title: Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level.
abstract: BACKGROUND
  The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.
  
  
  METHODS
  High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45(+) cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA(+), CD45(-), MART-1/gp100(+)). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis.
  
  
  RESULTS
  CTC (HMW-MAA(+), CD45(-), MART-1/gp100(+)) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient.
  
  
  CONCLUSIONS
  Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sakaizawa
authors.forename: Y
authors.initials: Y
authors.lastname: Goto
authors.forename: Y
authors.initials: Y
authors.lastname: Kiniwa
authors.forename: A
authors.initials: A
authors.lastname: Uchiyama
authors.forename: K
authors.initials: K
authors.lastname: Harada
authors.forename: S
authors.initials: S
authors.lastname: Shimada
authors.forename: T
authors.initials: T
authors.lastname: Saida
authors.forename: S
authors.initials: S
authors.lastname: Ferrone
authors.forename: M
authors.initials: M
authors.lastname: Takata
authors.forename: H
authors.initials: H
authors.lastname: Uhara
authors.forename: R
authors.initials: R
authors.lastname: Okuyama
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000097:blood; D001483:Base Sequence; D045744:Cell Line, Tumor; D002469:Cell Separation; D004252:DNA Mutational Analysis; D005260:Female; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D018189:Immunomagnetic Separation; D058965:MART-1 Antigen / Q000097:blood / Q000276:immunology; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000473:pathology*; D008875:Middle Aged; D058990:Molecular Targeted Therapy; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology; D048493:Proto-Oncogene Proteins B-raf / Q000097:blood / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D017422:Sequence Analysis, DNA; D059010:Single-Cell Analysis; D012878:Skin Neoplasms / Q000235:genetics; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; D009363:Neoplasm Proteins; D048493:Proto-Oncogene Proteins B-raf; D019009:Proto-Oncogene Proteins c-kit; D000099216:Chondroitin Sulfate Proteoglycan 4; C482119:BRAF protein, human