Hairy cell leukaemia (HCL) is a rare type of B-cell non-Hodgkin lymphoma (B-NHL), which is not known to be associated with any characteristic recurrent karyotypic abnormality. A recent study that used massively parallel whole exome sequencing identified an activating V600E mutation in BRAF, which appeared specific for HCL. Here, we confirm the specificity of BRAF V600E for HCL among low and intermediate grade B-NHL and describe a real-time polymerase chain reaction method for detecting this mutation in cases with low tumour burden. The V600E mutation does not appear to be associated with microsatellite instability, unlike the case in colorectal cancer. Thus, in conjunction with prior data, our results suggest incorporation of BRAF V600E mutation analysis in the diagnostic workup of HCL cases. Additionally, targeting the Ras-Raf-Mek-Erk-Map kinase pathway should be investigated as a potential therapeutic strategy for patients with this disease

Alobeid, Bachir

Bhagat, Govind

Ewalt, Mark

Mansukhani, Mahesh M.

Nandula, Subhadra

Phillips, Adrienne

Vundavalli, Murty V.

English

Columbia University Academic Commons

Hematological OncologyHematol Oncol 2012; 30: 190–193Published online 22 December 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/hon.1023Original Research ArticleReal-time PCR-based analysis of BRAF V600E mutation inlow and intermediate grade lymphomas confirms frequentoccurrence in hairy cell leukaemiaMark Ewalt1†, Subhadra Nandula1†, Adrienne Phillips2, Bachir Alobeid1, Vundavalli V. Murty1,Mahesh M. Mansukhani1* and Govind Bhagat1*1Department of Pathology and Cell Biology, Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA2Department of Medicine, Division of Hematology and Oncology, Columbia University Medical Center and New York Presbyterian Hospital, NewYork, NY, USA*Correspondence to:Govind Bhagat and Mahesh M.Mansukhani, Department ofPathology and Cell Biology,Columbia University MedicalCenter and New York PresbyterianHospital, 630 West 168th Street,VC 14–228, New York, NY, USA.E-mail: gb96@columbia.edu;E-mail: mm322@columbia.edu†These authors contributed equallyto this work.Received 5 October 2011Revised 4 November 2011Accepted 7 November 2011Copyright © 2011 John Wiley & SonAbstractHairy cell leukaemia (HCL) is a rare type of B-cell non-Hodgkin lymphoma (B-NHL),which is not known to be associated with any characteristic recurrent karyotypicabnormality. A recent study that used massively parallel whole exome sequencing identifiedan activating V600E mutation in BRAF, which appeared specific for HCL. Here, we confirmthe specificity of BRAF V600E for HCL among low and intermediate grade B-NHL anddescribe a real-time polymerase chain reaction method for detecting this mutation incases with low tumour burden. The V600E mutation does not appear to be associatedwith microsatellite instability, unlike the case in colorectal cancer. Thus, in conjunctionwith prior data, our results suggest incorporation of BRAF V600E mutation analysis inthe diagnostic workup of HCL cases. Additionally, targeting the Ras-Raf-Mek-Erk-Mapkinase pathway should be investigated as a potential therapeutic strategy for patientswith this disease. Copyright © 2011 John Wiley & Sons, Ltd.Keywords: BRAF V600E; hairy cell leukaemia; non-Hodgkin lymphomaIntroductionRecent advances in technology, especially high resolutionwhole genome analyses, have yielded clues to the molecu-lar pathogenesis of a variety of neoplasms that lacked recur-rent cytogenetic alterations, as ascertained by conventionalmethods [1]. In an early study analysing cancer genomesfor recurrent mutations, Davies et al. discovered activatingmutations of BRAF in exons 11 and 15, most frequently inmelanoma (up to 66% of cases harboured such a mutation),with the V600E (c.1799 T>A) mutation being the mostcommon [2]. Until recently, data regarding the presenceand frequency of BRAF mutations in lymphomas werelimited. Lee et al. identified somatic BRAF mutations in4 of 164 (2.4%) non-Hodgkin lymphomas (NHL), allrepresenting diffuse large B-cell lymphomas. The V600Emutation was not identified in this series [3]. BRAFmutations were recently detected in 4% of multiplemyeloma by a multi-institutional consortium on performingwhole genome and exome analyses of 38 samples andscreening an additional 161 cases, with four (2%) casesharbouring theV600E mutation [4]. More recently, Tiacciet al. also identified a V600E BRAF mutation in an indexcase of hairy cell leukaemia (HCL) with the use ofmassively parallel whole exome sequencing, and theauthors further showed that this mutation was present inall 47 additional HCL analysed with Sanger sequencing [1].These authors suggested that the BRAF V600E mutations, Ltd.may account for the activated phenotype of HCL, as a BRAFinhibitor could inhibit downstream ERK andMEK phosphor-ylation in vitro, and that targeting this pathway may be oftherapeutic value [1]. Recently, Boyd et al. verified the pres-ence of the V600Emutation in HCL patients with the use of ahigh resolution melting analysis assay [5].In this study, we sought to investigate the presence ofBRAF V600E mutations in a large panel of low and inter-mediate grade B-cell non-Hodgkin lymphoma (B-NHL)comprising different subtypes, and to assess any associationof this mutation with microsatellite instability (MSI). Weused a real-time polymerase chain reaction (PCR)-basedassay to detect the mutation in unpurified peripheral blood,bone marrow and tissue samples, obtained fresh and also af-ter fixative treatment. Limiting dilution analysis demon-strated utility of this assay in detecting V600E mutation insamples with a low tumour burden.Materials and methodsCase selectionOur departmental archive was searched for cases of lowand intermediate grade B-NHL diagnosed at our institutionbetween 1 January 2000 and 15 August 2011. The selectedB-NHL (n = 102) were classified according to the 2008World Health Organization Classification [6], as HCL(n=12 from eight patients), chronic lymphocytic leukaemia/191BRAF V600E mutations in B-cell lymphomassmall lymphocytic lymphoma (n=20), mantle cell lymphoma(n=19), marginal zone lymphoma (MZL, n = 24); repre-senting 18 extranodal MZL, stomach (n = 6), lung(n=4), eye/ocular adnexa (n=4), and thymus, kidney, ileumand rectum (n=1 each); four nodal MZL; and two splenicMZL, follicular lymphoma (n=20) and lymphoplasmacyticlymphoma (n=7). DNA was extracted with the use ofDNAzolW (Invitrogen, Carlsbad, CA) for samples fixed withCarnoy’s solution as previously described [7], or with theuse of the QIAampW tissue and blood kits (Qiagen, Valencia,CA) for fresh and formalin-fixed samples, as per manufac-turer’s instructions. Tumour percentagewas evaluated throughflow cytometry in nine HCL samples, and immunohistochem-istry for CD20, TRAP and DBA-44 in three HCL samples.This study was approved by our institutional review board.Detection of BRAF V600E mutationPresence of BRAF V600E mutation was determined withthe use of a real-time PCR assay and allele-specific hydro-lysis (‘Taqman’) probes. TaqmanW Universal PCR MasterMix (Applied Biosystems, Carlsbad, CA) was used toamplify a 136 bp DNA fragment. Wild type and mutantalleles were distinguished with the use of competingTaqmanW probes. The primers and probes used for PCRamplification (B-raf-51F and B-raf-176R) have beenpreviously described [8]. Briefly, the PCR assay was per-formed on an ABI 7500 (Applied Biosystems, Carlsbad,CA) standard real-time ddCT relative quantificationplate with the use of the BRAF-mutant—FAM-TAGCTACAGAGAAATC and BRAF-wild type—VIC-CTAGCTACAGTGAAATC \probes. A sample wasconsidered to harbour the BRAF V600E mutation if thedifference in the threshold cycle (dCt) between amplifica-tion of mutant and wild type sequence was less than orequal to eight cycles. (Figure 1). The detection limit (per-cent tumour cells) was determined by diluting DNA,which was extracted from a fresh HCL sample withknown neoplastic cell percentage (Patient 6, Table 1), withnormal high molecular weight DNA extracted using thesame method. The mutant allele was also detected throughthe dilution of the A2058 cell line DNA in normal DNA.AWild typeMutantFigure 1. Representative results from the real-time PCR assay for dmarginal zone lymphoma, stomach). (B) Positive case (hairy cell leuk(VIC), and blue curves represent mutant (c1799T> A pV600E) probCopyright © 2011 John Wiley & Sons, Ltd.Analysis for microsatellite instabilityMicrosatellite instability analysis was performed usingthe MSI Analysis System, version 1.2 (Promega Corp,Madison, WI). This assay entails a multiplex reaction offive quasi-monomorphic mononucleotide repeat markers(BAT-25, BAT-26, MONO-27, NR-21 and NR-24) andalso entails two highly polymorphic pentanucleotidemarkers (Penta C and Penta D) as sample identifiers. AfterPCR amplification, samples were analyzed with ABI 3100Avant™ (Applied Biosystems, Carlsbad, CA) for alteredalleles. In the absence of normal DNA for comparison, asample was considered microsatellite-high (MSI-H) ifgreater than two markers showed an altered pattern, inde-terminate if only two showed an alteration and microsatel-lite stable (MSS) if no marker showed an altered allele (orMSS with an allele polymorphism if only one markershowed an altered allele).ResultsThe clinical characteristics of hairy cell leukaemia cases arepresented in Table 1. Of note, one case (Patient 8) had anatypical immunophenotype, as detected by flow cytometry(CD103 negative), but it expressed CD25 and TRAP by im-munohistochemistry. Another case (Patient 7) presentedwith axillary lymphadenopathy without significant spleno-megaly. We purified DNA from tumour samples and usedthe real-time PCR assay to assess for the presence of BRAFV600E mutation (Table 2). The BRAF V600E mutationwas detected in all cases where the tumour percentage inthe analysed tissue exceeded 5%. In two patients (Patients2 and 5), no mutation was detected in the samples withlow tumour burden (≤5% tumour cells) but was detectedin additional samples containing a higher frequency of neo-plastic cells. We, thus, confirm the results of Tiacci et al. in-dicating a high prevalence of BRAF V600E mutation inHCL. The detection of the V600E mutation in an atypicalCD103 negative HCL case and a case with an atypical clin-ical presentation highlights the utility of this analysis to di-agnose challenging cases of HCL.BWild typeMutantetection of BRAF V600E mutation. (A) Negative case (extranodalaemia, Patient 6, spleen). Black curves represent wild type probeses (FAM)Hematol Oncol 2012; 30: 190–193DOI: 10.1002/honTable 2. BRAF V600E mutation detection in HCL samplesPatient Tissue type Tumour% WT Ct Mutant Ct dCt BRAF V600E1 Bone marrow 3.7%* 32.817 35.478 2.661 Mutant2 lung 5%** 29.932 39.83 9.898 Wild typeBone marrow 13.4%* 29.273 31.6 2.327 MutantLymph node 80%** 30.045 32.145 2.1 Mutant3 Bone marrow 35.3%* 31.135 33.78 2.645 Mutant4 Bone marrow 0.6%* 34.142 45 10.858 Wild type5 Peripheral blood 3.1%* 29.757 37.78 8.023 Wild typeBone marrow 23.0%* 31.089 33.531 2.442 Mutant6 Peripheral blood 42.9%* 28.341 30.298 1.957 MutantSpleen 78.4%* 24.81 24.968 0.158 Mutant7 Lymph node 70%** 27.819 31.116 3.297 Mutant8 Bone marrow 5.7%* 30.337 36.597 6.26 MutantdCt: difference in the threshold cycle; WT: wildtype; Ct: threshold cycle.*Tumour percentage based on flow cytometry.**Tumour percentage assessed by immunohistochemical staining for CD20, DBA-44 and TRAP.Table 1. Clinical characteristics of HCL patientsPatient Age at diagnosis Sex Presentation Treatment†1 58 M Pancytopenia Rituximab2 69 M Pancytopenia Cladribine3 46 M Pancytopenia Unknown4 41 M Pancytopenia Unknown5 53 M Pancytopenia Cladribine, Rituximab/Cladribine, Cladribine6 43 F Pancytopenia, splenomegaly Cladribine, Pentostatin (2 rounds),Rituximab/Pentostatin, Splenectomy7 61 M Pancytopenia,lymphadenopathy, weaknessNone8* 30 M Pancytopenia None†Sequential treatment regimens.*This case represents an HCL with an atypical phenotype.192 M Ewalt et al.To further evaluate the sensitivity of our real-time assay,we performed limiting dilution analysis with the use of thespleen sample from Patient 6 (Table 2), diluting theextracted DNA with wild type genomic DNA. We wereable to detect the BRAF V600E mutation at a dilution ratioof 1:8 (9.8% tumour cells) (Table 3). We were similarlyable to detect the mutant allele in a 10% dilution of theA2058 cell line DNA in normal DNA, or approximately5% mutant allele (data not shown). The absolute limit ofdetection may be improved with further refinement ofthis assay. At present, it provides sensitivity within therange of 5% and 10% tumour cells, demonstrating the valueTable 3. Limiting dilution analysis to determine mutationdetection sensitivity of the real-time PCR assayDilution Tumour% dCt BRAF V600E1:1 78.4% 0.3 Detected1:2 39.2% 0.6 Detected1:4 19.6% 3.1 Detected1:8 9.8% 5.2 Detected1:16 4.9% 11.4 Not Detected1:32 2.5% 13.1 Not DetecteddCt: difference in the threshold cycle.Tumour% based on flow cytometry.DNA was extracted from fresh splenic tissue (Patient 6).Copyright © 2011 John Wiley & Sons, Ltd.of real-time PCR in screening for the BRAF V600E muta-tion in HCL samples where the neoplastic cellular yield isoften limited because of underlying myelofibrosis.We next sought to evaluate the specificity of the BRAFV600E mutation for HCL among low and intermediateB-NHL by evaluating for the presence of this mutation in90 additional cases of B-NHL with a >20% tumourburden. We found that none of these additional casesharboured the BRAF V600E mutation, supporting thenotion that this alteration is specific to HCL (Table 4).Previous studies of mutations in the Ras-Raf-Mek-Erk-Mapkinase pathway have found an association between BRAFactivating mutations and MSI in colorectal neoplasms [9].able 4. BRAF V600E mutation detection in low-intermediaterade B-NHLeoplasm typeFrequency of V600Emutationairy cell leukaemia 9/12hronic lymphocytic leukaemia/smalllymphocytic lymphoma0/20antle cell lymphoma 0/19arginal zone lymphoma 0/24ollicular lymphoma 0/20ymphoplasmacytic lymphoma 0/7TgNHCMMFLThe assay failed for one lymphoplasmacytic lymphoma case.Hematol Oncol 2012; 30: 190–193DOI: 10.1002/hon193BRAF V600E mutations in B-cell lymphomasBRAF V600E mutations were also described in three ofnine (33%) immunodeficiency-related NHL displayingMSI, which consisted of EBV+ diffuse large B-cell lym-phomas, including a primary CNS lymphoma, and anEBV-T-cell post-transplant lymphoproliferative disorder[10]. We, thus, investigated whether HCL with BRAFmutations showed evidence of MSI. Interestingly, allcases exhibited an MSS phenotype with using a sensitiveassay.DiscussionHairy cell leukaemia is a rare and enigmatic B-NHL thatpredominantly involves the bone marrow and spleen. Itlacks characteristic chromosome abnormalities, and itsaetiology has remained elusive ever since its recognitionas a unique subtype of B-NHL [6,11–13]. The neoplasticsmall B-cells exhibit ‘hairy’ cytoplasmic projections andhave an activated phenotype, which may explain theirresponse to purine analogues and interferon therapy.The ubiquitous occurrence and high specificity of theBRAF V600E mutation for HCL among B-NHL, asrecently described [1,5], was confirmed in our series of HCLwith the use of a real-time PCR assay. We were able to iden-tify this mutation in seven of eight (87.5%) patients. We alsohave shown that this mutation was not present in 90 additionalsamples comprising a variety of low and intermediate gradeB-NHL, further supporting the notion that the BRAF V600Emutation is a specific genetic alteration in HCL.Tiacci et al. reported a sensitivity of approximately 30%tumour cells for the detection of BRAF V600E usingSanger sequencing [1]. Here, we describe a method thatdetects this mutation in cases with a lower tumour burdenby using either fresh or fixed archival tissue, within rangeof the method by Boyd et al. [5].Taken together, our data and other published datasuggest that the BRAF V600E mutation is specific forHCL among low to intermediate grade B-NHL. Priorstudies have shown that the BRAF V600E mutation leadsto activation of the MAP kinase pathway via sequentialphosphorylation of downstream signalling mediators.BRAF inhibition abrogates this activation, suggesting apotential role for BRAF inhibition in the treatment ofrefractory/resistant HCL [1,2]. Further studies will helpelucidate the exact pathogenic effects of this mutation.Given the high specificity of this mutation, it might alsobecome an integral part of disease classification in thefuture. Our study did not include cases of HCL variantsand only two cases of splenic MZL were evaluated, whichwere both negative. Analysis of additional such cases needsto be undertaken in order to confirm specificity of this mu-tation for HCL among low and intermediate grade B-NHL.ConclusionIn summary, we describe a sensitive method to specificallydetect the BRAF V600E mutation in patient samples downCopyright © 2011 John Wiley & Sons, Ltd.to a concentration of 5% to 10% tumour cells. The currentassay demonstrates an improved detection limit comparedto direct Sanger sequencing and could thus be useful for di-agnosis and therapy selection. Disease monitoring follow-ing treatment, however, may require an allele-specificPCR approach, or other sensitive methods that can detectlow level mutant alleles.Authorship contributionsGovind Bhagat and Mahesh Mansukhani designed thestudy and analysed data. Adrienne Phillips, GovindBhagat, Bachir Alobeidi and Mark Ewalt provided andcharacterized patient samples. Subhadra Nandula performedthe experiments and analysed data. Mark Ewalt and GovindBhagat wrote the manuscript. All authors reviewed themanuscript and provided critical comments.Conflicts of interestThe authors declare no conflict of interest.References1. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations inhairy-cell leukemia. N Engl J Med 2011; 364(24): 2305–2315.2. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF genein human cancer. Nature 2002; 417(6892): 949–954.3. Lee JW, Yoo NJ, Soung YH, et al. BRAF mutations in non-Hodgkin’s lymphoma. Br J Cancer 2003; 89(10): 1958–1960.4. Chapman MA, Lawrence MS, Keats JJ, et al. Initial genomesequencing and analysis of multiple myeloma. Nature 2011;471(7339): 467–472.5. Boyd EM, Bench AJ, van ’t Veer MB, et al. High resolutionmelting analysis for detection of BRAF exon 15 mutations inhairy cell leukaemia and other lymphoid malignancies. Br JHaematol 2011; 155(5): 609–612.6. Foucar K, Falini B, Catovsky D, Stein H. Hairy cell leukaemia.In WHO Classification of Tumours of Haematopoietic andLymphoid Tissues, (4th ed). Swerdlow SH, Campo E, HarrisNL, Jaffe ES, Pileri SA, Stein H, et al (eds.). InternationalAgency for Research on Cancer: Lyon, France, 2008; 188–190.7. Amorim MR, Vargas FR, Llerena JC Jr, Pombo-de-Oliveira MS.DNA extraction from fixed cytogenetic cell suspensions. GenetMol Res 2007; 6(3): 500–503.8. Benlloch S, Paya A, Alenda C, et al. Detection of BRAF V600Emutation in colorectal cancer: comparison of automatic sequencingand real-time chemistry methodology. J Mol Diagn 2006; 8(5):540–543.9. Domingo E, Espin E, Armengol M, et al. Activated BRAFtargets proximal colon tumors with mismatch repair deficiencyand MLH1 inactivation. Genes Chromosomes Cancer 2004; 39(2): 138–142.10. Borie C, Colas C, Dartigues P, et al. The mechanisms underlyingMMR deficiency in immunodeficiency-related non-Hodgkinlymphomas are different from those in other sporadic micro-satellite instable neoplasms. Int J Cancer 2009; 125(10):2360–2366.11. Cawley J. The biology of hairy cell leukemia. Leuk Lymphoma2009; 50(Suppl 1): 8–11.12. Cawley JC, Hawkins SF. The biology of hairy-cell leukaemia.Curr Opin Hematol 2010; 17(4): 341–349.13. Tiacci E, Liso A, Piris M, Falini B. Evolving concepts in thepathogenesis of hairy-cell leukaemia. Nat Rev Cancer 2006;6(6): 437–448.Hematol Oncol 2012; 30: 190–193DOI: 10.1002/hon

Real-time PCR-based analysis of BRAF V600E mutation in low and intermediate grade lymphomas confirms frequent occurrence in hairy cell leukaemia

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Real-time PCR-based analysis of BRAF V600E mutation in low and intermediate grade lymphomas confirms frequent occurrence in hairy cell leukaemia

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