title: Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.
abstract: BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum.
authors.affiliation: Clinical and Experimental Pharmacology Group, CRUK Manchester Institute, Manchester, United Kingdom.
authors.forename: Kyaw L
authors.initials: KL
authors.lastname: Aung
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Donald
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Gillian
authors.initials: G
authors.lastname: Ellison
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Bujac
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Lynn
authors.initials: L
authors.lastname: Fletcher
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Mireille
authors.initials: M
authors.lastname: Cantarini
authors.affiliation: Clinical and Experimental Pharmacology Group, CRUK Manchester Institute, Manchester, United Kingdom.
authors.forename: Ged
authors.initials: G
authors.lastname: Brady
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Orr
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom.
authors.forename: Glen
authors.initials: G
authors.lastname: Clack
authors.affiliation: Clinical and Experimental Pharmacology Group, CRUK Manchester Institute, Manchester, United Kingdom; Institute of Cancer Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Ranson
authors.affiliation: Clinical and Experimental Pharmacology Group, CRUK Manchester Institute, Manchester, United Kingdom. Electronic address: cdive@picr.man.ac.uk.
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom. Electronic address: andrew.hughes@astrazeneca.com.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hughes
mesh_terms: D004247:DNA / Q000097:blood / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000097:blood / Q000235:genetics* / Q000473:pathology; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D004273:DNA, Neoplasm; D004247:DNA; D048493:Proto-Oncogene Proteins B-raf