<scp>BRAF</scp> V600E mutation‐specific antibody, a sensitive diagnostic marker revealing minimal residual disease in hairy cell leukaemia (2013)

The proto-oncogene BRAF, found on chromosome 7q34, encodes a serine/threonine kinase that has a regulatory role in the activation of the mitogen-activated protein kinase (MAPK)/ERK signalling cascade, implicated in cancer induction, maintenance and progression (Ascierto et al, 2012). BRAF is one of the most frequently mutated protein kinase genes in human cancer, with the BRAF V600E mutant (a missense substitution of valine by glutamic acid in a mutational hotspot at amino acid position 600 of the BRAF protein) commonly present in metastatic melanomas, thyroid carcinoma and colon carcinomas (Cantwell-Dorris et al, 2011). Among haematological malignancies, recent studies have shown hairy cell leukaemia (HCL) and Langerhans cell histiocytosis (LCH) to harbour BRAF V600E mutations at a high frequency (Tiacci et al, 2011; Sahm et al, 2012). The fact that this mutation is found in virtually all cases of HCL suggests that it is a key underlying genetic defect in the pathogenesis of this leukaemia. Hairy cell leukaemia is a rare haematological disorder with distinct clinical and histopathological features (i.e. splenomegaly, pancytopenia, and infiltration of bone marrow, spleen and liver by leukaemic B-cells with abundant cytoplasm and hairy-like appearance), and low number of circulating leukaemic cells (Swerdlow et al, 2008). Although the diagnosis of HCL relies on its pathological and immunophenotypic features, in some cases it is difficult to differentiate this malignancy from other types of low grade B-cell lymphoma/leukaemia, such as hairy cell leukaemia variant (HCL-v) and splenic marginal zone leukaemia (SMZL). Several studies have shown the value of BRAF mutational analysis in making the diagnosis of HCL (Tiacci et al, 2011; Laurini et al, 2012) and one immunohistochemical study has shown that staining with a mutation-specific antibody may be an equally effective strategy (Andrulis et al, 2012). There are multiple treatment options available for HCL patients but purine nucleoside analogues are mostly used as front-line therapy. Although the initial response rate is high, 30–40% of patients relapse within 10 years and require further therapy (Grever, 2010). There may in future be a role for BRAF inhibitors, which have already been introduced into the clinic for the treatment of melanoma (Cantwell-Dorris et al, 2011; Dietrich et al, 2012). In the present investigation we aimed to validate the immunohistochemical utility of a monoclonal antibody raised against the BRAF V600E mutation for the diagnosis of HCL and to determine whether this approach is sufficiently sensitive to detect lower levels of disease than with the standard immunohistochemistry antibodies panel. A total of 101 samples (including normal and neoplastic haematopoietic and lymphoid tissues) were immunostained with specific antibodies raised against total BRAF (clone pBR1) and mutated BRAF V600E (clone VE1) proteins (Data S1). The relevant positive and negative controls were included throughout. Expression of total BRAF was found in all cell compartments of normal bone marrow (Fig 1A), spleen and tonsil (data not shown) whilst no VE1 labelling was observed in parallel sections of the same tissue samples. The high level expression of BRAF protein in human haemopoietic tissue is in accordance with documented mRNA expression data. (www.genecards.org). A similar scenario was observed among haematological neoplasms that showed diffuse staining with pBR1 antibody (data not shown) but failed to express mutated BRAF V600E protein. The only exception was HCL, which showed strong VE1 positivity in all analysed cases (26 out of 26) (Table 1 and Fig 1B) and by double immunostaining it was confirmed that the PAX-5 positive leukaemic cells co-expressed BRAF V600E protein (Fig 1B). Of the 11 post-treatment bone marrow trephine biopsies that were examined, all of which were deemed to show complete remission by conventional staining techniques, two revealed isolated cells or foci of cells expressing the BRAF V600E mutated protein (Fig 1C), whereas no staining was seen with normal marrow and the other nine cases. Reverse transcription polymerase chain reaction (RT-PCR; Qiagen, Manchester, UK) revealed the presence of BRAF V600 mutation in one of the HCL cases and seven melanomas, which all had shown strong BRAF V600E staining. Until recently, the diagnosis of HCL has been dependent on the demonstration of histopathological and phenotypic features using a panel of antibodies including CD11c, CD25, CCND1, DBA44, CD103, CD123 and CD20 (Grever, 2010). A breakthrough study by Tiacci et al (2011) identified the BRAF V600E mutation in virtually all cases of HCL using whole genome analysis and Sanger sequencing. The detection of point mutations is relatively straight forward on fresh leukaemic cells using molecular techniques, but they can be less efficient when formalin-fixed paraffin embedded tissue sections are used, particularly where the tumour burden is <15%. In particular, the circumstances of bone marrow biopsies with limited extractable haemopoietic tissue for PCR would benefit from this intact tissue approach. Blombery et al (2012) have suggested that high resolution of melting analysis (HRM) may be a more useful tool for detecting the BRAF V600E mutation at diagnosis but not all histopathology laboratories have experience with this technology. Immunohistochemistry is more readily available in the majority of diagnostic laboratories. This study has therefore assessed the sensitivity and specificity of immunohistochemical diagnosis of HCL using an antibody specific to BRAF V600E mutated cells. Our findings confirmed a previous analysis demonstrating the diagnostic utility of the VE1 antibody in HCL (Andrulis et al, 2012) by showing that all of our HCL cases strongly expressed BRAF V600E that was not seen in any other lymphoproliferative disorder studied. Moreover, we were able to demonstrate that immunocytochemistry with VE1 antibody could detect minimal residual disease (MRD), below the levels of detection with conventional histochemical reagents, in some patients following treatment. This raises the possibility that such patients would benefit from further therapy either with repeated cycles of purine analogues or with BRAF inhibitors. The value of such an approach can only be determined in a randomized trial which would need to be multi-centric if not multi-international, and the ability to detect MRD with a simple immunohistochemistry approach makes such a study more feasible. This work was supported by grants from the UCLH/UCL NIHR Comprehensive Biomedical Research Centre (TM), Lymphoma Research Trust (AUA) and UCL Cancer UK Development (VHS). TM and DL conceived the idea, designed the study, selected the clinical cases for inclusion and wrote the manuscript. TM compiled the results and created the figures. TM and ADR reviewed and interpreted the staining. AUA and VHS equally contributed to perform the immunostaining, collate the data, format and help editing the manuscript; TD performed the molecular analysis; MPF, HR and MC provided a substantial number of well-characterized clinical biopsies; TMG helped editing the manuscript. The authors do not have any financial interests to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.