Diagnostics & Imaging Week Contributing Writer

SINGAPORE – Pharmacogenetic testing is an emerging segment of the Asian in vitro diagnostics market, along with diagnostics employing microarrays and lab-on-a-chip microfluidics technology. Market trends in the region were discussed during the inaugural Diagnostics Asia 2006 conference, organized by IBC Asia Pte Ltd. (Singapore) and held here in mid-January.

Cancer diagnostics also is a major focus of diagnostic laboratories in Asia, as aging populations in the developed countries exhibit increased levels of incidence of neoplastic disease.

Pharmacogenetic testing is another application for molecular diagnostics that has the potential to drive expansion of the market in Asia. As discussed by Dr. Christoph Petry, PhD, head of diagnostics research in Germany for Bayer HealthCare AG (Leverkusen, Germany), today's diag- nostic assessment methods rely on a relatively small number of markers that have limited capability to determine if a particular patient will respond to a certain drug, or if that patient will have an adverse reaction.

For most diseases, existing diagnostic tests are not able to detect causative factors at an early stage. Responses to drug treatment vary widely between patients because information on individual biological variability is not readily available that could allow development of more specific, targeted therapies.

Future generations of molecular diagnostic tests will need to analyze a large number of markers for each patient in order to guide individualized therapy, outstripping the capabilities of existing testing platforms. With today's technologies, analysis of more than 10 markers in parallel creates a major challenge, whereas future applications in pharmacogenetic testing may require analysis of as many as 500 markers.

Bayer has analyzed a number of platforms for performing multiplex analysis, but has found most to be deficient. Liquid array technologies, employing microscopic beads and flow cytometric detection of fluorescent labels, typically exhibit low sensitivity, where polymerase chain reaction (PCR) offers high sensitivity but is difficult to multiplex.

Microarrays offer a means to efficiently multiplex, but only provide moderate levels of sensitivity. Coupling of PCR with microarray detection provides a possible solution, but that approach is very expensive, costing approximately $100 per sample, and can also be prone to errors.

To address the next generation of diagnostic testing technologies, with particular emphasis on high-sensitivity testing, Bayer is developing a new direct nucleic acid analysis system using planar waveguide technology. The system offers sensitivity that is 1,000-fold higher than confocal microscope scanning, the technology now used for detection in most microarray systems, along with a wide dynamic range, and the ability to perform multiplex analysis of large numbers of markers in parallel.

Waveguide technology employs fluorescence detection of molecules that are bound to the waveguide surface, typically using target-specific capture antibodies or nucleic acid probes to capture the analyte on the surface. Evanescent wave technology is used to achieve highly sensitive detection of the bound targets. Nucleic acid targets are detected directly, without amplification, by using antibodies that bind to DNA/RNA hybrids.

While the technology requires further development before it is usable clinically, it promises to provide a simple, fast and inexpensive method for performing highly parallel pharmacogenetic testing.

Dr. Israel Stein, CEO of Clinical Data (Newton, Massachusetts), described his company's programs in pharmacogenetic testing. Clinical Data recently announced a reorganization to focus on its core pharmacogenomics business. The company's new Clinical Data Molecular division consists of three businesses acquired in 2005, including Genaissance Pharmaceuticals (New Haven, Connecticut), Lark Technologies (Houston) and Icoria (Research Triangle Park, North Carolina). The division generated annualized revenues of $33 million for the 12 months ended Sept. 30, 2005, excluding revenue related to Icoria's agricultural genomic services. Clinical Data is one of the largest independent providers of pharmacogenomics and metabolomics services in the U.S.

The company's second division, Vital Diagnostics, markets small-volume clinical chemistry analyzers. According to Stein, the blockbuster model of drug development is broken. Increasingly, fewer patients will take a given drug, but treatment efficacy will be higher and the rate of adverse events will be lower.

Additional benefits of implementing pharmacogenomics may include reduction in the cost of medical care, retrospective analysis of factors involved in adverse drug reactions, and resurrection of drugs that have become stalled in development due to efficacy and/or safety issues.

Clinical Data now offers two pharmacogenetic tests through its diagnostic services laboratory, including the Familion test for long QT syndrome and a test for genetic mutations that result in an inability to metabolize thiopurine methyltransferase drugs. The company also is developing a new drug, Vilazodone, for use in the treatment of depression, along with a companion genetic test that identifies those patients most likely to benefit from therapy.

Another test is under development to identify the 1.6% of patients with schizophrenia who will have adverse reactions to clozapine. Clozapine has been available for 20 years, but is rarely used because of a low rate of major adverse reactions (agranulocytosis). Use of the new test, which is a "home brew" genetic assay, could allow clozapine to be offered to the majority of patients with schizophrenia as a new and effective treatment option.

Clinical Data has identified 74 candidate genes that are involved in clozapine metabolism or promyelocytic differentiation, or for which associations with clozapine-induced agranulocytosis have been discovered.

Pharmacogenetic testing can potentially be used for management of at least 50 million patients worldwide, a number that could expand significantly as advanced drug treatments become more widely used in developing countries such as China, India and others in the Asian region.

Cancer diagnostics was another topic highlighted at the Diagnostics Asia conference. Cancer is a large and growing public health issue in Asia. Cancer types with high incidence in the region include stomach, colorectal, liver and lung cancer. High rates of liver cancer are due in part to the high prevalence of infection with hepatitis C in parts of Asia. Breast cancer also is a common disease in some countries such as Singapore, where the number of new cases is increasing at 3% annually.

A number of research groups are investigating new biomarkers for use in cancer diagnostics, including Dr. Keli Ou of Agenica Research Pte Ltd. (Singapore), who has developed an integrated proteomic/genomic platform for breast cancer biomarker discovery. The platform employs 2-D gel electrophoresis or HPLC to separate proteins in a clinical sample followed by mass spectrometry analysis to identify proteins that are useful as cancer biomarkers.

A novel aspect of the Agenica approach is the use of cell lines as well as human tumor samples in the biomarker discovery process, as well as combined analysis of both sample types using genomics and proteomics. The method results in discovery of markers that are over-expressed both at the mRNA transcript level and at the protein level, providing increased confidence in the relevance of the markers.

A breast cancer biomarker panel developed using the Agenica discovery method has been shown to unambiguously segregate normal vs. tumor tissue samples using a test set of 100 specimens. Tissue microarrays are used for validation testing. One protein identified by the discovery method looks particularly promising as a breast cancer marker, since its expression is completely eliminated in tumor tissue.

Lance Miller, PhD, of the Genome Institute of Singapore, also presented results at the conference from studies conducted by his group on potential new breast cancer markers. Miller is using DNA microarray analysis to assess patterns of p53 oncogene expression in tumor tissue.

A p53 expression signature derived from analysis of 32 signature genes is used to classify breast tumors and correlated vs. histologic grade. In addition, the ability of the p53 signature to predict response to chemotherapy has been assessed, and has been shown to be an independent predictor that identifies patients who can be spared systemic adjuvant chemotherapy.

In comparison to the Nottingham Prognostic Index, which is based on tumor grade, nodal stage and tumor size and is the conventional index used to identify patients who do not need adjuvant therapy, an index that includes the genetic signature identifies twice as many patients who do not need added chemotherapy. The signature appears to be strongly related to tumor proliferation, and is superior to existing markers such as Ki-67 for assessing proliferative index.

Other studies conducted by Miller show that the genetic signature also can be used to characterize other types of cancer such as liver cancer, potentially making it useful in the management of therapy for a number of tumor types.

Cervical cancer is another important cancer type in Asia. It is the second-most-prevalent cancer among women in the developing world, with an incidence rate of 500,000 per year worldwide, resulting in 290,000 deaths.

The main causative agent of the disease is human papilloma virus (HPV). Only specific subtypes of HPV are associated with malignancy. For example, 50% of all cervical cancer cases are caused by HPV-16. However, the use of HPV testing to detect cervical cancer at an early stage is complicated by the fact that most women are infected with HPV at some point in life, typically at the stage when they become sexually active.

Dr. Vinay Badal of the Institute of Molecular and Cell Biology (Singapore) is investigating the use of DNA methylation analysis as a tool to detect transformation of HPV-16 from the latent state existing in most women to a tumorigenic state. A clinical study conducted by Badal has shown that the methylation status of the HPV-16 promoter region is negatively correlated with tumorigenesis, i.e., a low level of methylation is correlated with cancer progression.

Badal's methylation assay could potentially be used to identify the risk of cervical cancer development in women who are infected with one or more of the high-risk types of HPV. Badal speculated that exposure to carcinogens could be one of the mechanisms resulting in demethylation of HPV genes.

The market for products used in cervical cancer testing is one of the most rapidly growing sub-segments of the in vitro diagnostics market.

As shown in the accompanying table, worldwide sales of products used in cervical cancer screening, including HPV test kits sold by Digene (Gaithersburg, Maryland), as well as slide preparation and automated imaging products from Cytyc (Marlborough, Massachusetts) and TriPath Imaging (Burlington, North Carolina), are projected at well over $500 million in 2005, and have grown at an 18.6% compound annual rate over the past four years.

(Next week: Point-of-care testing is expanding rapidly in region.)

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