The Utility of BRAF V600E Mutation-Specific Antibody VE1 for the

AJCP / Original Article

The Utility of BRAF V600E Mutation-Specific Antibody VE1 for the Diagnosis of Hairy Cell Leukemia Guldeep Uppal, MD,1 Vandi Ly, MD,1 Zi-Xuan Wang, PhD,1 Renu Bajaj, PhD,1 Charalambos C. Solomides, MD,1 Peter M. Banks, MD,2 L. Jeffrey Medeiros, MD,3 Stephen C. Peiper, MD,1 and Jerald Z. Gong, MD1 From the 1Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA; 2Ventana Medical Systems, Tucson, AZ; and 3The University of Texas MD Anderson Cancer Center, Houston. Key Words: BRAF; Hairy cell leukemia; VE1; Immunohistochemistry Am J Clin Pathol January 2015;143:120-125 DOI: 10.1309/AJCPQLQ89VXTVWKN

ABSTRACT Objectives: BRAF V600E mutation is characteristic of hairy cell leukemia (HCL). A V600E mutation-specific antibody, VE1, has been recently developed. We studied the diagnostic utility of this antibody in HCL and compared it with other B-cell neoplasms. Methods: VE1 activity was assessed using immunohistochemistry in 90 mature B-cell neoplasms, including HCL (n = 17), HCL variant (n = 6), chronic lymphocytic leukemia (CLL) (n = 20), and 47 other B-cell lymphomas. Most (87/90) specimens were formalin-fixed, paraffin-embedded bone marrow (BM) biopsy specimens decalcified in either hydrochloric acid or formic acid. Results: VE1 was positive in 15 (88%) cases of HCL and two (10%) cases of CLL and was negative in all other tumors assessed. The VE1-positive HCL cases showed uniform staining in all tumor cells, but intensity was variable. The two VE1-negative HCL cases had BRAF V600 mutations proven by molecular analysis. The two CLL cases positive with VE1 showed an atypical staining pattern with expression in a minority of lymphoma cells. Immunohistochemistry using the VE1 antibody had a sensitivity of 88% and a specificity of 97% for HCL. Conclusions: VE1 immunohistochemistry is a useful and convenient surrogate for detecting BRAF V600E mutation in BM biopsy specimens decalcified with hydrochloric or formic acid–based solutions.

120 Am J Clin Pathol 2015;143:120-125 DOI: 10.1309/AJCPQLQ89VXTVWKN

Hairy cell leukemia (HCL) is an indolent, mature B-cell leukemia that primarily involves the bone marrow (BM) and spleen, with low-level leukemic involvement. The disease most commonly presents with pancytopenia and splenomegaly in middle-aged to elderly individuals.1 HCL in the BM is typically associated with fibrosis, often resulting in a “dry tap” with sampling of blood rather than BM cells when BM aspiration is performed. HCL cells have characteristic small round or kidney-shaped nuclei without nucleoli and relatively abundant cytoplasm with “hairy” cytoplasmic projections on the surface. The diagnosis of HCL requires immunophenotyping with flow cytometry and/or immunohistochemistry. The neoplastic lymphocytes express monotypic surface immunoglobulin light chain, pan–B-cell antigens, and CD11c, CD25, CD103, DBA.44, TRAP, and annexin A1. The correct diagnosis of HCL is critical for optimal treatment. In 2011, Tiacci et al2 described an activating mutation of BRAF, V600E, in all cases of HCL (n = 48) using a whole-exome sequencing-based approach followed by Sanger sequencing. Somatic mutations in the BRAF gene have been frequently described in human cancers. The most frequent mutation in BRAF is c.T1799A (V600E). The mutation involves a single base change in exon 15 of the BRAF gene, resulting in substitution of valine for glutamic acid at amino acid residue 600 (BRAF V600E). The mutant BRAF has increased kinase activity, leading to the constitutive activation of the downstream mitogen-activated protein kinase/extracellular signal–regulated kinase (MAPK/ ERK) pathways, increased cell proliferation, and eventually cancer.3 Since the seminal report by Tiacci et al, various studies have confirmed that BRAF V600E is present in

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nearly all HCL and is present at a very low frequency or is entirely absent in other mature B-cell lymphoproliferative disorders.4-7 The discovery of BRAF V600E mutation in HCL has opened a new era in which the diagnostic and therapeutic paradigms of HCL have shifted. Recently, a BRAF V600E mutation-specific antibody, clone VE1, became available for immunohistochemical analysis. Andrulis et al8 from Germany recently reported high efficacy of immunohistochemical staining using the VE1 antibody to detect BRAF V600E mutation and to distinguish HCL from other mimics of HCL, such as HCL variant (HCLv) and splenic marginal zone lymphoma, using EDTA-decalcified BM tissue. Akarca et al9 from England performed immunostaining using the same BRAF antibody (VE1) in 26 cases of HCL and found 100% concordance with molecular results. Since BM specimens in North America are often processed differently from those of European countries, in an attempt to confirm their results, we evaluated a cohort of BM biopsy specimens representing HCL and various other lymphomas that were processed in hydrochloric acid or formic acid–decalcified solutions. In this study, we found that VE1 antibody had 88% sensitivity and 97% specificity in detecting BRAF V600E mutation in HCL when applied to such specimens.

Materials and Methods Case Selection This study was approved by the institutional review boards where the experiments were conducted. All cases included in this study were classified according to the criteria of the 2008 World Health Organization classification.1 Diagnoses were confirmed by review of morphologic, immunohistochemical, and flow cytometry findings. All cases except three were formalin-fixed, paraffinembedded (FFPE) BM biopsy specimens decalcified in either hydrochloric acid or formic acid–based solutions. The remaining three cases included a lymph node biopsy specimen (n = 1), a splenectomy specimen (n = 1), and a BM clot section (n = 1). Immunohistochemical Staining Immunohistochemistry for BRAF was performed using an automated immunohistochemical stainer according to the manufacturer’s specifications (Ventana Medical Systems, Tucson, AZ). The immunohistochemistry stains were performed using BRAF V600E mutation-specific antibody (clone VE1; Ventana Medical Systems). The anti–BRAF V600E antibody is a mouse monoclonal antibody produced


against a synthetic peptide representing the mutated BRAF sequence from amino acids 596 to 606 (GLATEKSRWSG). This antibody differentiates the BRAF V600E mutated protein from wild-type BRAF protein and BRAF mutated proteins at other codons (other than codon 600). The antigen-antibody reaction was detected using a streptavidinbiotin-peroxidase system with diaminobenzidine (DAB) as the chromogen. The cases were evaluated with the OptiView DAB IHC detection kit (Ventana Medical Systems). Appropriate positive and negative controls were included. The positive controls were obtained from cases of papillary thyroid carcinoma in which BRAF V600E mutation was confirmed by polymerase chain reaction (PCR) and sequencing. The VE1 antibody exhibits a cytoplasmic staining pattern with variable staining intensity ranging from weak to strong. Immunohistochemistry for other relevant markers (TRAP, annexin A1) was performed using standard methods according to the manufacturer’s instruction (Ventana Medical Systems). Flow Cytometry Analysis BM aspirate or peripheral blood samples were analyzed by four-color flow cytometry within 24 hours. The neoplastic cells were first identified by a CD45/ forward-scatter gating strategy, and abnormal B cells were identified by expression of pan–B-cell antigens and monotypic immunoglobulin light chain expression. The abnormal cells were further analyzed using antibodies specific for CD11c (HL3), CD25 (M-A251), and CD103 (M290) (BD Biosciences, San Jose, CA). The stained cells were acquired on a benchtop flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA) and analyzed using Kaluza software (Beckman Coulter, Fullerton, CA). The fluorescence intensity was measured using a logarithmic scale with a signal intensity ranging from 100 to 104. The biologically negative cells within each tube were used as negative controls. A negative population was defined as no significant difference of mean fluorescence intensity from the negative control, while a positive population was defined as significant shift of mean fluorescence intensity from the negative control (dimly positive: greater than but significantly overlaps with the negative control; moderately positive: distinct from or only slightly overlaps with the negative control; brightly positive: 1.5- to 2-log shift from the negative control). BRAF Mutational Analysis BRAF mutational analysis was performed in selected HCL cases using the Ventana-Roche platform (cobas 4800 BRAF V600 Mutation Test; Roche Diagnostics,

Am J Clin Pathol 2015;143:120-125 121 DOI: 10.1309/AJCPQLQ89VXTVWKN

Uppal et al / BRAF Immunohistochemistry in Hairy Cell Leukemia

Indianapolis, IN). The cobas 4800 BRAF V600 Mutation Test is a real-time PCR assay that uses DNA from FFPE tissues and detects the presence of BRAF V600E mutation, as well as other V600 mutations, including V660D and V600K. After PCR amplification, detection of target DNA was performed by using a complementary primer pair and two oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wildtype BRAF V600 sequence, and the other probe is designed to detect the mutated BRAF V600 sequence. The presence or absence of mutation was determined based on the ratio of the fluorescent signals of each probe.

Results A total of 90 cases of mature B-cell neoplasms were selected, which included HCL (n = 17), HCL variant (n = 6), splenic diffuse red pulp small B-cell lymphoma (SDRPL; n = 1), chronic lymphocytic leukemia (CLL; n = 20), follicular lymphoma (n = 12), lymphoplasmacytic lymphoma (n = 8), mantle cell lymphoma (n = 7), marginal zone lymphoma (splenic, n = 4; nodal, n = 2; mucosa-associated lymphoid tissue, n = 2; total, n = 8), and diffuse large B-cell lymphoma (n = 11). All HCL cases showed a B-cell immunophenotype by flow cytometry, with surface monotypic immunoglobulin light chain expression, pan–B-cell antigens, and expression of CD11c (bright), CD25, and CD103. In addition, all HCL cases were positive for TRAP (17/17) and annexin A1 (15/15 tested). Fifteen cases of HCL showed positive staining for the VE1 (BRAF V600E specific) antibody and exhibited a distinct cytoplasmic positive staining pattern. Two cases of HCL were negative for VE1 staining. Of the VE1-positive cases, four showed weak staining, four showed moderate staining, and the remaining seven cases showed strong staining ❚Image 1❚. Staining was frequently observed in mast cells and megakaryocytes, but this staining did not interfere with the interpretation. All the remaining background hematopoietic cells were negative for VE1 staining. Both cases of HCL negative for VE1 had BRAF V600 mutation proven by molecular analysis. With the exception of two CLL cases, all other tested lymphomas, including HCLv and SDRPL, were negative for VE1. Two cases of CLL showed positive signals using VE1, but both cases had an atypical, granular, cytoplasmic staining pattern unlike HCL. One CLL case showed moderate staining in a subset of neoplastic B cells, and the other CLL case showed focal weak staining (Image 1). The overall sensitivity of VE1 antibody in this HCL cohort using hydrochloric acid or formic acid decalcification was 88%, and the overall specificity was 97%.


Discussion BRAF belongs to the RAF-kinase family and encodes for serine/threonine protein kinase involved in MAPK/ ERK pathway signaling. In normal cells, wild-type BRAF (BRAFwt) serves as a transponder for mitotic signals in the ERK pathway and is activated by binding of mitogen to its membrane ligand. Mutated BRAF (BRAFmut) induces constitutive activation of the kinase activity, resulting in mitogen-independent activation of the ERK pathway.3 The most common mutation of BRAF occurs in exon 15 with a T to A substitution at position 1799, leading to exchange of amino acids valine to glutamic acid.3 BRAF mutation has been reported in many neoplasms, including cutaneous malignant melanoma,10 papillary thyroid carcinoma,11 colorectal carcinoma,12 non–small cell lung carcinoma,13 pleomorphic xanthoastrocytoma,14 Langerhans cell histiocytosis,15 Erdheim-Chester disease,16 and HCL.2 After the 2011 discovery of BRAF mutation in HCL by Tiacci et al,2 multiple studies have confirmed that BRAF V600E mutation is present in essentially all cases of HCL. Although BRAF V600E mutational analysis can be achieved with a relatively simple sequencing or allele-specific PCR method, it requires the presence of sufficient numbers (percentages) of leukemic cells in the test specimens. In many cases, both peripheral blood and BM aspirate samples may contain insufficient HCL cells for these tests to be adequate. The peripheral blood of patients with HCL often shows a very small number of circulating neoplastic cells, which may often be below the detection limits of molecular assays. BM aspiration frequently yields a “dry tap” due to BM fibrosis. With these limitations, single-gene mutational analysis may not be an ideal approach for testing BRAF V600E mutation in some patients. Next-generation sequencing approaches that are more sensitive may overcome some of these limitations. Alternatively, testing can be performed on BM core biopsy specimens. Immunohistochemistry using the VE1 antibody for detecting BRAF V600E mutant protein has been previously shown to be highly reliable in the evaluation of melanoma and thyroid carcinoma specimens.17,18 However, the value of VE1 in the diagnosis of HCL has not been reported extensively in the literature, especially from institutions in North America. Two recent studies, both analyzing European cohorts, have shown 100% sensitivity in detecting mutated BRAF in HCL. Andrulis et al8 studied BRAF expression in 32 cases of HCL and 20 HCL mimics using the VE1 antibody. VE1 was positive in all HCL cases and in none of the HCL mimics. Andrulis and colleagues subsequently screened an additional 228 mature B-cell neoplasms using tissue microarrays, and only one case showed BRAF expression. All the HCL specimens in their



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❚Image 1❚ Representative VE1 staining patterns in hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and hematopoietic cells (×500). A, Weak BRAF expression in HCL. B, Moderate BRAF expression in HCL. C, Strong BRAF expression in HCL. D, BRAF expression in a subset of lymphocytes in CLL. E, Nonspecific BRAF staining in mast cells. F, Nonspecific BRAF staining in megakaryocytes. © American Society for Clinical Pathology


Uppal et al / BRAF Immunohistochemistry in Hairy Cell Leukemia

cohort were fixed in buffered formalin and processed after decalcification in EDTA.8 A study from England by Akarca et al9 tested BRAF expression in 26 cases of HCL and 75 other lymphomas. The authors concluded that VE1 reactivity was 100% sensitive and specific for the diagnosis of HCL. In the current study, as expected, most of our HCL cases showed positive VE1 staining. However, two HCL cases were negative. We have subsequently performed molecular studies on these two cases using the cobas 4800 platform and confirmed the presence of BRAF V600 mutations in both cases. Unfortunately, Sanger sequencing of the BRAF gene in these two cases could not be attempted due to the limitations of decalcified specimens. The reasons for negative VE1 staining in two cases of HCL with confirmed BRAF V600 mutation are not clear, and several possible scenarios may have contributed to the negative results. The VE1 reactivity in HCL, as observed in our series and in others, is variable, ranging from strong to weak from case to case. It is possible that the mutational protein is not sufficiently expressed in these cases to be detected by immunostaining. The decalcification solution used in our series was either hydrochloric acid or formic acid, which differs from the EDTA solution commonly used in European countries. The different types of decalcification may have contributed to the slightly discrepant results from our series compared with the European cohorts. Alternatively, the lack of VE1 reactivity in the two HCL cases could be attributable to variant mutations in these cases, such as V600D or V600K, which are not detectable using the VE1 antibody. Molecular study using the cobas 4800 platform confirmed V600 mutation in these two cases. However, this method is not specific for V600E mutation and may detect other V600 mutations such as V600D and V600E. Confirmation by Sanger sequencing was not attempted because both specimens had been subjected to decalcification, which is known to have an insufficient result by Sanger sequencing. To date, all the reported BRAF mutations in HCL in codon 600 were the V600E mutation.2,6,19,20 Rare variant BRAF mutations have been reported only in exon 11 in HCL21 but not in exon 15 codon 600.2,22 Since codon 600 mutations other than V600E were not reported in HCL, it seems unlikely that a variant mutation contributed to the lack of VE1 reactivity in these two cases. Nevertheless, one should be aware of the presence of rare V600E mutation-negative HCL,20-22 and these cases may yield negative results by VE1 antibody staining. We included 72 cases of B-cell tumors, some of which can closely mimic HCL, to compare with VE1 reactivity in HCL. Only two cases of CLL showed staining of BRAF in tumor cells. The remaining 70 cases were negative for BRAF expression. The two CLL cases showed a variant pattern of VE1 staining with only a subset of the tumor


cells being positive. One case showed moderate staining in a fraction of the CLL cells. The second case showed weak staining in only rare lymphocytes. The variant staining pattern by VE1 in CLL can be readily distinguished from the uniform diffuse expression pattern in HCL. Several authors have studied BRAF mutation in CLL and prolymphocytic leukemia (PLL), and rare cases had BRAF mutations. Jebaraj et al23 reported BRAF V600E mutation in 2.8% of CLL after screening of 138 CLL samples. Langabeer et al5 studied 163 patients with CLL and PLL for evaluation of BRAF V600E mutation by allele-specific PCR and found BRAF mutation in one CLL and one PLL. These studies indicate that BRAF V600E mutation is not completely specific for HCL and is present at low frequency in CLL. Furthermore, other studies have suggested that BRAF V600E mutation may exist in only a small subclone of tumor cells when identified in rare B-cell tumors other than HCL. Arcaini et al19 performed allele-specific PCR for BRAF V600E analysis and studied 240 cases of mature B-cell neoplasms. They detected BRAF V600E in only two patients with B-cell chronic lymphoproliferative disorder. Despite PCR positivity, the mutation could not be detected by Sanger sequencing, suggesting that it was associated with a small subclone. This may explain the immunohistochemical staining pattern seen in our two cases of CLL, in which only a subset of leukemic cells was positive. This hypothesis in CLL has yet to be proved in future studies. HCLv and SDRPL are two mature B-cell leukemias with overlapping features with HCL. HCLv resembles HCL with “hairy” leukemic cells and expresses some of the common HCL-associated markers such as CD11c, DBA-44, and CD103. HCLv is typically negative for CD25, TRAP, and annexin A1. Several studies have shown that BRAF V600E mutation was absent in HCLv using various molecular approaches.20,21 Andrulis et al8 reported negative VE1 results in two cases of HCLv by immunohistochemistry. We have included six cases of HCLv in our series. All six cases had no staining by VE1 in the leukemic cells. Our results further confirm that VE1 immunohistochemical analysis is a useful marker in distinguishing HCL from HCLv. SDRPL is a very rare B-cell lymphoma involving primarily splenic red pulp, BM, and peripheral blood. The leukemic cells typically have a villous cytology, resembling HCL and HCLv. The single case of SDRPL in our study showed complete lack of VE1 reactivity, suggesting the absence of BRAF V600E mutation in this disease. Additional cases need to be assessed to draw a more definitive conclusion with regard to the utility of VE1 to exclude this rare entity. In summary, we demonstrated that BRAF V600E mutation-specific antibody VE1 can successfully detect overexpression of the mutated BRAF protein in most cases of HCL (15/17; 88%) and is an effective marker to



distinguish HCL from common mimics such as HCLv and SDRPL. VE1 antibody can be included as a first-line diagnostic marker in HCL. One needs to be aware that VE1 does not stain all HCL when BM specimens are processed with hydrochloric acid or formic acid decalcification. VE1negative HCL cases may be confirmed by BRAF mutational analysis when adequate specimens are available. Rare cases of CLL may show positive VE1 reactivity, but the pattern of staining is usually focal and weak. Overall, our results demonstrate that the VE1 antibody is a useful adjunct to the diagnostic armamentarium for the recognition of HCL. Address reprint requests to Dr Gong: Thomas Jefferson University Hospital, 117 South 11th St, Suite 301, Philadelphia, PA 19107; [email protected]. This work is partially supported by a grant from Ventana Medical Systems. Dr Banks is currently an employee of Ventana Medical Systems. The remaining authors reported no conflicts of interest (both financial and personal).

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