Received on September 18, 2008
Accepted on January 12, 2009

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

¹ Department of Molecular Oncology, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA
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

^* To whom correspondence should be addressed. 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 a 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.