title: Quantitative analysis of the BRAFV600Emutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR.
abstract: BACKGROUND
  BRAFV600Eis a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA.
  
  
  METHODS
  The level of BRAFV600EctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients.
  
  
  RESULTS
  The castPCR assay was performed using a mixture of BRAFV600EDNA and BRAFwildDNA and found to be able to detect BRAFV600Eat a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAFV600Emutation. BRAFV600EctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAFV600EctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAFV600EctDNA preceded exacerbation of clinical symptoms.
  
  
  CONCLUSIONS
  The castPCR assay can detect and quantitate small amounts of BRAFV600EctDNA in samples containing large amounts of BRAFwildcell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Ashida
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Sakaizawa
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Mikoshiba
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Uhara
authors.affiliation: Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. rokuyama@shinshu-u.ac.jp.
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Okuyama
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D014408:Biomarkers, Tumor / Q000097:blood*; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000097:blood / Q000235:genetics* / Q000556:secondary; D008875:Middle Aged; D009154:Mutation; D009360:Neoplastic Cells, Circulating; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012878:Skin Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D047368:Tumor Burden; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Circulating tumor-derived DNA; Melanoma; Therapeutic monitoring; castPCR