Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia

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 pa...

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Bibliographic Details
Published in:Blood 2012-03, Vol.119 (13), p.3151-3154
Main Authors: Schnittger, Susanne, Bacher, Ulrike, Haferlach, Torsten, Wendland, Nicole, Ulke, Madlen, Dicker, Frank, Grossmann, Vera, Haferlach, Claudia, Kern, Wolfgang
Format: Article
Language:English
Subjects:
Adult
Aged
Aged, 80 and over
Biological and medical sciences
Biomarkers, Tumor - analysis
Biomarkers, Tumor - genetics
DNA Mutational Analysis - methods
Early Detection of Cancer - methods
Female
Follow-Up Studies
Glutamic Acid - genetics
Hematologic and hematopoietic diseases
Humans
Leukemia, Hairy Cell - diagnosis
Leukemia, Hairy Cell - genetics
Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis
Male
Medical sciences
Middle Aged
Monitoring, Physiologic - methods
Mutation, Missense - physiology
Proto-Oncogene Proteins B-raf - genetics
Real-Time Polymerase Chain Reaction - methods
Real-Time Polymerase Chain Reaction - standards
Valine - genetics
Young Adult
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Summary: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.
ISSN:0006-4971
1528-0020
DOI:10.1182/blood-2011-10-383323