mika Tuomola
Raisio Benecol Ltd
Raisio, Finland
130
FP7 PROJECTS
Biomedical imaging
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
rant Agreement No
G
Project type
EC contribution
Website
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.
HEALTH-F5-2008-200820
Collaborative Project
e 3 091 129
1 March 2008
36 months
http://www.carsexplorer.eu
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.
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;
132
Diagnostics - FP7 Projects - Biomedical Imaging
DIAGNOSTICS – FP7
•
•
WP3, photonic crystal fibres for NLO
imaging;
WP4, signal recovery and imaging
processing.
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.
Partners
Hervé Rigneault
Centre National
de la Recherche Scientifique (CNRS)
Institut Fresnel
Marseille, France
Benoît Van den Eynde
De Duve Institute
Brussels, Belgium
Andreas Volkmer
Universität Stuttgart
Stuttgart, Germany
Jonathan Knight
University of Bath
Bath, UK
Coordinator
Didier Marguet
Institut National de la Santé
et de la Recherche Médicale (INSERM)
18 Avenue Mozart
13276/09 Marseille, France
François Lacombe
Mauna Kea Technologies
Paris, France
Christiane Dascher-Nadel
Inserm Transfert SA
Marseille, France
133
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
Grant Agreement No
Project type
EC contribution
HEALTH-F5-2008-201837
e 4 197 774
1 July 2008
42 months
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.
134
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.
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
DIAGNOSTICS
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
Partners
Hans Wiksell
VibraTech AB
Stockholm, Sweden
Gert Auer
Stockholm, Sweden
Stefan Hell
Max-Planck-Gesellschaft zur Förderung der
Wissenschaften e.V.
Institute for Biophysical Chemistry
Goettingen, Germany
135
FLUODIAMON
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 Hänninen
University of Turku
Turku, Finland
Sampsa Hautaniemi
Helsinki, Finland
Silvere van der Maarel
Academisch Ziekenhuis Leiden
(Leiden University Medical Center)
Leiden, Netherlands
136
DIAGNOSTICS
FUN OCT
Functional Optical Coherence Tomography
Grant Agreement No
Project type
EC contribution
Website
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
‘morphofunctional’performance, 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.
HEALTH-F5-2008-201880
e 5 411 720
1 April 2008
48 months
www.funoct.eu
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.
This novel platform fills an important gap in today’s 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 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:
137
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
1 Anker Engelunds Vej
2800 Kgs. Lyngby, Denmark
Partners
Gereon Hüttmann
Universität zu Lübeck
Luebeck, Germany
Wolfgang Drexler
Cardiff University
Cardiff, UK
Robert Huber
Ludwig-Maximilians-Universität München
Munich, Germany
Christoph Hitzenberger
and Rainer Leitgeb
Medizinische Universität 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
rant Agreement No
G
Project type
EC contribution
Website
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
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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.
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
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
Charité - Universitätsmedizin Berlin
Berlin, Germany
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Ewa Mayzner-Zawadzka
Medical University of Warsaw
Warsaw, Poland
Alvin Kienle
Institut für Lasertechnologien in der Medizin und
Meßtechnik – UlmUniversität
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
DIAGNOSTICS
FMT-XCT
Hybrid Fluorescence Molecular Tomography – X-ray Computed Tomography
method and system
Contract No
Project type
EC contribution
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.
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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.
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
Partners
Bertrand Tavitian
Laboratory for Experimental Molecular Imaging
Paris, France
Philippe Rizo
LETI, Commissariat à l’Energie Atomique
Grenoble, France
Jorge Ripoll
Foundation for Research and Technology Hellas
Heraklion, Crete, Greece
Simon Arridge
London, UK
Manuel Desco
Fudacion para la Investigacion
Biomedica del Hospital Gregorio Marañon
Madrid, Spain
Markus Rudin
Universität Zürich
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
Website
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.
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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.
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
Partners
Peter Fischer
University of Heidelberg
Mannheim, Germany
Claudio Piemonte
Fondazione Bruno Kessler-IRST
Trieste, Italy
Tobias Schaeffter
London, UK
Julia Redondo
Fundación Centro Nacional de
Investigaciones Cardiovasculares, Carlo III
Madrid, Spain
Gijsberta Krenn
Netherlands Cancer Institute
Gerhard Adam
Universitätsklinikum 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
Website
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
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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.
The partners will develop an MEG-MRI scanner
for brain studies. The MRI mode will function
at low field with an initial resolution of 3×3×3
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
Partners
Jari Sakari Penttilä
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 D’Annunzio
Department of Clinical Sciences and
Bioimaging
Chieti, Italy
Claude Fermon
Commissariat à l’Energie Atomique
Paris, France
Antti Ahonen
Elekta AB
Stockholm, Sweden
Paolo Maria Rossini
Assiociazione Fatebenefratelli per la Ricerca
Biomedica e Sanitaria
Rome, Italy
Jyrki Mäkelä
Hospital District of Helsinki and Uusimaa
Helsinki, Finland
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Antonello Sotgiu
Imaging Technology Abruzzo
L’Aquila, Italy
Lutz Trahms
Physikalisch-Technische Bundesanstalt
Braunschweig, Germany
Giacomo Rizzolatti
University of Parma
Parma, Italy
Panu Helistö
VTT Technical Research Centre of Finland
Espoo, Finland
DIAGNOSTICS
SKINSPECTION
Multimodal skin inspection with hybrid acoustic
and optical spectroscopic imaging
Grant Agreement No
Project type
EC contribution
Website
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.
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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.
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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SKINSPECTION
Coordinator
Volkmar Schulz
Robert Lemor
Fraunhofer IBMT
48 Ensheimer Strasse
66386 Sankt Ingbert, Germany
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
Website
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 Aβ, the hallmark of
Alzheimer’s disease (AD). Impaired multi-drug
transporter function with reduced clearance of
Aβ 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
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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.
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).
150
•
•
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 7:
P-gp
in AD
C-VPM
C-PIB
WP 2:
Biological
evaluation of
new P-gp tracer
animal PET
studies
WP 6:
P-gp
in
epilepsy
C-VPM
New
PgP
tracer
WP 5:
P-gp
in
healthy
controls
C-VPM
New
PgP
tracer
WP 3:
In-tro
inhibition
and induction
of P-gp
WP 4:
C-FMZ
F-MPPF
WP 4:
C-FMZ
F-MPPF
human
PET
studies
animal
PET
studies
WP 8:
Ex-vivo studies
WP 10:
Project Management
DIAGNOSTICS
Coordinator
Matthias Johannes Koepp
33 Queen Square
London, WC1N 3BG, UK
Email: [email protected]
Rob Voskuyl
Stichting Epilepsie Instellingen Nederland
Heemstede, Netherlands
Alexandra Varvarigou
National Center for Scientific Research
“Demokritos”
Aghia Paraskevi, Athens, Greece
Partners
Adriaan Lammertsma
VU medisch centrum
Ekaterina Patraia
Medizinische Universitaet Wien
Vienna, Austria
Gitte Moos Knudsen
Rigshospitalet
Copenhagen, Denmark
Birgit Fuchs
GABO.mi (Gesellschaft für Ablauforganisation:
milliarium mbH & Co. KG)
Munich, Germany
Oliver Langer
Austrian Research Centres GmbH
Seibersdorf, Austria
Wolfgang Loescher
Stiftung Tierärztliche Hochschule Hannover
Hannover, Germany
Heidrun Potschka
Ludwig-Maximillians-Universitaet
Munich, Germany
Philippe Ryvlin
Hospices Civils de Lyon
Lyon, France
Karl Herholz
Manchester, UK
Munir Pirmohamed
University of Liverpool
Liverpool, UK
Elizabeth De Lange
Universiteit Leiden
Leiden, Netherlands
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ENCITE
European Network for Cell Imaging and Tracking Expertise
Grant Agreement No
Project type
EC contribution
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;
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•
•
•
•
•
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.
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.
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
Mathias Hoehn
Max Planck Gesellschaft zur Foerderung der
Wissenschaften E.V.
Colgone, Germany
Gil Navon
Tel Aviv University
School of Chemistry
Tel Aviv, Israel
Partners
Monique Bernsen
Erasmus Medical Center
Department of Radiology
Mike Modo
Centre for the Cellular Basis of Behaviour & MRC
Centre for
Neurodegeneration Research
London, UK
Silvio Aime
Università di Torino
Turin, Italy
Milan Hájek
Institute for Clinical and Experimental Medicine
Department of Diagnostic and Interventional
Radiology
Juergen Henning
University of Freiburg
Freiburg, Germany
Michal Neeman
Weizmann Institute of Science
Rehovot, Israel
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ENCITE
Robert Muller
University of Mons-Hainaut
General, Organic and Biomedical Chemistry
Mons, Belgium
Oliviér Clément
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
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, Immunité et cancer
Paris, France
Pierre-Alix Dancer
BioSpace
Paris, France
154
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
FP7 PROJECTS
Molecular testing
SPIDIA
Standardisation and improvement of generic pre-analytical tools
and procedures for in vitro diagnostics
rant Agreement No
G
Project type
EC contribution
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 Alzheimer’s 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
156
Diagnostics - FP7 Projects - Molecular Testing
HEALTH-F5-2008-222916
e 9 000 000 (proposed)
To be confirmed
48 months
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.
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;
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 Oelmüller
QIAGEN GmbH
1 Qiagen Strasse
40724 Hilden, Germany
Partners
Kurt Zatloukal
Medical University of Graz
Graz, Austria
Ivano Bertini
Consorzio Interuniversitario Risonanze
Magnetiche di Metalloproteine
Paramagnetiche
Sesto Fiorentino, Italy
Robert Sjöback
TATAA BIOCENTER AB
Gothenburg, Sweden
Christian Lenz
PreAnalytiX GmbH
Hombrechtikon, Switzerland
157
SPIDIA
Anders Lönneborg
DIAGENIC ASA
Oslo, Norway
Mogens Kruhoffer
AROS Applied Biotechnology AS
Aarhus, Denmark
Nanna Christensen
Dako Denmark AS
Glostrup, Denmark
Bénédicte Charrin
ACIES
Lyon, France
Mikael Kubista
Institute of Molecular Genetics
Czech Academy of Sciences
Anna von Groote
European Committee for Standardization
(CEN)
Brussels, Belgium
Sébastien 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
158
Heinz Höfler
Institute of Pathology of the Technical
University Munich
Munich, Germany
DIAGNOSTICS
EURO-GENE-SCAN
European Genetic Disease Diagnostics
rant Agreement No
G
Project type
EC contribution
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
HEALTH-F5-2008-223293
To be confirmed
36 months
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.
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.
159
EURO-GENE-SCAN
Coordinator
Edvard Smith
5 Nobels väg
Email: [email protected]
Partners
Bodo Grimbacher
London, UK
Johannes Regenbogen
GATC Biotech AG
Konstanz, Germany
Ewa Bernatowska
Children’s Memorial Health Institute
Warsaw, Poland
Mats Nilsson
Uppsala University
Uppsala, Sweden
160
DIAGNOSTICS
NMD-Chip
Development of targeted DNA-Chips for High Throughput Diagnosis
of NeuroMuscular Disorders
Grant Agreement No
Project type
EC contribution
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,
HEALTH-F5-2008-223026
e 2 907 735
To be confirmed
36 months
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-
161
NMD-Chip
term perspective, this will probably contribute to
decreasing the global NMD prevalence by developing an appropriate diagnostic counselling.
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.
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
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
Clinical
consulation
Clinical
diagnostic
NMD patient
Biopsy
Biochemical
characterisation
Blood
Molecular
diagnostic
Test the
most
pertinent
gene 1
Diagnosed
patient
Participation
to clinical trials
NO
Clinical consulation
Up to 10
genes tested
Test most
pertinent
gene X
Mutation in untested gene
Undetected mutation
Unknown mutation
Undiagnosed patient
(40% of NMD patients)
2 weeks to 1 year
Tomorrow with NMD Chips
Clinical
consulation
Clinical
diagnostic
Blood
NMD CHIP
(Test 2,000,000
in one shot)
Diagnosed
patient
Participation
to clinical trials
NMD patient
Unknown gene mutated
72 hours to 1 week
162
Undiagnosed patient
(YY% NMD patients)
Therapy
Therapy
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 patient’s 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
163
NMD-Chip
Coordinator
Nicolas Levy
Institut National de la Santé et de la
Recherche Médicale (INSERM)
101 Rue de Tolbiac
75654/13 Paris, France
Partners
Pascal Soularue
PARTNERCHIP
Evry, France
Thomas Sejersen
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
164
Alessandra Ferlini
University of Ferrara
Ferrara, Italy
Johan Den Dunnen
Leiden University
Leiden, Netherlands
Francesco Muntoni
London, UK
Angela Huebner
University Dresden
Dresden, Germany
DIAGNOSTICS
TECHGENE
High throughput molecular diagnostics in individual patients
for genetic diseases with heterogeneous clinical presentation
Grant Agreement No
Project type
EC contribution
HEALTH-F5-2008-223143
To be confirmed
36 months
of-principle for its implementation in selected
model disorders.
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-
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
WP2
WP3
WP4
Hemoglobinopathies
Breast Cancer Few Genes
with Multiple Mutations
Sensory D isorders
1 Major Gene &
Multiple Minor Genes
Ataxia & Paraplegia
Multiple Equally
Important Genes
Mental Retardation
Rare Mutations in
Rare Genes
Long PCR Enrichment
Validation
Array Enrichment
Read-out & Data Anal
WP5
WP6
WP7
Ethical Issues
Economic Issues
Dissemination & Training
165
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.
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
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
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
Anne Cambon Thomsen
University Paul Sabatier
INSERM U558
Toulouse, France
Katherine Payne
Manchester, UK
Partners
Gert Matthijs
Katholieke Universiteit (KU) Leuven
Leuven, Belgium
166
Katrin Sak
Asper Biotech Ltd
Tartu, Estonia
DIAGNOSTICS
167
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
168
Diagnostics – Index of Projects
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
DIAGNOSTICS
INDEX OF
COORDINATORS
SME-Specific Targeted Research Project
NETWORK OF EXCELLENCE
Cassiman, Jean-Jacques (EuroGentest)
Hughes, Kate (SAFE)
Jacobs, Andreas H. (DiMI)
16
24
80
Integrated Project
Gress, Thomas M. (MolDiag-Paca)
49
Aguzzi, Adriano (TSEUR)
Arndt-Jovin, Donna J. (FLUOROMAG)
Borsella, Elisabetta (BONSAI)
Enfors, Sven-Olof (eBIOSENSE)
Gijs, Martin (DETECTHIV)
Holthöfer, Harry (ADDNET)
Ikonomopoulos, John (NANOMYC)
Isaksson, Olle (NACARDIO)
Kutter, Jörg 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
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)
93
99
113
63
96
68
61
46
31
111
53
29
Kent, Alastair (EUROGENGUIDE)
Moss, David (DASIM)
21
87
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)
Oelmüller, Uwe (SPIDIA)
Smith, Edvard (EURO-GENE-SCAN)
Levy, Nicolas (NMD-Chip)
Scheffer, Hans (TECHGENE)
Diagnostics – Index of Projects
131
133
136
138
140
142
144
146
149
151
155
158
162
164
169
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
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 EU’s 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 - CORDIS

Transcription

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