title: Enhanced detection of BRAF-mutants by pre-PCR cleavage of wild-type sequences revealed circulating melanoma cells heterogeneity.
abstract: OBJECTIVE
  Characterisation of circulating melanoma cells (CMC) for BRAF status can provide a strategy for non-invasive serial genotype monitoring in patients receiving BRAF inhibitors. We aimed to establish a method for BRAF mutation analysis at CMC level and we applied it in a cohort of CMC-positive patients.
  
  
  METHODS
  We established a sensitive method for detection of BRAF mutations at codon 600 in whole blood samples of patients with melanoma, positive for presence of CMC. The method based on selective cleavage of wild-type sequences by taking the advantage of the presence of a TspR1 enzyme restriction site located at the site of mutation.
  
  
  RESULTS
  In a serial dilution experiment spiking BRAF mutated cDNA into BRAF wild-type cDNA the method allowed to detect mutated cDNA till a dilution of 1:10⁴. Peripheral blood (PB) samples resulting positive for CMC and matched tissue specimens from 21 different AJCC stage IV melanoma patients were analysed. A 91% (19/21) correspondence between BRAF status in tissue and PB specimens was observed. In a patient (whose melanoma showed to bear the BRAF V600E mutation in blood, but not at tissue level) our analysis showed that blood samples with PCR evidence for CMC were heterogeneous for BRAF status under limiting-dilution conditions, suggestive of heterogeneity of CMC.
  
  
  CONCLUSIONS
  The method reported here represents a rapid approach for determination of BRAF status in the blood of patients with CMC.
authors.affiliation: Department of Hematology and Medical Oncology, Charité, Campus Benjamin Franklin, Berlin, Germany.
authors.forename: Alberto
authors.initials: A
authors.lastname: Fusi
authors.forename: Rebecca
authors.initials: R
authors.lastname: Berdel
authors.forename: Swantje
authors.initials: S
authors.lastname: Havemann
authors.forename: Anika
authors.initials: A
authors.lastname: Nonnenmacher
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keilholz
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008545:Melanoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009154:Mutation; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D048493:Proto-Oncogene Proteins B-raf / Q000097:blood / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf