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Feb 15, 2011 (Vol. 31, No. 4)

Value of PCR Amplified as It Moves to Clinic

Polymerase Chain Reaction Proving Its Mettle In Yet Another Arena: Clinical Tests

  • Quantifying Predictive Biomarkers

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    Researchers at the Innsbruck Medical University have developed an assay strategy for CYP2D6 genotyping that combines PCR, ion-pair reversed-phase HPLC, and electrospray ionization TOF mass spec.

    Compendia Bioscience recently introduced the first of its Tumor Segregation Panels™—the Breast Cancer Segregation Panel™ Assay—in partnership with Althea Diagnostics (www.altheadx.com). The companies also plan to launch Colon Segregation Panel™ and Lung Segregation Panel™ assays later in 2011.

    The breast cancer assay uses quantitative RT-PCR to measure the expression of a panel of 96 genes selected to represent key areas of molecular variability in the breast cancer genome. The test is performed on FFPE tissue samples. Analytical algorithms used to evaluate the results can identify correlations between gene-expression variability in individual breast tumors and clinical endpoints such as drug response. The goal is to translate this information into predictive biomarkers that can form the basis for companion diagnostics for standard-of-care therapies and new, more targeted drugs in development.

    In designing the Tumor Segregation Panels, Compendia’s aim is “to build a multipurpose tool that measures all the core modules [gene-expression variables for a particular tumor type] and has a clear path to market as a companion diagnostic,” says Daniel Rhodes, Ph.D., CEO and co-founder of Compendia.

    These “core modules” provide comprehensive coverage of the complexity and variability of breast cancer, according to Dr. Rhodes, who believes the company’s RT-PCR assays can replace custom gene signatures used to predict parameters such as prognosis and drug efficacy. The assays yield quantitative results that can be used to define, more precisely, characteristics such as tumor grade, proliferative risk, and drug response, and to quantify biomarkers such as estrogen receptor or Her2 positivity. Dr. Rhodes will describe the technology underlying the tumor segregation panels at the upcoming “Molecular Medicine TriConference.”

    The 25 core gene modules selected for Compendia’s breast cancer panel represent patterns of genes that showed variation across a cohort of more than 5,000 breast tumors. Each module is represented in the assay by two to three marker genes. The 96-gene panel can be assayed against four samples simultaneously. Althea performs the test in its CLIA laboratory on FFPE biopsy or surgical specimens collected during a clinical trial. Compendia and Althea then analyze the results and correlate the data for individual modules or combinations of modules depending on the clinical endpoints defined in the trial.

    At the “San Antonio Breast Cancer Symposium” late last year, Beate Beer and colleagues from Innsbruck Medical University presented work focused on the cytochrome P450 enzyme designated as 2D6, which has a role in metabolizing about 25% of the prescription drugs presently on the market.

    Tamoxifen, an anti-estrogen drug used to treat breast cancer, is one example of a drug metabolically activated by CYP2D6. Risk of adverse drug reactions or of absent or enhanced drug activity may affect patient populations that have variations in the CYP2D6 gene resulting in a nonfunctional, slow-acting, or super-fast acting form of the enzyme. Eighty CYP2D6 alleles have been identified.

    The Austrian group of researchers has developed an assay strategy for CYP2D6 genotyping that combines PCR, ion-pair reversed-phase HPLC, and electrospray ionization time-of-flight mass spectrometry. Their goal is to validate the clinical relevance of CYP2D6 genotyping and its use in tailoring more effective, personalized treatment regimens to combat breast cancer.

    The PCR component of the assay isolates the gene of interest and amplifies targeted regions in the CYP2D6 gene using a two-step protocol. The first step generates a long-PCR copy of the entire gene. An aliquot of the long-PCR copy then serves as a template for the second PCR step, a 20 µL multiplexed reaction that amplifies nine variable regions of the gene, which cover 604 nucleotide positions (approximately 12% of the entire gene sequence) and 27 known polymorphic sites, enabling the resolution of at least 37 known CYP2D6 alleles. HPLC-MS analysis of the PCR amplicons yields molecular mass information that serves as a sensitive measure of the nucleotide composition of each amplicon.

    “By determining the nucleotide composition of an amplicon, nucleotide variations occurring within the amplified region can be detected”—including unknown variations in vicinity to the polymorphic site of interest, “which enables the resolution of subvariants of common alleles,” says co-author Herbert Oberacher.

    The researchers described the use of their trimodal strategy for CYP2D6 genotyping in 199 individuals: 106 patients with breast cancer who were taking tamoxifen, and 93 healthy subjects. The method yielded “successful amplification of DNA isolated from blood and buccal swabs,” says Oberacher.

    They identified 14 CYP2D6 alleles, forming 39 different genotypes, and used the information to categorize the samples according to predicted enzyme activity. Statistical evaluation of the observed frequencies revealed no significant differences between patients and healthy individuals.

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