BBI Contributing Editor

LOS ANGELES – Clinical diagnostics comprises one of the largest segments of the worldwide medical products market, with sales exceeding $24 billion in 2003 as shown in Table 1. However, when viewed at the provider level, total spending on diagnostic testing accounts for only about 2% to 3% of healthcare costs in the U.S. and most other countries, in spite of the significant role played by diagnostics in healthcare. The importance of diagnostics within the healthcare system is expected to increase in the emerging era of personalized medicine, with technologies such as nucleic acid diagnostics and proteomics playing a key role in market expansion. As discussed by presenters at the late-July annual meeting of the American Association for Clinical Chemistry (AACC, Washington), clinical diagnostics is growing in part because of the expansion of overall healthcare spending, which has continued to increase at double-digit rates for the past few years. But developments in areas such as pharmacogenetic testing, genetic screening, cancer diagnostics and cardiac marker testing are expected to fuel additional growth in the diagnostics sector, and perhaps result in the clinical laboratory playing a central role in the management of patient data.

Emerging applications of clinical diagnostics that appear particularly promising include new and more effective markers for cardiovascular disease, including stroke markers, which will provide more a definitive diagnosis and help to select the optimal therapy for individual patients, as well as developments in highly sensitive molecular testing that could greatly enhance the ability of physicians to detect disease, including applications in prenatal testing and the detection of cancer at an early stage. Informatics also is expected to play an increasingly important role in the diagnostics market, as the amount of data that can be brought to bear on a patient's condition expands and becomes readily available through integrated electronic networks.

As discussed by Jeff Goldsmith, PhD, of Health Futures (Charlottesville, Virginia), advances in image- based information technology are already fundamentally changing the way in which pathology is practiced, and in a few years the initial analysis of cellular images may be performed exclusively by computerized systems, with follow-up evaluation performed remotely by expert pathologists using telepathology. Goldsmith predicts continued advances in noninvasive imaging technologies, including molecular imaging, that may begin to compete with laboratory-based diagnostics, as well as intelligent implantable devices that provide an improved ability to track patient condition in diseases such as diabetes or heart disease and deliver therapy in real time. Point-of-care testing also is likely to become more widely used, according to Goldsmith, as advances in noninvasive diagnostics allow a wider range of parameters to be remotely monitored.

Expanding role for molecular diagnostics

Molecular diagnostics undoubtedly will play a major role in the clinical diagnostics market of the future, as applications of the technology begin to expand beyond the current focus on infectious disease testing into areas such as cancer diagnostics, prenatal testing and pharmacogenetic testing. The worldwide market for molecular diagnostic testing products for clinical use grew 24% in 2003, to more than $1.4 billion. As shown in Table 2, key molecular diagnostics suppliers, many of which reported strong growth in their molecular diagnostics businesses for their most recent fiscal year, include Roche Diagnostics (Indianapolis), Digene (Gaithersburg, Maryland), Gen-Probe (San Diego), Bayer Diagnostics (Tarrytown, New York), BD (Franklin Lakes, New Jersey) and Celera Diagnostics, (Alameda, California), a unit of Applera (Norwalk, Connecticut). Other major suppliers include Abbott Molecular Diagnostics (Des Plaines, Illinois), Innogenetics (Gent, Belgium), bioMerieux SA (Marcy l'Etoile, France) and Third Wave Technologies (Madison, Wisconsin).

One of the newest applications of molecular diagnostics is testing for DNA and RNA in plasma and serum from cancer patients, and noninvasive prenatal testing performed via the analysis of fetal DNA in maternal plasma. As described by Phillippe Anker, PhD, of the Extracellular Biology Research Laboratory (Geneva, Switzerland), at the AACC meeting, the analysis of circulating nucleic acids in plasma and serum (CNAPs) is showing considerable promise as a new type of tumor marker. One of the most promising markers is human telomerase reverse transcriptase (hTERT) catalytic subunit mRNA.

Anker described studies using polymerase chain reaction (PCR)-based analysis of hTERT in patients with pancreatic, head and neck, colorectal and breast cancer, in which the sensitivity for cancer detection ranged from 81% to 100%. For the detection of colorectal cancer, for example, hTERT analysis of plasma samples exhibited 82% sensitivity with a specificity of 90% in a study of 50 patients. While the studies are at an early stage, with less than 100 patients assessed so far with any one cancer type, the technique offers a new approach to early detection of cancer that may have broad applicability. Telomerase is an enzyme involved in cell death, and its activation is associated with uncontrolled cell proliferation.

In addition, studies have demonstrated a correlation between hTERT expression and tumor progression. While data on a much broader spectrum of patients is needed to validate the use of hTERT as a new type of cancer marker, the exquisite sensitivity of nucleic acid target amplification methods holds promise for use in early detection of cancer. Other studies discussed by Anker have assessed monitoring of oncogenes such as k-ras in plasma samples of colorectal cancer patients, as described recently by researchers using the PamChip 3-D microarray developed by PamGene International (Hertogenbosch, the Netherlands), as well as monitoring of Epstein-Barr Virus (EBV) in patients with nasopharyngeal cancer. As described by Anker, one hospital in Hong Kong already is using plasma DNA-based EBV monitoring via real-time quantitative PCR techniques to monitor patients with nasopharyngeal cancer, and has achieved 98% sensitivity as well as correlation of the measured levels with the development of bone metastasis and failure of therapy.

Roche Diagnostics is the leading supplier of real-time PCR diagnostic products, on its LightCycler testing platform. Other suppliers of real-time PCR testing systems include Stratagene (La Jolla, California) and Bio-Rad Laboratories (Hercules, California). Cepheid (Sunnyvale, California) is developing the GeneXpert system that will use real-time PCR analysis, and bioMerieux offers the NucliSens EasyQ that combines NASBA amplification and real-time molecular beacon detection.

Exact Sciences (Marlborough, Massachusetts) discussed its development of a new molecular test for colorectal cancer that is performed on stool samples and employs pcr amplification plus capillary electrophoresis, both widely used and relatively mature molecular testing technologies, to detect gene modifications associated with progression to colorectal cancer. Exact has chosen to commercialize its test via a partnership with Laboratory Corporation of America (LabCorp; Burlington, North Carolina) as a reference laboratory assay. The test was introduced in mid-2003 and is priced at $795. So far, adoption has been rather slow, with a total of about 2,000 tests performed by LabCorp as of the end of Q2 2004, although recent month-to-month growth has been strong.

Studies have shown that the Exact PreGen-Plus test has significantly better performance than the occult blood test, now the most widely used test for colorectal cancer screening, with an overall sensitivity of 67.3%, and specificity of 97.2% for colorectal cancer detection when used in combination with a DNA Integrity Assay and a microsatellite instability marker (BAT-26). A randomized controlled trial comparing the original version of the Exact molecular test to occult blood testing using the Hemoccult II assay from Beckman Coulter Primary Care Diagnostics (Brea, California), a unit of Beckman Coulter (Fullerton, California), found a four-fold improvement in sensitivity. The PreGen-Plus test measures 23 molecular markers associated with colorectal cancer, including 21 point mutations in the APC, k-ras, and p53 oncogenes.

Analysis of fetal DNA in maternal plasma was described by Diana Bianchi, MD, of Tufts University School of Medicine (Boston). The technique offers a noninvasive approach to testing for fetal abnormalities that, unlike amniocentesis, requires only a simple blood draw with no increased risk to the fetus. The method is based on the discovery that there is an unexpectedly high level of DNA trafficking between the mother and the fetus during pregnancy, producing significant levels of fetal DNA in maternal plasma. Using real-time PCR, Bianchi has been able to detect fetal DNA in maternal plasma as early as 32 days post-gestation. Because of the large amount of DNA liberated from the fetus, and the lack of increase in the amount present once the plasma is isolated, it is likely that the DNA is of tissue origin, perhaps making it highly representative of the genetic characteristics of the fetus.

One application that already is offered in certain countries in Europe is fetal screening for Rhesus D factor, and testing for Congenital Adrenal Hyperplasia also appears quite promising. Yet another potential application is the detection of pre-eclampsia, a condition that affects 5% to 7% of all pregnancies, and which can be life-threatening for both mother and fetus. Recent studies have demonstrated a five-fold increase in circulating fetal DNA in women with pre-eclampsia, potentially allowing the condition to be detected up to one month prior to the development of symptoms. The test could be combined with routine second trimester screening, according to Bianchi, requiring no additional visits on the part of the mother. Analysis of fetal DNA in maternal blood also may have applications in improving the ability to detect Down's syndrome, which is missed in 30% to 40% of cases with existing screening tests, and to detect Trisomy 13, a genetic disorder resulting in severe mental retardation for which there is currently no effective prenatal screening test.

Molecular diagnostic methods also are being developed to improve the ability to test for genetic disorders using amniotic fluid. While amniocentesis involves some risk to the fetus, the procedure can be beneficial in high-risk pregnancies by allowing early detection of a number of genetic abnormalities. Bianchi is using technology from the Vysis (Downers Grove, Illinois) unit of Abbott Laboratories (Abbott Park, Illinois) in the development of a new microarray-based molecular karyotyping test using Comparative Genomic Hybridization, a Vysis technique that offers the ability to increase the resolution of standard karyotyping. In addition to allowing genetics labs to pre-screen for wider range of genetic diseases, the technique may prove useful as a screen for certain complications of pregnancy.

Companies highlight new testing systems

New molecular diagnostic testing systems were described at the AACC conference by a number of companies, as shown in Table 3 below. Privately held Iquum (Allston, Massachusetts) is developing the Lab in a Tube (Liat) system, which consists of a single closed tube containing all assay consumables and a small tabletop analyzer. Samples ranging in volume from 10 ul to 400 ul are collected directly into the tube; a fingerstick sample can be used in the case of blood. Other types of fluid samples also can be used. After the user scans a barcode on the tube which designates the test to be performed, the tube is inserted in the analyzer and a series of manipulations are automatically performed, including target enrichment, target amplification and detection in a four-channel photometer. Turnaround time is 30 to 60 minutes.

According to the company, the system has the capability to perform rapid PCR, real-time PCR and transcription-mediated amplification (TMA), an amplification technology developed by Gen-Probe. Sensitivity of 10 copies of DNA has been demonstrated using PCR, and the assays have a dynamic range of seven logs. The 3 kg analyzer can connect to various types of electronic information networks, including AUTO3-A networks used in clinical point-of-care testing. Initial applications include a test for detection of the C282Y mutation associated with hemochromatosis, and a cytomegalovirus assay that uses a 50 ul plasma or urine sample and provides a result in 50 minutes.

Another new molecular diagnostics system was announced at AACC by Nanosphere (San Diego). Its technology consists of nucleic acid probes labeled with gold nanoparticles, a disposable hybridization unit containing the assay slide, an Auto Processing System containing a reagent pack that uses integrated fluid processing, and an automated imaging/analysis unit to read the assay slide and report results. The system has the capability to perform DNA, RNA and protein detection and does not require the use of target amplification to achieve the sensitivity required for tests such as SNP assays. A next-generation device, the Verigene Mobile, is under development that will incorporate all assay steps into a single hand-held device. Nanosphere has raised a total of $23.4 million in funding, and is led by William Moffitt, the former president of i-STAT (East Windsor, New Jersey).

Microarrays are another technology platform being developed for applications in molecular diagnostics. Gene microarrays from Affymetrix (Santa Clara, California) and Nanogen are being used in numerous research studies to assess their ability to accurately identify cancer types with a goal of improving therapy selection, while proteomic arrays from suppliers including Ciphergen Biosystems (Fremont, California) and BD Biosciences are being used to analyze changes in protein expression in cancer patients. As discussed by Eleftherios Diamandis, MD, PhD, of Mount Sinai Hospital (Toronto), at an AACC workshop, some laboratories in Europe are using proteomic microarray tests to decide which patients will receive adjuvant chemotherapy based on their individual protein expression patterns. Another recent example is studies of mutations in the genotype of the epidermal growth factor receptor (EGFR) that correlate with a patient's response to the drug Iressa, a drug manufactured by AstraZeneca Pharmaceuticals (Wilmington, Delaware) used in the treatment of non-small cell lung cancer. Ultimately, both genotype and phenotype (proteomic) data is likely to be required to adequately characterize cancer patients. Considerable improvement is needed in microarray performance, however, particularly in the areas of reproducibility and standardization of results, before the devices are likely to become widely used in cancer testing.

Roche Diagnostics, the leading supplier in the clinical diagnostics market, is partnering with Affymetrix, the leading microarray supplier, in the development of chips with diagnostic applications, and has recently developed a 2000-element chip for the early detection of colon cancer. Roche also has developed a $425 chip in collaboration with Affymetrix for use in pharmacogenetic testing which analyzes changes in the CYP450 gene that controls the metabolism of a wide range of therapeutic drugs, including antiemetic agents used in cancer therapy.

Another application of microarrays in cancer diagnostics, discussed by Glenn Miller, PhD, of Genzyme Genetics (Cambridge, Massachusetts), at an AACC EduTrak session, is in the diagnosis of pediatric acute lymphocytic leukemia (ALL), that will both identify the specific cancer type present as well as check for mutations in the thiopurine methyl transferase gene that signal an inability to metabolize the drug most commonly used to treat ALL.

Proteomics is attracting considerable interest within the clinical diagnostics community, although at present it is not clear how significant its impact on the clinical diagnostics market will be. New tools for proteomic analysis are under development that will allow measurement of the expression of hundreds of proteins simultaneously. Denis Hochstrasser, MD, of Geneva University Hospital (Geneva, Switzerland) said researchers have calculated that there are at least 300,000 different proteins in human blood, and there may be considerably more than 1 million, according to Hochstrasser's estimates. So far, only 289 proteins have been documented in the scientific literature, and only 117 are clinical analytes. Thus it is likely that there are a large number of additional protein markers remaining to be discovered which will prove to be useful in clinical diagnostics.

GeneProt, a Geneva-based proteomics company involved in drug and biomarker discovery, is using mass spectrometry in a systematic effort to identify new proteins with diagnostic and therapeutic applications, and now has approximately 1,800 different proteins in its database. Among the areas of focus for GeneProt are new markers for stroke and sepsis. By analyzing post-mortem cerebrospinal fluid from stroke victims, the company has identified human fatty acid binding protein as a new stroke marker that is superior to S-100 or Neuron Specific Enolase (NSE) used alone. Proteomics also is being used to develop new tests for cancer, as exemplified by a microarray-based protein profiling method for ovarian cancer detection described by researchers at the National Cancer Institute (Bethesda, Maryland) last year. However, the technology is proving to be more difficult to commercialize than originally anticipated.

Daniel Chan, PhD, of Johns Hopkins Medical Center (Baltimore), quoted the Wall Street Journal as predicting that a proteomic panel for ovarian cancer would become available at a price of $125 in 2003, but the test has yet to be introduced. Issues with proteomics technology for clinical diagnostic applications include artifacts introduced by specimen processing, lack of reproducibility of results among different laboratories, the extremely wide (10¹⁰) concentration range of proteins in serum, and post-translational modifications of proteins that increase the complexity of the analysis. In addition, bioinformatics technology needed to analyze protein expression patterns and elucidate changes that can be correlated with disease traits (e.g., early-stage tumor development) is lagging behind the ability to generate large protein profiles.

Nevertheless, some progress is being made in developing clinically useful proteomics methods, as described by Sam Hanash, MD, PhD, of the University of Michigan (Ann Arbor, Michigan) at an EduTrak session on clinical proteomics. Hanash discussed studies using a system called IPAS (Intact Protein Analysis System) that employs an initial depletion of high-abundance proteins such as albumin followed by fluorescence labeling and chromatographic fractionation of the remaining protein constituents. By incorporating reference markers in the sample, Hanash has studied quantitative expression of proteins in patients with graft-versus-host disease. About 200 proteins have been detected that exhibit changes in expression level in the presence of GVHD, and four proteins have been detected that are only expressed in GVHD patients. Related studies are evaluating the use of proteomics to identify tumor-related proteins that induce immune responses in cancer patients. Such proteins could potentially be used to develop a proteomic biosensor for cancer detection, Hanash said.

New markers for stroke

One application of proteomics that appears to be moving closer to commercialization is new diagnostic markers for stroke. Biosite (San Diego) has identified a test panel for stroke that consists of six protein markers (including S-100b, B-type neurotrophic growth factor, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1), and is planning to file for a premarket approval application to market the test in the U.S. in 2005. The use of protein marker panels represents a new approach to diagnostic marker utilization, which involves using the combined levels of a panel of markers to compute an index, rather than analyzing the levels of individual markers relative to a decision threshold. Biosite's studies have found that, while none of the individual markers used in the panel provide an adequate degree of sensitivity and specificity for stroke diagnosis, a stroke index computed from the combined expression pattern of the five markers in the initial panel studied by the company provides a sensitivity of 96.6% and specificity of 97.3%, vs. 33% specificity and 100% specificity for a CT scan.

The company initiated a multi-center trial about a year ago that is now ongoing at 11 sites, analyzing samples from stroke patients presenting within 24 hours of onset, and tracking the patients at defined intervals over a 48-hour period. The trial is designed to include a total of 900 patients, and 637 had been enrolled as of July, including patients with ischemic and hemorrhagic stroke, transient ischemic attack, head injury, and stroke mimic. Since beginning the study, Biosite has expanded the stroke panel to six markers to achieve improved performance of the test.

Other companies developing stroke marker panels include Ischemia Technologies (Arvada, Colorado) and Nanogen (San Diego). As shown in Table 4 below, a substantial market opportunity exists for stroke markers, with the number of patients who are candidates for stroke testing estimated at well over 1 million in the U.S.

The introduction of proteomic test panels into clinical practice may prove challenging, as discussed by Robert Christenson, PhD, of the University of Maryland Medical Center (Baltimore) at an AACC session on stroke markers. New markers have typically been introduced as individual tests in the past, with an associated decision threshold that clinicians use to help guide triage and treatment decisions. In the emerging era of proteomic diagnostics, barriers may arise in obtaining regulatory approval for multiple markers that must be used in combination, along with issues related to reimbursement. For example, labs now are reimbursed for performing individual tests, and not for computing indices based on a panel of markers. Quality control issues also may present a barrier to commercialization for proteomic tests. New tests such as the Biosite stroke panel represent an initial phase in the introduction of proteomics for clinical diagnostics that should provide valuable experience to guide the development of future proteomic tests that may involve much larger panels of protein markers.

Ciphergen Biosystems is one of the leading companies pursuing development of proteomics technologies for clinical diagnostics. The company recently formed a diagnostics division that is developing products based on its ProteinChip platform. The ProteinChip system, which is sold for research use in two different configurations (Enterprise and Personal edition), provides analysis of 16 samples on each chip, and allows up to 168 chips to be loaded on the analyzer. One approach to using the ProteinChip for clinical applications under evaluation by Ciphergen could use antibodies on the chip to capture protein fractions of interest, followed by analysis of the captured proteins using SELDI mass spectrometry. Initial studies with that configuration have demonstrated precision ranging from 10% to 15%. Existing ProteinChips used in research applications costs about $80 each, or between $5 and $10 per sample. While one possibility is development of an automated version of the ProteinChip system for diagnostics applications, Ciphergen also has reported a collaboration with Biosite under which Ciphergen will use its technology for discovery of new biomarkers to be commercialized by Biosite on its Triage immunoassay platform.

Researchers at Johns Hopkins, for example, are using the Ciphergen chip to identify proteins that are released during myocardial infarction. Correlogic Systems (Bethesda, Maryland), a second player in the proteomics testing segment, has introduced the OvaCheck ovarian cancer detection test, which is being offered via a testing service through Correlogic's reference laboratory, Quest Diagnostics and LabCorp. The OvaCheck test analyzes patterns of protein expression for early detection of ovarian cancer in high-risk women.

Another company that may emerge as a player in the clinical proteomics market is Dade Behring (Deerfield, Illinois), the leading supplier of protein analyzers used in today's clinical lab. At an AACC press conference, Dade said it has initiated development of a next-generation protein analyzer that will be derived from its development-stage VISTA platform and will use the company's proprietary LOCI detection technology. At present, however, given the early stage of development of proteomics, Dade has not yet determined the size of the market opportunity.

Strong growth in CV testing

New tests for stroke comprise an emerging segment of the now well-established market for tests used in the management of patients with cardiovascular (CV) disease, which encompasses conditions such as myocardial infarction (MI), coronary artery disease, hemostasis disorders, peripheral vascular disease and stroke. The cardiac testing segment is one of the highest-growth segments of the clinical diagnostics market, driven by rapid uptake of new markers such as BNP for congestive heart failure, continued growth in the use of cardiac markers including troponin for the diagnosis of MI patients, and increased testing for risk assessment in cardiovascular disease. The market opportunity is substantial, with the U.S. market for BNP alone estimated by Biosite at $500 million to $1 billion.

Cardiac risk assessment tests are used to identify patients who can benefit from preventive therapy, such as statin drug treatment, and comprise a growing segment of the clinical diagnostics market. High-sensitivity C-reactive protein (hs-CRP) is the most important new marker to be added for risk assessment, complementing the traditional lipid profile by identifying patients with vascular inflammation who represent an independent risk group. Hs-CRP also has been recently shown to provide additive risk stratification in patients with metabolic syndrome. Key suppliers of hs-CRP tests include Dade Behring, Diagnostic Products (Los Angeles), Beckman Coulter and Roche Diagnostics. Based on proficiency testing data presented at the AACC conference by Gary Myers, PhD, of the Centers for Disease Control and Prevention (Atlanta), Dade Behring is the dominant supplier of systems for hs-CRP testing, followed by Diagnostic Products and Roche Diagnostics. Because hs-CRP is not recommended for use in population screening, test volume likely will not reach the levels for traditional lipid profiles such as total cholesterol, HDL, LDL and triglycerides, but leading providers of hs-CRP testing services such as Quest Diagnostics have reported rapid growth in utilization of the test.

New markers that are now under study for use in cardiac risk assessment, as discussed by David Morrow, MD, of Brigham and Women's Hospital (Boston), include CD-40 ligand and metalloproteinases, as well as some novel markers. According to Morrow, cardiac risk assessment markers that have proven utility include hs-CRP, sCAM-1, IL-6, IL-18, myeloperoxidase and CD40 ligand. Biosite has licensed rights to myeloperoxidase, a putative marker for risk of heart attack, from the Cleveland Clinic Foundation (Cleveland). Risk markers that have utility in selected groups of patients include PAI-1, fibrinogen, D-dimer and homocysteine, while markers whose role in risk assessment remains controversial include Lp(a) and LDL particle size. Some additional new markers that show promise, but for which additional research is needed, include Lp-PLA2 and adiponectin. As discussed by Jesse Adams, MD, of Louisville, Kentucky, while a number of diagnostic tests are now available for cardiac risk assessment and therapies to reduce risk are also available, physicians nationwide have done rather poorly so far in implementing risk reduction strategies. While 95% of physicians understand the latest guidelines for risk assessment and risk reduction therapy, only 18% treat to goal.

New markers also are being introduced to aid in diagnosis, prognosis and therapy selection in patients with acute coronary syndromes. One of the newest markers for acute coronary syndromes is Ischemia Modified Albumin, or IMA, a test developed by Ischemia Technologies and now available on analyzers from Roche Diagnostics and Beckman Coulter. IMA is potentially a very powerful tool, since it detects ischemia that could precede development of an overt myocardial infarction. Consequently, it could provide early warning of an impending event, allowing preventative or prophylactic measures to be taken to minimize or eliminate tissue damage to the heart. At present, IMA usually is used in combination with ECG and troponin measurements to improve the detection of myocardial infarction in chest pain patients. IMA alone gives 90% sensitivity and 80% to 90% specificity for MI detection, vs. 50% sensitivity and specificity for ECG. A panel of tests including troponin, IMA and ECG gives a 97% to 98% negative predictive value for MI, and IMA detects almost twice as many patients at risk for MI as other tests. The IMA test is priced at $30, and so far has been used mainly in the 40% to 45% of chest pain patients who are determined to be at high risk. The test was introduced in February 2003, and so far about 20 U.S. hospitals have adopted it.

A new test that may compete with IMA for early detection of MI is under development by Biosite in partnership with DMI Biosciences (Englewood, Colorado). The test detects cysteinylated albumin, which is a marker of ischemia. As the number of markers that prove useful in the management of patients with acute coronary syndromes or at risk for such conditions expands, diagnostics is expected to play an increasingly important role in the healthcare system.