Dear Editor,
Differentiation of classic hairy cell leukemia (HCL-c) from HCL-variant (HCL-v) or
splenic marginal zone lymphoma (SMZL) is important owing to their different treatment
strategies and prognostic implications. Recently, testing for BRAF V600E mutations
was suggested as an important diagnostic option for HCL-considering that it was exclusively
detected in almost all cases [1]. The BRAF V600E mutation has been reported to be
absent in most cases of immunoglobulin variable heavy chain rearrangements 4-34 (IGHV4-34)-positive
HCL-c, HCL-v, and SMZL [2]. However, it was recently reported that high prevalence
of MAP2K1 mutation is observed in IGHV-34-positive HCL-c (5/7, 71.4%) [3].
We investigated the presence of BRAF V600E and MAP2K1 mutations in four HCL-c, two
HCL-v, and four SMZL cases involving the bone marrow that were diagnosed between June
2005 and June 2014 at our hospital. HCL and SMZL was diagnosed in accordance with
the 2008 WHO classification of tumors of hematopoietic and lymphoid tissues [4]. HCL-c
was defined as the expression of Annexin A1, CD20, CD22, CD11c, CD103, and CD25. HCL-v
was defined as the negative expression of CD25 and Annexin A1 [4]. Real-time PCR was
performed by using the Real Q BRAF V600E Detection Kits (BioSewoom Inc., Seoul, Korea)
on the 7500 Fast Real-Time System (Applied Biosystems, Foster City, CA, USA), according
to the manufacturer's instructions [5]. Mutant enrichment 3'-modified oligonucleotide
(MEMO)-PCR and sequencing analysis for the BRAF V600E mutation were performed as previously
described [6]. We designed the sequencing primers for MAP2K: exon 2 (forward) 5'-TTCTCTGGTGACAGTATTGACTTG-3',
(reverse) 5'-CCCTGAGAAATAATCCAATTACC-' and exon 3 (forward) 5'-CATCCCTTCCTCCCTCTTTC-3',
(reverse) 5'-CTCTTAAGGCCATTGCTCCA-3'. Sequencing was performed by using the Big-Dye
Terminator Cycle Sequencing Ready Reaction Kit on the ABI Prism 3130 Genetic Analyzer
(Applied Biosystems). The DNA extracted from bone marrow aspirate slide was used for
sequencing analysis.
We detected the BRAF V600E mutation in all HCL-c cases either by real-time PCR or
by the MEMO-sequencing method (Table 1). All SMZL and HCL-v cases were negative for
BRAF V600E on both real-time PCR and MEMO-sequencing analyses. The MAP2K1 mutation
analysis of three HCL-c cases, one HCL-v case, and three SMZL cases revealed negative
results.
The most common types of BRAF mutation involve exon 15, and the substitution from
valine to glutamate at the 600th amino acid (V600E) constitutes >90% of all reported
cases of mutation. BRAF mutations in hematological malignancy are relatively rare
[1, 7, 8]. In 2011, Tiacci et al. [1] reported that nearly all cases of HCL-c harbored
the BRAF V600E mutation, although this mutation was not detected in any other B cell
lymphomas including SMZL; this finding is in agreement with that of other studies
[8, 9, 10]. Presently, BRAF V600E mutation analysis is considered to be the most useful
diagnostic tool for differentiating HCL from related lymphomas. We also confirmed
the presence of BRAF V600E in all HCL-c cases, but not in HCL-v or SMZL cases.
Recently, MAP2K1 mutation was identified in a subset of HCL patients: 6/15 of IGHV-34-negative
HCL-v, 4/9 of IGHV-34-positive HCL-v, and 5/7 of IGHV-34-positive HCL-c cases [3].
MAP2K1 encodes mitogen-activated protein kinase kinase 1, which is a component of
the MAP kinase signal transduction pathway. Somatic mutations were detected in HCL-v-
and IGHV-34-positive HCL clusters in exons 2 and 3, which encode the N-terminal autoregulatory
domain [3]. We detected negative results in all cases, which may be attributed to
the small sample size and the low incidence of MAP2K1 mutation.
In conclusion, we analyzed BRAF V600E and MAP2K1 mutations in a small series of HCL-c,
HCL-v, and SMZL cases. The BRAF V600E mutation was detected in all cases of HCL-c,
but in none of the HCL-v or SMZL cases. This observation is consistent with that of
a previous study, confirming the diagnostic utility of BRAF testing in HCL. Considering
the rarity of HCL cases, further studies are needed for drawing conclusive observations
from a sufficiently large sample size.