Molecular Diagnostics

By Louise Lazear

At an ever-accelerating pace, scientists are identifying which genes and variations direct the production of proteins that cause or forestall disease. These discoveries are changing the practice of medicine: from predisposition to diagnosis to treatment, we now have a blueprint for understanding disease at the molecular level. Not surprisingly, molecular diagnostics continues to be one of the fastest growing segments within the genomic testing market. According to industry sources, in 2001 the market for NAT diagnostic testing alone represented approximately half of the $2.1 billion worldwide market for genomic and proteomic testing. The market for molecular diagnostics is expected to increase 50 percent annually to $5.5 billion in 2005. Other segments – including drug discovery and pharmacogenomics, resistance testing, forensics and food production – will propel the worldwide genomic testing market to grow to $40 billion by the year 2010.

The molecular diagnostics market is often hard to encompass due to the multitude of players and technologies. The market has been dominated by Roche Diagnostics and PCR technology, which in the year 2001 earned approximately half of all revenue generated by molecular diagnostics for the clinical diagnostics market. However, smaller firms with innovative and proprietary testing methods, and partnerships between reference labs and vendors of all sizes, have added to the mix of products and services involved in this segment, accelerating the availability of technology. And the laboratory is now positioned to become a key player not only in diagnosis, but also in the treatment and management of all types of disease in the not so distant future. But the road to implementation is rife with challenges, including which tests to implement, and how.

“Molecular diagnostics is the characterization of human disease by examination of nucleic acids, which are the templates for proteins that mediate disease,” said Ronald McGlennen, M.D., associate professor at the University of Minnesota and the founder of Access Genetics, Inc. Current applications for molecular diagnostics include assessment of inherited disorders, such as cystic fibrosis, and acquired diseases such as cancer, which may be caused by both inherited and environmental mechanisms, as well as assays for infectious disease, which can be more sensitive and faster than conventional

culture techniques. Beyond these applications, the laboratory can also test for genetic predispositions for disease, which according to McGlennen, dramatically impacts the role of the laboratory in patient care. “In the classic sense, the laboratory has been responsible for the management and analysis of patient samples,” he said. “Now, we are testing for diseases that might occur. This means we now must also deal with issues of informed consent and patient counseling, and an increasingly important role in how the patient is managed.”

While applications have increased and improvements in technology have reduced complexity, the number of laboratories offering genetic testing has remained relatively stagnant. According to the College of American Pathologists, there are 190 DNA diagnostics laboratories in the U.S., in a field of over 10,000 clinical labs. The number of labs performing genetic tests has increased by only 30 since 1998. “The reason that more labs are not providing this service is the perceived complexity of the genetic testing process,” said McGlennen. In order to help clinical laboratories enter this arena, Access Genetics offers labs that do as few as 20 tests per month the opportunity to get into the business of providing genetic tests with a turnkey system of prepackaged kits, a compact equipment suite, and expert test interpretations and consultative services for patients and physicians.

According to McGlennen, HIV and HCV quantitative, qualitative, and genotyping assays, as well as conversion to HPV testing as a complement to monolayer PAP preparations are short-term growth opportunities for laboratories. He also sees testing of disease predisposition for treatable conditions and drug metabolism likely areas of growth due to their impact on therapeutic decisions. McGlennen uses mutational analysis of Factor V Leiden, the most common cause of familial predisposition to venous thrombosis, as a model of how a relatively simple genetic test can significantly impact patient management. “If I test you for FVL mutation, and you are positive, then one can directly infer your risk of developing thrombosis,” he said. This information is especially important for women considering oral contraceptives, for targeting patients for anticoagulant therapy, and for postoperative treatment of surgical patients at risk for thrombosis. In addition, healthcare providers can extrapolate from sample populations the incidence of risk for venous thrombosis, and use this data to predict costs associated with this disease and to justify the expense of testing.

Targeting diseases for which clinical genetic testing results may affect the diagnosis, therapy or both, is an emphasis at Third Wave Technologies, Inc. The company currently offers over a dozen ASRs for laboratories in the U.S. While PCR is based upon the mechanism by which DNA copies itself, Third Wave’s proprietary Invader technology capitalizes on the cellular mechanism for DNA repair. “PCR harnessed the inherent biology involved in copying DNA. DNA repair is also a basic biological process, and is inherently much more accurate. We analyzed how DNA repair works, and developed this mechanism into a method of genetic analysis,” said Lance Fors, Ph.D. and CEO at Third Wave. “With the Invader probe and signal probe, our method mimics what a cell sees if specific DNA is present. This is a very digital method. If the sequence is not exactly correct, no signal is emitted,” he explained. Invader assays utilize fluorescence resonance energy transfer (FRET) cassettes with probes and reagents for specific genetic sequences. Results may be read on a variety of currently available fluorescent instruments, which can minimize a laboratory’s capital outlay.

Third Wave’s initial product offering includes ASRs for Factor II and V, homocysteine metabolism, cardiovascular risk assessment with ASRs for apolipoproteins and glycoproteins, and cytochrome p450 for drug metabolism analysis, including adverse reactions and drug efficacy. The company currently markets ASRs for Tay-Sachs disease, and is planning to launch ASRs for CF and Connexin by the end of the year. According to Fors, the company is planning to move these ASRs into the IVD market, and to offer homebrew assays for individual laboratories. While the current market for Third Wave technology is high-complexity labs, with the move into IVD applications, the company intends to target medium and small clinical laboratories.

In addition to new molecular assays, the industry is also focusing on new analyzers suited for use in the clinical laboratory. AutoGenomics, Inc. is developing the Infiniti benchtop analyzer to perform both genomic and proteomic assays on one platform. This continuous flow, microarray system automates sample handling, reagent dispensing, hybridization and process management, with minimal operator interface. The system uses BioFilmChip microarrays, which are composed of a polyester film coated with a proprietary multi-layered technology. The system can process 24 microarrays simultaneously, and has the ability to perform single nucleotide polymorphisms, short tandem repeats, gene expression using hybridization and primer extension assays, as well as competitive/sandwich immunoassays.

“With the availability of new technology, more hospitals will be able to provide routine molecular diagnostic testing. We are going to launch our system with markers that will demonstrate to the market that hospitals can and should be providing these tests to their communities,” said Ram Vairavan, vice president at AutoGenomics. Planned applications include assays for Connexin 26, CF, cytokines, coagulation, cytochrome p450, breast cancer, HPV and cardiovascular disease. The company expects the Infiniti analyzer and assays to become available the first half of 2003.

Because many of the new molecular assays are offered as ASR or RUO products, this burgeoning market has created opportunities for companies to provide controls and standards to both manufacturers and laboratories. “We saw a gap in the marketplace for third party independent controls for this type of assay,” said Michael Eck, CEO at AcroMetrix, Inc. “There seemed to be a lack of understanding on the part of manufacturers on how to offer controls for NAT. What we do is work with manufacturers to ensure that once their assays are available, laboratories have quality control products in place to streamline validation and training.” AcroMetrix currently provides NAT controls for HIV-1 RNA, HCV RNA and HBV DNA. Both hepatitis controls are calibrated to WHO standards, which can help bolster physicians’ use of laboratory results in making therapeutic decisions. The company also provides the HCV Genotyping panel, HIV drug resistance genotyping controls, and LabQC.Org, a web-based portal for proficiency testing programs.

Molecular applications continue to be an area of high growth potential for the traditional IVD companies as well, including Abbott, Bayer, BD and Roche. Roche Diagnostics, which has been the market leader in molecular testing with PCR technology and infectious disease assays, is expanding beyond HIV and HCV testing with numerous products for other applications in the pipeline. At Roche, the development of molecular assays also involves an assessment of the impact of this new information to clinicians. “Molecular diagnostics is changing the way that healthcare is provided, and justification for this technology is being shifted to outside of the laboratory,” said Jim Floberg, marketing director for transitional diagnostics at Roche. “For example, in the case of infectious disease, more sensitive, faster molecular assay are available, but may cost more than traditional assays. However, the financial windfall typically occurs outside of the lab, where the downstream impact of reduced length of stay and therapeutic optimization is significant.”

Molecular assays currently available from Roche run the gamut from IVD to RUO applications on Cobas, LightCycler and TaqMan platforms. A qualitative assay for HCV is FDA cleared for use on microwell plate and Cobas platforms. Additionally, quantitative assays for HCV are available as RUO on Cobas and microwell. Other FDA-cleared assays for the Cobas platform include Chlamydia trachomatis, Neiserria gonorrhea, Mycobacterium tuberculosis and HIV. According to Floberg, molecular assays in the pipeline include risk assessment panels for cardiovascular disease, arthritis, diabetes and inflammation. Roche is also investigating other platforms including microarrays for genes that impact drug metabolism including cytochrome p450, as well as the use of quantitative expression in oncology to assist with therapy monitoring in leukemia and lymphoma.

Roche is also developing a number of ASRs for assays traditionally performed with cell culture techniques in microbiology labs. The company recently launched an ASR for group A strep that can deliver results in less than one hour on the LightCycler. ASRs in the pipeline for this platform include tests for herpes simplex 1 and 2, Epstein-Barr virus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococcus (VRE), group B streptococcus, varicella zoster virus (VZV), and Bordetella pertussis. “We have a family of products under development for the speciation of bacteria, and a next generation of products that will allow you to screen for these bacteria, and if positive, to follow-up with additional testing to determine which bacteria are present,” said Floberg.

Bayer Diagnostics’ product line for the molecular diagnostics has recently expanded with FDA clearance of the Bayer System 340, an automated platform for the Versant family of branched DNA (bDNA) signal amplification assays. This benchtop system, which is configured with a reagent module, a bDNA analyzer and Windows-based data management software is designed for medium to large hospitals. The System 340 simplifies workflow by automating plate processing, and can accommodate from 12 to 168 specimens per run. Hands-on time is also reduced by the inherent simplicity of the bDNA amplification method employed in the Versant assays. “With PCR and TMA, you multiply the target to obtain your signal,” explained Peter Knueppel, senior vice president for nucleic acid diagnostics at Bayer. “These techniques are very susceptible to contamination. With the Versant bDNA method, you isolate the target and then create many signals around the target. This reduces the risk of contamination, and means that sample preparation, which is much less complex than for PCR or TMA methods, does not require a clean room.”

The system was launched with the Versant HIV-1 RNA 3.0 assay for the quantification of HIV-1 in plasma. “The Versant HIV-1 assay has a high degree of precision, providing detection of a three-fold change in viral load across the assay range,” said Knueppel. “This has a direct impact on patient management, because now a clinician can be assured that changes in results over time are related to changes in patient status, not variation in the assay.” According to Knueppel, Bayer anticipates the availability of a HCV bDNA assay for the 340 platform the first quarter of 2003, to be followed by the development of a HBV assay to complement the Versant product line. Bayer is also continuing development of qualitative hepatitis assays for the TMA-based Gen-Probe platform, and will be offering HIV-1 genotyping through the integration of Visible Genetics technology into the Bayer portfolio.

With the plethora of new assays and the rapid development of new technologies, it is often a challenge to determine which assays to implement within the clinical laboratory, especially in light of the increased cost associated with these assays. “Laboratory managers and clinicians depend upon thought leaders in academic settings to determine the impact of laboratory testing on practice patterns. Industry also has a role in test acceptance and utilization. Ultimately, the laboratory manager must form a partnership with other healthcare providers in a team approach to provide these services to their communities,” suggested Roche’s Floberg. “We are on the verge of a paradigm shift where the laboratory’s role will expand from disease diagnosis to include evaluation of results, the identification of risks and symptoms, and therapy management,” agrees Access Genetics’ McGlennen.

For now, he suggests that laboratories look to implement molecular techniques that have clear and significant clinical impact within their medical communities, and to streamline both access and interpretation. “Laboratories should strive to develop very accessible and straightforward methods to place this information into the hands of practitioners.”

Louise Lazear is a freelance writer based in Charlotte, N.C.