title: Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia.
abstract: The BRAFV600E mutation was recently detected in hairy cell leukemia (HCL) by whole exome sequencing. To make use of this new marker for diagnosis and follow-up of HCL, we developed a BRAFV600Emut-specific quantitative real-time PCR assay and validated it in 117 HCL patients and 102 non-HCL/BRAFwt patients. The cut-off level to discriminate BRAFV600E-positive/-negative cases was set at 0.023% BRAFV600E/BRAFwt. A total of 115 of 117 HCL (98.3%) demonstrated percentage BRAFV600E/BRAFwt above the cut-off (mean, 29.6 ± 41.1). The remaining 2 of 117 HCL with lower percentage BRAFV600E/BRAFwt ratios were also BRAFwt by deep-sequencing technology. Sixteen HCL-variant patients showed percentage BRAFV600E/BRAFwt values corresponding to "non-HCL." Follow-up studies in 19 HCL cases demonstrated a decrease of percentage BRAFV600E/BRAFwt during therapy. The log-reductions as determined by RT-PCR and immunophenotyping correlated significantly (P < .001). In conclusion, we confirmed BRAFmut as a useful marker in HCL, its absence in HCL variant, and developed an RT-PCR-based assay to monitor minimal residual disease in HCL.
authors.affiliation: MLL Munich Leukemia Laboratory, Munich, Germany. susanne.schnittger@mll.com
authors.forename: Susanne
authors.initials: S
authors.lastname: Schnittger
authors.forename: Ulrike
authors.initials: U
authors.lastname: Bacher
authors.forename: Torsten
authors.initials: T
authors.lastname: Haferlach
authors.forename: Nicole
authors.initials: N
authors.lastname: Wendland
authors.forename: Madlen
authors.initials: M
authors.lastname: Ulke
authors.forename: Frank
authors.initials: F
authors.lastname: Dicker
authors.forename: Vera
authors.initials: V
authors.lastname: Grossmann
authors.forename: Claudia
authors.initials: C
authors.lastname: Haferlach
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kern
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D055088:Early Detection of Cancer / Q000379:methods*; D005260:Female; D005500:Follow-Up Studies; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007943:Leukemia, Hairy Cell / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008991:Monitoring, Physiologic / Q000379:methods; D020125:Mutation, Missense / Q000502:physiology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D014633:Valine / Q000235:genetics; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D018698:Glutamic Acid; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D014633:Valine