Diagnostics & Imaging Week Contributing Editor

New technologies for molecular diagnostics were highlighted in a number of presentations at the recent Oak Ridge Conference.

The market for clinical molecular diagnostic products now exceeds $2.2 billion worldwide and is one of the most rapidly growing segments of the worldwide clinical diagnostics market.

The focus at this year’s conference was on nucleic acid sequencing technologies for diagnostic applications. Determination of nucleic acid sequences is already an important application in molecular diagnostics, for analysis of viral genotype in patients with infectious diseases such as AIDS and hepatitis, as well as in genetic testing to detect mutations associated with drug response (pharmacogenetic testing) and genetic diseases.

A variety of new technologies are under development for nucleic acid sequencing with potential applications in clinical diagnostics. Charles Cantor, PhD, of Sequenom (San Diego), described the use of mass spectrometry in DNA sequencing, using the company’s MassArray system and associated iPLEX and MassEXTEND assay technologies.

Mass spectrometry has so far been used mainly in protein analysis, as well for detection of toxic metals in blood samples. However, the technology is now showing promise in nucleic acid sequencing, where its ability to perform high precision measurements of molecular mass allows direct determination of a nucleic acid sequence.

Although the equipment needed for mass spectrometry is complex and expensive, fully automated systems are now available for sequencing which are suitable for the clinical lab setting. Cantor discussed applications in cystic fibrosis screening, screening for beta-thalassemia, detection of cancer-related mutations, genotyping of human papilloma virus, and quantification of SNPs in DNA extracted from maternal plasma for prenatal diagnosis of Trisomy 21 (Down’s Syndrome).

The versatility of mass spectrometric analysis is demonstrated by analysis of DNA methylation in patients with cancers such as acute myelogenous leukemia. Nevertheless, mass spectrometric sequencing requires use of complex and expensive equipment, and several hours are typically required for pre-processing of samples, pcr, and processing of samples prior to mass spectrometry analysis.

Other new technologies for nucleic acid sequencing include nanopores, Sequencing by Synthesis, and nanofluidics. Amir Meller, PhD, of Boston University discussed development of nanopore array technology and its applications in sequencing. Nanopore arrays are being developed by Meller in partnership with LingVitae (Sk yen, Oslo) using that company’s Design Polymer technology.

Nanopores have been studied for a number of years as a potential vehicle for single-molecule analysis, including sequencing of nucleic acids. Nanopores can be formed using a variety of methods, such as by creating protein structures with naturally occurring channels having nanoscale dimensions, or by the use of a variety of fabrication techniques employed in the semiconductor industry such as e-beam cutting of nanopores in silicon nitride.

Meller is now using e-beam fabrication after finding that protein nanopores did not provide adequate discrimination of single bases. Miller is employing Design Polymer tags, which are 20-base nucleic acid polymers attached to each base in the sequence and providing added discrimination of bases, along with nanopore arrays to increase throughput relative to single nanopores. The goal is to reach a throughput of greater than 4 megabases per second using a 100x100 array, with a total cost for genome sequencing of under $1,000.

Helicos BioSciences (Cambridge, Massachusetts) is developing a new sequencing technology using single-molecule analysis. The assay involves immobilization of primers on the surface of a chip, followed by hybridization of sample genomic DNA fragments, and then by labeling one base at a time with a fluorescent tag.

An image of the chip surface is collected during each cycle, allowing the sequence of the target to be determined by monitoring the sequence of attachment of labeled bases. The method offers simple sample preparation methods, high throughput, and low cost. However, control of errors is an issue, necessitating the use of two passes to obtain an acceptable error rate of 0.01%.

The technology is being applied in oncology to guide treatment of lung cancer with agents such as Gefininib, which target specific oncogenes; and in personalized medicine to identify patients at risk for adverse drug reactions. Jerzy Olejnik, PhD, of Intelligent Bio-Systems (Waltham, Massachusetts), described his company’s program to commercialize Sequencing by Synthesis technology licensed from Columbia University (New York).

The technique is implemented in a high-density array on a chip having dimensions similar to a microscope slide. Four-color fluorescent labels are incorporated one base at a time, and fluorescent microscopy is used to determine the base binding pattern during analysis. The company’s goal is to develop a sequencer capable of a throughput of 3 gigabases per day at a cost of $1,500 per gigabase.

Cell analysis is another expanding area within clinical diagnostics, having moved beyond cell counting and flow cytometry to use of advanced automated imaging methods to determine cellular properties and assess response to drugs or other stimuli.

Products for cell analysis represent a significant and rapidly growing market. In addition to products for analysis of cellular specimens such as PAP smears, the field has advanced to include assessment of circulating tumor cells for prediction of cancer recurrence, analysis of cellular response to drugs or other stimuli for therapy guidance, and use of cells as sensors for sensitive detection of biological pathogens.

Advances in cell analysis techniques for cancer were described by Garry Nolan, PhD, of Stanford University (Palo Alto, California). Nolan’s team has developed new multiparameter flow cytometry techniques that allow interrogation of cell signaling pathways in complex diseases such as Acute Myelogenous Leukemia, Follicular Lymphoma, and Systemic Lupus Erythematosus. Analysis of the flow cytometric data presents a new challenge due the very large amount of data generated in a typical analysis and the complexity of the signaling pathways.

The Stanford researchers developed a new electronic architecture for a statistics supercomputer which is used to analyze the data. The project is now focused on development of a comprehensive network topology map of signaling in all primary immune subsets, creating a generalized tool for assessment of a wide range of autoimmune disorders and malignancies. The technology may also have applications in monitoring of stem cell therapy. One near-term application involves assessment of B cells in SLE patients to rapidly predict response to drug therapy.

Douglas Malinowski, PhD, of BD Diagnostics Tripath Imaging (Durham, North Carolina), described new markers for applications in cell-based diagnostics for breast and cervical cancer.

While PAP smear screening has resulted in a significant reduction in the death rate from cervical cancer in the U.S., there is a 15%-25% incidence of false negatives, and current methods are unable to predict disease progression. There are similar issues with breast cancer screening using mammography and with prediction of relapse of breast cancer using lymph node analysis, and with the diagnosis of ovarian cancer.

Tripath has analyzed clinical specimens from patients with breast and cervical cancer to identify a panel of six protein biomarkers that can be used to improve detection and prediction of recurrence. Expression of any two or more markers indicates an increased risk for cancer or cancer recurrence.

Tripath has developed the VIAS Imager, an interactive histology imaging system, to perform quantitative analysis of protein biomarker expression patterns in cells, improving the detection of CIN2+ lesions within atypical cells in PAP specimens, and improving the predictive capability of tests used in early stage breast cancer.

Another cell imaging technology with applications in cancer diagnostics was described by Richard Bruce of Scripps-PARC Institute for Advanced Biomedical Research (Palo Alto, California). The Fiber Array Scan Technology (FAST) is used to detect rare circulating tumor cells that serve as an early indicator of recurrence in breast and lung cancer.

A system for detection of circulating tumor cells in breast cancer patients developed by the Veridex unit of Johnson & Johnson (New Brunswick, New Jersey) has already received FDA clearance and is in clinical use. FAST technology promises to decrease analysis time, improve predictive value, and extend the applications of circulating tumor cell analysis to include diagnosis of lung cancer.

Because the FAST technology does not magnetically capture circulating cells, disruption of cell morphology doe not occur as with other systems. The analyzer employs a scanning laser beam to interrogate the cells, and a fiber optic detector to capture the emitted light. Positive cells are presented to the pathologist for verification following an automated scan. Initial studies indicate the VIAS system is more sensitive than the Veridex analyzer.

Two companies described development-stage systems that employ cells as sensors for sensitive detection of biological markers.

ACEA Biosciences (San Diego) described an integrated cell processing system, the BT-CES System, which measures changes in cell impedance to determine their response to various environmental stimuli using an electronic cell sensor array. The system has applications in cancer chemotherapy, immune system therapy, and infectious disease testing.

A drug resistance assay for use in predicting response to chemotherapy has been developed which measures cell impedance vs. time following exposure to a drug such as paclitaxel. The system has also been used to assess response to cancer immunotherapy using effector cells. In infectious disease diagnostics, the BT-CES system can be used to assess response to exposure to infectious agents such as West Nile Virus, and to assess the degree of protection provided by neutralizing antibodies.

Innovative Biosensors is developing the Cellular Analysis and Notification of Antigen Risks and Yields (CANARY) system for rapid detection of pathogens such as those involved in sexually transmitted diseases and respiratory diseases.

CANARY technology employs B cells engineered to express cell surface receptors that are specific for a pathogen of interest. Upon exposure to the pathogen and binding to the receptor, internal cell signaling pathways are activated resulting in stimulation of reactions that generate luminescence. The cells can be used to detect the presence of a specific pathogen rapidly, with a response time of one to three minutes depending on whether dry or liquid samples are tested.

A model system has been developed for detection of Chlamydia in urine with a sensitivity of 200 elementary bodies. CANARY requires minimal sample handling, making it ideal for applications in which rapid identification in non-laboratory settings is important.

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