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Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 62-65
V-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in hairy cell leukaemia


1 Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Pathology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Immunohaematology and Transfusion Medicine, Christian Medical College, Vellore, Tamil Nadu, India

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Date of Web Publication11-Feb-2015
 

   Abstract 

Introduction: Hairy cell leukemia (HCL) is a B-cell non-Hodgkin lymphoma with distinct clinical, morphological and immunophenotypic features; however, there are many other B-cell lymphomas, which closely mimic HCL. Accurate diagnosis of HCL is important as treatment with 2-chloro-2'-deoxyadenosine (cladribine) is associated with >80% chance of complete cure. The recent description of BRAF p.V600E mutations in almost all HCL cases in various studies has not only improved the pathogenetic understanding of this entity but also increased the diagnostic accuracy of this disorder. Aim: The aim of the study was to standardize a molecular test for diagnosis of HCL and compare with standard established morphological, cytochemical and immunophenotypic parameters for HCL diagnosis. Materials and Methods: The incidence of this mutation was sought in 20 patients with either classical HCL or HCL variant (HCLv) by Sanger sequencing and allele-specific polymerase chain reaction. BRAF p.V600E mutation was present in all HCL cases and absent in the only HCLv case. Results: A high degree of correlation was noted between the presence of BRAF p.V600E and established diagnostic criteria in 20/20 patients with HCL/HCLv. Our data supports the observation that this mutation is present in all cases of HCL and is absent in HCLv. Hence, detection of the BRAF p. V600E mutation can be a useful adjunct in the diagnostic algorithm.

Keywords: BRAF mutations, BRAF mutations in HCL, hairy cell leukemia

How to cite this article:
Arora N, Nair S, Pai R, Nair S, Ahmed R, Abraham A, Viswabandya A, George B, Balasubramanian P, Srivastava A, Mathews V. V-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in hairy cell leukaemia. Indian J Pathol Microbiol 2015;58:62-5

How to cite this URL:
Arora N, Nair S, Pai R, Nair S, Ahmed R, Abraham A, Viswabandya A, George B, Balasubramanian P, Srivastava A, Mathews V. V-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in hairy cell leukaemia. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Apr 8];58:62-5. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/62/151190



   Introduction Top


V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) a proto-oncogene of the Raf kinase family encodes a serine/threonine-specific protein kinases and is known to play an important role in regulating the mitogen-activated protein kinase/ extracellular-signal-regulated kinase signaling pathway. [1] Of the various BRAF mutations documented in hematolymphoid as well as in nonhematolymphoid disorders, (1799T→ A) in the exon 15 of the BRAF gene, resulting in an amino acid change from valine to glutamic acid at codon 600 (V600E) is the commonest documented mutation. [2],[3] After the initial description of this mutation in 100% cases of hairy cell leukemia (HCL) by Tiacci et al., subsequent studies have demonstrated this mutation in almost 100% of HCL cases using various platforms. [4],[5],[6],[7],[8] Though HCL has a distinct immunomorphology, there are many B-cell lymphomas, which immunomorphologically mimic HCL. [9],[10] It is always crucial to differentiate HCL from its mimics, including splenic lymphoma/leukemia unclassifiable, HCL variant (HCLv), and splenic marginal zone lymphoma, as only HCLs respond to purine antagonist therapy. [9],[10],[11]

The aim of this study was to standardize a molecular test for diagnosis of HCL and compare with standard established morphological, cytochemical and immunophenotypic parameters for HCL diagnosis. We also wanted to evaluate the incidence of this mutation by direct sequencing and allele-specific polymerase chain reaction (AS-PCR) approach and compare the findings to already existing diagnostic criteria.


   Materials and Methods Top


This was a partly retrospective and partly prospective study carried out in the Department of Haematology, Christian Medical College, Vellore, covering a period of 3 years from 2010 to 2013, and included 24 patients identified with a clinical, morphological, cytochemical and immunophenotypic diagnosis of HCL (n = 19), HCLv (n = 1), chronic lymphocytic leukemia (CLL), (n = 2), peripheral T-cell lymphoma (PTCL) NOS (n = 1) and intravascular large B-cell lymphoma (n = 1) [Table 1] according to WHO 2008 classification. [12] The 4 cases of CLL, PTCL, diffuse large B-cell lymphoma were included as a negative control for AS-PCR. Immunohistochemistry (IHC) was performed using fixed paraffin embedded tissues as described earlier. [13] Immunophenotyping was performed by multicolor flow cytometry (FACS Caliber; Becton Dickinson, Oxford, UK) with a panel of four-color conjugated monoclonal antibodies that included detection of CD5, CD10, CD11c, CD19, CD20, CD25, CD103, CD123 and FMC7 antigen expression in addition to the routine lymphoma panel antibodies. Genomic DNA was extracted from either fresh or archival bone marrow (BM) aspirates using commercial kit (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany). The BRAF V600E mutation was screened using a previously reported AS-PCR protocol. [8] In addition, exon 15 of BRAF gene of each sample was sequenced using an ABI 3130 DNA Analyzer (Applied Biosystems, Foster city, California, USA) as described by Tiacci et al. [4] The sequencing electropherograms were screened for nucleotide changes using SeqScape software (Applied Biosystems, Foster city, California, USA). Only samples with good quality amplifiable DNA were included in this study.
Table 1: The details of the cases included in this study

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   Results Top


This study included 19 cases of HCL (median age; 52 years: Range 38-72 years). More than 50% of the patients were seen in fifth to sixth decade and almost all were males (n = 18). In 14 cases of HCL where cytochemistry was performed, tartrate resistant acid phosphatase (TRAP) activity was detected in 11/13 (84.6%) and was inconclusive in one case. IHC for Annexin A1 (the most specific marker of HCL) was performed in 16 HCL cases and was positive in 13/15 (86.7%) cases and was inconclusive in a single case. BRAF V600E mutation was detected in 14/19 (73.7%) cases using Sanger sequencing. This included seven cases where the mutant peak was very small and was labeled as suspicious. In other five cases (5/18; 27.8%) no mutation could be detected by Sanger sequencing. BRAF V600E mutation was detected in all 18/18 (100%) patients of HCL and absent in all five cases of HCLv, CLL, T-chronic lymphoproliferative disorders and intravascular large B-cell lymphoma using a sensitive AS-PCR. There was a case of HCL (UPN 10) where the mutation was demonstrated on Sanger sequencing but AS-PCR did not work. This could be due to the poor quality of DNA. The only case of HCLv was negative both for TRAP stain and Annexin A1 and no mutation was detected by Sanger sequencing or AS-PCR.


   Discussion Top


There are many investigations currently available for evaluation of HCL and its mimics. This includes morphology, cytochemistry and immunophenotyping both by flow cytometry and IHC. [9],[12] All these investigations are not always necessary, but in cases where the results are equivocal (availability of the tissue, subjectivity, faulty technique, fixation issues) additional tools are helpful. Characterization of this new BRAF V600E mutation in HCL has implications not only for the diagnosis, but it also is a potential target for targeted therapy currently investigated in other BRAF mutated diseases. [1],[14] In this study, we evaluated BRAF V600E mutation in HCL cases and compared with established diagnostic criteria. The molecular analysis for this mutation was done by 2 methods, the gold standard Sanger sequencing and AS-PCR. BRAF V600E mutation was seen in 100% (18/18) of patients with HCL when screened by AS-PCR. All five non-HCL/HCLv cases were negative for this mutation by AS-PCR. These results are consistent with the recently reported 100% prevalence of BRAF mutations in HCL and absence of BRAF V600E in other types of lymphoma and leukemia. [2],[4],[5],[6],[7],[15] Sanger sequencing with its inherent limitations of sensitivity was able to detect BRAF V600E only in 14/19 (73.7%) and hence cannot be reliably used for the detection of BRAF mutations in routine diagnosis. Annexin A1 was evaluated in 16 HCL cases and was positive in 13/15 (86.7%) cases. This is similar to an earlier study by Shao et al. where Annexin A1 positivity was seen only in 74% cases of the HCL. [16] Expression of both TRAP with DBA.44 has been shown to have high sensitivity and specificity for the diagnosis of HCL. [17] In this study, TRAP activity was detected by cytochemistry in 11/13 (84.6%). This is similar to published literature. [9],[16]

One patient (UPN 6) initially diagnosed and treated as HCLv based on morphology and immunophenotype, when reanalyzed at relapse (after 8 months) had a distinct HCL morphology and was TRAP positive on cytochemistry. The BM biopsy demonstrated an Annexin A1+ interstitial B-cell lymphoid infiltrate and a classic HCL immunophenotype on flow cytometry. BRAF V600E mutation was detected both by AS-PCR and sequencing based analysis.

Another case (UPN 22), a 51-year-old male presented with asymptomatic lymphocytosis. His complete blood count (CBC) profile revealed, hemoglobin (Hb) 12.7 g/dL, white blood cell (WBC) 52 × 10 9 /L and platelets 90 × 10 9 /L. BM biopsy showed an interstitial DBA.44-ve, CD5-ve, CD10-ve, Annexin A1-ve B-cell lymphoid infiltrate. Immunophenotyping by flow cytometry revealed a classic HCL phenotype. There was confusion regarding the diagnosis as IHC profile was not suggestive of HCL whereas flow cytometry was consistent with classical HCL.

A similar diagnostic dilemma was seen in a 49-year-old male (UPN 23), who presented with asymptomatic lymphocytosis. CBC profile showed Hb 10.1 g/dL, WBC 54 × 10 9 /L and platelets 15 × 10 9 /L. BM biopsy showed an interstitial DBA.44+ve, CD5-ve, CD10-ve, annexin A1-ve B-cell lymphoid infiltrate. Immunophenotyping by flow cytometry revealed a classic HCL phenotype. Again the IHC was not suggestive of HCL whereas flow cytometry was very much suggestive of HCL. In both these cases, the BRAF V600E mutation was detected by AS-PCR but was negative by sequencing analysis. Hence in equivocal cases the presence of the BRAF V600E by a sensitive technique like AS-PCR can be a useful adjunct in the differential diagnosis of HCL and HCLv.


   Conclusion Top


A high degree of correlation was noted between the presence of BRAF V600E and established diagnostic criteria in all 20/20 patients with HCL/HCLv analyzed in this study using the AS-PCR. Sanger sequencing has a limited role for the detection of this mutation in a diagnostic laboratory. We also demonstrate that testing for BRAF V600E adds to the current diagnostic algorithm of morphology, cytochemistry, IHC and flow cytometry, especially where not all investigations are performed, or the results are equivocal. This study is limited by the availability of good quality DNA and all not all cases of HCL diagnosed at our center could be included. Only those cases with good quality DNA, which could be amplified, were included. This pilot study lays the foundation for further analysis in prospective cases.

 
   References Top

1.
Cantwell-Dorris ER, O'Leary JJ, Sheils OM. BRAFV600E: Implications for carcinogenesis and molecular therapy. Mol Cancer Ther 2011;10:385-94.  Back to cited text no. 1
    
2.
Ping N, Wang Q, Wang Q, Dong S, Wu L, Xue Y, et al. Absence of BRAF V600E mutation in hematologic malignancies excluding hairy-cell leukemia. Leuk Lymphoma 2012;53:2498-9.  Back to cited text no. 2
    
3.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949-54.  Back to cited text no. 3
    
4.
Tiacci E, Trifonov V, Schiavoni G, Holmes A, Kern W, Martelli MP, et al. BRAF mutations in hairy-cell leukemia. N Engl J Med 2011;364:2305-15.  Back to cited text no. 4
    
5.
Arcaini L, Zibellini S, Boveri E, Riboni R, Rattotti S, Varettoni M, et al. The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms. Blood 2012;119:188-91.  Back to cited text no. 5
    
6.
Blombery PA, Wong SQ, Hewitt CA, Dobrovic A, Maxwell EL, Juneja S, et al. Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders. Haematologica 2012;97:780-3.  Back to cited text no. 6
    
7.
Langabeer SE, O'Brien D, Liptrot S, Flynn CM, Hayden PJ, Conneally E, et al. Correlation of the BRAF V600E mutation in hairy cell leukaemia with morphology, cytochemistry and immunophenotype. Int J Lab Hematol 2012;34:417-21.  Back to cited text no. 7
    
8.
Tiacci E, Schiavoni G, Forconi F, Santi A, Trentin L, Ambrosetti A, et al. Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation. Blood 2012;119:192-5.  Back to cited text no. 8
    
9.
Goodman GR, Bethel KJ, Saven A. Hairy cell leukemia: an update. Curr Opin Hematol 2003;10:258-66.  Back to cited text no. 9
    
10.
Naik RR, Saven A. My treatment approach to hairy cell leukemia. Mayo Clin Proc 2012;87:67-76.  Back to cited text no. 10
    
11.
Matutes E, Wotherspoon A, Catovsky D. The variant form of hairy-cell leukaemia. Best Pract Res Clin Haematol 2003;16:41-56.  Back to cited text no. 11
    
12.
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri S, Stein H, et al. WHO Classification: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. 4 th ed. Lyon: IARC; 2008.  Back to cited text no. 12
    
13.
Arora N, Manipadam MT, Nair S. Frequency and distribution of lymphoma types in a tertiary care hospital in South India: Analysis of 5115 cases using the World Health Organization 2008 classification and comparison with world literature. Leuk Lymphoma 2013;54:1004-11.  Back to cited text no. 13
    
14.
Ziai J, Hui P. BRAF mutation testing in clinical practice. Expert Rev Mol Diagn 2012;12:127-38.  Back to cited text no. 14
    
15.
Trifa AP, Popp RA, Cucuianu A, Coada CA, Urian LG, Militaru MS, et al. Absence of BRAF V600E mutation in a cohort of 402 patients with various chronic and acute myeloid neoplasms. Leuk Lymphoma 2012;53:2496-7.  Back to cited text no. 15
    
16.
Shao H, Calvo KR, Grönborg M, Tembhare PR, Kreitman RJ, Stetler-Stevenson M, et al. Distinguishing hairy cell leukemia variant from hairy cell leukemia: Development and validation of diagnostic criteria. Leuk Res 2013;37:401-9.  Back to cited text no. 16
    
17.
Dunphy CH. Reaction patterns of TRAP and DBA.44 in hairy cell leukemia, hairy cell variant, and nodal and extranodal marginal zone B-cell lymphomas. Appl Immunohistochem Mol Morphol 2008;16:135-9.  Back to cited text no. 17
    

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Correspondence Address:
Dr. Neeraj Arora
Department of Laboratory Haematology and Molecular Genetics, TATA Medical Center, Kolkata, West Bengal
India
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Source of Support: This study was supported by an intramural grant from Christian Medical College, Vellore IRB Min no 7932 dated 02.08.2012., Conflict of Interest: None


DOI: 10.4103/0377-4929.151190

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