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Published: August 20, 2010
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15th Anniversary Essay: Molecular Diagnostics: A 15-Year Perspective
The field has matured since 1995. The challenge that remains is to improve clinical utility.
The era of clinical genomics began well before the announcement by private and federally funded scientists that a draft detailing the sequence of the human genome was complete. Using what now would appear to be crude tools in the laboratory, molecular biologists made the initial transition from research to clinical diagnostics using the Southern Blot transfer analysis. This technique was used for the following purposes: (1) to identify the presence of any particular sequence in DNA isolated from a patient sample, (2) to identify gene rearrangements associated with B and T cell lymphoma, (3) to track chromosomal inheritance patterns through linkage analysis, and (4) to identify gene targets by size variations in repetitive sequences. And, incidentally, scientists had to make and pH their own buffers. There was plenty of excitement built around the information provided by the Southern Blot assays, but in hindsight we realize that most clinical molecular applications require technologies with much better performance characteristics. The Southern Blot carried the field for at least fifteen years, from the mid-1970s thru the 1980s as the “gold standard” for molecular diagnostics.
While we have experienced many changes in the technologies used routinely by clinical molecular diagnostic laboratories, we have also experienced an overwhelming increase in the number of clinical applications that have been accepted as standards of practice. This era of clinical genomics has exceeded expectations of the initial promises for better diagnostic testing, more-accurate risk assessments, expanded preventive medicine practices, and better therapeutics. Since 1995, the field of molecular diagnostics has exploded on all fronts including technology, clinical applications, and social implications.
Technology
In Vitro Amplification. Initial blotting technologies suffered from relatively poor analytical sensitivity, long turnaround time, and extensive hands-on time. In the early 1990s the few clinical laboratories that were performing molecular diagnostic testing began the transition to the in vitro amplification technology identified as the polymerase chain reaction (PCR). This new technology demonstrated improved performance characteristics with end-point detection that included much better turnaround times and unprecedented sensitivity. It became apparent, however, that post-PCR manipulations of amplicon for detection purposes were prone to contamination if strict precautionary procedures were not practiced.
Other in vitro sequence amplification technologies were developed, such as the ligase chain reaction (LCR), transcription mediated amplification (TMA), and strand displacement amplification (SDA), which were more closed technologies that required specific instrumentation for detection. In addition, signal amplification technologies such as hybrid capture and branched-chain DNA chemistries were implemented for specific clinical applications. During the next 15 years, the traditional or end-point PCR was almost completely replaced in the clinical laboratory by real-time PCR.
Real-Time Amplification. The introduction and acceptance of real-time PCR in molecular diagnostics was stimulated by its offer of better sensitivity and specificity and increased quantitative capabilities. Turnaround times were even further improved by eliminating the need for separate post-PCR detection, and closed reactions helped reduce and even eliminate issues of contamination. The initial use of DNA binding dyes allowed for rapid identification of amplicon. However, these dyes were nonspecific in that they would bind to any double-stranded DNA molecule.
The development of more robust detection chemistries through the use of specific probe technologies (e.g., Taqman, etc.) helped to revolutionize the use of real-time PCR. By labeling probes with different fluorescent dyes, one could perform multiplex reactions and quantify targets with relative ease. During this same time frame, we saw the first turnkey system from Cepheid, the GeneXpert, that combined sample prep along with real-time PCR in a single-use, disposable cartridge.
Post-PCR Detection. Although real-time PCR had very distinct advantages over end-point PCR, there was still a need to perform traditional PCR for certain clinical applications. For example, real-time PCR has not been able to compete with the detailed information provided by post-PCR sequencing, and the multiplexing capabilities of real-time PCR is normally limited to a few targets. Due to those limitations, post-PCR detection systems were never completely replaced by real-time technologies, and in recent years there has been a resurgence of post-PCR analysis methods in the clinical lab. The increasing demands of traditional PCR in clinical laboratories has been made possible by the many advances in capillary electrophoresis instrumentation for DNA fragment size analysis and Sanger sequencing. Array technologies, from microchip arrays (Affymetrix, Nanosphere) to bead arrays (Luminex) to linear (LipA) and liquid arrays (microfluidic cards), were introduced to the field at unprecedented speed. Many were designed for specific clinical applications, while others could be used in discovery mode with hopes of penetrating the clinical market. That market share soon developed with the use of these microchips for array CGH studies (e.g., Affymetix, NimblGen, Agilent, Illumina).
Automated Sample Prep. Perhaps the most significant bottleneck for clinical laboratories has been the extremely labor-intensive methods needed for the extraction of nucleic acids from various specimen types. The past 15 years saw an enormous dedication of industrial resources to provide highly automated platforms for DNA and/or RNA isolations with small, medium, and high throughput capabilities. The transition from manual to automated extractions was made possible not only by the investment of numerous companies in automated platforms, but also by extraction chemistries, often using magnetic particles to aid in automated processing, that were also developed, providing the highest quality and quantity of nucleic acid from various source specimens.
Automation for High-Volume Testing. The power of molecular technologies was gaining acceptance in the clinical setting, and IVD manufacturers realized that with the introduction of automated platforms, this was not to be limited to small-volume tests. The race was on for manufacturers to develop automated systems for specific high-volume infectious disease targets. In 1996, Abbott Laboratories (Abbott Park, IL) received the first FDA approval for an in vitro amplification technology. The agency approved Abbott’s ligase chain reaction (LCR) technology for detecting Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae). Not only did Abbott introduce a new amplification chemistry, it also set the stage for the introduction of semiautomated platforms for nucleic acid detection with its LCx instrument.
Soon afterward, Roche Diagnostics (Indianapolis) launched the Cobas Amplicor for semiautomated PCR, followed several years later by the ProbeTec by Becton Dickinson (Sparks, MD) for strand-displacement amplification analysis. Gen-Probe Inc. (San Diego) also developed an in vitro amplification technology and transcription-mediated amplification, and launched the Tigris instrument and its successor, the Panther, which fully automate high-volume C. trachomatis and N. gonorrhoeae testing.
Chiron Corp. (Emeryville, CA) developed the first HIV-1 viral load assay, a new technology that was not PCR-based and could quantify the amount of virus in patient specimens. This branched-chain DNA (bDNA) signal-amplification technology accurately measured levels of HIV-1 in patient plasma. Roche followed with a reverse transcriptase–PCR assay for quantifying viral load. In addition to the development of several generations of such assays, newer chemistries with automated instruments (e.g., the System 440 by Siemens [Tarrytown, NY] and the Roche Ampliprep Taqman) have resulted in viral-load assays that can detect as few as 50 copies of a virus per ml of patient plasma.
Tissue-Based Molecular Diagnostics. With new advances in probe technology, we also saw a surge in fluorescence in situ hybridization (FISH), where fluorescent labeled probes could detect target sequences in interphase cells most commonly found in paraffin embedded tissue sections. The need to routinely make chromosome spreads as in the earlier days of FISH chemistries was no longer necessary, and this opened a whole new arena for this technology. This ability to localize probe hybridizations to specific cell types became a method of choice for the pathologist when searching for the presence or absence of certain genetic variants, including translocations and abnormal gene copy number.
Clinical Applications
The past 15 years have been an era of rapid assay development for the many clinical applications of these technologies. It seems as if the only limitation to further development of applications is one’s imagination and, of course, the clinical utility of the particular molecular diagnostic. What follows are some highlights of new applications that came about in the Molecular Pathology Laboratory at the Dartmouth Hitchcock Medical Center (Lebanon, NH) during this time.
Genetics. During this time frame, we saw the completion of the identification of the 6 billion bases that comprise the Human Genome. This accomplishment catapulted the field of molecular genetics by making known the many benign and potentially disease-causing variants in our estimated 25,000 genes. Molecular genetics was introduced nationwide with the first population-screening program for cystic fibrosis (CF). In 1997, the NIH convened a consensus conference to address the need for a national CF screening program. The American College of Medical Genetics, in conjunction with the American College of Obstetricians and Gynecologists, recommended that all pregnant women should be offered screening with a minimum panel of (now 23) CF transmembrane conductance regulator gene mutations. As a result of these newly developed guidelines, laboratories were obligated to develop both diagnostic and carrier-screening mutation panels.
Another first for molecular genetics was the introduction of microchip array technologies for the determination of copy-number variants and deletions associated with developmental delay and other disorders. While many laboratories have not yet brought this testing in house, the availability of this technology through reference laboratories has gained routine acceptance in the clinical setting.
Hematopathology. Hematopathology was the first clinical discipline to truly embrace molecular technologies. In the early 1980s, gene-rearrangement studies for the determination of clonality in B and T cell lymphomas were performed by Southern Blot transfer analysis. This was quickly replaced by more modern PCR technologies and capillary electrophoresis in many laboratories. Over the years, hematopoietic malignancies have been reclassified, and most recently the WHO classifications include various forms of genetic abnormalities, including gene mutations and translocations as critical biomarkers for diagnosis of, prognosis for, and therapeutic monitoring of these diseases.
Infectious Diseases. The past 15 years have exploded with new clinical questions and applications for infectious disease testing. We saw the development of rapid tests with unprecedented performance characteristics become available for many pathogens, and molecular diagnostic technologies rose to the occasion for several major pandemic outbreaks. Molecular surveillance programs sprung up at institutions around the country for methicillin-resistant Staphyloccocus aureus (MRSA), which was heralded as the “Superbug” in the lay press. Advances in HIV and HCV viral-load testing became more automated and the need for viral genotyping became apparent with the introduction of novel therapies. While many laboratorians spoke of the need for rapid molecular respiratory viral testing, the need came to fruition during several flu seasons that resulted in FDA’s granting emergency-use authorization for reagents once designated as analyte-specific reagents (ASRs). We also saw a rapid development of infectious disease testing for specific patient populations, in particular the transplant patients. New assays for viruses associated with transplant patients, such as CMV and BKV, were developed on various platforms using a number of different reagents for both qualitative and quantitative assessments.
The association of the human papillomavirus (HPV) with cervical cancer was established and new guidelines for screening were established using the liquid cytology specimens collected for Pap smears. During this time, two assays received FDA-approval for the detection of high-risk HPV genotypes in cervical samples: the Hybrid Capture II assay (Digene Corp. [now Qiagen], Gaithersburg, MD) and the Cervista assay (Third Wave Technolgies [now Hologic], Madison, WI).
Oncology. As molecular testing was becoming common and influential in other fields of medicine, new insights into the mechanisms of human cancer and novel therapeutics designed to target specific cancer pathways emerged. Similar to the HIV situation in which new therapeutics led to the need for viral-load assays, the development of the anticancer therapeutic Herceptin (Transtuzumab) required a clinical laboratory test to determine a patient’s Her-2 receptor status. While many techniques in clinical labs could accomplish this task, FISH became the method of choice for evaluating Her-2 gene amplification and determining patient eligibility for Herceptin treatment. This example was heralded as the poster-child of novel targeted therapies. Soon after we saw a similar revelation with Gleevec (Imatinib) which targeted the tyrosine kinase created by the fusion of the BCR and ABL genes in Chronic Myelogenous Leukemia (CML). Eventually we would see these novel therapies target either a receptor or tyrosine kinase in numerous other tumors including EGFR in lung cancer. This required testing for the mutation status of certain genes in these tumors to determine the likelihood of response.
Pharmacogenomics. The clinical application of pharmacogenomics (or “pharmacogenetics”) has the potential to be the next big development in molecular diagnostics. The excitement over pharmacogenomics stems from being able to evaluate common genetic variants in genes involved in the activation, metabolism, or elimination of various classes of drugs in order to help physicians choose the most appropriate drug and dose for a specific patient. There have been many obstacles to overcome, however, in making clinical pharmacogenetic testing a reality as a standard of care. These obstacles vary depending on the specific pharmacogenetic application, but they often include financial concerns, uncertainty among physicians about what to do with pharmacogenetic information, and a lack of clinical data proving the benefit of such testing.
Pharmacogenetic testing to predict warfarin metabolism and to help determine its proper dosing created much excitement in molecular diagnostics, especially after FDA changed the labeling of warfarin to point out genetic variants that could contribute to the variability in response seen among patients. The well-established methods of correcting a patient’s warfarin dose along with the previously mentioned obstacles impeded the use of pharmacogenetic testing for warfarin, but more recent changes in warfarin labeling that give specific dosing recommendations based on genotype may lead to increased acceptance and usage of this testing.
Another pharmacogenetic target that has drawn attention in the past year is the antiplatelet medicine Plavix (clopidogrel). Testing for variants in the p450 gene CYP2C19 may be a valuable tool in determining how patients might respond to Plavix treatment. The verdict is still out on the clinical utility of pharmacogenetic testing to aid in prescribing Plavix and for pharmacogenetics in general. However, as the benefits of pharmacogenetic testing become more apparent and more recognized by a larger audience, usage of pharmacogenetic testing is likely to increase, especially as drug manufacturers begin to incorporate this type of testing in their original clinical trials of new drugs entering the market in the years to come.
Conclusion
The molecular diagnostics field has certainly matured over the past 15 years. Technologies and clinical applications have been introduced to our healthcare providers at record speeds. Research will continue to produce an increased understanding of disease processes, and manufacturers will continue to expand and refine the technology and automation needed for clinical testing. As a result, we will continue to push these technologies to their limits in hopes of further developing better clinical utility in molecular diagnostic testing. 
Joel A. Lefferts, PhD is a post-doctoral fellow in the Translational Research Laboratory and Molecular Pathology Laboratory at the Dartmouth Medical School and Dartmouth Hitchcock Medical Center, Lebanon, NH. He can be reached at Joel.A.Lefferts@hitchcock.org.Gregory J. Tsongalis, PhD is an associate professor at the Dartmouth Medical School and Director of the Molecular Pathology Laboratory at the Dartmouth Hitchcock Medical Center. He can be reached at Gregory.J.Tsongalis@hitchcock.org.
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