BRAF mutations in hairy-cell leukemia

Abstract

Background: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure.

Methods: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL.

Results: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Figures

Figure 1. Bioinformatics pipeline for the identification of somatic mutations

After aligning the Illumina reads from the HCL and the normal cell DNA to the human reference genome a series of filters were applied to discard reads not usable for the downstream purpose of somatic mutation discovery. Sequence variants fulfilling the further 5 criteria indicated in the bottom rectangle were subjected to Sanger sequencing validation. See Supplementary Materials for details.

Figure 2. BRAF V600E mutation in HCL but not in other peripheral B-cell neoplasms

Top panels. Flow cytometry analysis of a HCL patient shows peripheral blood mononuclear cells partly with high forward and side scatter features (black events in the dot plot on the far left), expressing CD19 together with CD11c and with CD103 (red events in the middle and right dot plot, respectively). Direct DNA Sanger sequencing of purified leukemic cells reveals a heterozygous T->A mutation (far right; arrow) Upper middle panels. Another HCL case analyzed in paraffin sections from a bone marrow biopsy (x 400). Diffuse marrow infiltration by HCL cells (far left; hematoxylin & eosin staining) that display positivity for CD20 (stain in the middle) and for ANXA1 (stain on the right). Purified HCL cells harbor a homozygous/hemizygous T->A mutation (far right; arrow). Lower middle panels. Splenic Lymphoma/Leukemia Unclassifiable (paraffin sections from a bone marrow biopsy; x 400). Marked marrow infiltration by leukemic cells (far left; hematoxylin & eosin staining), that express CD20 (stain in the middle) but not ANXA1 (stain on the right, where positive myeloid cells act as internal control). Purified leukemic cells do not carry the T->A mutation (far right; arrow). Bottom panels. Splenic marginal zone lymphoma. Leukemic cells in the peripheral blood smear show the typical morphological features with polar villi (far left; May-Grünwald-Giemsa staining, x 630). At flow cytometry, leukemic cells express CD19 but not CD103 (red events in the left dot plot). These cells only weakly express CD11c and are negative for CD25 (red events in the right dot plot). Leukemic cells do not carry the T->A mutation (far right; arrow).

Figure 3. Expression of phospho-MEK and phospho-ERK by HCL cells

Top panels: Double immunofluorescence staining for CD20 (green) and phospho-ERK (red) in paraffin sections from a bone marrow trephine. The panels show the expression of phospho-ERK in CD20-positive leukemic hairy cells at lower (left) and higher magnification (middle). The inset shows a 3D reconstruction of confocal slices of a representative cell with the “isosurface” tecnique. The right panel shows that staining for phospho-ERK is completely blocked by pre-incubation of the antibody with the specific phospho-ERK peptide. Bottom panels: Western blot analysis on purified HCL cells from 3 representative patients showing phosphorylation of both MEK and ERK kinases under basal conditions (vehicle treatment) and their dose-dependent dephosphorylation after 2, 6 and 24 h incubation with the specific active BRAF inhibitor PLX-4720 at 250 nM, 500 nM or 1000 nM concentrations. Membranes were probed with antibodies against phospho-ERK, phospho-MEK, total ERK and total MEK, as indicated on the far right. Protein lysates from either serum-starved or unstarved human leukemic Jurkat T cell line cells were used as negative (−) and positive (+) control, respectively, for ERK and MEK phosphorylation. Numbers on the left indicate molecular weight markers (in kDa).