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 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. (Blood. 2012;119(13):3151-3154)