Presence and Absence of the BRAF V600E Mutation in Hairy Cell Leukemia and Its Variants

Hairy cell leukemia (HCL) is a B-cell malignancy with distinctive immunophenotype. Purine analog therapy achieves durable complete remissions in 65–90% of patients. HCL variant (HCLv), recognized by the World Health Organization (WHO) as a different disease, lacks CD25, annexin A1 and/or TRAP, and responds poorly to purine analogs with only partial responses (PR) in <50% and lower overall survival (OS) from diagnosis. The recently described HCL variant expressing the immunoglobulin rearrangement IGHV4-34 also has poor response to purine analogs and OS, but can resemble HCL or HCLv immunophenotypically. The V600E BRAF mutation was recently reported present in 100% of 48 patients with HCL and absent in 16 with related disorders including at least 1 case of HCLv. We wished to confirm these results and test well-characterized cases of HCLv and IGHV4-34+ HCL.

DNA was prepared from the blood of 70 patients with HCL and HCLv, 64 of whom were molecularly characterized with respect to IGHV gene usage. The mutation analysis of BRAF c.1799T>A (V600E) and other variants among codons 599–601 within exon 15 was performed using a target-specific mutant allele enriching COLD-PCR technique followed by pyrosequencing. The apparent percentage of mutant versus wild-type alleles was calculated with allele quantification (AQ) mode using PyroMark Software. The threshold AQ value for classifying samples as positive as a mutation was calculated as 3 standard deviations above the mean value of 24 normal blood samples.

Out of 70 total patients tested, 16 (23%) were diagnosed as HCLv based on WHO criteria, and the other 54 were classic HCL. Thirteen (19%) of the 70 cases expressed IGHV4-34, 5 classic HCL and 8 HCLv immunophenotypically. All 6 cases not characterized for IGHV gene usage were classic HCL. The analytic sensitivity of the pyrosequencing assay using cell line controls containing BRAF mutations was <5% tumor cells, and all cases were required to have ≥10% of total white blood cells as HCL. As shown in the table, 28 (40%) of the cases were wild-type with respect to BRAF, including all cases of HCLv. In addition, all 13 cases of IGHV4-34+ HCL, including 5 with classic immunophenotype, were negative for the V600E mutation. Moreover, 7 classic HCL cases were wild-type at V600 of BRAF, including 1 with unknown IGHV and 6 expressing IGHV2-70, IGHV3-15, IGHV3-23, IGHV3-48, IGHV4-39 and IGHV4-59. These 7 cases were relatively resistant to purine analog therapy although numbers were too few for statistical comparisons. In one of these 7 classic HCL cases, CD25 expression had decreased over time.

The V600E BRAF mutation is not present in HCLv or in HCL cases with typical immunophenotype expressing IGHV4-34. A significant minority of other classic HCL cases, 7 (14%) of 49, were negative for the V600E BRAF mutation. It is possible that the V600E BRAF mutation is related to factors other than those affecting immunophenotype, including those influencing prognosis. Additional studies will be needed to better understand the role of V600E-mutated BRAF in HCL and the molecular basis of variants of this disease (Supported in part by NCI, intramural research program, NIH).

No relevant conflicts of interest to declare.