Molecular methods have revolutionized the way clinical labs perform testing. Here's what IVD manufacturers need to know about this game-changing field. (This essay appears as the introduction to the "Tools for Molecular Diagnostics" section of the 2012-2013 annual buyers guide.)
Molecular methods have revolutionized the way clinical labs identify the presence of microorganisms in patient samples, monitor viral responses to therapy, and characterize genetic disorders. Challenges with traditional techniques include difficulty growing organisms with prepared media or in cell culture, slow or poor growth kinetics in vitro, limited sensitivity, and the labor and technical expertise requirements associated with manual methods. The molecular diagnostics industry continues to grow rapidly, thanks to improvements in technology, automation, and assay design, and the increasing availability of methods with FDA approval or clearance.
As IVD manufacturers make molecular diagnostic tests more accessible for labs with low to moderate testing volumes, it puts successful implementation of sophisticated technology within their reach, providing faster turnaround times, expanded menus, and opportunities for growth.
Many molecular diagnostic methods evolved in the research laboratory from natural processes facilitated by eukaryotes, viruses, and bacteria to replicate nucleic acids. Polymerase chain reaction (PCR) technology, the current gold standard for molecular diagnostic tests, is an in vitro method of amplifying and detecting very specific, short sequences of nucleotides. Similar to an accurate postal address, which ensures that a letter is delivered to a single exact location, the targets for PCR assays are cross-matched and evaluated with databases packed with sequence information from years of clinical and life science research studies, guiding assay developers to unique and appropriate sequences within the genome. Infectious agents, cancers, and inherited disorders can frequently be identified through amplification and detection of these sequences, which are carefully selected through rigorous evaluation. The minimal essential components for this process have been well characterized:
• purified recombinant polymerase,
• nucleotides in molar excess,
• primers and probes specific to the target sequence of interest,
• buffers and cofactors to stabilize the reaction,
• the patient sample/nucleic acid of interest.
These PCR-based techniques are rapid, highly sensitive, and easily developed with widely available tools for designing and developing custom applications.
The clinical diagnostics community has adapted these technologies for a diverse array of applications, from detecting the presence of an infectious disease to evaluating gene mutations associated with cancer. Molecular diagnostics tools are providing laboratories and clinicians with information that can be used to personalize the management and treatment of patients, leading to better outcomes, minimizing the impact and side effects of unnecessary treatment, and improving quality of life.
Innovative technology has yielded tremendous progress on the system side, as well. Fully automated systems for PCR deliver reliable results, minimize handling and processing errors, and help improve confidence in reporting patient results. Consolidation of multiple applications on a single test platform allows for streamlined operation and facilitates staff familiarity with the system, reducing the need to train operators on each system for a single application.
In addition, more intuitive software is simplifying training and providing end users with a better working environment. Remote diagnostics offers new tools for troubleshooting, allowing a technical support representative direct access to the laboratory system for real-time assessment and problem solving without delays associated with onsite evaluation. Laboratory efficiency improves as these evolving technologies are implemented in clinical laboratory systems.
Concerns with previous systems included a lack of flexibility, requirements to perform large batches, and loss of reagents when suboptimal runs were performed, significant investment of labor for daily and weekly system maintenance, long turnaround times, and the generation of excessive liquid and plastics waste with each run. New technologies, while still focused on the fundamental chemistries that established the field, have incorporated innovative features such as positive sample ID tracking, enzymatic and engineered contamination control, precision pipetting, dramatically reduced system maintenance, and reduction in plastics and consumables.
Another area that has traditionally posed challenges for molecular testing is genetic evolution and the associated variability of target sequences. For example, as human immunodeficiency virus (HIV) evolves within the host, it can lead to mutations in the target sequences used for diagnostic tests. Monitoring for the evolution of such mutations requires a robust effort in global surveillance and monitoring of public and proprietary databases. Accumulating mutations in HIV have been shown to affect the ability of previous-generation assays to accurately quantify and detect the virus. In 2006, a Swedish strain of Chlamydia trachomatis was found to go undetected by commercial molecular diagnostic screening assays, attributed to a 377 base pair deletion in the target region, the cryptic plasmid. We now have innovative molecular applications that target multiple regions of the genome in a multiplex reaction, a so-called dual target approach. This method involves amplifying and detecting signals from more than one unique target within the sequences of interest.
Companion diagnostics, or qualifier tests for specific biotechnology/pharmaceutical products, are transforming healthcare because they provide a more effective alternative to the one-size-fits-all approach to drug therapy. Instead, companion diagnostics target specific populations where drug efficacy is already known. The advent of companion diagnostics will forever change the way clinical trials are developed for the pharmaceutical industry. Where some drugs had severe adverse side effects on subpopulations, pharmaceutical companies can now exclude those demographics through diagnostic selection. While this can dramatically shrink the potential market size for the drug, it can also increase the number of drugs that previously had been suspended because of side effects. A nonprofit advocacy group called the Personalized Medicine Coalition reports that there are currently about 40 drugs in the United States that have companion diagnostic tests associated with them, indicating that this new approach—sometimes called theranostics—is here to stay.
There are two major developmental approaches for companion diagnostics:
• tests that are developed in conjunction with a pharmaceutical,
• tests developed after the drug has been commercialized.
In additional to developmental factors, reimbursement/cost, regulatory approval, and clinical utility are some of the other factors that affect adoption of companion diagnostics.
Typically, the cost is higher for each companion diagnostic test than for the average diagnostic, because the reimbursement structure has not yet matured to provide single codes for specific companion diagnostic tests. Regarding regulatory constraints, direction from many global regulatory groups is unclear on the definitions and inclusion of companion diagnostic tests. Additionally, many diagnostic labs had already developed their own tests for some of the recent companion diagnostic IVDs. In order to receive reimbursement for the drugs, the prescribing provider is required to use an FDA-approved companion diagnostic test. The requirement has been untested so far, but as regulation becomes more stringent, this may change.
During the past few years, next generation sequencing (NGS) has seen widespread growth into expanded applications. Its growing popularity has paralleled advancements in technology, particularly in the areas of benchtop sequencing and high-throughput systems, bringing increased automation, ease of use, and decreased cost. This trend is facilitating access to sequencing for laboratories of all sizes, enabling leaps in genetic research across varied fields of biology, from agriculture to energy to human disease research.
Perhaps most poised to benefit from the latest advances in sequencing technology are applications in translational research, the process of translating scientific discoveries into routine applications, including clinical diagnostics. The ability to examine consequential portions of an individual’s genome, or the genomes of the bacteria and viruses in and on them, in order to identify individualized risk predictions and treatment decisions, is finally within reach and has the potential to affect clinical decision making globally.
Although whole genome sequencing (WGS) shows promise as a diagnostic tool for the future, challenges remain in establishing its clinical utility. One of the largest hurdles is not generating sequence data, but interpreting them. While next-generation sequencing can efficiently generate myriad data, additional innovation is needed to improve our ability to make sense of these data so the technology is feasible for routine use. Current postanalytical software programs that would potentially offer clinical relevance only provide rudimentary data interpretation and can carry a hefty price tag. The mainstream adoption of clinical sequencing will require software with a user-friendly interface, a stronger focus on clinical results, and a high degree of flexibility, because each institution has its own specialties/capabilities and will potentially be looking for different markers.
There are other cost considerations as well, especially related to managing genomic data. While some sequencing instruments offer data acquisition prices of less than $1000/genome, this is just one part of the cost. With patient genome data reaching the terabyte file size, it is no longer practical to warehouse the data at each hospital/clinic. New markets are being created for this purpose by companies such as Amazon (Amazon S3) and Google (DNAnexus). To store and have full accessibility to the data for a genome, the cost is roughly $800.
Perhaps most importantly, there are several issues that must be addressed before next-generation sequencing technology can become a part of routine clinical practice. Manufacturers, laboratorians and regulatory agencies first need to reach greater consensus in areas such as the criteria for selection and quality of samples, data quality and reproducibility, workflow standardization, bioinformatics handling, regulatory guidance and the clinical significance of variants. Several of these issues were raised at an FDA meeting in June 2011, but the agency has not yet identified a clear path or timeline for providing definitive direction from a regulatory perspective.
In the meantime, one sequencing approach that shows promise for future clinical applications is gene panels/targeted amplification. The technology for this application uses long reads, which are capable of resolving areas of biological complexity by correctly identifying large insertions and deletions, distinguishing multiple variations, and allowing haplotyping over a biologically meaningful distance.
It is clear from recent advances in sequencing and companion diagnostics that technological innovation will continue to drive changes in the field of molecular diagnostics. System improvements and the consolidation of platforms are already paving the way for short-term gains in efficiency for the clinical lab. In the near future, the evolution likely will involve a shift from a strong focus on PCR-based assays, with one test yielding one result, to technologies that generate more information, such as sequencing and other multiplex assays. Other likely developments include more centralized platforms with random access testing and the capability to work in concert with other laboratory technologies with full automation on the front and back end. Molecular test results will continue to play an increasingly important role in the overall diagnosis of disease, individualization of patient management, and value-based medicine.—John Osiecki, PhD, manager, medical and scientific affairs, and Chris Bird, PhD, strategic business development manager, Molecular Centers of Excellence, Roche Diagnostics (Indianapolis)