title: Validation of standards for quantitative assessment of JAK2 c.1849G>T (p.V617F) allele burden analysis in clinical samples.
abstract: The substitution of valine with phenylalanine at amino acid 617 of the Janus kinase 2 (JAK2) gene (JAK2 p.V617F) occurs in a high proportion of patients with myeloproliferative neoplasms (MPNs). The ability to accurately measure JAK2 p.V617F allele burden is of great interest given the diagnostic relevance of the mutation and the ongoing clinical evaluation of JAK inhibitors. A main hurdle in developing quantitative assays for allele burden measurement is the unavailability of accurate standards for both assay validation and use in a standard curve for quantification. We describe our approach to the validation of standards for quantitative assessment of JAK2 p.V617F allele burden in clinical MPN samples. These standards were used in two JAK2 p.V617F assays, which were used to support clinical studies of ruxolitinib (Jakafi(®)) in myelofibrosis, a real-time polymerase chain reaction assay for initial screening of all samples, and a novel single-nucleotide polymorphism typing (SNaPshot)-based assay for samples with less than 5% mutant allele burden. Comparisons of allele burden data from clinical samples generated with these assays show a high degree of concordance with each other and with a pyrosequencing-based assay used for clinical reporting from an independent laboratory, thus providing independent validation to the accuracy of these standards.
authors.affiliation: Incyte Corporation, Route 141 and Henry Clay Road, Experimental Station E336/237B, Wilmington, DE 19880, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Collier
authors.forename: Keyur
authors.initials: K
authors.lastname: Patel
authors.forename: Paul
authors.initials: P
authors.lastname: Waeltz
authors.forename: Mark
authors.initials: M
authors.lastname: Rupar
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
authors.forename: Phillip C C
authors.initials: PC
authors.lastname: Liu
authors.forename: Gregory
authors.initials: G
authors.lastname: Hollis
authors.forename: Reid
authors.initials: R
authors.lastname: Huber
authors.forename: Srdan
authors.initials: S
authors.lastname: Verstovsek
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Burn
mesh_terms: D000483:Alleles; D019046:Bone Marrow Neoplasms / Q000175:diagnosis / Q000235:genetics*; D045744:Cell Line, Tumor; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D015203:Reproducibility of Results
chemical_list: D053614:Janus Kinase 2
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