title: Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.
abstract: A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Roza
authors.initials: R
authors.lastname: Bidshahri
authors.affiliation: Department of Statistics, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Dean
authors.initials: D
authors.lastname: Attali
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Kareem
authors.initials: K
authors.lastname: Fakhfakh
authors.affiliation: Department of Genetics and Molecular Diagnostics, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: Kelly
authors.initials: K
authors.lastname: McNeil
authors.affiliation: Department of Genetics and Molecular Diagnostics, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: Aly
authors.initials: A
authors.lastname: Karsan
authors.affiliation: Canadian Immunohistochemistry Quality Control Unit, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Won
authors.affiliation: Department of Pathology, Lion's Gate Hospital, North Vancouver, British Columbia, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Wolber
authors.affiliation: Department of Statistics, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bryan
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: curtish@msl.ubc.ca.
authors.forename: Curtis
authors.initials: C
authors.lastname: Hughesman
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: israels@chbe.ubc.ca.
authors.forename: Charles
authors.initials: C
authors.lastname: Haynes
mesh_terms: D000483:Alleles; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D006801:Humans; D057230:Limit of Detection; D016612:Paraffin Embedding; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D000090063:Proto-Oncogene Mas; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D057188:Workflow
chemical_list: D015342:DNA Probes; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf