BBI Contributing Editor

ORLANDO – Molecular diagnostics is one of the fastest-growing segments of the clinical diagnostics market, having expanded from research-use-only testing over two decades ago to a market that is expected to approach $2 billion worldwide this year for products used in clinical testing. The market encompasses infectious disease testing products used to detect pathogens in a wide range of clinical specimens, blood screening products, tests for the diagnosis of genetic disorders, cancer tests, and the emerging area of pharmacogenetic testing.

New technologies for specimen processing, ampli- fication and detection have played an important role in driving market growth, including technologies for automation of nucleic acid testing, which have become particularly important as test volumes have increased. Applications of molecular testing in the clinical lab are expanding rapidly, and were a key topic addressed at the 2005 annual meeting of the American Association for Clinical Chemistry (AACC; Washington), held here in late July jointly with the International Federation of Clinical Chemistry (IFCC).

A number of new products were described at the conference that promise to expand the range of applications of molecular diagnostics, including new tests for cancer diagnosis and genetic testing. Pharmacogenetic testing is beginning to emerge as an important segment of the market, helping to usher in the era of personalized medicine. Applications of nanotechnology and microfluidics in molecular diagnostics, also described at the AACC gathering, may play a key role in determining how molecular testing will be performed in the lab of the future.

Continued rapid growth for sector

As shown in Table 1, the worldwide market for molecular diagnostics products is continuing to expand rapidly, and is by far the fastest-growing segment of the total diagnostics market. The market exceeded $1.6 billion in 2004, with about three-fourths of sales attributable to infectious disease testing products. Blood-screening products account for the next-largest segment, followed by products for cancer and genetic/pharmacogenetic testing. The market is projected to approach $3 billion worldwide by 2009.

As discussed by Heinrich Dreisman, president and CEO of Roche Molecular Diagnostics (Pleasanton, California), at the AACC conference, the molecular diagnostics market emerged about 20 years ago with the launch of the first nucleic acid-based tests from Gen-Probe (San Diego). Roche entered the market around 1992, introducing products based on the PCR nucleic acid amplification technology. For many of the molecular diagnostic products that are now widely used in clinical labs, there was no established market at the time they were launched. HIV viral load testing, for example, was not feasible until the development of amplified molecular assays, but now is a standard of care in the management of AIDS patients.

That characteristic of the market continues today, as exemplified by the newest molecular diagnostic products introduced by Roche, which include the P450 AmpliChip, a new microarray-based pharmacogenetic test that tests for 8,000 genetic markers simultaneously; a new molecular sepsis assay; and a nucleic acid blood screening test for West Nile virus launched in 2003. While there is consequently some risk associated with competing in the molecular diagnostics segment, the rewards have in most cases justified the investment.

Dreisman identified key growth segments in the molecular diagnostics market as virology, blood screening, women’s health (human papilloma virus testing), microbiology/infectious disease testing, oncology and genomics. An indication of the relative opportunity in those segments is provided by the distribution of molecular diagnostics R&D investment for Roche: 35% in blood screening, 18% in oncology, 14% in genetics, 13% in women’s health, 11% in virology and 9% in microbiology.

The market is far from saturated. Dreisman cited estimates from Roche that only 6% of all clinical laboratories worldwide are performing molecular diagnostic testing in 2005, including 9% to 10% of the labs in the U.S., where adoption is highest. That statistic indicates a need for new automated systems, according to Dreisman, that integrate all aspects of the testing process and thus reduce operator requirements. In response, one product under development by Roche is an instrument that will be based around the AmpliChip technology and will provide full automation of the testing process. Among the assays that may be available on that system are oncology-related tests, such as a p53 AmpliChip that will be the first cancer gene sequencing diagnostic device, with applications in six different cancer types. Other versions of the AmpliChip will have applications in a wide range of other disciplines including psychiatry, cardiology, and pain management.

Roche also is developing a fully integrated (all-in-one) real-time PCR system that is scheduled for launch in 2008. One application under development is a real-time PCR assay for bladder cancer recurrence, and Roche also has seen promising results with a new molecular marker for breast cancer. Test automation is a key trend in the molecular diagnostics market at present. Roche, for example, is planning development of a new system to fully automate HPV testing, as well as a high-throughput automated system for blood screening that will allow single-unit testing to be performed, as opposed to pooled testing that is the norm now.

Another segment of the molecular diagnostics market that has remained essentially untouched so far is point-of-care molecular testing. Roche has begun development of a new product called ICAD that will integrate sample lysis, target capture, amplification and detection in a single device, providing the capability to perform nucleic acid testing at the bedside or in the physician’s office.

A similar product, the Liat analyzer, is being developed by Iquum (Allston, Massachusetts). The Iquum analyzer is a highly compact, self-contained analyzer that uses a specialized tube that integrates all testing processes from sample preparation to amplification to real-time detection. A single test can be performed in one hour, and only requires the operator to collect a sample into the Liat tube, scan the tube’s barcode, and insert the tube into the analyzer. Sample types can include whole blood, urine, plasma, and swab samples.

A number of existing nucleic acid testing technologies, such as silica bead-based nucleic acid ex-traction, immunomagnetic sample enrichment, and PCR have already been adapted to the Liat system. Assays under development or under consideration include infectious disease, oncology, drug metabolism, hemostasis, genotyping and workplace screening tests. Viral load assays are one potential application of the technology. Iquum planned to start clinical trials in late summer for its first diagnostic test, a molecular Leishmaniasis assay that is being developed for the U.S. Army.

POC molecular diagnostic devices could represent a new high-growth segment of the market, allowing the technology to penetrate the large number of testing sites outside of central laboratories, which at present outnumber central labs by a factor of more than ten to one in the U.S.

Many of the applications of molecular diagnostics envisioned by experts at the AACC conference will require the use of new labeling and detection technologies in order to achieve the sensitivity and throughput demanded by the clinical laboratory.

Nanotechnology is one promising option being explored by a number of companies for next-generation applications in clinical diagnostics, including both laboratory-based testing as well as POC testing. One example is a nanoparticle probe technology used in the Verigene system from Nanosphere (Northbrook, Illinois). Nanosphere’s detection technology uses nanoparticles labeled with oligonucleotides, and allows analysis of both proteins and nucleic acids. It achieves detection sensitivity of tens to hundreds of molecules by employing nanoparticles that are each labeled with approximately 100 oligonucleotides, each of which can in turn be labeled with multiple fluorophores or other types of detectable labels, such as electrical or magnetic labels. One nanoparticle can produce a fluorescence signal equivalent to half a million individual fluorescent labels.

Nanosphere also has developed Bio-barcodes technology, which combines nanoparticle detection with magnetic microparticles designed to capture and enrich the nucleic acid or protein target. The company initially is focusing on opportunities in the life science market with the Verigene system, but plans development of clinical diagnostic systems for applications in infectious disease, oncology, pharmacogenetics, blood screening, neurological, cardiology and sepsis testing.

As discussed by Larry Kricka, PhD, of the University of Pennsylvania School of Medicine (Phila-delphia) at the AACC conference, nanotechnology now is widely employed in a variety of applications outside of clinical diagnostics, including cosmetics, clothing and fabrics, sunglasses and sports equipment such as tennis rackets.

The technology to manufacture nanoparticles and structures such as carbon nanotubes in large quantities is now available, and researchers are finding that such materials can be used for advanced, high-sensitivity labels, and as structures for manipulating nucleic acids to perform processes such as DNA sequencing using carbon nanotubes, or for separation of target nucleic acids. For example, a team led by Efthimios Kaxiras of the department of physics and division of engineering and applied science at Harvard University (Cambridge, Massachusetts) has fabricated an array of carbon nanotubes that periodically fit into the major groove of DNA, and that may enable ultrafast electrical sequencing of DNA. Nanochannels also are being investigated for use in DNA sequencing by researchers including Schmidt at Carnegie Mellon University (Pittsburgh), and by Albert Van Den Berg, PhD, of Mesa Institute of Technology at the University of Twente (Twente, the Netherlands).

While such nanostructures so far cannot provide single-base discrimination, they can produce signatures that may prove useful for rapid genotyping, or for detection of target sequences without the need for labeling. They also have applications in nucleic acid separation, to provide a highly purified sample of a specific population of DNA or RNA molecules for subsequent sequence-based analysis. Companies active in the development of nanotechnology-based analytical technologies include Agilent (Palo Alto, California), Nanosphere, Access Bio (Monmouth, New Jersey) and Oxonica (Oxfordshire, UK). Oxinica’s Serrcode Detector uses silver colloid nanoparticles to enhance the optical response of Surface Enhanced Resonant Raman Serrcode reagents. The technology provides a 100-fold to 1,000-fold improvement in detection limits over fluorescence techniques using low-cost LED laser excitation. In addition, the Raman-active labels are protected by a polymer shell that avoids inactivation by substances in the sample matrix and also provides a convenient surface for bioconjugation.

As shown in Table 2 below, a number of new molecular diagnostic systems were described at the AACC meeting that will provide an increased level of test automation for the central laboratory and that also are expected to allow molecular testing to be performed in new sites, including the point of care. Bayer Diagnostics (Tarrytown, New York) is developing a new totally automated analyzer, the Versant 440, which will replace the System 340 in the company’s product line in 2006. The new system will provide walk-away automation, as opposed to the current System 340, which requires the operator to return to the instrument at four points during a test, resulting in a two-day turnaround time for existing tests run on the System 340. The new analyzer will run the company’s bDNA infectious disease tests initially, and will offer a significantly higher daily test throughput as compared to existing technologies.

Roche is developing the CAP/CTM, which will provide full automation of nucleic acid testing using PCR amplification technology. Roche also recently received a CE mark for a new Linear Array HPV genotyping test that identifies 37 high and low-risk human papillomavirus genotypes. AutoGenomics (Carlsbad, California) is developing the Infiniti analyzer for test automation in genomic/proteomic profiling as well as infectious disease testing. The company now offers ASR tests for certain analytes, and will eventually develop markers for use in personalized medicine (i.e., pharmacogenetic testing).

An emerging application of molecular testing highlighted at the AACC conference is analysis of circulating nucleic acids, including testing for nucleic acids in urine. Circulating nucleic acids, i.e., nucleic acids that have been released from cells and are circulating in the blood stream or passing into the urine, were first discovered in 1947, prior to the discovery of the structure of DNA by Watson and Crick. Circulating nucleic acids were found in patients with cancer and autoimmune diseases such as lupus in the 1960s and 1970s, but the first evidence for tumor-derived circulating nucleic acids was not uncovered until 1994, opening up the possibility of non-invasively analyzing the genetic make-up of a tumor using a sample of the patient’s blood.

Subsequently, researchers have identified tumor-derived DNA in patients with lung, colorectal, liver, breast and nasopharyngeal cancer. The level of circulating nucleic acid can be quite high, up to a million-fold higher in patients with nasopharyngeal cancer compared to normal. The level also is correlated with tumor load, increasing as the disease progresses and dropping exponentially if effective therapy is initiated, including surgical resection.

RNA also can be found in the circulation of patients with certain diseases, in part because it becomes associated with particles that help protect it from degradation. In addition to potential uses for diagnosis and monitoring of patients with disease, circulating nucleic acids also have possible applications in prenatal diagnosis, since fetal DNA can be detected in maternal plasma as early as seven weeks post-gestation. In a normal pregnancy, fetal DNA comprises 3% to 6% of total DNA in plasma. Pre-natal applications include detection of Rhesus D factor, detection of genetic diseases such as beta-thalassemia and detection of conditions such as pre-eclampsia. At present, pre-natal testing using circulating nucleic acids is limited by the need to use the presence of a Y-chromosome as an indicator of fetal-derived DNA. As discussed at the AACC gathering by Dennis Lo, MD, of the Chinese University of Hong Kong, hypomethylated maspin can serve as another marker of fetal DNA, providing a universal indication of fetus-derived DNA in circulation.

Other potential applications include detection of organ rejection in transplant patients, since DNA is released into the circulation in rejection, probably due to cell death, as well as detection of tissue damage in stroke and trauma. In the latter conditions, the level of circulating nucleic acid is correlated to the degree of tissue damage. At present, development of commercial assays for circulating nucleic acids hinges on the availability of test automation, standardization, characterization of nucleic acids in various disease states, and determination of the function and biological role of circulating nucleic acids. However, Lo said he believes a noninvasive molecular test for detection of Down syndrome based on circulating nucleic acid analysis could be available in as little as five years.

Pharmacogenetic testing applications expand

One of the major emerging segments of the molecular diagnostics market is pharmacogenetic testing, which has important applications in personalized medicine to identify patients who will have adverse reactions to specific drugs, and to identify patients who will respond to specific drugs, particularly to targeted drugs that rely on the presence of a molecular target in the patient in order to act. Richard Weinshilboum, MD, of the Mayo Clinic (Rochester, Minnesota), addressed the promises and challenges of pharmacogenomics and pharmacogenetic testing at an AACC plenary session. Pharmacogenetics is the study of the role of inheritance in individual variations in drug response. Drug response can vary for a variety of reasons, including age, disease state, and presence of other drugs, and is a complex function of drug absorption, drug distribution, drug-target interaction, drug metabolism and drug excretion. Every protein involved in drug response can exhibit variation.

The first pharmacogenetic tests to be introduced were based on analysis of simple markers, such as detection of mutations in the thiopurine methyltransferase gene that affect the metabolism of azothioprine, used in the treatment of Crohn’s disease; as well as tests for molecular markers such as Her2/neu, to select patients who would respond to Herceptin therapy. Those applications required detection of a relatively small number of mutations. However, broader applications of pharmacogenetic testing require genome-wide scans to detect all relevant mutations. In some instances cited by Weinshilboum, the metabolic pathway for a drug can involve as many as 30 different enzymes. As a result, there is a need for enhanced test methods.

The need for improved methods to detect drug non-responders is demonstrated by the data shown in Table 3 below, which shows that poor or no response to a drug is a significant problem in a wide range of common diseases having a collective U.S. prevalence of well over 100 million. In addition to improving response rates for drug therapy, pharmacogenetic testing also provides a tool to reduce or eliminate adverse drug reactions (ADRs). An estimated 3% to 11% of hospital admissions have been attributed to ADRs, and it is estimated that there are about 2 million ADRs in the U.S. annually, including 110,000 fatal episodes.

Initially, pharmacogenetic testing such as assays for TPMT variants was performed using test methods developed by a few specialized labs, and was limited to major institutions such as the Mayo Clinic. Now, pharmacogenetic tests are available as commercial kits from suppliers such as Roche Diagnostics. The CYP450 AmpliChip incorporates a test panel of 8,000 probes, a significant advance in terms of test complexity, and a number of other companies are developing such tests. For example, TmBioscience (Toronto) has developed a research-use CYP450 assay, and AutoGenomics is developing a test for epidermal growth factor receptor (EGFR) mutations with applications in guidance of chemotherapy. Third Wave Technologies (Madison, Wisconsin) also is developing ASR pharmacogenetic tests.

The FDA’s interest in fostering commercialization of pharmacogenetic tests is indicated by the fact that approval of the Roche AmpliChip P450 ASR required only five months. Future development of the market for pharmacogenetic tests will be dependent on test reimbursement, regulation and competition.

Lab-in-a-cell concept holds promise

Another emerging segment of the diagnostics market highlighted at the IFCC/AACC conference dealt with lab-in-a-cell technology. As discussed by Helene Andersson, PhD, of Silex Microsystems AB (Jarfalla, Sweden), which is a company involved in the development of lab-in-a-cell technologies, individual cells provide perhaps the ultimate platform for miniaturization of analytical processes. Advantages of the technology include lower sample and reagent volume, faster turnaround time, a high degree of parallelization, improved test performance, a high level of integration and automation, and portability.

Lab-on-a-chip technology under development at Silex provides a 128 x 128 sensor array on a 1 mm² chip that can perform 2,000 readings per second. Single biological cells are used as the test platform, as opposed to attempting to fabricate all of the micro- and nano-scale features comprising a cell. Between 1,000 and 10,000 different chemical operations can be performed simultaneously in a one picoliter volume, a density that would be difficult to reproduce using existing microscale fabrication procedures.

The system under development by Silex, called NanoScan, includes the use of autofluorescence to track and sort cells, nanoneedles for sampling and injection of sample and reagent, single-cell electroporation, and single-cell mass spectrometry analysis. So far, experiments performed on the individual system components have demonstrated proof of principle for cell isolation, single-cell mass spectrometer analysis, and electroporation to infuse reagents. A sub-microliter electrochemical pump is in development to provide controlled delivery of reagents and samples.

In one configuration of an analytical system based on the lab-in-a-cell technology, approximately 50,000 cells are distributed over the surface of a disc that contains an ordered array of needles and detectors. Individual cells can then be accessed via a simple scanning process, and reagents can be applied either in a gradient across all the cells or to individual cells, with the response analyzed via single cell mass spectroscopy. Studies have shown that cells remain viable after being punctured with nanoneedles.

While such technologies are far from ready for use in the routine clinical laboratory, they offer the promise of a new generation of analytical devices that can provide direct analysis of cellular events. For example, single-cell studies have been performed to track apoptosis in samples from patients with cancer, AIDS, autoimmune disorders and stroke, and different behavior has been observed based on the disease present. Upon exposure to therapeutic agents, such as oncogene inhibitors, cell responses have been shown to track tumor behavior.

Another example of lab-in-a-cell technology is the B cell-based CANARY sensor for rapid identification of pathogens described by a group led by Todd Rider at the Massachusetts Institute of Technology’s (Cam-bridge, Massachusetts) Lincoln Laboratory (Lexington, Massachusetts). In that application, B lymphocyte cells are genetically engineered to express cytosolic aequorin, a bioluminescent protein, as well as membrane-bound antibodies specific for various pathogens including Yersinia pestis (the bacteria causing plague), anthrax spores and E. coli O157:H7. The individual cells emit light within seconds of exposure to the target pathogen, providing speed and sensitivity that cannot be achieved using immunoassays or molecular PCR-based methods.

The technology has recently been commercialized for research use by Innovative Biosensors (College Park, Maryland) as the BioFlash rapid diagnostic system for the ultra-sensitive detection of pathogens. Results can be obtained in less than five minutes. One example of single-cell analysis technology that has begun to penetrate the clinical market is the CellSearch system from Veridex (Warren, New Jersey). While this system does not manipulate single cells to perform diagnostic tests, it detects small numbers of cells with specific markers in a large volume of blood, and is presently being used for detection of recurrence in cancer patients. Veridex has placed 15 to 20 systems so far for research use. The primary application being investigated is detection of metastatic cells in breast cancer patients. The system can detect as few as one circulating tumor cell in 7.5 ml of whole blood with a specificity of 99.99% at greater than or equal to 5 cells.