Molecular strategy for detecting metastatic cancers with use of multiple tumor-specific MAGE-A genes

Background: The human melanoma-associated antigen family A (MAGE-A) has hig h specificity and expression in various malignancies, but individual family members are expressed at low frequency in any one particular type of cance r. We therefore developed a method to detect mRNAs from multiple MAGE-A gen es in a single reaction. Methods: Universal MAGE-A (MAGE-A) primers and probe were designed to rever se-transcribe, amplify, and detect by electrochemiluminescence (ECL) MAGE-A mRNAs on the Origen Analyzer. The assay was performed on total RNA of mela noma (n = 9 cell lines and 24 tumors), breast cancer (n = 7 and 26), and co lorectal cancer (CRC; n = 5 and 12). We also evaluated blood from melanoma (n = 50), breast cancer (n = 16), and CRC (n = 21) patients. Results: The uMAGE-A mRNA was detectable in 0.01-1 ng of cell line RNA. The identity of the uMAGE-A cDNA products was confirmed by sequencing and poly acrylamide gel electrophoresis. The uMAGE-A assay increased detection of me lanoma, breast cancer, and CRC tumor by 13%, 31%, and 25%, respectively, co mpared with a MAGE-A1 assay, and by 17%, 19%, and 25%, respectively, compar ed with a MAGE-A3 assay. The uMAGE-A assay detected circulating tumor cells in the blood of melanoma (24%), breast cancer (25%), and CRC (29%) patient s. Conclusions: The uMAGE-A reverse transcription-PCR/ECL assay provides a pra ctical and sensitive approach for detection of various metastatic cancers i n tissues and blood. (C) 2001 American Association for Clinical Chemistry.