title: mRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with high molecular weight melanoma-associated antigen-specific monoclonal antibody beads.
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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular Oncology, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA 90404, USA.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kitago
authors.forename: Kazuo
authors.initials: K
authors.lastname: Koyanagi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Goto
authors.forename: Mark
authors.initials: M
authors.lastname: Faries
authors.forename: Steven J
authors.initials: SJ
authors.lastname: O'Day
authors.forename: Donald L
authors.initials: DL
authors.lastname: Morton
authors.forename: Soldano
authors.initials: S
authors.lastname: Ferrone
authors.forename: Dave S B
authors.initials: DS
authors.lastname: Hoon
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D007962:Leukocytes; D008545:Melanoma / Q000097:blood / Q000235:genetics* / Q000276:immunology / Q000473:pathology*; D009154:Mutation / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D048493:Proto-Oncogene Proteins B-raf; D012333:RNA, Messenger; D000099216:Chondroitin Sulfate Proteoglycan 4; C482119:BRAF protein, human