Clinical Chemistry
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Clinical Chemistry 55: 757-764, 2009. First published February 20, 2009; 10.1373/clinchem.2008.116467
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(Clinical Chemistry. 2009;55:757-764.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

mRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen-Specific Monoclonal Antibody Beads

Minoru Kitago1, Kazuo Koyanagi1, Takeshi Nakamura1, Yasufumi Goto1, Mark Faries2, Steven J. O'Day3, Donald L. Morton2, Soldano Ferrone4 and Dave S.B. Hoon1,a

1 Department of Molecular Oncology; and 2 Division of Surgical Oncology, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, CA; 3 The Angeles Clinic and Research Institute, Santa Monica, CA; 4 Departments of Surgery, Immunology, and Pathology, University of Pittsburgh Cancer Institute, Pittsburgh, PA.

aAddress correspondence to this author at: Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404. Fax 310-449-5282; e-mail hoon{at}jwci.org.

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 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.






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Copyright © 2009 by the American Association for Clinical Chemistry.