BRAFV600E mutations are found in Richter syndrome and may allow targeted therapy in a subset of patients
2015-03-10

BRAFV600E mutations result in activation of the RAS-ERK pathway independently of RAS activation and are found in a variety of tumour types (Davies et al, 2002). They were initially identified in approximately 50% of patients with malignant melanoma and subsequently identified in almost all cases of hairy cell leukaemia (HCL) (Tiacci et al, 2011; Arcaini et al, 2012). Conventionally, BRAFV600E mutations are identified by genetic analysis, but an antibody specific for the BRAFV600E mutation has become available which permits sensitive and reliable detection of BRAFV600E protein using immunocytochemistry (Akarca et al, 2013). BRAFV600E inhibitors were shown to increase survival in patients with melanoma (Chapman et al, 2011) and there are reports of responses to such inhibitors in cases of HCL refractory to conventional therapy (Dietrich et al, 2012; Follows et al, 2013). Rarely BRAFV600E mutations are found in other haematological malignancies, including chronic lymphocytic leukaemia (CLL). Jebaraj et al (2013) examined 138 cases of CLL and found four cases with BRAFV600E mutation. Langabeer et al (2012) described a single patient with a biclonal disorder with composite HCL and CLL. We detected a single patient with CLL exhibiting BRAFV600E mutation, and this patient had a Richter transformation at the time of analysis. We therefore carried out a more extensive study with a particular focus on Richter syndrome (RS). RS transformation occurs at a frequency of approximately 0·5% per year (Parikh et al, 2013) and the results of therapy after transformation are poor. It would therefore be important to identify any recurrent genetic abnormalities associated with transformation that are amenable to targeted therapy. In this multi-institutional study, we assessed 259 cases of CLL where no transformation to RS had occurred. In addition we assessed 43 patients with RS. Paired patient samples showing CLL preceding transformation to RS were also analysed. Diagnosis of CLL and RS was made by expert haematopathologists following the World Health Organization classification criteria. Detection of BRAFV600E by immunocytochemistry was performed using the previously validated specific mouse monoclonal antibody VE1 was performed as previously described (Akarca et al, 2013). Cases were assessed independently by at least two haematopathologists without knowledge of the BRAF mutational status. To confirm the presence of BRAFV600, DNA was extracted from formalin-fixed, paraffin-embedded tissues using Qiagen DNeasy kit according to manufacturer's instructions. The sequences of allele-specific oligonucleotides and method to detect the wild type and BRAF T1860A mutation alleles have been described previously (Arcaini et al, 2012). These were used in a noncompetitive polymerase chain reaction (PCR) before separation of PCR products on 2·5% agarose gel electrophoresis. The mutation status of samples positive by allele-specific oligonucleotide (ASO) PCR was confirmed by PCR amplification of exon 15 (forward 5′-TCATAATGCTTGCTCTGATAGGA-3′; reverse 5′GGCCAAAAATTTAATCAGTGGA-3′) and Sanger sequencing of the 224 bp product. Comparison between groups was performed using a 2-tailed Fisher's exact test. Of the 259 patients with CLL without transformation, 4 were found to have a BRAFV600E mutation by immunocytochemistry (1·5%). Three of these patients have been followed up for 1, 3 and 7 years without transformation. The other patient was lost to follow-up. Of the 43 patients with RS, 3 (7%) were found to have a BRAFV600E mutation. This apparently higher frequency after RS is not statistically significant (P = 0·1) but even though this is a large study of a rare disorder such as RS, the power of the analysis is limited. More detailed review of the three cases of RS with BRAFV600E is informative. The samples relating to these cases are summarized in Table 1. Patient 1 presented at the age of 56 years with classical CLL. Eight years later he developed RS, with a lymph node biopsy showing diffuse large B cell lymphoma (DLBCL) of the non-germinal centre type by Hans algorithm and virtually all of the large cells were positive for BRAFV600E by immunocytochemistry (Fig 1B). The mutation was confirmed by ASO-PCR and Sanger sequencing (Fig 1D and F). Bone marrow sections from the CLL phase were available and the leukaemic cells were all BRAFV600E mutant by immunocytochemistry (Fig 1A). Patient 2 is a female with a history of CLL who presented at the age of 67 years with pulmonary lesions, which, when biopsied, revealed infiltration by CLL, with a classical phenotype and no large cells. Only 2 months later, new pulmonary nodules appeared and a repeat biopsy from a site different from the original biopsy showed DLBCL. The DLBCL was analysed for BRAFV600E mutation by ASO-PCR and was positive. The BRAFV600E mutation was also present in the original untransformed CLL. Patient 3 presented at the age of 63 years with lymphadenopathy and lymphocytosis. A bone marrow biopsy showed 50% infiltration with classical CLL. There was no evidence of BRAFV600E by immunocytochemistry or molecular analysis at this time. Two years later he developed RS in a lymph node that demonstrated widespread positive staining with the BRAFV600E mutation-specific VE1 antibody. BRAFV600E was confirmed by ASO-PCR and Sanger sequencing. Following treatment with six cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) chemotherapy he achieved remission from his RS with persistence of the original CLL cells. At this time both bone marrow and lymph node were negative for BRAFV600E by immunocytochemistry, ASO-PCR and sequencing. In Patients 1 and 2, the BRAFV600E mutation was clearly present in the untransformed cells and the mutation cannot be considered as the cause of the transformation. In Patient 3, the BRAFV600E mutation was only seen at the time of transformation and may thus be relevant to the transformation process. The fact that the BRAFV600E mutation can be acquired during the evolution of CLL suggests that the apparently higher frequency of BRAFV600E mutations in RS compared to untransformed CLL may be a real phenomenon and further large cohort studies are required to confirm this. The presence of BRAFV600E mutations in some patients with RS raises the possibility of targeted therapy in such patients. R.S.S. is a Cancer Research UK Clinical Research Fellow. This work was supported by grants from the UCLH/UCL NIHR Comprehensive Biomedical Research Centre (TM), Lymphoma Research Trust (AUA) and AIRC 5 × 1000 n. 10007 (SAP). R.S.S. performed genetic analysis. F.F., C.A., LQ-M, H.S., and S.A.P., diagnosed and provided well-characterized cases of CLL and Richter Syndrome. A.U.A., V.S., and J.W., performed immunohistochemistry. R.S.S., D.L and T.M. designed the study, interpreted the data and wrote the manuscript. The authors declare no competing financial interests.