Hairy cell leukaemia with unusual BRAF mutations

Abstract Hairy cell leukaemia (HCL) diagnosis is based on the morphologic detection of circulating abnormal hairy cells in the peripheral blood and/or bone marrow, an HCL immunological score of 3 or 4 based on the expression of the CD11c, CD25, CD103 and CD123 and also the presence of a BRAF V600E activating mutation in the B‐raf proto‐oncogene (BRAF gene) (7q34). When using new generation sequencing of 21 targeted genes in 124 HCL patients, we identified a cohort of 6/124 (2%) patients with unusual BRAF mutations: two patients presented non‐V600 mutations (BRAF F595L, BRAF W604L respectively) and four other patients silent BRAF mutations. When using droplet digital PCR (ddPCR) three of the four patients with concomitant BRAF V600E and silent mutation were negative. The respective role of these mutations in the occurrence of HCL or its progression remains to be clarified, but BRAF sequencing is necessary in case of negative BRAF V600E by ddPCR.

(Bio-rad) with ddPCR BRAF V600 screening kit (#12001037) in accordance with the manufacturer's specifications. Oral informed consent was made by physicians, and non-opposition consents was obtained. The study was conducted in accordance with and approved by the Institutional Review Board of CCTIRS and CNIL (protocol code 16

| RE SULTS AND D ISCUSS I ON
We report for the first time a patient with HCL associated with a BRAF F595L mutation. The patient (UPN-50), a 57-year-old man, was hospitalized in September 2011 and presented moderate splenomegaly without peripheral lymph node involvement. The blood cell count showed thrombocytopenia (platelets count: 87 × 10 9 /L), monocytopenia (<0.01 × 10 9 /L), moderate lymphocytosis (6.20 × 10 9 /L) without anaemia (13.4 g/dL) and neutropenia (2.79 × 10 9 /L). The peripheral blood smear analysis detected 33% of typical hairy cells (2.97 × 10 9 /L) ( Figure 1A) and the peripheral blood flow cytometry analysis, performed after isolating mononuclear cells by density gradient centrifugation (e.g. peripheral blood mononuclear cells PBMC), showed a clonal B-cell proliferation (evaluated at 42% of the total number of leucocytes), CD20 bright , CD79b bright with a kappa light chain restriction. CD5, CD10 and CD38 were negative. The four specific markers of HCL (CD11c, CD25, CD103 and CD123) were brightly expressed ( Figure 1B). The bone marrow biopsy was not performed and Annexin A1 staining was not tested. However, the bone marrow infiltration by typical hairy cells and the peripheral blood and bone marrow immunophenotypic analysis were sufficient to establish HCL diagnosis. The activating missense mutation BRAF V600E was not detected; however, BRAF F595L (c.1783T>C) mutation ( Figure 1C, Figure S1) was identified with a variant allele frequency (VAF) of 33%. Copy number analysis of BRAF zygosity showed no significant variation suggesting no loss of heterozygosity ( Figure S2). Two mutations of Krüppel-like Factor-2 (KLF2; 19p13) were also associated ( Table 1). The immunoglobulin heavy-chain variable region genes (IGHV) were mutated (95.7%) with VH3-7 rearrangements. The patient was treated with Cladribine (CDA) in monotherapy but relapsed  Figure S4). When using specific BRAF V600E ddPCR assay, three of the four patients with BRAF V600E associated with silent mutation were negative ( Only three HCL patients with non-V600 mutations were previously reported: two patients with mutations in exon 11 (F468C, D449E) and one patient presenting a BRAF V600E mutation associated with BRAF S602T. 12 The BRAF F595L mutation, disrupting the D 594 F 595 G 596 motif of the BRAF kinase domain essential for catalysis, was reported in a patient with histiocytic sarcoma. The authors demonstrated the mutation was associated with an intermediate kinase activity and the cooperation with HRAS Q61 allowed the promotion of oncogenic signals. 13 The HCL patient with a BRAF F595L mutation located in exon 15 responded to CDA in monotherapy. Note also that the patient does not present any associated poor prognostic factors. The IGHV profile was mutated (95.7%) and VH3-7 rearrangements were used. In HCL, an unmutated (UM) IGHV profile and the VH4-34 usage is usually associated with resistance to CDA and shorter progression-free survival and overall survival. 14,15 The second HCL patient we reported presented a BRAF V600E mutation associated with W604L. The patient was treated with CDA plus rituximab and a CR was achieved.
The role of synonymous mutations remains to be studied in HCL.
In a study of 659,194 synonymous (silent) mutations from mainly solid tumour samples, including melanoma, the silent mutations were the second most frequent type of point mutations (23.4%) after missense mutations (64.1%). 16 Synonymous mutations do not change the amino acid sequence and most of them are expected to be functionally neutral. The clinical impact of synonymous mutations remains to be specified but they can change protein levels or protein conformation by altering splicing regulatory sites, mRNA stability, miRNA binding sites or translation efficiency. 17 Either in or out of the coding regions, they can affect gene expression and may be contribute to tumorigenesis and cancer-cell fitness. 18 Note that in our cohort, synonymous mutations were associated in all cases with a BRAF V600E mutation. When using ddPCR, alternative BRAF mutations could be misdetected due to impaired hybridization and the specificity of the test, which is not able to screen for non-V600 additional BRAF mutations. The respective role of these mutations in the occurrence of HCL or its progression remains to be clarified. -review and editing (equal). Xavier Troussard: Conceptualization (equal); data curation (equal); formal analysis (supporting); investigation (supporting); methodology (equal); project administration (lead); resources (equal); supervision (lead); visualization (supporting); writing -original draft (lead); writing -review and editing (equal).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.