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Mar 1, 2009 (Vol. 29, No. 5)

Personalized Medicine Grows in Fits and Starts

Researchers Are Focusing on Biomarker Identification and Clinical Development

  • pMRM Assay

    Speaking at CHI’s “Molecular Medicine Triconference” in San Francisco, Michael Pisano, president and CEO of NextGen Sciences (NGS), described work his company is collaborating on with the University of Michigan’s Proteomic Alliance for Cancer Research on “Biomarker Validation in Translational Medicine: In-vitro Model to Human.” Dr. Pisano notes that NGS’s peptide multiple  reaction monitoring (pMRM) assay is specific for targeted proteins, including post-translational modifications, isoforms, and fragments, and provides absolute or relative quantification based on mass spectrometry. 

    The technology directly measures the protein, not the reaction of antigen and antibody as done in ELISA and immunoassays. pMRM requires small sample sizes and is applicable to biofluids, tissues, cells, and FFPE tissue. NGS’s biomarker technology reportedly decreases timelines and increases success rates traditionally associated with the various stages of biomarker development. “The way we do things,” Dr. Pisano states, “allows us to progress very quickly from biomarker discovery to assay. The assay is flexible and can be used for biomarker validation without spending lots of time and money.”

    In the current work, a model system exhibiting the development of metastatic cancer was employed for the discovery of biomarkers indicating the efficacy of new compounds, as well as putative biomarkers for various stages of cancer in humans.

    Quantitative protein profiling was performed on conditioned media obtained from A549 lung adenocarcinoma cells undergoing TGF-α-induced epithelial-mesenchymal transition, (EMT), which results in changed cell morphology and acquisition of a migratory and invasive phenotype. Cells undergoing EMT would mimic circulating tumor cells or cells in the process of metastasis. Therefore, secreted proteins from these cells may represent protein secreted by tumor cells in the early stages of metastasis.

    The discovery phase yielded a panel of about 20 proteins as biomarker candidates for metastatic disease. A pMRM multiplexed assay was developed for the quantitation of all of the proteins. The assay was utilized to validate the marker proteins in the cell model system for testing compounds. The assay was also applied to human plasma samples obtained from normal volunteers and patients with various stages of lung cancer.

    “Our goals were twofold,” Dr. Pisano stated. “First, we used the cell-based system as an assay of EMT to test drugs that can block this mechanism of metastasis.  Our second goal was to look for marker proteins being secreted by metastatic cells into media that might also show up in serum and plasma as putative markers for metastasis.”

    A literature search added three additional biomarkers to the panel and all were tested for correlation with Stage I–IV lung cancer and metastatic disease. Predictably, some are “falling off the panel and others correlate,” as Dr. Pisano puts it. “We hope to have a good, solid panel of confirmed markers for metastatic lung cancer with data from 50 to 60 patients.” The next step will be to validate results in a larger sample set.

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