125
02
Nucleic acid diagnostics market - Unmet needs and product potential
Nucleic acid diagnostics market
Unmet needs and product potential
Technology Review 125/2002
For more information, please contact
Dr Harri Siitari
Senior Lecturer, University of Turku
Tel. +358-400-784 517
[email protected]
Ms Auli Pere, Senior Technical Adviser
Tekes
Tel. +358-10 521 5799
Fax +358-10 521 5905
[email protected]
Technology Review
National Technology Agency
Kyllikinportti 2
P.O.Box
FIN-00101 Helsinki
Finland
Tel. +358-105 2151
Fax +358-9-694 9196
[email protected]
www.tekes.fi/eng/
April 2002
ISBN 952-457-077-7
ISSN 1239-758X
Nucleic acid diagnostics market
Unmet needs and product potential
Harri Siitari
Technology Review 125/2002
Nucleic acid diagnostics market
Unmet needs and product potential
Harri Siitari
National Technology Agency
Technology Review 125/2002
Helsinki 2002
Tekes – your contact for Finnish technology
Tekes, the National Technology Agency, is the main financing organisation
for applied and industrial R&D in Finland. Funding is granted from the state
budget.
Tekes’ primary objective is to promote the competitiveness of Finnish industry and the service sector by technological means. Activities aim to diversify production structures, increase productivity and exports, and create a
foundation for employment and social well-being. Tekes supports applied
and industrial R&D in Finland to the extent of some EUR 390 million, annually. The Tekes network in Finland and overseas offers excellent channels for
cooperation with Finnish companies, universities and research institutes.
Technology programmes – part of the innovation chain
The technology programmes for developing innovative products and processes are an essential part of the Finnish innovation system. These programmes have proved to be an effective form of cooperation and networking
for companies and the research sector. Technology programmes promote
development in specific sectors of technology or industry, and the results of
the research work are passed on to business systematically. The programmes also serve as excellent frameworks for international R&D cooperation.
Currently, a total of about 50 extensive national technology programmes are
under way.
ISSN 1239-758X
ISBN 952-457-077-7
Cover: LM&CO
Page layout: DTPage Oy
Printers: Paino-Center Oy, 2002
Foreword
Nucleic acid diagnostics is expected to be the fastest growing area of diagnostics in the years ahead.
However, only few Finnish diagnostics companies have so far entered this interesting field. Some
reasons for this may become evident through the reading of this report, which provides an overview
of the present nucleic acid world market and describes the technologies of the main players in the
field.
The report has been compiled as part of the half-way strategic work of the Tekes Diagnostics 2000
technology programme. Hence it includes also an evaluation of the ongoing programme projects
dealing with nucleic acid diagnostics. The author also gives recommendations for the future Tekes
investments in the field.
We wish to thank the persons who have through interviews or in other ways contributed to the work
accomplished. It is our wish that the report will help scientists and enterprises to plan their possible
future activities in the field of nucleic acid diagnostics.
Helsinki, April 2002
Tekes, the National Technology Agency
Executive summary
The overall in-vitro diagnostic (IVD) market was estimated to be worth USD 18-19 billion in 2000. Part of this
market, the nucleic acid-based testing (NA) market was
thought to represent some USD 660 million. It is estimated
that the NA market will grow by 20% annually, reaching
some USD 2 billion in 2006, whereas the overall IVD market will grow only by approx. 4%.
Geographically, the US market accounts for 40%, but it has
been forecast that the European market will be the dominating one by 2006. The most vigorous growth is expected
to take place in Latin America and China.
Most of the NA testing market consists of microbial testing
including bacterial and viral detection, genotyping, viral
load assays and blood screening. One growth area, although small today, is the area of disease predisposition
studies and pharmacogenomics including drug efficacy,
toxicology and metabolism studies. Common diseases in
Western populations, such as cardiovascular diseases, diabetes and certain cancers, are typical areas in which possibilities for intervention using new targetted drugs are being
studied. Research will provide a basis for disease predisposition markers and for better, more effective and safer treatment procedures that presuppose specific diagnostic procedures.
From the technological point of view, the limited access of diagnostic companies to the polymerase chain reaction (PCR)
method owned by F. Hoffmann-La Roche Ltd (Roche) has
forced the players to seek alternative strategies for target or
signal amplification. Today, there are a number of alternative ways of amplifying targets isothermally, e.g. transcription mediated amplification or the use of signal amplification as in the bDNA technology of Bayer AG.
In addition to the large IVD companies, a number of smaller
biotechnology companies are active in the field. The business strategy can rely on technological innovations (e.g.
Affymetrix Inc., Digene Corp., IGEN International Inc.,
Myriad Genetics,.) or clinical know-how in the chosen disease area (e.g. Tibotec-Virgo, Myriad Genetics, EXACT
Sciences Corp.) or both (e.g. Orchid Biosciences Inc.,
Vysis, Innogenetics, Gen-Probe). In most cases, the novel
technologies are applied to the testing of, e.g., HIV, HBV
and HCV or single nucleotide polymorphism (SNP) genotyping in collaboration with other companies. Typically,
cross-licencing aggreements or collaboration projects are
used to facilitate the development efforts needed in the NA
testing process.
Biochips or microarrays possess an interesting technology
platform for future IVD products. As of today, their main
use is in expression studies or genome wide sequencing
(e.g. Affymetrix) consisting of thousands or hundreds of
thousands of spots. In the future, however, once the relevant markers in human disease are identified (e.g. defined
set of SNPs), one can envision a system where only a limited number of sequence variations are to be determined.
As there are still technological bottlenecks to be resolved in
the NA testing process, opportunities exists for new players with innovative solutions. For example, such companies as Visible Genetics, Genetic Vectors, Genaissance
Pharmaceuticals and Sequenom are in a development
phase with an interesting technological or market niche to
be exploited. However, with the coming years in mind,
there is still a need to simplify the process, make the analysis more robust, rapid and reproducible. In order to be able
to deliver the quality results provided by the present IVD
products, NA-based methods need to be further automated
and standardised.
Finally, by looking the NA testing market from the Finnish
perspective certain competitive advantages can be seen.
We have a well-developed health care structure providing a
basis for for high quality clinical research. Finland has
been recognized being the number one in overall global
competitiveness, our technological know-how is at the cutting edge, and a number of Finnish researchers in the NA
field are recognized internationally. By combining these,
the companies working in the field can be well positioned
in their respective business niches.
Purpose of the report
The objective of the report is to provide a market overview of NA-based testing today, of the present trends in the field of NA testing, and to make recommendations for focusing the future Tekes
contributions in the field. A further goal is to position the current research projects launched under
the Diagnostics 2000 Programme within the NA testing process, to aid the strategic discussions
about the focus of the programme during its advanced stages, and to make related recommendations and to propose new projects, when appropriate. Ethical issues related to genetic testing are not
discussed in this summary.
This summary is based on the work and discussions that have been carried out in the planning phase
of the Diagnostics 2000 Programme since the late 1998 and since then at the steering and follow-up
group meetings. The chosen projects in the DNA diagnostics sector form the skeleton of the practical work funded by Tekes. Most of the project leaders and researchers of these projects were interviewed. In addition, many Finnish companies, with existing products or products under development for NA testing, were interviewed (Appendix 1).
The Internet, various market reports and news have been followed and used in the summary, when
appropriate.
Contents
Foreword
Executive summary
Purpose of the report
Summary of the world market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Competition and technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Patent issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Diagnostics 2000 projects and the NA testing market . . . . . . . . . . . . . . . . . . . . . . . . . 17
Sample handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Clinical knowledge; genotyping, disease predisposition studies . . . . . . . . . . . . . . . . . . . . . . . . 18
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Clinical laboratory routine and business potentials for Finnish industry. . . . . . . . . . . . . . . . . . . 22
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Appendix 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Technology Reviews from Tekes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Summary of the world market
During the last two decades, a large number of global market reports have been published (e.g. from Frost &Sullivan,
Clinica, Theta). Several common points arise:
• they contain comprehensive descriptions of various
technologies used in NA testing
• the sales figures are small at the beginning of the period
• there is substantial growth potential over the time period
studied
• the overall technological development (synergies from
other fields) is not fully exploited.
Thus, one needs to interpret the sales figures with care, especially when it comes to growth potential and time scales.
However, it is evident, that there are a number of drivers
that bring the growth phase of the market closer every day.
Today, the following market drivers can be identified,
among others:
• results from the publicly funded HGP (human genome
project) and CELERA sequencing projects
• overall NA technology developments
• blood screening guidelines, and guidelines for treatment
of HIV, HCV and HBV
• results from studies toward individualised disease
predisposition markers
• investments in pharmacogenomics
• price pressure issues in health care aiming to more
efficient managed care strategies.
From the estimated USD 18–19 billion IVD market in
2000, NA-based testing accounts for 3.7% or USD 660
million (Table 1). This can be divided by application as
shown in the table.
An example of a rapid growth area in the NA testing market is provided by the blood born viruses. The driving
forces have been 1) direct detection of infectious agents, 2)
potential for quantitation of the agent, 3) treatment potential with modern drugs and, 4) the guidelines provided by
the regulatory agencies. The sales figures have increased
by 20% annually during the last few years.
It has been estimated, that the most vigorous growth rates
are in Latin America and Europe. In this respect, it is interesting that Europe will be the widest NA testing market
globally (Table 2.).
Table 1. Nucleic acid testing market by application.
Application
2000
2006
CAGR¹
MUSD
MUSD
%
Infectious diseases (70%)
– Bacterial tests
– Viral detection & load
– Viral &bacterial genotyping
– Blood screening
460
1470
125
175
10
390
540
88
21
21
44
150
450
22
Cancer (10%)
– Cervical cancer screening
– Chromosomal aberrations
40
30
10
190
170
18
34
10
Genetic (20%)
– Genetic testing
– Forensic & identity
– Disease predisposition
160
360
50
90
20
90
170
100
10
11
30
TOTAL
660
2020
21
(HPV screening)
Modified from Nucleic acid testing: technologies and markets, Clinica 2001. ¹Compound annual growth rate.
1
Table 2. Nucleic acid testing market by geography (MUSD).
Geographical regions
2000
2006
CAGR
Sales
Share
Sales
Share
%
US
Canada
260
13
39
2
650
20
32
1
16
10
Europe
205
31
790
39
25
Japan
Latin America
80
26
12
4
140
150
7
8
10
34
ROI
73
11
270
13
20
660
100
2020
100
21
TOTAL
Modified from Nucleic acid testing: technologies and markets, Clinica 2001
It is evident that the NA testing market will face a vigorous
growth phase over the coming years. This is especially true
for infectious diseases. It is also estimated that Europe will
become the main market over the next few years, and this
will provide the Finnish companies with an important role
to play.
In summary, although exponential growth of the NA testing market has been forecast in many commercial reports
earlier, the expectations have not really been fulfilled. Today, it seems likely that the market will grow by 20%. The
growth is supported by the completed Human Genome projects and the continued projects, that provide additional understanding in, e.g., the function of the growing number of
genes and mutations affecting the outcomes of these genes.
Especially the post genomic research will increase the
knowledge about metabolic disorders and aid in finding the
2
potential intervention therapies at the molecular level. Furthermore, the overall publicity has increased the awareness
and potential of the public and private bodies providing
funding for NA testing.
In addition, the market is driven by the investments that
pharmaceutical companies have made in R&D (including
acquisitions) while utilising the genetic knowledge gained
from target identification. An increasing number of human
diseases with a genetic component are being studied to find
better genetic markers. Individuals identified as carriers of
these markers form the basic resource for clinical studies
on various populations and the basis for new treatment
strategies. In order to exploit the potentials of the targetted
drugs, specific, robust diagnostic procedures needs to be
available.
Competition and technologies
The companies in the NA testing field can be divided into
three categories. Today, the market is dominated by large
diagnostic companies. These companies have internally
dedicated R&D resources and their own technologies to the
analysis of NAs.
Roche is the market leader. Since the acquisition of PCR
technology (Figure 1) from Chiron for USD 300 million,
the company has made investments in other methods, too.
Roche Molecular Diagnostics is developing tests for blood
screening, STDs and tuberculosis. The COBAS AMPLICOR analyser automates both the amplification and detection steps of PCR in one instrument. Roche is working with
Affymetrix to develop array-based products for diagnostics. Other collaborators include e.g., IGEN (Origen technology), Innogenetics (rapid microbiology tests) and Applied Biosystems.
Double-stranded DNA
separate strands
and anneal primers
5'
1. cycle
primers
3'
Temperature
95 – 55 °C
3'
5'
Tag polymerase
5'
3'
3'
5'
3'
new primers
5'
5'
3'
Abbott Laboratories utilise their Ligase chain reaction
(LCR) in their LCx analyser (Figure 2). The test menu consists of STDs (chlamydia, N. gonorrhoea) and respiratory
infections. Abbott’s marketing and distribution partners include e.g. Digene (HBV and CMV) and Vysis (PathVysion
HER-2 and UroVysion).
new
strands
Bayer Diagnostics acquired Chiron Diagnostics in 1998,
and the company now offers an extensive portfolio of products for clinical chemistry, haematology, diabetes, immunodiagnostics and nucleic acid diagnostics. In the NA testing
market, Bayer has access to three amplification technologies; bDNA (Figure 3), HPA and TMA (Figure 4). The
VERSANT HIV-1 RNA 3.0 assay is a third generation assay, which can measure viral loads as low as 50 copies/ml.
It runs on an automated DNA system (Bayer System 340,
bDNA). The assays for HCV and HBV are based on the
same technology and are meant for research use, whereas
HIV is also for diagnostic use, at least in Europe.
Desired
fragment
strands
After selling its IVD division to Bayer in 1998, Chiron
Corporation has focused on the blood testing market. Since
that time Chiron has licenced their HCV and HIV IPs to
Bayer, Abbott and Roche and concentrated on NA-based
testing. The company is working with Gen-Probe while developing a series of Procleix products. Chiron provides a
simultaneous test for HIV and HCV and has shown that the
window period (the period between infection and detection) can be shortened at least by 50%.
Figure 1. Principle of polymerase chain reaction
(PCR, Roche Molecular Diagnostics). The cycle is repeated 30–40 times leading to more than 1 billion copies of the original segment of DNA.
Gen-Probe is a subsidiary of Chugai Pharmaceutical and
was the first company to receive an FDA clearance to market a clinical diagnostic kit using a DNA probe. The company has five core technologies; ribosomal RNA targeting,
TMA, hybridisation protection assay, target capture and
dual kinetic assay. Gen-Probe has obtained a number of
FDA clearances for NA-based tests to detect STDs, tuberculosis, strep., pneumonia and fungal infections. They collaborate with Chiron (Procleix HIV-1/HCV), Bayer (TMA)
and BioMerieux (microbiology).
3
Base to be analyzed
Normal
Sickle
+ Thermostabile
ligase
Repeat cycle e.g. 30 times
Figure 2. Ligase chain reaction (LCR, Abbott Laboratories). In LCR, only oligonucleotides
hybridised next to each other are ligated. The strands are separated by heating and the ligation reaction is repeated.
Target
probes
Hybridize probes
to target and
solid phase
Hybrizine enzyme
labeled probes
to amplifier
Target probes
Microwell coated
with capture probes
Add dioxetane
substrate
Hybrizine
bDNA
amplifier
Measure chemiluminescence
Figure 3. Branched DNA technology (bDNA, Bayer Diagnostics). In this signal amplification technology, target probes are used to catch the target sequence from the sample onto a
solid phase. The target probes are also used to catch the bDNA amplifier probes onto the
solid phase. The final signal is obtained by hybridising the enzyme-conjugated probed to
amplifier probes.
4
Promoterprimer
rRNA target
reverse transcriptase (RT)
DNA
RNA
RNAse H
activities
DNA
RT
primer 2
DNA template
Promoter primer
RNA
POL
RNA amplicon
RNAse H
activities
100–1000 copies
primer 2
Figure 4a. Transcription mediated amplification (TMA, Gen-Probe Inc.)
Each DNA template can make 100–1,000 copies of RNA amplicon. This
expansion can result in the production of 10 billion amplicons an hour.
RNA amplicon
Probe
AE AE
1.
60 °C
AE
AE
15'
hybridization
AE
AE
Select
AE
2. a)
b)
AE
AE
AE
Detect
Light
+
No light
+
AE = Acridinium ester
a) Hybridized probe
b) Unhybridizes probe
Figure 4b. Detection of RNA amplicons with DNA probes and hybridisation protection
assay (HPA, Gen-Probe Inc). Acridinium ester (AE) labelled DNA probes are added.
Separation of hybridised probes from unhybridised ones is done by the addition of selection reagent, which hydrolyses the AE on the unhybridised probes.
5
Table 3. Examples of companies active in nucleic acid testing.
Company
Abbott Laboratories
Bayer Group
Chiron Corp.
Quest Diagnostics
F.Hoffmann-la Roche
Affymetrix Inc.
Cepheid Inc.
Digene Corp.
Enzo Biochem
Gen-Probe (Chugai Pharm.)
IGEN Int.
Innogenetics
Myriad Genetics
Orchid Biosciences Inc.
Sequenom
Visible Genetics
Vysis
CyGene
Exact Sciences
Genaissance Pharmaceuticals
Genetic Vectors
Revenue
13750¹
26700
972
3421
17791
Profit
Technology/products
2790
1590
8.5
102
5362
LCR amplification, LCx Analyser
bDNA signal ampl./Versant tests, Analyser
Procleix HIV/HCV/(Gen-Probe collaboration)
Service testing/Nichols Institute products
COBAS anal./AMPLICOR HIV-1, PCR kits
200
4.4
23
50
150
21
36
34
18
10
13
24
-54
-15
-6.8
6.6
?
-32
11
-8.7
-48
-33
-32
-0.5
Semiautomatic system/GeneChip products
Portable autom. DNA analyser/SmartCycler
Hybrid Capture tests
IF reagents, microtitration plate pathogen kits
TMA amplification system for DNA or RNA
ORIGEN technology
INNO-Lipa HIV, HBV, HCV, CF
Sequencing, proteomics, BRCA1 & 2
SNP-IT, LifeMatch HLA- and genotyping
MassArray, SNP portfolio
CLIP single step seq., OpenGene system
FISH probes, CGH microarrays, Q-beta replic.
TPA (Target Protection Assay)
Colorectal Cancer kits
HAP markers, software system
EasyID oligoplates for genotyping, risk evaluation methods
¹MUSD, Modified from Nucleic acid testing: technologies and markets, Clinica 2001, and other sources
In addition to these big players in the NA testing market,
there are numerous medium-sized and small companies,
whose business ideas are based mainly on proprietary technologies. These technologies offer certain improvements
over those presently used, but it remains to be seen how
widely they will be spread (Table 3) in the future. A few of
these companies are presented below.
Orchid Biosciences has commercialized an interesting
SNP scoring technology (with comprehensive IP rights),
based on single nucleotide extension method (Figure 11).
The SNP-IT platform has been used at high- and lowthroughput applications in pharmaceutical industry and in
agricultural and academic research. The technology can be
used on a wide variety of readout platforms, including instruments already present in laboratories. Recently, Orchid
obtained key patents from Affymetrix for array-based
genotyping, aiming towards ultra high-throughput SNP
scoring applications. In addition to SNP Consortium, a
number of biotechnology and pharmaceutical companies
have collaboration projects with Orchid. In March 2002,
they launched the LifeMatch platform for HLA genotyping
and antibody detection in Europe. The system runs on the
Luminex xMAP platform allowing over 300 samples to be
tested with up to 100 different probes or antigens in a signle
reaction.
6
IGEN International is developing a universal platform for
IVD that can be used in central laboratory settings, POC,
and in home testing. The ORIGEN technology is based on
electroluminescence, which utilises labels that emit light at
a particular wavelength when attached to a biological substance. By measuring the light emission, the amount of
analyte in the sample can be quantified. By including
Organon Teknika’s NASBA, the two companies have developed a series of NA testing products. The main area of
application is in cancer. IGEN works with Roche, too.
Digene Corporation has developed a line of Hybrid Capture tests for screening women’s cancers and infectious
diseases. The technology uses RNA probes that bind to
specific DNA sequences. The created RNA:DNA hybrid is
then detected with antibodies (Figure 5). The assay format
has been applied to 96- and 384-well microtitration plates
and to DNA chips. The main test is for the follow-up
screening of HPV after equivocal Pap smears (marketed
via Roche in Europe). In addition, Digene markets tests for
chlamydia, gonorrhea, HBV and CMV.
Sequenom is developing genetics-based diagnostics and
therapeutic products by exploiting its MassArray technology for DNA analysis. The company claims to have the
widest SNP assay portfolio in the industry. Unlike tradi-
RNA probe
RNA-DNA
hybrides
Target DNA
Capture hybrids onto
a solid phase coated
with specific antibodies
to RNA-DNA hybrids
phosphate
+ Alkaline
substrate
Figure 5. Hybrid capture system (Digene Corp.). Captured hybrids are detected with multiple antibodies conjucated to ALP.
The chemiluminescence from dioxetane substrate is defected
on a luminometer.
tional research, which is focused on small genetic regions
for a specific disease, the technology allows screening of
human genes in relation to diseases in large numbers of individuals. The MassArray technology uses mass spectrometry and a miniaturised chip, eliminating the need for labels
or tags. As an example, the ACE Inhibitor Responder Assay is based on genetic variations in the renin-angiotensin-aldosterone system. This is used to identify and preselect individuals who would experience a significant reduction in blood pressure when treated with ACE inhibitors
alone. Other examples include assays for predicting the
risk of osteoporosis (COL1A1 gene), and identification of
DNA polymorphism patterns affecting the rate of drug metabolism (CYP2D6 and CYP 2C19 genes), as well as predispositions to hypertension and elevated serum LDL-cholesterol.
Genaissance Pharmaceuticals uses its proprietary pClasper
technology to organise numerous SNPs in a gene-specific
region into haplotypes. They offer an integrated solution
where informatics, a high through-put process for analysing clinical samples and patients’ genomic variation are
combined. For example, they have demonstrated for the
first time that a response to a drug can be predicted from an
individual’s haplotype markers (Drysdale et al., 2000). In a
study of 121 asthmatic patients, the response to albuterol/
salbutamol was clinically effective in only 40% of the patients.
Genetic Vectors’ EasyID screening products are designed
to study disease risks, drug resistance and responsiveness.
Today, three areas are on focus, yeast detection and genotyping, assessment of predisposition to juvenile diabetes
and the genes involved in cardiovascular disease. The technology utilises standard laboratory equipment and OligoPlates.
CyGene has patented a Target protection assay (TPA),
where the sequence of interest has been protected by triplex
forming oligonucleotide and the rest of the DNA is digested. After this, a signal amplification step can be performed.
FDA-approved products based on real-time amplification
and detection technology are sold by Becton Dickinson
(BD ProbeTec). The strand displacement amplification
(SDA) method is based on DNA polymerase and restriction enzyme (Figure 6).
ID Biomedical has developed a cycling probe technology
that utilises unique chimeric DNA-RNA-DNA probe sequence providing a RNase H sensitive scissile link. When
hybridised to target, DNA cleaved fragments accumulate
and are detected (Figure 7).
New real-time amplification technologies are used in e.g.
LightCycler from Roche Diagnostics corp., in TaqMan instrument from Applied Biosystems and in Invader Assays
from Third Wave Technologies. Today, these technologies
are used in research applications, but an increasing number
of ready-to-use kits are sold. It remains to be seen, what
role the methods are going to have in diagnostics in the fu-
7
5'
3' – DNA polymerase
– primer
5'
3'
3' – restriction enzyme
5'
– DNA polymerase
Figure 6. Strand displacement amplification (SDA, Becton Dickinson
Corp.). DNA polymerase, restriction enzyme and specific primer are included in this FDA-approved realtime amplification and detection technology (BD Probetec).
RNase H
Cleavage of RNA
portion of hybridizated
probe
+
Hybridization of
probe to target
DNA-RNA-DNA
Probe molecules
Target DNA
Assumalation
and detection
Figure 7. Cycling probe technology (CPT, ID Biochemicals Corp.). CPT
utilises unique chimeric DNA-RNA-DNA probe sequence providing a
RNase H sensitive scissile link when hybridized to target DNA. Cleaved
fragments accumulate and are detected.
8
Oligo 2 (LC red 640)
Oligo 1
(flourescein)
1.
Amplification
product
excitation
1. Stand displacement
R
Q
3'
5'
3'
5'
5'
3'
emission
2.
R
2. Cleavage
excitation
transfer
Q
3'
3'
5'
5'
3'
emission
3.
Figure 8. PCR monitoring with hybridisation probes
(LightCycler, Roche Diagnostics Corp.). Two different oligonucleotides are labelled with (1) fluorescein
and (2) LCred 640. When the probes are hybridised
next to each other (1-5 bp), the fluorescence energy
transfer can take place (FRET). The fluorescence
measured is proportional to the amount of DNA generated.
ture. It is evident that they offer a higher level of automation
and shorten the time needed for the amplification and detection steps. Equally important, quantitative results can be obtained with the new real-time methods. However, sample
handling and the price per assay are among the factors to be
resolved prior to a wider use in clinical laboratories.
In the Lightcycler system (Figure 8), fluorescence energy
transfer (FRET) between two adjacently hybridised
oligonucleotide probes is measured. If the probes are hybridised to the target sequence, the FRET signal can be detected.
Another approach, the Taqman assay, is sold by Applied Biosystems. Here, fluorescence quenching and 5’-exonuclease
activity of DNA polymerase is used (Figure 9). During every amplification cycle, the polymerase cleaves the label
from the hybridised oligonucleotide probe and the
quencher is no longer active and a fluorescent signal is ob-
Figure 9. PCR monitoring with 5’ exonuclease activity (TaqMan, Applied Biosystems Corp). The probe
has two labels, a fluorescent label and a quencher label. When the labels are attached to the probe, no
signal is obtained. During the amplification reaction,
the 5’ exonuclease activity of the polymerase enzyme cleaves the fluorescent label (R=reporter) and a
fluorescent signal is obtained.
tained. The signal increase can be followed in real time.
The quantification of the specific target sequence in the
sample is based on the cycle number when a positive signal
exceeding a treshold value is obtained.
Third Wave Technologies has developed an interesting
new technology, where proprietary cleavage enzymes are
used. In the Invader assays (Figure 10), linear amplification is obtained via a hybridisation of two short DNA
probes forming a structure that is recognised by the cleavage enzyme. The enzyme cuts one of the probes to release a
short DNA ”flap”. Each target can induce the release of
several thousand flaps per hour. In practice, however, the
assay is often combined with PCR, especially when the target sequence is present at low concentration in the sample.
Today, there are already a number of potential technologies offering homogeneous formats, making the amplification and detection steps a single entity, although the sample
preparation step is still a separate entity. Many of the homogeneous assays are based on FRET with labeled primers, for SNP scoring allele specific primers are used. In
spite of the fact that plenty of resources are used, there is
still room for innovation and improvement here.
9
5'
5'
3'
invader
oligo
cleavage
site
C
G
target
Release of
probe arm
3'
5'
5'
e
vag
cleasite
F
F
Release of
fluorescent molecule
Signal
5'
3'
C
Q
C
Signal probe
Figure 10. In the Invader assay (Third Wave Technologies Inc), two
short DNA probes hybridise to target to form a structure recognised by
the cleavage enzyme. The enzyme cuts one of the probes to release a
short DNA ‘flap’. Each target can induce the release of several thousand flaps per hour.
Table 4. Amplification technologies.
Type
Enzymes
Sensitivity
/target mol.
Time-to-result
Cost
/USD
Temperature cycling
PCR
LCR
Thermostabile DNA-pol.
Thermostabile DNA ligase
<10
app. 200
<2h
<2h
2.8¹
Isothermal
TMA, NASBA
SDA
Cycling probe
RT, Rnase H, RNA-pol.
DNA-pol. Restriction enz.
Rnase H
<100
App.500
<10
20’
2h
30’
Signal amplification
bDNA
Hybrid capture
No
No
10-100
<1h
0.8²
¹Cost of consumables/assay (159 samples/run), ²Cost of consumables/assay (168 samples/run), (Elbeik, T. et al. 2000).
One example of the first generation fully automated DNA
analysing system is provided by Cepheid. The Smart Cycler
XC has 16 independently controlled, real-time DNA testing sites. The system is based on PCR amplification and
oligos labeled with fluorescent labels. Furthermore, because the system has a four-colour detection capacity, as
many as four targets can be detected simultaneously in a
single reaction. Cepheid has developed the system over the
last decade mainly for field applications by the U.S. Department of Defense. They have also provided systems for
Centres for Disease Control to test pathogens in field conditions.
10
Microarrays
DNA-chip based technologies have attracted both the scientific and financial communities in recent years. Several
new companies have entered the field (Table 5) and many
acquisitions have been made and partnerships set up by the
newly established biotechnology companies. The common
theme is miniaturisation and the potential to perform a
large number of tests in a small device. The main application area has been in the expression profiling, where the
chips have offered means for for simultaneous analysis of
thousands hybridization reactions at a time.
Table 5. Examples of microarray companies and products.
Company
Product
Array
Capacity
Signal
Application
Affymetrix Inc
GeneChip
20-25b oligos
10–260.00
Fluorescence
E, P, D
In silicon wafer
spots
Hyseq Inc
Incyte pharm.
HyChip
GEM
5-mer oligos
PCR fragments
1024 spots
1–10.000
Fluor.+isotopes
Fluor.+isotopes
S, E
E, P, D
NEN¹ LS Inc
Micromax
cDNA
2400 spots
Fluorescence
D, E
Synteni Inc
UniGEM
0.5-5kb cDNAs
10.000
Fluorescence
E, I
Brax Ltd
Digene Inc
1000
HC ExpressArr.
short oligo
cDNA
Mass spectrometry D, E, I
Antibodies-Fluor.
E
Nanogen
NanoChip
20bp oligos
25–400
Fluorescence
Motorola LS
eSensor
cDNA
36
Electronic current
D
Sequenom
Caliper Inc
MassArray
LabChip
20-25bp oligos
Microchannels
250
96
Mass spectrometry I, D
Fluorescence
I, D
D
electroactive spots
D=diagnostics, E=expression profiling, I=gene identification, P=polymorfism analysis, S=sequencing
¹ Now part of Perkinelmer Life Sciences
The strategies chosen are based on oligonucleotide arrays
using in-situ synthesis (e.g. Affymetrix Inc.), cDNA libraries (Digene Corp., PerkinElmer Life Sciences, Motorola
Life Sciences), in-jet deposition of presynthesised oligos
(e.g. Agilent Technologies Inc., Qiagen Operon, Hyseq
Inc., Mergen Ltd, MWG Biotech AG ), electronic paths to
accelerate hybridisation (e.g. Nanogen) or microchannels
(e.g. Caliper).
In the field of microarray technology, Affymetrix is the
market leader. The company was one of the first to commercialse chip technology in 1996. Their IP relies to a great
extent on the photolitographic production method of the
chips. However, there are several patent disputes under
way, relating to the issue; who owns what? (see the patents
below). Affymetrix applies GeneChips to three areas; gene
expression, analysis of polymorphism and disease management. The company has acquired Genetic MicroSystems,
and 15% of EOS Biotechnology, Neomorphic and Perlegen. The goal is to link array data with polymorfism studies and collect data for disease association studies. Other
collaborators include; Roche, Beckman Coulter, Gemini
Genomics and BioMerieux-Vitek.
Microarray-technology categories can also be based on capacity; high-density arrays, low-density arrays and microfabricated arrays (Table 5). A newer strategy is to offer focused arrays, e.g. for certain pathways, where the spotted
gene sequences are known to interact (e.g. Origene Technologies Inc., SuperArray Inc.). In addition, a number of
companies have introduced protein arrays, aiming at
proteomic and protein functional analyses (e.g. Ciphergen
Biosystems Inc, Phylos Inc.), but these arrays are outside
the scope of this summary. However, protein arrays will be
important in diagnostics, employing e.g. antibodies or
affibodies (Affibody AB), which will be employed in a
large project funded by Wellcome trust to produce
affibodies recognizing all human proteins.
However, miniaturisation raises a host of questions about
quality control, sample preparation, sampling error and
sensitivity. With regard to sensitivity, a few examples can
be given here. First, when detecting low organism load in a
disease, e.g. Candida albicans in blood stream, where today
one needs to have a sample volume of 10–30 ml. With
microarrays, the sample volumes vary between 5 and a few
dozen µl. The same volume-sensitivity problem can be
seen in HIV tests. Earlier, the tests were optimised to use
25µl, but due to the improved efficacy of treatment with
drug combinations, the small number of HIV particles are
being looked for in volumes of 0.5–1.0 ml. Another example is the detection of rare events in circulation. These include the detection of fetal cells in maternal blood, where
typically 20 ml of blood is needed to find potentially a few
dozen fetal cells for analysis. In this respect, the sample
handling plays a key role.
It can be concluded that microarrays for infectious disease
testing (especially in the cases presented above) might become available in the market later. Testing for genetic diseases and disease susceptilities can be earlier accomplished. The human genetic material (approx. 100,000 copies in 5–10 ml blood) is easier to isolate and detect from
various sample matrices.
11
When considering the role of microarrays in diagnostics,
there are still additional issues to resolve. Sample handling
cannot be overlooked and the “garbage in, garbage out”
principle holds here, too. The key factor in the successful
introduction of the new technologies in clinical laboratories is the automation of procedures. Hence it is important
to automate sample handling, integrate the system further,
cut the analysing time significantly, and finally, to cut the
overall costs to a level that clinical laboratories can accept.
12
Patent issues
PCR
Microarrays
Roche’s Native Taq patent was revoked (Promega etc) by
EPO in May 2001. The recombinant Taq’s used today are
still valid. In the early methodology patent, the amplification and detection steps were carried out separately. In
March 2001, Roche was granted a US patent for combining
these steps. However, it became evident during the dispute
process concerning the thermo stabile Taq polymerases
used in the PCR process that the applicants had left out relevant prior- art data from the application. For the time being, this is being considered by the appeal court, whose decision may change the present situation. E.g. the value of
the PCR method patent may be changed, which would lead
to a new situation in NA testing as a whole (the patent coverage, licensing, etc. issues would be affected).
Affymetrix and Oxford Gene Technology settled their patent dispute concerning the chemistry used in the preparation of microarrays. Affymetrix is still in litigation against
Hyseq and Incyte Genomics. These cases are expected to
proceed to trials, which could clarify the situation. Also,
Incyte Genomics and Affymetrix are in litigation over
non-PCR based amplification. However, for arrays with a
smaller number of spots (approximately hundreds), the situation is much easier.
In 2000, Chiron and Roche settled their patent dispute concerning the HCV and HIV sequences used in the test kits.
Roche obtained a licence in clinical diagnostics.
With regard to microarrays, it is probably fair to state that it
will take a few years before the patent situation is clarified
to a greater extent, if the cases proceed to trials. On the
other hand, it is common in the bioindustry that the parties
negotiate a satisfactory agreement that gives each party an
opportunity to concentrate its resources on marketing and
R&D efforts. Furthermore, when using microarrays with
less spots, e.g. in diagnostics with a limited number of sequences to be tested, the patent cases mentioned do not
constitute a limitation.
13
Trends
The pharmaceutical industry is the main driving force behind the growth seen on the NA testing market. In viral
testing, e.g., the treatment protocols of HIV-infected individuals are based on viral load and genotyping results. Owing to the significant genetic variation of the virus even
during the course of the infection, the best therapeutical
drug combination is determined according to the genetic
variant of the virus present in the patient. In principle, the
same strategy has been recommended also for the treatment of HBV and HCV. And other viruses are expected to
follow.
In addition, a great deal of effort is being put into the identification of relevant genetic markers, which could be used in:
• pharmacogenomics
• disease risk predictions
• disease predisposition studies.
In the case of pharmacogenomics, information about genetic variation at the individual level is used to predict drug
responses. This includes mutations in e.g. enzymes participating in drug metabolism (CYP2D6 and CYP 2C19 polymorphism), drug toxicity and efficacy. Personalised medicine has been shown to make a difference in treatment
schemes already today, as was shown in a study concerning
albuterol/salbutamol responses in asthmatic patients. The
drug was effective in only 40% of patients, and these could
be predicted beforehand (Drysdale et al., 2000). Other examples include individual markers for statins, ACE inhibitors (genetic variation in renin-angiotensin-aldosterone
pathway) and a product named CardiaRisk, which detects
the mutation in angiotensin gene, where a low salt diet and
antihypertensive drug therapy are effective.
A growing number of examples are being presented, in
which a certain genetic make-up predisposes individuals to
diseases such as cancer. Examples include mutations in
BRCA1 and BRCA2 genes, p53 and colorectal cancer.
A study of type 1 diabetes offers examples of the risk prediction principle. Juvenile diabetes has a strong genetic
component consisting of certain high risk HLA alleles. In
future, as treatments become available, it will be important
to screen susceptible individuals and plan the treatment
schemes accordingly. This applies not only to diabetes, but
also to other common polygenic diseases that significantly
impact the overall health care systems. Predisposition to
osteoporosis, hypertension or early onset Alzheimers’ are
examples of these.
In the near future, DNA testing will potentially see an increased demand at individual level. This is driven by the
new knowledge provided by mapping of the genetic risk
factors behind many diseases, where life style has an important role to play. Here the willingness to follow the clinically proven life style recommendations, in order to decrease the risk or postpone the outbreak is probably going
to take place. However, the targetted genetic testing with
risk assessment and good quality counselling needs to be
provided.
In conclusion, NA testing is facing a new era with the advent of targetted drugs. Of course, a great deal of new biological knowledge of various pathways is needed before effective treatments for important diseases are available. In
addition, the technological bottlenecks affecting the price
per test need to be overcome. And finally, the decision-makers in the health care sector should understand the
overall benefits of the new possibilities and support the
clinical research phase we are facing.
15
Diagnostics 2000 projects and
the NA testing market
The Tekes-funded public NA-related research projects
within the Diagnostics 2000 Programme are listed below.
At the time of the drawing up of this summary, these projects have continued for two years. One important goal of
this summary is to provide a global framework of the NA
testing market and technologies to aid the strategic planning of the Tekes programme in the NA testing field.
laboratories, the starting point is of utmost importance.
Project 4. addresses one demanding application, where
cells very rarely present in the blood are searched for. If
successful, the principle of the method could be used for
any type of cell, which would create a basis for a continued
analysis at the NA or protein level.
The following conclusions can be drawn:
Detection
Sample handling
Research projects 1 and 2 are very basic in their nature.
However, it is important that also in Finland resources are
used for the basic scientific research of fluorescence and
nucleic acid chemistry. These could be strengthened by;
Sample handling represents one of the main bottlenecks. In
order to produce the high-quality results needed in clinical
Table 6. Research projects within the Tekes Diagnostics 2000 Programme.
1.
Synthesis of fluorescent nucleoside derivatives and their incorporation in Oligonucleotides.
Rainer Sjöholm, Åbo Akademi University, Turku
2.
Improving the selectivity of oligonucleotide hybridisation assays.
Alex Azhayev, University of Kuopio, Kuopio
3.
Active solid support for DNA diagnostics.
Ari Hokkanen, VTT Technical Research Centre of Finland, Espoo
4.
Isolation of fetal cells from maternal blood for prenatal diagnosis.
Jim Schröder, University of Helsinki, Helsinki
5.
Development of a laboratory method for the detection of antimicrobial drug-resistance
associated mutations.
Mika Salminen, National Public Health Institute, Helsinki
6.
Identification of novel molecular markers for the early diagnosis of type 1 diabetes.
Riitta Lahesmaa, Centre for Biotechnology, Turku
Table 7. Tekes-funded projects in the NA testing process.
Project
Place in process
Goal
1.
Detection
Fluorescent nucleosides, sensitive for environmental
changes
2.
Detection
New nucleoside structures improving hybridisation
selectivity
3.
Amplification/detection
Silicon-based disposable device for amplification
and detection
4.
Sample handling
Isolation of rare cells in blood
5.
Clinical research
Non-isotopic measurement of known mutations from blood
6.
Clinical research
New early markers for prediction of predisposition
17
1. forming a research group with enough critical mass and
by intensifying international collaboration,
2. support funding from the Academy of Finland or from
the EU (6th Frame work, Genomics), or both. In the preparation of the content of such a programme, interested
companies in the field should be actively involved in the
planning.
Project 3 has the goal to miniaturize the amplification and
detection steps of the process in a single device. In part, the
project relies on the experiences obtained during the ’Finn
chip’ project and on the experiences at the Microelecronics
centre (VTT Information technology) in other biological
applications. This is a demanding project from the technological part, but especially from the ’wet’ part, too. Attention should be paid to quarantee enough resources in the
chemistry part including sample introduction, amplification and detection and preliminary production process issues. Results from this project might play a central role in
the planning of the proposed project 2.
Clinical knowledge; genotyping,
disease predisposition studies
Projects 5 and 6 can be grouped here. They provide good examples of the utility of Finnish know-how in important clinical areas. In project 5, the overall process is streamlined and
the detection of HIV drug resistance is improved by a new
method and the non-isotopic labels employed. If successful,
the method will be very interesting from the global perspective, too. Furthermore, it will provide a good basis for the
next generation of automation of genotyping assays.
In project 6, new and early predisposition markers of type 1
diabetes (juvenile diabetes) are sought. Type 1 diabetes is a
disease of importance not only from the point of view of
the individual, but also from that of the overall western
health care system. In western countries, the number of
new cases is increasing, Finland and Sardinia are the leading locations in this respect, but the reason is still unknown.
Type 1 diabetes has a genetic component, where children
with certain HLA risk alleles are more suscebtile to the disease than others. Screening programmes have been initiated, where the children with high risk HLA alleles are followed, and the appearance of islet cell antibodies (ICA) is
measured (Kupila A., et al., 2002). The appearance of these
(and antibodies to certain other antigens) are indicative of
the progress of the disease. Today, a number of various
treatment studies are under way, and new treatment protocols will be available in a fairly near future.
The project serves well as an example of how the microarray technology is used to find new potential early markers at the NA level. The microarray technology, including
the developments proposed below, could form a universal
platform applicable to a variety of needs present also in
clinical laboratories.
In Finland, there are companies that are active in the NA
testing market. The NA products either support the basic
business (Thermolabsystems, PerkinElmer Life Sciences,
Wallac), form the backbone of sales (FinnZymes,
Labmaster to some extend) or form a basis for future sales
(Mobidiag, Jurilab). It can be concluded, that all the companies would benefit from a future national investment in
the NA testing field. Issues raised during the interviews included e.g:
1. improvements needed in sample treatment
2. the future ’microarray’ technology base
3. additional funding possibilities
4. a common patent survey.
Table 8. Companies interviewed for the summary.
Company
Business area
NA products
Comments/NA strategy
Arctic Diagnostics ltd
Technology dev.
None
Techn.dev./applic., licencing
FinnZymes Oy
Research
Reagents, kits
Widen offering to NA mkt
FIT Biotech Oyj
Drug devel.
None
DNA vaccines, diagn.kits
Innotrac Diagnostics Oy
POC testing
None
Focus on POC / kits, system
Jurilab ltd
Disease pred.
Chips, information
Combine clin. data with SNPs
Labmaster Oy
Res.kits
PCR reagents
Kits for new diagn.niche areas
Thermolabsystems
Research
KingFisher family
Automation of sample hand.
Mobidiag Oy
Infect.diseases
Chip development
Bacterial, fungal dis., service
PerkinElmer LS, Wallac
Screening
TRF reagents
Risk prediction products/SW
Note! Labsystems is part of Thermo Corp., Wallac is part of PerkinElmer Life Sciences (SW=software products)
18
Synergies have not been fully exploited. This does not only
apply to the relations between research groups, research
groups and companies, but also to those between companies. There is work to be done to find the most effective
ways of exploiting synergies. The Programme concept
launched by Tekes has been well received. In addition, the
follow-up group meetings have promoted the exchange of
knowledge and information about interesting topics between the attending parties. However, an even more active
forum or body is needed. The present structure leaves out
the companies not attending the meetings of the group con-
cerned, the high level research groups not participating in
the programmes and other industries not working directly
on the theme of the programmes (e.g. microfluidistics,
micromachining, optoelectronics). The flow of information could also be promoted and potential technological
synergies between programmes could be made more efficient (e.g. Lääke 2000, Diagnostiikka 2000). It can be
learned from the history, that many of the world class innovations have emerged from the intersection of different scientific areas.
19
Conclusions
Market
The NA testing market has grown vigorously in the last
few years and it is expected grow by 20% (CAGR) over the
next 5-year period. One growth area is the area of disease
predisposition studies and theranostics including drug efficacy, toxicology and metabolism studies. Common diseases in Western populations, such as cardiovascular diseases, diabetes and certain cancers, are examples in which
possibilities for intervention using new targetted drugs are
being studied.
L
dd T
5'
3'
The incorporation assays offer one alternative for homogeneous analysis (employing e.g. fluorescence polarisation
end point measurement, figure 11) and also for solid phase
methods (see a comprehensive review by Syvänen, A.-C.,
2001). Since the invention of the method by A.-C. Syvänen
and H. Söderlund, while working at Orion Corp., the principle has been applied to many fields and new formats, e.g.
at the National Public Health Institute (KTL). However, no
Finnish company has yet commercialised any products
based on the technology. The European method patent has
since been sold to Sangtec Medical AB (Sweden) and they
have collaborated with F. Hoffman La Roche to market the
products in Europe. Mostly Sangtec collaborated on quantification by sandwich hybridization in microbial diagnosis, not in the area of analyzing SNPs or point mutations. In
the US, Orchid Biosciences (see above) has bought the
Söderlund-Syvänen patent and two others, so they have almost all the rights for the method. Today, they are selling
products and licenses in the US.
G
5'
5'
3'
5'
C
G
A
5'
L
L
3'
As there are still unresolved technological bottlenecks in
the NA testing process, opportunities exist for new players
with innovative solutions. There is still a need to simplify the
testing process, and to make it more robust, rapid and reproducible. In order to be able to deliver as high-quality results
as those obtained by means of the present IVD products, the
NA-based methods need to be further automated and standardised. PCR is still the major bottleneck (e.g. in terms of
throughput, multiplexing for simultaneous amplifications).
L
dd C L
dd G
dd A
This is particularly interesting from the Finnish perspective, where there are good potentials to perform high quality clinical research projects.
Technology
labeled
dideoxynucleotides
L
5'
3'
T
A
Figure 11. Incorporation assays; minisequencing,
template-directed incorporation and single base extension assays. Incorporation assays can be performed in one tube or on a solid phase, and end-point reading can be based on e.g. fluorescence polarisation.
Biochips or microarrays possess an interesting technology
platform for future IVD products. As of today, their main
use is in expression studies consisting of thousands or hundreds of thousands of spots. In the future, however, once
the knowledge of the identification of relevant markers in
human disease is available (using e.g. a defined set of
SNPs), one can envisage a system where only a limited
number of sequence variations are to be determined. Still,
amplification is needed both for the specific and sensitive
detection of SNPs. Furthermore, it is important to automate
sample handling, integrate the system further, cut the analysing time significantly, and finally, cut the overall costs
to a level that clinical laboratories can accept.
The bottlenecks to be resolved include such issues as;
1. price of systems and reagents used in measurements,
2. low speed of reaction, (hybridization based methods,
not so for primer extension methods)
3. relevant sequence content on the chips,
4. standardisation of formats,
5. quality of results (clinical lab. diagnostics point of view)
and
6. patent situation.
21
There is still a need for improvements in combining the
technology and biological know-how. A new open technology platform addressing the points above can be globally successful in the future. This is well supported by the
potential for high-quality clinical research in Finland. In
addition, the Finnish centre for microarrays (Centre for
Biotechnology, Turku), among the other centers, has now
established the technology and production of microarrays
for research applications. This, together with the other
microarray centres in Finland, provides a good starting
point for the future development needs.
Patents
The NA testing field is an IP intensive business area. Several litigations are presently under way and will probably
lead to trials, which will give an indication of the strengths
of the patents in question. The PCR method patent situation, for example, might change, depending on the decision
of the appeal court. IP issues are most relevant also in the
area of microarrays. On the other hand, the settlement of
disputes is often a better choice, especially for smaller
companies which do not have unlimited resources and
could use the money saved for continued R&D. From the
Finnish point of view, however, a conclusion to be drawn
from this survey is that there certainly is a need to clarify
the situation further. A common patent study to be funded
and coordinated by Tekes was also proposed by some of
the companies interviewed. This can be started by having
the companies concerned define the products and technologies to be included in the study.
As a whole, the IP issues are an important part of research
work. For the ongoing projects, the potential to apply for a
patent should (as has been done in some projects) be consider always prior to any kind of publication. For the new
projects, patenting costs should be included in the project
plan. The patenting process can also help the inventors to
materialize the whole innovation and give them a feed back
about what already exists in the patent databases.
Clinical laboratory routine and
business potentials for
Finnish industry
If we consider the clinical laboratory routine work load and
the ever-increasing cost cutting demands of today, we can
state that NA testing doesn’t very well fit the picture. In addition to what has been stated below, the number of the
tests potentially needed (e.g. number of genetic diseases
>4000) is high, whereas the number of the tests to be carried out per disease and per laboratory is small.
22
Today, from the clinical laboratory system point of view,
there are a number of bottlenecks in the process. Areas of
improvement addressing the bottlenecks include;
• automated and integrated sample preparation
• integrated amplification and detection steps
• robust technnology producing results with high
precision, and
• technology easy to use and new analytes easy to add.
From the clinical laboratory perspective, the systems available today are still far from optimal. For NA testing, there
are global drivers, such as ever-increasing biological knowledge leading to more precise genetic markers, and technological improvements in robotics, liquid handling, system
integration etc. The Finnish companies should introduce a
new business model simultaneously with the technology
development programmes. We have learned from history
that building successful marketing and sales (S&M) channels is also economically a demanding task. For companies
that do not have an existing S&M channel, the choise of a
new business model is important at an early stage. Today, a
number of choises are available, and depending on the
business goals set, the work should be started early on. In
clinical laboratories, the consolidation has been taking
place for several years already. This concerns also suppliers, which means that the well-known present suppliers
have a marketing benefit on their side. Thus, for example,
an interesting technology and a product range with proven
clinical relevance could be best marketed via the present
suppliers to obtain the market share they deserve in the
clinical laboratory branch.
Finally, the current developments in many fields of research will add to the existing knowledge and provide a basis for better, more effective, and safer treatment procedures presupposing specific diagnostic procedures. The location of the Finnish companies provides them with certain
competitive advantages. Finland ranks first in overall
global competitiveness, we have a well-developed heath
care structure providing a basis for high-quality clinical research. In Finland, technological know-how is on the top
level, and a number of Finnish researchers in the NA field
are also recognised globally. By combining these facts
with well-documented clinical research, the companies operating in the field can be well positioned on the global NA
business map.
The practical recommendations for the Diagnostics 2000
Programme are presented in the form of project proposals
in the Recommendations section. One cannot expect
break-through results within a short period of time, but it is
important to launch certain key projects in a timely manner.
Recommendations
The recommendations below are based on the NA testing
market, the current projects included in the Programme and
the comments obtained during the interviews. It is proposed that if the participating companies and research
groups are sufficiently interested, specified work should be
started. This could create a basis for new applications and
promote the competitive edge of the Finnish industry in the
NA testing field. Hence the following projects are proposed. In order to address the sample treatment issues (especially for microbial diagnostics, not so for human genetics), the project one is proposed.
Project/main goals, Time frame
1. Sample treatment, Short term (1–3 years)
• faster process
• robust, various sample matrices
• easy to perform and automate
• high-quality NA for testing.
2. Combined sample treatment, amplification
and detection, Long term (3 years onward)
• faster process
• amendable for automation
• single-step, homogeneous
• easy-to-add new targets.
The projects above should be defined so that the clinical
importance is one of the driving forces, i.e. that the clinical
areas used as models for the projects are of interest to the
Finnish companies involved. In addition, it would be important to involve parties specialized in bioinformatics, instrumentation, micromechanics etc to exploit the system
approach. In order to gain the best possible technology leverage in the future, other NA application areas (in addition to human diagnostics) should be included.
In addition to blood, other important sample matrices are
used in biosciences. These can include cultivated cells, tissues, stool and NPS specimens, food and other materials. A
method which can be applied to various matrices and is
amendable to automation (simple enough) is still needed.
The miniaturisation trend (microarrays) has brought up an
additional development need. The small volumes used in
microarrays will need a more efficient and automated
means for extracting and concentrating NAs for further
analysis (new means to avoid PCR, should also be looked
for).
23
References
Cortesse, J.D., 2000. Array of options, instrumentation to
exploit the DNA microarray explosion. The Scientist
14 (11): 26.
Drysdale C.M., McGraw D., Stack C.B. et al., 2000. Complex promoter and coding region beta-2-adrenergic receptor haplotypes alter receptor expression and predict
in vivo responsiveness. Proceeding of the National
Academy of Sciences, 97;10483-10488.
Elbeik, et al., 2000, Quantitative and cost comparison of ultra-sensitive human immunodeficiency virus type 1
RNA viral load assays: Bayer bDNA Quantiplex versions 3.0 and 2.0 and Roche PCR amplicor monitor
version 1.5. J. Clin. Microbiol., 38, no 3;1113-1120.
Kupila A., Keskinen P., Simell T., Erkkilä S., Arvilommi
P., Korhonen S., Kimpimäki T., Sjöroos M., Ronkainen M., Ilonen J., Knip M., and Simell O. Genetic risk
determines the emergence of diabetes-associated autoantibodies in young children. Diabetes 2002; 51:646651.
Nucleic Acid testing: technologies and markets. Clinica
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Syvänen, A.-C. 2001. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nature Genetics, 2; December 930-942.
Web sites
www.abbottdiagnostics.com
www.affymetrix.com
www.agilent.com
www.apbiotech.com
www.appliedbiosystems.com
www.bayerdiag.com
www.bdbiosciences.com
www.biochem.roche.com
www.chiron.com
www.ciphergen.com
www.clontech.com
www.digene.com
www.genomicsolutions.com
www.gen-probe.com
www.sbh.com
www.incyte.com
www.mergen.com
www.nen.com
www.orchidbio.com
www.origene.com
www.packardinst.com
www.perkinelmer.com
www.sequenom.com
www.superarray.com
www.twt.com
www.vysis.com
25
Acknowledgements
All the researchers and industrial partners participating in the interviews are acknowledged. Also
valuable comments given by professors Ann-Christine Syvänen and Timo Hyypiä are greatly appreciated, as well as is appreciated the productive discussions with Dr Auli Pere, Tekes, during the
preparation of the summary.
26
Abbreviations
ACE inhibitor
angiotensin converting enzyme inhibitor
AE
acridinium ester
Alleles
alternative forms of the same gene
ALP
alkaline phosphate
bDNA
branched deoxyribonucleic acid
BRCA
breast cancer gene
CAGR
compound annual growth rate
CGH
comparative genomic hybridisation
CML
chronic myelogeneous leukaemia
CMV
cytomegalovirus
COLIA1
collagen type 1 osteoporosis gene polymorphism
CPT
cycling probe technology
DNA
deoxyribonucleic acid
ECL
electrochemiluminescence
FDA
Food and Drug Administration
FISH
fluorescence in situ hybridisation
Genotyping
determination of the polymorhism of individuals’ genomes
with respect to a specific set of markers
HAP
haplotype technology
Haplotype
set of closely linked markers at one locus which is inherited as a unit
HBV
hepatitis B virus
HCV
hepatitis C virus
HGP
human genome project
HIV
human immunodeficiency virus
HLA
human leucocyte antigen
HPA
hybridization protection assay
HPV
human papillomavirus
IP
intellectual property
IVD
in vitro diagnostics
KTL
national public health institute
LCR
ligase chain reaction
LDL
low density lipoprotein
LiPA
Line probe assay
27
28
NASBA
nucleic acid sequence based amplification
NA testing
nucleic acid testing
PCR
polymerase chain reaction
POC
point-of-care
R&D
research and development
RNA
ribonucleic acid
RT
reverse transcriptase
SDA
strand displacement amplification
SNP
single nucleotide polymorphism
STD
sexually transmitted disease
SW
software
Taq polymerase
polymerase from Thermus aquaticus
TB
tuberculosis
TMA
transcription mediated amplification
TPA
target protection assay
Appendix 1
Interviewed companies and researchers:
Companies
Researchers
•
•
•
•
•
•
•
•
•
• Azhayev A., University of Kuopio
• Hokkanen A. & Leppihalme M.,
VTT Microelectronics, Espoo
• Lahesmaa R., Centre for Biotechnology, Turku
• Lönnberg H., University of Turku
• Mononen I., Turku University Hospital, Turku
• Salminen M. & Zetterberg V.,
National Public Health Institutre, Helsinki
• Sjöholm R. & Kronberg L.,
Åbo Akademi University, Turku
• Söderlund H., VTT Biotechnology, Espoo
Arctic Diagnostics Oy, Erkki Soini
FinnZymes Oy, Pekka Mattila
FIT Biotech Oyj, Pekka Sillanaukee
Innotrac Diagnostics Oy, Antti Iitiä
Jurilab Ltd, Jukka T. Salonen, Veli-Pekka Korhonen
Labmaster Oy, Teppo Laaksonen, Jarkko Eskola
Mobidiag Oy, S. Nikkari, M. Richarson, J. Ylikoski
PerkinElmer Life Sciences, Wallac Oy, I. Hemmilä
Thermo Labsystems, K. Käpyaho, M. Partanen
29
Technology Reviews from Tekes
125/2002 Nucleic acid diagnostics markets, Unmet needs and product potential. Harri Siitari. 29 p.
124/2002 Polttopuun pientuotannon ja -käytön kehitystarpeet. Satu Helynen, Heikki Oravainen. 26 s.
123/2002 US Corporate Wellness Study. 88 p.
122/2002 Benchmarking Innovation Systems: Government Funding for R&D. Draft Final Report. 62 p.
Erik Frinking, Mari Hjelt, Irma Essers, Päivi Luoma, Sami Mahroum
121/2002 Government innovation support for commercialisation of research, new R&D performers and
R&D networks. 128 p. Jari Kuusisto, Erik Arnold (editors)
120/2002 Yritysten innovaatioympäristöt – Tutkimus yritysten innovaatiotoiminnasta ja alueellisesta innovaatiopolitiikasta Pirkanmaalla ja Keski-Suomessa. 215 s. Mika Kautonen, Jari Kolehmainen,
Pasi Koski
119/2001 Teollisen muotoilun teknologiaohjelma, Esiselvitys. 29 s. Eija Nieminen, Juha Järvinen
118/2001 Digitalisoituvan viestinnän monet kasvot. 131 s. Kuluttajatutkimukset-hanke (Kultu)
117/2001 Ympäristömittausten automatisointi- ja kehittämistarpeet Suomessa. 118 s. Lauri Hietaniemi,
Ari Lehto
116/2001 From Periphery to Center: Emerging Research Topics on Knowledge Society. Ilkka Tuomi
115/2001 Terveysvaikutteisten elintarvikkeiden kansainvälinen kaupallistaminen. 23 s. Kari Salminen
114/2001 Global Networking in Wireless Teletechnology Business. Lasse Baldauf, Michael Lovejoy,
Jarmo Karesto, Laura Paija
113/2001 Critical Success Factors in Biopharmaceutical Business: A Comparison Between Finnish and
Californian Businesses. 23 p. Tanja Rautiainen
112/2001 Finnish Pharma Cluster – Vision 2010.
111/2001 Uuden tietotekniikan vaikutukset liiketoimintaan. 60 s. Jyrki Ali-Yrkkö, Kim Jansson, Iris Karvonen,
Veli-Pekka Mattila, Juha Nurmilaakso, Martin Ollus, Iiro Salkari, Pekka Ylä-Anttila
110/2001 Digitaalinen verkostotalous – Tietotekniikan mahdollisuudet liiketoiminnan kehittämisessä. 86 s.
Juha Luomala, Juha Heikkinen, Karri Virkajärvi, Jukka Heikkilä, Anne Karjalainen, Anri Kivimäki,
Timo Käkölä, Outi Uusitalo, Hannu Lähdevaara
109/2001 Ohjelmistoalan tutkimustoiminta Yhdysvalloissa. Veikko Seppänen, Timo Käkölä, Olli Pitkänen,
Reijo Sulonen, Markku Sääksjärvi
108/2001 Software Business Models, A Framework for Analyzing Software Industry. Risto Rajala,
Matti Rossi, Virpi Kristiina Tuunainen and Santeri Korri
107/2001 State of Mathematical Modelling and Simulation in the Finnish Process Industry, Universities
and Research Centres. 95 s. Kimmo Klemola, Ilkka Turunen
106/2001 Research and technology programme activities in Finland. 54 s. Ellen Tuomaala, Satu Rask,
Erkki Kaukonen, Jyrki Laaksonen, Mika Nieminen, Pekka Berg
105/2001 Tutkimus- ja teknologiaohjelmatoiminta Suomessa. 50 s. Ellen Tuomaala, Satu Raak,
Erkki Kaukonen, Jyrki Laaksonen, Mika Nieminen, Pekka Berg
104/2001 Matemaattiset menetelmät suomalaisten yritysten t&k-toiminnassa. Heikki Haario, Matti Heiliö,
Jari Järvinen, Pekka Neittaanmäki
103/2001 Hyvinvointi- ja terveysalan teknologia- ja palvelutuotteet. 64 s. Niilo Saranummi
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