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Sep 15, 2010 (Vol. 30, No. 16)

Finding Promising Protein Biomarkers

Discovery Aided by Availability of Large Patient Databases and Robust Data-Mining Practices

  • Multireaction Monitoring

    Click Image To Enlarge +
    In the iMALDI technique, antibody-coated beads capture peptides (red) and a known amount of an isotopically labeled version of the peptide (blue), both of which are then analyzed in a high-throughput manner by MALDI mass spec. The immuno-affinity enrichment of peptides increases the sensitivity of the assay, which allows researchers to detect and quantify low abundant proteins of interest. [Analyst/Image reproduced by permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/COAN00105H ]

    Christoph Borchers, Ph.D., facility director of the University of Victoria Genome BC Proteomics Centre, presented his concept for developing biomarkers that predict risk for inflammatory bowel disease (IBD) and cardiovascular disease (CVD) using multireaction monitoring (MRM) for quantitative measurement of proteins.

    The range of clinically significant proteins in human blood and serum spans as much as 12 orders of magnitude and is a serious challenge to most protein biomarker screening technologies. MRM can provide accurate quantitation of proteins over a large dynamic range within a single sample. Absolute quantitation of proteins is made possible by the use of isotopically coded peptides as standards.

    According to Dr. Borchers, each protein can have many peptides being used for MRM analysis associated with it. By developing an assay that includes the standard peptide spiked in at a ratio of approximately 1:1 to the endogenous peptide, it is possible to cover a large portion of the range of abundance of proteins in the human blood or serum proteome. Dr. Borchers and his colleagues then created cocktails of these standards tailored to specific diseases for large-scale proteomic analysis.

    In the case of cardiovascular disease, Dr. Borchers identified approximately 80 proteins that are known to be potential cardiovascular risk biomarkers. In a preliminary study using a standard cocktail, he narrowed the list down to five markers that could stratify patient groups with and without CVD with 80–90% accuracy.

    Taking these techniques into IBD, Dr. Borchers is looking at approximately 100 proteins that are potential markers of risk for the disease. Diagnosis of IBD is complicated by the flare and remission cycle, which obscures endpoints and at times generates large placebo effects in clinical trials. Reliable biomarkers and companion diagnostics would eliminate much of the uncertainty in IBD therapeutic discovery and development.

    Progress in biomarker discovery in areas such as CVD and IBD depends on rapid, accurate methods for screening samples. “We’re talking about more than three million people being screened every year,” said Dr. Borchers.

    “We need to have high throughput in addition to absolute quantitation. For this purpose we are now setting up an assay based on the immuno MALDI (iMALDI) technology we have pioneered. iMALDI combines immuno-affinity enrichment of peptides, MALDI mass spectrometry, and MRM. It is capable of analyzing a sample in a few seconds, or thousands of samples in one day, with absolute quantitative results for proteins.”

  • Breast Cancer

    Ginette Serrero, Ph.D., CEO of A&G Pharmaceutical, talked about A&G’s quest to find a breast cancer biomarker that could be used as a theranostic target with diagnostic and therapeutic application, similar to Genentech’s Her-2 and Herceptin.

    A wide-scale biological screen to mine for functional targets turned up GP88, which is a secreted protein that is overexpressed by breast cancer tumors. It is also involved in breast tumorigenesis and resistance to therapy. GP88 is not expressed in normal tissue. Because it is secreted, it can be found in the extracellular environment, making it a good biomarker candidate.

    Dr. Serrero has studied GP88 expression in tumor biopsies and correlated results with breast cancer patient survival. Researchers at A&G are developing two tests for GP88. One is a tissue test to be used in tumor biopsies. The second is a serum test. The firm also has an ongoing prospective study measuring GP88 in breast cancer patients that includes correlation with clinical outcomes.

    The GP88 marker has been shown in biological studies to be associated with resistance to anti-estrogen therapy. “That means that this biomarker could have application in the current standard of care not just as a companion diagnostic to its own therapy,” said Dr. Serrero.

    One of the biggest challenges in discovering GP88 was in tracking down quality clinical samples. “You have a lot of banked tissue samples that don’t necessarily have clinical outcomes associated with them. It  took an enormous amount of time to find the right samples to establish the clinical utility of GP88.”

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