by Catherine Shaffer
There is a great deal of momentum gathering around the field of Molecular Diagnostics, which encompasses a broad range of technologies and therapeutic areas; nevertheless, a disconnect exists between the potential of molecular diagnostics and the number of available diagnostic tests on the market. At this point, the molecular diagnostics market is dominated by infectious disease testing, particularly three types of tests that have been around for a number of years. These tests are for human immunodeficiency virus (HIV), hepatitis C (HCV) and chlamydia-gonorrhea (CT-NG). According to Jonathan Witonsky, a healthcare practice industry manager for Frost and Sullivan, the top three infectious disease tests represent a market of approximately $1.8 billion globally. But it's a market that is saturated, with relatively flat growth potential.
Emerging, largely unaddressed, markets for molecular diagnostics include HPV and MRSA (under the infectious disease umbrella) and cancer diagnostics. A dizzying array of technologies are available for meeting this demand, including DNA microarray technology, proteomics methods, real-time PCR, also known as quantitative PCR, or qPCR, and more. Yet, particularly in the case of cancer diagnostics, it has been difficult to find good biomarkers that provide a clear differential between a healthy and diseased state, with good specificity and sensitivity. Much of the interest and expectation in molecular diagnostics has settled on gene expression studies via qPCR, which has shown some exciting early results. Gene expression provides access not only to infectious disease testing, but may provide the key to accurate early testing for high-stakes cancers such as breast, colorectal, and prostate. qPCR can also be used to study microRNA, a molecule that has turned the biotechnology world upside down in the short time it has been known to exist. Says Witonsky, "A lot of microRNAs in humans have still to be characterized ... on the life sciences tool side, these technologies have only been on the market for three years, but are quickly moving from academic research to the diagnostic forefront in a matter of years."
qPCR: a handy solution
A number of qPCR systems are now available for diagnostic laboratories. However, these systems tend to be extremely large and costly, making them impractical for laboratories with space and budget restrictions. Another limitation when investing in these room-sized qPCR systems: you are committing to a suite of proprietary diagnostic tests available from that particular company. For a large diagnostic laboratory with well-defined testing needs, this may be the perfect solution—but another option now exists in the Jaguar system by Handylab. Jaguar is a benchtop system based on microfluidic technology, with the ability to run twenty-four tests in about two hours. Further, the twenty-four slots are customizable, and can even accommodate the user's home-brew assays, as well as assays provided by Handylab. Currently, they only offer gonorrhea testing, one of the "big three" in the infectious disease market, but plans are in the works for more, including a group B strep test. Says Sundaresh Brahmasandra, PhD, vice president of product development for Handylab: "Customers are implementing their own assays onto the system. A lot of flu assays have been implemented by two or three of our customers ... we have about fifteen assays in development right now that we hope to commercialize over the next two or three years."
Getting a head start with kits and reagents
A qPCR expression profiling experiment can be compromised by poor assay parameters, if the experimenter does not do their "homework," which is usually a set of validation assays, titrating primer-probe concentration. One way to be certain of having the best possible qPCR is to do a set of serial-fold dilutions of template DNA. The dilutions can help diagnose unwanted primer-dimers and other problems in the assay that can hamper efficiency, but this step does take time. In Qiagen's QuantiFast Multiplex RT-PCR and Rotor Gene Multiplex RT-PCR kits, this homework is done in advance, and users can set up a standard or fast-cycle qPCR experiment in one or two steps. RNA purification steps are eliminated, and up to four targets can be analyzed in one tube. Annette Tietze, senior global product manager, says: "As little as 10 copies of target DNA can be detected in just 60 minutes." Qiagen's proprietary technologies, Q-bond and Factor MP, permit fast cycling and reliable multiplexing without PCR optimization, respectively. Q-bond is a non-protein molecule that increases the binding affinity of DNA polymerase to single-stranded DNA. Factor MP increases the local concentration of primers at template DNA and stabilizes primers bound to template.
Profiling the trees, not the forest
TATAA Biocenter is one of Europe's leading contract research organizations in the area of real-time PCR analysis and clinical molecular diagnostics of circulating tumor cells in the blood. One of the unique strategies that TATAA has taken is zooming down the focus on gene expression profiling. As an alternative to profiling populations of cells, TATAA has profiled single cells, and subcellular sections. Cells can respond quite differently to stimuli. Even though some genes are activated by stimuli, only some of these genes have correlated expression on the cellular level. Characterizing individual responses of cells can yield important information about the mechanism that regulates expression and expression pathways. In a 2005 publication in Genome Research, Mikael Kubista, PhD, of TATAA Biocenter, and colleagues at the University of Lund did single cell expression profiling of pancreatic islet cells, and found that there was a lognormal distribution of mRNA (Genome Research 15, 1388-1392, 2005; Nature Reviews Genetics 6, 1, 2006). This helped them to develop a technique for measuring intracellular mRNA gradients by qPCR. These advances are important in the development of a "smarter" new generation of molecular diagnostics.
Kubista explains: "So far we have only shown this for a very small system. This is where the future is. Essentially assume that you are adding some stimuli to a sample. If you measure the entire sample ... maybe fifty or one hundred genes are affected by the drug. If you go down to the single cell levels, not all of the genes are altered. What you will find, most likely, is that groups of genes are always affected the same way in individual cells. If five genes are always changed the same way in every cell, you can conclude the expression of these genes is controlled by a common mechanism."
Innovative approaches and improved technologies are necessary to solve the difficult problems in molecular diagnostics. Taking a systems approach that encompasses an understanding of biological mechanisms at all levels, from molecule to organism, will yield the greatest breakthroughs in diagnosing "silent killer" cancers and other diseases that have resisted conventional biomarker identification. This requires both rapid and efficient assay technologies, such as high throughput qPCR and optimized assay kits, and an understanding that disease is more than the presence or absence of a marker molecule, but a set of stochastic changes in gene expression in cell populations in response to disease stimulus.