title: Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3-Positive Tumors.
abstract: Melanoma antigen A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti-MAGE-A3 immunotherapy treatment. Herein, we describe the development and validation of a quantitative real-time RT-PCR (RT-qPCR) assay for the determination of MAGEA3 gene expression in tumor tissues. In the early phases of development, the designed primers and probe were not able to distinguish between MAGE-A3 and MAGE-A6. To ensure the specificity for MAGE-A3 over MAGE-A6, our strategy was to use a 5'-nuclease probe (or hydrolysis probe). The final assay was shown to be specific and linear within the analytical range, with an acceptable CV for repeatability and intermediate precision. When compared with a reference semiquantitative RT-PCR assay, the two methods were in good agreement, with only 4.23% of the samples giving discordant results. In conclusion, we have developed a MAGE-A3-specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials.
authors.affiliation: Immunotherapeutics, GSK Vaccines, Rixensart, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Gruselle
authors.affiliation: Immunotherapeutics, GSK Vaccines, Rixensart, Belgium. Electronic address: thierry.coche@gsk.com.
authors.forename: Thierry
authors.initials: T
authors.lastname: Coche
authors.affiliation: Immunotherapeutics, GSK Vaccines, Rixensart, Belgium.
authors.forename: Jamila
authors.initials: J
authors.lastname: Louahed
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger