| 19233913 |
Kitago M, Koyanagi K, Nakamura T, Goto Y, Faries M, O'Day SJ, Morton DL, Ferrone S, Hoon DS: mRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with high molecular weight melanoma-associated antigen-specific monoclonal antibody beads. Clin Chem. 2009 Apr;55(4):757-64. Epub 2009 Feb 20.
BACKGROUND: The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. METHODS: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanoma-associated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. RESULTS: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 x 10 (6) PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression. |
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