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Minoru Kitago, Kazuo Koyanagi, Takeshi Nakamura, Yasufumi Goto, Mark Faries, Steven J O'Day, Donald L Morton, Soldano Ferrone, Dave S B Hoon, mRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen-Specific Monoclonal Antibody Beads, Clinical Chemistry, Volume 55, Issue 4, 1 April 2009, Pages 757–764, https://doi.org/10.1373/clinchem.2008.116467
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Abstract
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 × 106 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.
