title: Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors.
abstract: BACKGROUND
  Around 50% of cutaneous melanomas harbor the BRAF(V600E) mutation and can be treated with BRAF inhibitors. DNA carrying this mutation can be released into circulation as cell-free BRAF(V600E) (cfBRAF(V600E)). Droplet digital PCR (ddPCR) is an analytically sensitive technique for quantifying small concentrations of DNA. We studied the plasma concentrations of cfBRAF(V600E) by ddPCR in patients with melanoma during therapy with BRAF inhibitors.
  
  
  METHODS
  Plasma concentrations of cfBRAF(V600E) were measured in 8 controls and 20 patients with advanced melanoma having the BRAF(V600E) mutation during treatment with BRAF inhibitors at baseline, first month, best response, and progression.
  
  
  RESULTS
  The BRAF(V600E) mutation was detected by ddPCR even at a fractional abundance of 0.005% in the wild-type gene. Agreement between tumor tissue BRAF(V600E) and plasma cfBRAF(V600E) was 84.3%. Baseline cfBRAF(V600E) correlated with tumor burden (r = 0.742, P < 0.001). cfBRAF(V600E) concentrations decreased significantly at the first month of therapy (basal median, 216 copies/mL; Q1-Q3, 27-647 copies/mL; first response median, 0 copies/mL; Q1-Q3, 0-49 copies/mL; P < 0.01) and at the moment of best response (median, 0 copies/mL; Q1-Q3, 0-33 copies/mL; P < 0.01). At progression, there was a significant increase in the concentration of cfBRAF(V600E) compared with best response (median, 115 copies/mL; Q1-Q3, 3-707 copies/mL; P = 0.013). Lower concentrations of basal cfBRAF(V600E) were significantly associated with longer overall survival and progression-free survival (27.7 months and 9 months, respectively) than higher basal concentrations (8.6 months and 3 months, P < 0.001 and P = 0.024, respectively).
  
  
  CONCLUSIONS
  cfBRAF(V600E) quantification in plasma by ddPCR is useful as a follow-up to treatment response in patients with advanced melanoma.
authors.affiliation: Department of Medical Oncology.
authors.forename: Miguel F
authors.initials: MF
authors.lastname: Sanmamed
authors.affiliation: Department of Biochemistry, and.
authors.forename: Sara
authors.initials: S
authors.lastname: Fernández-Landázuri
authors.affiliation: Department of Biochemistry, and.
authors.forename: Carmen
authors.initials: C
authors.lastname: Rodríguez
authors.affiliation: Centro de Investigación Médica Aplicada (CIMA) Lab-Diagnostic, Pamplona, Navarra, Spain.
authors.forename: Ruth
authors.initials: R
authors.lastname: Zárate
authors.affiliation: Department of Pathology, University Clinic of Navarra, Pamplona, Spain;
authors.forename: María D
authors.initials: MD
authors.lastname: Lozano
authors.affiliation: Department of Medical Oncology.
authors.forename: Leyre
authors.initials: L
authors.lastname: Zubiri
authors.affiliation: Department of Medical Oncology.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Perez-Gracia
authors.affiliation: Department of Medical Oncology.
authors.forename: Salvador
authors.initials: S
authors.lastname: Martín-Algarra
authors.affiliation: Department of Biochemistry, and agonzaleh@unav.es.
authors.forename: Alvaro
authors.initials: A
authors.lastname: González
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000627:therapeutic use; D004247:DNA / Q000097:blood* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D018572:Disease-Free Survival; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage / Q000627:therapeutic use; D007211:Indoles / Q000008:administration & dosage / Q000627:therapeutic use; D053208:Kaplan-Meier Estimate; D008297:Male; D008545:Melanoma / Q000097:blood / Q000188:drug therapy* / Q000235:genetics / Q000401:mortality; D008875:Middle Aged; D009154:Mutation; D010091:Oximes / Q000008:administration & dosage / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics; D013449:Sulfonamides / Q000008:administration & dosage / Q000627:therapeutic use; D016896:Treatment Outcome; D000077484:Vemurafenib
chemical_list: D000970:Antineoplastic Agents; D007093:Imidazoles; D007211:Indoles; D010091:Oximes; D013449:Sulfonamides; D000077484:Vemurafenib; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C561627:dabrafenib