BBI Contributing Editor

BALTIMORE, Marylamd Almost four years have elapsed since the announcement of the sequencing of the human genome, a feat first accomplished by privately funded researchers at Celera Genomics (Rockville, Maryland), led by Dr. Craig Venter, in June 2000, with a more detailed analysis published in February 2001 based on research conducted by Celera and the National Human Genome Research Institute (NHGRI), a unit of the National Institutes of Health (both Bethesda, Maryland). So far, the impact of that breakthrough has been limited in areas such as clinical diagnostics and drug development, but advances in the understanding of the genetic basis of disease have begun to lay the groundwork for a fundamentally new approach to disease management. As discussed by Christopher Austin, MD, of the NHGRI at a mid-February conference on new developments in therapeutic drug management (TDM), sponsored by the American Association for Clinical Chemistry (AACC, Washington), some of the first new diagnostic tests for common diseases based on genomics are expected to emerge within the next two to five years. New developments in therapeutics are likely to follow in four to 10 years, Austin said.

By 2010, experts such as Francis Collins, MD, PhD, director of the NHGRI, expect genomics to begin to enter the mainstream of medicine, with primary care physicians beginning to practice genetic medicine, and the genetic basis of many common diseases elucidated. Collins predicts that at least 25 common diseases will be addressed using genetic diagnostics, and that gene therapies for certain diseases will begin to appear. One aspect of genomics now beginning to be used in the clinic is analysis of genes involved in drug metabolism to improve the guidance of drug therapy, or pharmacogenetic testing.

As shown in Table 1 below, the number of patients who are candidates for pharmacogenetic testing is estimated to be at least 125 million worldwide in 2004, excluding those taking CYP450 drugs, with the latter group representing at least another 7 million to 8 million patients in the U.S. alone (assuming monthly dosing of drugs). The estimates are probably conservatively low since they are based on demonstrated clinical applications of the technology. The total could expand significantly if all chemotherapy patients as well as additional categories such as patients with neurological diseases (other than epilepsy and Alzheimer's disease) and cardiovascular disease are included.

Clinical applications of pharmacogenetic testing described at the AACC conference include management of drug therapy for infectious disease, cardiovascular disease, cancer and epilepsy. To address those applications, a number of emerging analytical technologies are under development that promise to make testing practical in the routine clinical laboratory. As products have begun to enter the market, regulatory approval and reimbursement have become more important issues for manufacturers. In addition, while pharmacogenetic tests are now becoming available to the laboratory, adoption by clinicians has been very limited. Reasons for the slow adoption of pharmacogenetic testing, and approaches to removing the barriers to adoption, are a key focus for laboratories and test developers at present, and were a major topic of discussion at the conference.

New analytical technologies drive growth

Table 2 below provides examples of some important new technologies for pharmacogenetic testing, including some that are in the development stage as well as those that already are in use for research applications. Nanosphere (Northbrook, Illinois) is developing a new clinical diagnostic system based on the emerging field of nanotechnology, with applications in the detection of RNA, DNA and proteins with very high sensitivity (1,000 copies) without the need for PCR amplification. The technology, called Verigene, uses particles having a diameter ranging from 13 nanometers to 15 nanometers that are densely labeled with nucleic acid oligomers (about 100 oligos per particle). The high binding capacity of the particles along with a silver labeling technique provides high sensitivity. A research use analyzer has already been introduced, and a compact automated Verigene analyzer for point-of-care use in clinical testing is under development. The instrument employs a 10-well disposable plastic hybridization cartridge. Initial applications will focus on coagulation factor tests including a combination Factor V/Factor II test. Total assay time is under a half-hour. Future applications include assays for 5-methyltetrahydrofolate reductase, methicillin-resistant staph aureus, biotoxins, cancer markers, multi-agent infectious disease markers and single nucleotide polymorphism (SNP) detection. The company is beginning discussions with the FDA to plan its program for regulatory clearance of the Verigene analyzer.

Roche Diagnostics (Indianapolis, Indiana), the leading supplier in the global in vitro diagnostics market and the leader in the molecular diagnostics segment, has developed a microarray product in partnership with Affymetrix (Santa Clara, California). The Roche AmpliChip CYP450 employs a large array of oligomers to detect multiple markers simultaneously. Regulatory approval of the product has proven challenging for Roche, primarily because of the emerging nature of microarray and pharmacogenetic testing technology, resulting in a lack of clear guidelines from the FDA regarding the requirements for approval. Roche initially submitted the product as an analyte-specific reagent, allowing it to be used for clinical testing provided the lab performs its own validation of the assay. However, the FDA took issue with that classification for the device, and Roche now has re-released the AmpliChip as a research-use-only product, while also initiating the process for obtaining de novo regulatory clearance as an IVD product. Applications for the AmpliChip CYP450 include drug development and discovery, guidance of drug therapy, disease diagnosis and disease screening/preventative medicine. In the area of drug therapy guidance, the goal is to develop a chip-based assay that will allow patients to be pre-classified into five categories of dose requirement based on their CYP450 genotype. The AmpliChip contains all known variants of the CYP450 enzyme system, allowing a comprehensive assessment to be performed in a single test. The test uses a fingerstick blood sample or a bucchal swab, and uses the Roche polymerase chain reaction (PCR) technology to amplify the target DNA from the specimen. The test is performed using Roche equipment for the initial sample prep and amplification steps, and Affymetrix equipment for hybridization and readout.

Mass spectrometry, often coupled with front-end processing using liquid chromatography, is being applied to pharmacogenetic testing by a number of companies. The technology, while requiring highly complex and expensive instrumentation, offers the capability to analyze a very large number of proteins in a single assay. In one embodiment under study at the National Cancer Institute (Bethesda, Maryland), both tissue and plasma specimens are being analyzed using pattern analysis of proteins by mass spectrometry for early detection of diseases such as cancer and cardiovascular disease. The technology allows a molecular profile to be created for the patient, which can in principle be used to provide a highly specific diagnosis and identify the most effective combination of therapies to use. A protein microarray is used in tandem with the mass spectrometry analysis to create a proteomic pattern derived from a tissue biopsy, while mass spectroscopy is used to perform a pattern analysis of the patient's plasma proteins. A bioinformatics learning algorithm is then employed to correlate observed patient outcome with the measured proteomic patterns, and to develop rules that can be used for therapy guidance, early diagnosis and early warning of drug toxicity.

Other companies that have developed mass spectrometry technologies for applications in clinical therapeutic drug management include Waters Micromass (Manchester, UK) and Cohesive Technologies (Franklin, Massachusetts). The Micromass business unit of Waters Corp. has developed a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/ MS) system for performing therapeutic drug monitoring assays, including assays for monitoring of immunosuppressive drugs. The Micromass Quattro Micro 2795 instrument represents a new generation of mass spectrometry technology that is highly compact and that is equipped with user-friendly software making the system practical for the routine clinical lab. The use of MS/MS helps to minimize effects that plague other assay technologies such as cross-reactivity that often occurs with immunoassays, and implementation of advanced techniques such as ion suppression and an internal standard help to further reduce interferences. The technology typically exhibits high precision (2% to 3%), a wide dynamic range (0 ug/ml to 5,000 ug/ml for cyclosporin), and allows use of a small (10 ul) blood sample that can be collected via a fingerstick. More advanced applications, however, such as simultaneous analysis of multiple drug levels in patients on combination therapy, require a larger sample volume of 50 ul in order to achieve adequate sensitivity at the lower doses typically employed. A sample can be analyzed in five to six minutes after preparation.

Cohesive Technologies is developing a high-throughput LC-MS/MS method for the simultaneous analysis of cyclosporin A, tacrolimus and sirolimus, which are widely used for immunosuppression in transplant patients. In addition, a newer TDM serum panel has been developed that provides results for 14 drugs in a single assay. The Cohesive method provides typical precision of 3% to 6%, and a three-minute analysis time following a 10-minute sample preparation step (centrifugation).

Biotage AB (formerly Pyrosequencing; Uppsala, Sweden) is marketing its Sequencing by Synthesis pyrosequencing system for applications in pharmacogenetic testing (CYP analysis) as well as for a number of other applications in hemostasis testing, neurological disease diagnosis and drug interaction assessment. Applications also exist in the areas of infectious disease, drug resistance, mutation detection and oncology testing (analysis of DNA methylation). The Biotage research-use system eliminates the need for gels, labels, and nucleic acid probes, and can perform multiplex testing, with the capability to analyze for up to six target gene markers simultaneously demonstrated in a CYP assay.

Clinical applications emerging

The use of pharmacogenetic testing to guide clinical decisions on drug therapy is not widespread at present, although there are certain applications, such as testing for thiopurine methyltransferase (TPMT) deficiency in patients undergoing 6-mercaptopurine chemotherapy for acute lymphoblastic leukemia, that have gained a significant level of acceptance. Patients with gastrointestinal disorders and neoplasms also are treated with 6-MP, with a total of more than 650,000 prescriptions written annually in the U.S., and up to 10% of the population has reduced or no TPMT activity, putting them at risk for a severe adverse reaction to the drug. Testing for mutations in the CYP450 enzymes is another application addressing a large number of patients. As shown in Table 3, the number of prescriptions written annually for drugs that are metabolized by three of the major CYP450 enzyme variants total more than 500 million. In addition, 30% of all approved drugs are metabolized by CYP2D6. In total, there are an estimated 2.2 million hospitalizations a year in the U.S. for adverse drug reactions involving commonly prescribed drugs. While there is in principle a strong case for testing of patients prior to giving them a CYP450 drug in order to avoid potentially life-threatening toxic reactions, many pharmaceutical suppliers have been hesitant to recommend testing to doctors who use the drugs. The FDA also has not yet implemented new drug labeling requirements that stipulate testing before administration of drugs metabolized by CYP450, in part because of a lack of FDA-approved pharmacogenetic tests. For example, patients who are candidates for treatment with Straterra, a new drug from Eli Lilly and Co. (Indianapolis, Indiana), recently approved for the treatment of attention deficit/hyperactivity disorder, could benefit from testing, but the FDA decided not to require strong language on the label regarding the use of testing prior to administration, mainly because of the lack of an approved test at the time the drug was approved.

Experts presenting at the AACC conference, such as Michael Murphy, PhD, of Gentris (Morrisville, North Carolina), predict that the situation will change, perhaps later this year, as approved tests enter the market. Gentris expects to file a premarket approval application for a CYP450 pharmacogenetic test in about two months. The Gentris system will have applications in dose management of more than 50 drugs, including analgesics, neuroleptics, cardiovascular drugs, antidepressants, anticonvulsants and anticoagulant drugs. Gentris said it does not expect a major push for adoption to come from physicians, but rather from patients (and perhaps their lawyers) who will be willing to pay for testing. One barrier to market penetration for CYP2D6 tests is a complex web of overlapping patents, 20 in all, that may result in multiple stacking royalties of 5% for a supplier who commercializes such a test. Regulatory issues also may present a hurdle, according to Murphy, since at present the approval process for pharmacogenetic tests is not well defined, and such tests are categorized as genetic tests, raising concerns about legal and ethical issues surrounding genetic testing that are for the most part not highly relevant for pharmacogenetic testing.

Nevertheless, the trend is likely to favor the increased use of pharmacogenetic testing based on the ability of the technology to improve patient outcome and avoid serious adverse reactions. In addition, combining such testing with monitoring of therapeutic drug levels can create a very powerful tool for optimizing drug therapy. For example, researchers at the conference quoted an improvement in survival rate from 30% to 80% for cancer patients undergoing methotrexate therapy when treatment is optimized using therapeutic drug monitoring.

Pharmacogenetic testing also may have important applications in the management of cardiovascular disease. As discussed by Gualberto Ruano, MD, PhD, chief scientific officer of Genomas (New Haven, Connecticut), at the AACC conference, genomics data can be used to predict an individual's response to various preventative strategies employed for patients at high risk for myocardial infarction. Genomas, founded in September of last year, is focusing initially on the treatment of obesity using both genomics and classical physiology to help identify the optimal therapeutic strategy. Quoting statistics showing that more than 50% of the U.S. population is overweight, Ruano described methods under development that may allow doctors to determine which form of therapy (diet, exercise, drug treatment, or others) will have the greatest benefit. In collaboration with Dr. Paul Thompson at Hartford Hospital (Hartford, Connecticut), Genomas has shown a correlation between changes in oxygen consumption and exercise that also are correlated with ApoE genotype. One ApoE type benefits from exercise, showing improved oxygen consumption, while another type does not. The relationship between LDL cholesterol levels and exercise has also been studied, and a population of patients has been identified who derive as much benefit from exercise as from statin treatment. While genotype is not completely predictive of response, since environmental factors and past patient history also come into play, Ruano predicts that genetic testing, coupled with a database of clinical study results and expert system software, will prove to be a valuable tool for development of personalized medicine programs for disease prevention.

Another growing clinical application for pharmacogenetic testing is the management of anti-epileptic drug therapy. As discussed by Charles Pippenger, PhD, of Grand Valley State University (Allendale, Michigan), at the AACC conference, opportunities exist for using pharmacogenomics to improve the selection of anti-epileptic drugs, as well as for development of immunoassays to monitor drug levels during treatment. Anti-epileptic drug therapy has become increasingly challenging as a large number of new drugs have come on the market, and as treatment with multiple drugs has become commonplace. A number of new-generation drugs have been introduced within the past few years that, while providing increased effectiveness, also can produce serious adverse effects in some patients if drug levels are not properly controlled. Examples include vigabatrin, lamotrigine, gabapentin, topiramate, oxcarbazepine, levetiracetam, falbamate and zonisamide. At present, immunoassays are available only for topiramate and zonisamide, while the other drugs are measured by HPLC or mass spectrometry, limiting availability of testing and limiting the ability to obtain results rapidly for dose adjustment based on patient symptoms. Pippenger believes there is an urgent need for new immunoassays to allow rapid assessment of drug levels. That information can then be combined with data from pharmacogenetic testing to allow better selection of drugs as well as optimal determination of dose.

MedTox (St. Paul, Minnesota) is one of the leading commercial labs providing anti-epileptic drug testing services. As described by Jennifer Collins, PhD, of MedTox, measurement of blood levels of anti-convulsant drugs is largely performed in reference labs such as MedTox. However, patient condition can change rapidly, creating a demand for improved access to testing and more rapid turnaround. MedTox is evaluating a novel specimen collection method that is designed to address the problem of lab access, employing collection of blood samples on filter paper. The method requires a small sample volume, allowing collection of a sample at the time a seizure occurs, if desired, and making it useful for pediatric and geriatric patients. Acceptable accuracy (3% to 12%) has been demonstrated in a pilot study, and MedTox now is collaborating with a local hospital to perform a clinical trial of the method.

One of the largest opportunities for pharmacogenetic tests lies in the area of oncology, for the identification of biomarkers that serve as targets for anti-cancer drugs, as well as for identifying patients who are at high risk for cancer who can then be screened at more frequent intervals. One of the first successful pharmacogenetic tests is the HER2 assay marketed by Dako A/S (Glostrup, Denmark). The Vysis (Downer's Grove, Illinois) unit of Abbott Diagnostics (Abbott Park, Illinois) also has recently obtained FDA clearance for a HER2 assay employing in situ hybridization technology. Based on the high incidence of cancer worldwide 10 million cases annually, according to the World Health Organization (Geneva, Switzerland) and about five times that many individuals alive with a history of cancer there is a very large target population for cancer-related testing. Genomic tests also can play a role in the development of new anti-cancer drugs, by helping to identify patients who carry certain molecular targets. As discussed by Michael Stocum of GlaxoSmithKline (GSK; Research Triangle Park, North Carolina), immunohistochemistry (IHC) assays are the most widely used methodology for target identification in drug development. One tool used by Stocum to improve the efficiency and precision of IHC testing is an automated image analysis system from the Quality Diagnostic Labs unit of Ventana Medical Systems (Tucson, Arizona). A limitation of IHC testing, however, is that it typically is practical to measure only one or two markers per assay, creating a risk of missing some important markers that may allow the tumor to escape chemotherapy. As a result, GSK is evaluating technologies such as drug resistance assays and ex vivo tumor testing to enhance their ability to identify drugs that will be effective in a broad spectrum of patients. It also is planning to develop methods to monitor combination therapies for cancer, such as multiple drug regimens and combined drug-radiation therapy.

Another important application for pharmacogenetic testing is for guidance of infectious disease treatment, as in the treatment of HIV infection with protease inhibitors. Most molecular diagnostic testing today is for infectious disease screening, diagnosis and drug resistance assessment. An important difference between pharmacogenetic testing for infectious disease and other types of pharmacogenetic testing is the need to analyze not only human genes but also viral, bacterial and mitochondrial genes. In addition, the genomes of infectious agents such as HIV are highly dynamic, with the potential to change every base in their genome in a period of only 24 hours. As a result, the goal of drug therapy is to suppress replication of the infectious agent's genome, in order to avoid the emergence of mutant strains that are drug-resistant. FDA-approved HIV genotyping assays are marketed by suppliers including Bayer Diagnostics (Tarrytown, New York) and Celera Diagnostics (Alameda, California). Genotyping allows identification of mutations that are linked to drug resistance, enabling the clinician to switch to an alternative drug sooner, and avoiding further proliferation and development of additional resistant strains of the virus. Genotyping in AIDS patients is proving very cost-effective, as described by Debs Payne, PhD, of the University of Texas Medical Branch (Galveston, Texas). Her lab performs a large number of molecular diagnostic tests on HIV-infected prison inmates in its role as a reference lab for the state of Texas, and has demonstrated documented annual savings of $1.5 million, based on testing costs of $500,000 and savings to the pharmacy of $2 million as a result of improved drug selection.

New regulatory, reimbursement strategies

The rapid emergence of pharmacogenetic testing has in some respects outpaced the development of a regulatory and reimbursement framework for the technology. As noted earlier, Roche Diagnostics was forced to reclassify its AmpliChip CYP450 from an ASR to a research-use-only device, having encountered a disparity between its interpretation of the regulatory requirements for such new devices and the FDA's subsequent judgment on their status. However, the agency is now beginning to make progress in developing more specific guidelines for regulatory approval of pharmacogenetic tests, and, perhaps more importantly, in implementing new labeling guidelines for pharmaceutical products that include requirements for testing prior to administration of the drug when appropriate.

An important new aspect of genomic testing is the use of multiplex analysis, as exemplified in the Roche AmpliChip and other microarray-based devices. Conventional TDM tests provide only one result per sample, whereas genomic tests may provide 10, 100 or 1,000 results per sample. The FDA has had to develop a new guidance document pertaining to regulatory approval requirements for multiplex IVD devices, which is now available on the Center for Devices and Radiological Health's web site. The guidance document addresses study design, analytical validation, comparison studies and clinical validation, but sections dealing with expression arrays, GMP adherence, calibration and standardization, and bioinformatics remain to be developed. The regulatory pathway for new pharmacogenetic testing products has consequently become more clearly defined, but suppliers will still need to work closely with the FDA and perhaps take the step of submitting a pre-IDE application and consider narrow claims, at least initially, to achieve timely regulatory approval of new products.

Reimbursement remains a key issue for pharmacogenetic testing products. One negative factor is that programs such as Medicare, which establish reimbursement precedents that become the de facto standard for most other health care benefit programs, do not cover most screening tests as a matter of policy. To the extent that pharmacogenetic testing performed prior to beginning therapy is viewed as a screening test by the Centers for Medicare & Medicaid Services (CMS, Baltimore, Maryland), there may be issues in obtaining reimbursement. However, one tactic available to labs performing pharmacogenetic tests is to submit for reimbursement using existing codes that apply to generic steps required for most molecular diagnostic tests, such as DNA extraction and amplification. According to Murphy, a total reimbursement of $250 is potentially available for CYP2D6 analysis based on the use of generic molecular diagnostic procedure codes. In the future, test manufacturers are expected to lobby CMS to change its policy on reimbursement to allow pharmacogenetic tests to be classified as a highly recommended component of the drug therapy management process.