|Year : 2018 | Volume
| Issue : 4 | Page : 532-536
|BRAFV600E mutation in hairy cell leukemia: A single-center experience
Asma Bibi1, Shrutika Java1, Shruti Chaudhary1, Swapnali Joshi1, Russel Mascerhenas1, Nikhil Rabade1, Prashant Tembhare1, Papagudi Ganesan Subramanian1, Sumeet Gujral1, Hari Menon1, Navin Khattry1, Manju Sengar1, Bhausaheb Bagal1, Hasmukh Jain1, Nikhil Patkar2
1 Tata Memorial Centre, Hematopathology Laboratory, Mumbai, Maharashtra, India
2 Molecular Division, Tata Memorial Centre, Hematopathology Laboratory, Mumbai, Maharashtra, India
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|Date of Web Publication||10-Oct-2018|
| Abstract|| |
Background: BRAFV600E mutation has been reported as a unique genetic lesion of hairy cell leukemia (HCL), a subset of which lacks this lesion and shows adverse outcomes. Aims: To determine the prevalence of BRAFV600E in HCL from our center and derive clinicopathological correlation, if any. Materials and Methods: A 9-year retrospective analysis of 46 consecutive cases of HCL diagnosed on morphology and immunophenotyping was done. Stained smears were used as samples for amplification refractory mutation system polymerase-chain reaction using fluorescent primers for mutation detection. Results: BRAFV600E mutation was detected in 41/46 patients (89.1%) while absent in control samples of chronic lymphocytic leukemia. Cases mimicking HCL-variant clinically or immunophenotypically too showed the presence of this mutation. HCL with mutated BRAF presented at a younger age. No statistical difference in blood counts, tumor load, and immunophenotype patterns existed among BRAF mutated and unmutated group. Nine patients (45%) with mutated BRAF had residual disease following treatment with cladribine. Conclusion: BRAFV600E mutation analysis has a definitive role in the diagnosis of HCL.
Keywords: Amplification refractory mutation system-polymerase chain reaction, BRAFV600E, hairy cell leukemia
|How to cite this article:|
Bibi A, Java S, Chaudhary S, Joshi S, Mascerhenas R, Rabade N, Tembhare P, Subramanian PG, Gujral S, Menon H, Khattry N, Sengar M, Bagal B, Jain H, Patkar N. BRAFV600E mutation in hairy cell leukemia: A single-center experience. Indian J Pathol Microbiol 2018;61:532-6
|How to cite this URL:|
Bibi A, Java S, Chaudhary S, Joshi S, Mascerhenas R, Rabade N, Tembhare P, Subramanian PG, Gujral S, Menon H, Khattry N, Sengar M, Bagal B, Jain H, Patkar N. BRAFV600E mutation in hairy cell leukemia: A single-center experience. Indian J Pathol Microbiol [serial online] 2018 [cited 2019 Jun 16];61:532-6. Available from: http://www.ijpmonline.org/text.asp?2018/61/4/532/242988
| Introduction|| |
Classical hairy cell leukemia (HCL) is a rare indolent B-cell chronic lymphoproliferative disorder (B-CLPD) characterized by massive splenomegaly, pancytopenia and typical-appearing “hairy cells” infiltrating bone marrow, spleen, liver, and circulating in peripheral blood (PB). These cells show abundant cytoplasm with fine hairy projections and a characteristic reniform nucleus without a nucleolus. Lymphadenopathy is rare. Immunophenotype (IPT) shows characteristic bright coexpression of CD19, CD20, CD22, CD11c and uniformly positive CD25, CD103, and CD123 along with highly prevalent lambda-chain restriction.,, HCL diagnosis till date relies on the morphological and immunophenotypic features which allow its distinction from other CD5neg, and CD10neg HCL-like splenic lymphomas. Although MAP kinase and activated MEK-ERK pathway was implicated in the pathogenesis of HCL, driver mutations remained unknown.
The impact of genome-wide massive parallel sequencing is evident in various hematological malignancies. While mutations in NPM1, FLT3, and CEBPA are already a part of AML classification and risk-stratification, mutations in TET2, SF3B1, ASXL1, DNMT3A, and RUNX1 portend newer prognostic models in MDS. Mutated SF3B1 also showed a strong association with the presence of ring sideroblasts in MDS., Lymphomas such as SMZL, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, and mantle cell lymphoma have been reported to show frequently mutated NOTCH2, MYD88, and NOTCH1 genes respectively, whereas CLL harbors recurrent mutations in NOTCH1, XPO1, MYD88, and KLHL6.
In a landmark paper by Tiacci et al., BRAFV600E mutation (BRAF c. 1799T > A p. Val600Glu) was described as a disease-defining genetic marker present in all cases of HCL and absent in other B-cell neoplasms using whole exome sequencing. The high frequency of this mutation in HCL has been corroborated by few other groups.,
In view of limited data from Indian subcontinent, the aims of our study were to document the baseline frequency of BRAFV600E in a large series of HCL from our center and to correlate the same with the clinical and pathological findings.
| Materials and Methods|| |
This is a retrospective study of 46 consecutive patients with HCL seen over a period of 10 years from 2005 to 2014 at our institution. The diagnosis was based on morphological and IPT findings wherever available. Based on the electronic medical records and case files retrieved from the medical record department, clinical information included symptomatology, laboratory data at diagnosis and follow-up, treatment received, and response to treatment. Giemsa-stained bone marrow aspirate (BMA) slides were used for morphological assessment. The BMA sample was subjected to processing, staining with a CLPD antibody panel on a three-color BD FACS Calibur and after 2008 on an 8-color BD FACS Canto II (BD Biosciences, San Jose, CA) up to November 2013. In 2014, the list mode data were acquired in Navios (Beckman Coulter Inc). Clonal B cells were characterized by their expression of CD5, CD10, CD20, CD22, CD11c, CD23, CD38, and surface kappa and lambda light chains. HCL-specific panel including CD25, CD103, and CD123 was added. All antibodies were purchased from BD Biosciences, USA. The intensity of expression was determined as bright, intermediate (int), and dim in comparison to their normal counterparts. H- and E-stained sections of the BM biopsy were also studied wherever available to understand the extent of bone marrow infiltration by leukemic cells.
Genomic DNA was extracted from stained BMA slides using a magnetic bead-based extraction technique that has been validated for use in forensic criminology (ChargeSwitch® Forensic DNA Purification Kit, Invitrogen™, CA, USA). This DNA was subjected to an amplification refractory mutation system (ARMS)-based polymerase-chain reaction PCR, an allele-specific PCR technique that was modified using fluorescent primers to increase the sensitivity of the assay, followed by capillary electrophoresis on ABI3500 genetic analyzer (Applied Biosystems®, CA, USA). The PCR was designed to amplify a 200 bp control product, a 144 bp BRAFV600E-specific amplicon and a 97 bp wild-type specific amplicon. As a part of assay validation, we simultaneously tested DNA from fresh PB samples of 10 cases of chronic lymphocytic leukemia (CLL). The gold standard used for the diagnosis of HCL was a combination of immunophenotyping, hairy cell-like morphology, and the demonstration of BRAF V600E by a mutation-specific PCR. At follow-up, recovery of the complete blood count (CBC) parameters was noted as response to treatment. BRAFV600E mutation status was correlated with clinicopathological features and treatment outcomes.
Statistical analysis was done on SPSS 21 [IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.]. Statistical analysis was done using 2-tailed Student t-test and Fisher exact test of independence.
| Results|| |
A total of 46 patients were studied. The median age at diagnosis was 51.5 years (range 26–67 years) with a striking male preponderance (M: F ratio-10.5:1). At presentation, splenomegaly was seen in 73.1% of cases, whereas 30.8% cases and 8.6% cases had hepatomegaly and lymphadenopathy, respectively. Nine patients had infections at onset owing to the immune dysfunction and pancytopenia.
CBC revealed pancytopenia and bicytopenia in 50% and 36.1% of cases, respectively. Thrombocytopenia (82.9%) and anemia (80.6%) were most common, followed by neutropenia (74.3%), monocytopenia (70.6%), and leukopenia (58.3%). Leukocytosis was observed in 4 cases, 2 of which also showed absolute monocytosis.
Giemsa-stained BMA smears showed tumor cells with the classical “hairy-cell” morphology in all cases. Mean tumor load in BM was 55% (range: 3%–97%). Trephine biopsies were available in 37/46 cases. The various patterns of bone marrow infiltration were noted; the most common was diffuse interstitial in both mutated and unmutated cases accounting for 25/37 (67.6%) cases. Lymphoid aggregates, sheets of neoplastic cells, and scattered atypical cells were seen in 5/37, 4/37 and 3/37 cases, respectively.
Multicolor flow cytometry data were available in 43 cases. The combined panel of CD20+, CD11c+, CD25+, and CD103+ was observed in 40/43 cases (93%). All but two cases showed uniform bright coexpression of CD20 and CD11c. Aberrant IPT was seen in 13 cases. Single marker deviation such as CD10int (4/13), CD5dim (1/13), CD38bright (1/13), CD23int (2/13), and CD25neg (1/13) was seen in cases with an otherwise typical IPT of HCL. Multiple abnormalities coexisted in four other patients. The IPT of leukemic B cells is summarized in [Table 1] and [Figure 1].
|Figure 1: Flow cytometry immunophenotyping of classical hairy cell leukemia. HCL cells have bright CD45, bright CD19 and CD20; homogenous bright CD11c, moderate CD25, moderate CD103 and CD123. The HCL cells show kappa light chain restriction and are negative for CD5 and CD10|
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All patients were treated with cladribine and demonstrated hematologic remission. The median follow-up was 30 months (range: 0–118 months). Out of the 46 patients, 31 were alive at the last follow-up. Six of them are dead; out of which two had succumbed during treatment. Nine patients were lost to follow-up. Residual disease was assessed in twenty patients by morphology or IPT and found to be present in nine patients (45%) with mutated BRAF. All of these nine patients were alive at last follow-up (duration 6–113 months). Of the five patients with wild-type BRAF, one succumbed, two followed up for 78 and 85 months respectively without relapse while two patients were lost to follow-up.
Molecular testing for BRAFV600E mutation
BRAFV600E mutation was present in 41/46 cases (89.1%), which included the three referred cases which were diagnosed solely on the basis of morphology (as sample for IPT was not available). Three out of the five cases showing wild-type BRAF had a classical HCL IPT of CD11c+/CD25+/CD103+ on flow cytometry analysis whereas the remaining two were CD11c+/CD25-/CD103+. In the CD25-cases, CD123 was consistently positive.
Comparison of BRAFV600E mutated and unmutated cases
The positive and negative predictive values of BRAF mutation for hairy-cell leukemia were both 100% since there were no false-positive or false-negative values. Patients with mutated BRAF tend to present a decade earlier than those with wild-type BRAF (P < 0.05). There was no significant difference between the two groups with respect to CBC findings and prevalence of aberrant phenotypes [Table 2].
|Table 2: Clinicopathological correlation of BRAF-mutated versus unmutated cases|
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| Discussion|| |
BRAFV600E mutation has been recently designated as the disease-defining genetic lesion in HCL, whereas it is not seen in HCL-v cases.,,,, On the other hand, there are occasional reports of presence of this mutation in other B cell neoplasms, thereby pointing to a potential diagnostic pitfall.,, For analysis of BRAFV600E mutation, we used an ARMS-PCR technique which was validated for formalin-fixed, paraffin-embedded tissue with a reported sensitivity of 0.5% for the mutant allele. This method proved to be specific in our analysis as the mutation was not detected in consecutive samples of CLL. We documented a prevalence of 89.1% of BRAFV600E mutation in our cohort, which is comparable to 79%, 76%, and 70.6% reported in other studies. However, the detection rate of 100% as published by various groups,, was not seen in our cohort. DNA in these studies was extracted from either fresh PB, BM biopsy, or fresh PB/BMA and unstained BMA slides selected for tumor load of >30%, >15%, and >10%, respectively. We had five cases of HCL which lacked BRAFV600E mutation. We propose the following explanations. Although PCR is the gold standard for genetic diagnosis of HCL, assay sensitivity in our study could be confounded by DNA integrity in material scraped from archival BMA smears, especially having low-tumor load. Xi et al. described a subset of HCL expressing IgVH4-34 and lacking BRAFV600E mutation. This subset is reported to harbor mutations in other genes such as MAP2K1, ARID1A, and TTN. Recently, novel mutations were described in exon 11 of BRAF gene in HCL negative for V600E (which occurs in exon 15). Immunogenetic analysis was not done in our study.
HCL is reported to have immunophenotypic variations. Lennerz et al. had reported CD5bright HCL having wild-type BRAF and proposed it to be a new variant in HCL lacking BRAFV600E mutation. Our case with CD5dim expression had tested positive for V600E. One case with dim (instead of typical bright) expression of CD20/CD11c showed mutated BRAF and achieved remission on cladribine although later showed residual disease on BMA. BRAF was also mutated in another case with leukocytosis, absence of monocytopenia, and CD25neg (mimicking HCL-v). This patient was in sustained remission at last follow-up (54 months). Clasically, HCL has been identified by the characteristic features of pancytopenia, splenomegaly and monocytopenia whereas HCL-v has shown leukocytosis, absence of monocytopenia, and lack of marrow fibrosis at presentation., CD25 is reported to be typically absent in HCL-v.,, Thus, BRAFV600E mutation analysis can be useful to substantiate the diagnosis of HCL when clinical or IPT features deviate from typical findings.
Residual disease is known to persist despite hematological remission after standard treatment with cladribine, with prevalence ranging from 13% to 50%. Our data is comparable with these reports. Refractoriness to cladribine has been documented in a subset of HCL with IgVH4-34 expression,,, which was later shown to also lack BRAFV600E mutation. Recent reports highlight the effectiveness of targeted therapy against BRAFV600E mutation in relapse/refractory HCL.,,
Except for age at diagnosis, patients with unmutated BRAF did not differ significantly from those harboring the mutation, at least with respect to laboratory parameters. Our findings have certain implications. One, although BRAFV600E mutation is present in a majority of HCL cases, its absence does not rule out the diagnosis. Two, this mutation is also positive in those HCL cases with features overlapping with HCL-v and thus can aid to establish the diagnosis of former. This distinction is significant as therapy differs in both cases.
| Conclusion|| |
BRAF mutation analysis does find a place in diagnostic armamentarium of HCL. Select cases can be further investigated for IgVH4-34 expression, novel mutations in BRAF, and mutations in additional genes such as MAP2K1, ARID1A, and TTN. The presence of BRAFV600E mutation paves way for targeted therapy and minimal residual disease monitoring in patients of HCL.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Foucar K, Falini B, Catovsky D, Stein H. Hairy cell leukemia. In: Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al
., editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th
ed. Lyon, France: International Agency for Research on Cancer (IARC); 2008. p. 188-90.
Stetler-Stevenson M, Tembhare PR. Diagnosis of hairy cell leukemia by flow cytometry. Leuk Lymphoma 2011;52 Suppl 2:11-3.
Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008;111:3941-67.
Chen YH, Tallman MS, Goolsby C, Peterson L. Immunophenotypic variations in hairy cell leukemia. Am J Clin Pathol 2006;125:251-9.
Tiacci E, Liso A, Piris M, Falini B. Evolving concepts in the pathogenesis of hairy-cell leukaemia. Nat Rev Cancer 2006;6:437-48.
Arber DA, Brunning RD, Le Beau MM, Falini B, Vardiman JW, Porwit A, et al.
Acute myeloid leukemia with recurrent genetic abnormalities. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th
ed. Lyon, France: International Agency for Research on Cancer; 2008. p. 110-23. Available from: http://www.apps.who.int/bookorders/anglais/detart1.jsp?codlan=1&codcol=70&codcch=4002
. [Last accessed on 2016 Mar 13].
Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, et al.
Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014;28:241-7.
Malcovati L, Karimi M, Papaemmanuil E, Ambaglio I, Jädersten M, Jansson M, et al.
SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood 2015;126:233-41.
Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D, et al.
Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med 2011;365:1384-95.
Kiel MJ, Velusamy T, Betz BL, Zhao L, Weigelin HG, Chiang MY, et al.
Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma. J Exp Med 2012;209:1553-65.
Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al.
MYD88 L265P somatic mutation in Waldenström's macroglobulinemia. N Engl J Med 2012;367:826-33.
Kridel R, Meissner B, Rogic S, Boyle M, Telenius A, Woolcock B, et al.
Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma. Blood 2012;119:1963-71.
Puente XS, Pinyol M, Quesada V, Conde L, Ordóñez GR, Villamor N, et al.
Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 2011;475:101-5.
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.
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.
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.
Huang T, Zhuge J, Zhang WW. Sensitive detection of BRAF V600E mutation by amplification refractory mutation system (ARMS)-PCR. Biomark Res 2013;1:3.
Xi L, Arons E, Navarro W, Calvo KR, Stetler-Stevenson M, Raffeld M, et al.
Both variant and IGHV4-34-expressing hairy cell leukemia lack the BRAF V600E mutation. Blood 2012;119:3330-2.
Raess PW, Mintzer D, Husson M, Nakashima MO, Morrissette JJ, Daber R, et al.
BRAF V600E is also seen in unclassifiable splenic B-cell lymphoma/leukemia, a potential mimic of hairy cell leukemia. Blood 2013;122:3084-5.
Langabeer SE, Quinn F, O'Brien D, McElligott AM, Kelly J, Browne PV, et al.
Incidence of the BRAF V600E mutation in chronic lymphocytic leukaemia and prolymphocytic leukaemia. Leuk Res 2012;36:483-4.
Jebaraj BM, Kienle D, Bühler A, Winkler D, Döhner H, Stilgenbauer S, et al.
BRAF mutations in chronic lymphocytic leukemia. Leuk Lymphoma 2013;54:1177-82.
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.
Boyd EM, Bench AJ, van 't Veer MB, Wright P, Bloxham DM, Follows GA, et al
. High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies: Short Report. Br J Haematol 2011;155:609-12.
Tiacci E, Schiavoni G, Martelli MP, Boveri E, Pacini R, Tabarrini A, et al.
Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia. Haematologica 2013;98:635-9.
Waterfall JJ, Arons E, Walker RL, Pineda M, Roth L, Killian JK, et al.
High prevalence of MAP2K1 mutations in variant and IGHV4-34-expressing hairy-cell leukemias. Nat Genet 2014;46:8-10.
Tschernitz S, Flossbach L, Bonengel M, Roth S, Rosenwald A, Geissinger E, et al.
Alternative BRAF mutations in BRAF V600E-negative hairy cell leukaemias. Br J Haematol 2014;165:529-33.
Lennerz JK, Klaus BM, Marienfeld RB, Möller P. Pyrosequencing of BRAF V600E in routine samples of hairy cell leukaemia identifies CD5+ variant hairy cell leukaemia that lacks V600E. Br J Haematol 2012;157:267-9.
Matutes E. Immunophenotyping and differential diagnosis of hairy cell leukemia. Hematol Oncol Clin North Am 2006;20:1051-63.
Grever MR. How I treat hairy cell leukemia. Blood 2010;115:21-8.
Tallman MS, Hakimian D, Kopecky KJ, Wheaton S, Wollins E, Foucar K, et al.
Minimal residual disease in patients with hairy cell leukemia in complete remission treated with 2-chlorodeoxyadenosine or 2-deoxycoformycin and prediction of early relapse. Clin Cancer Res 1999;5:1665-70.
Sharpe RW, Bethel KJ. Hairy cell leukemia: Diagnostic pathology. Hematol Oncol Clin North Am 2006;20:1023-49.
Arons E, Suntum T, Stetler-Stevenson M, Kreitman RJ. VH4-34+ hairy cell leukemia, a new variant with poor prognosis despite standard therapy. Blood 2009;114:4687-95.
Forconi F, Sozzi E, Cencini E, Zaja F, Intermesoli T, Stelitano C, et al.
Hairy cell leukemias with unmutated IGHV genes define the minor subset refractory to single-agent cladribine and with more aggressive behavior. Blood 2009;114:4696-702.
Arons E, Kreitman RJ. Molecular variant of hairy cell leukemia with poor prognosis. Leuk Lymphoma 2011;52 Suppl 2:99-102.
Forconi F, Cencini E, Sozzi E, Sicuranza A, Raspadori D, Lauria F, et al.
Insight into the behavior of hairy cell leukemia by immunogenetic analysis. Leuk Lymphoma 2011;52 Suppl 2:103-7.
Pettirossi V, Santi A, Imperi E, Russo G, Pucciarini A, Bigerna B, et al.
BRAF inhibitors reverse the unique molecular signature and phenotype of hairy cell leukemia and exert potent antileukemic activity. Blood 2015;125:1207-16.
Tiacci E, Park JH, De Carolis L, Chung SS, Broccoli A, Scott S, et al.
Targeting mutant BRAF in relapsed or refractory hairy-cell leukemia. N Engl J Med 2015;373:1733-47.
Dietrich S, Glimm H, Andrulis M, von Kalle C, Ho AD, Zenz T, et al.
BRAF inhibition in refractory hairy-cell leukemia. N Engl J Med 2012;366:2038-40.
Molecular Division, Tata Memorial Centre, Hematopathology Laboratory, KS-231, Khanolkar Shodika, ACTREC, Mumbai - 410 210, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]