BRAF(V600E) mutation together with loss of Trp53 or pTEN drives the origination of hairy cell leukemia from B-lymphocytes.

Hairy cell leukemia (HCL) is a B-lymphoma induced by BRAF(V600E) mutation. However, introducing BRAF(V600E) in B-lymphocytes fails to induce hematological malignancy, suggesting that BRAF(V600E) needs concurrent mutations to drive HCL ontogeny. To resolve this issue, here we surveyed human HCL genomic sequencing data. Together with previous reports, we speculated that the tumor suppressor TP53, P27, or PTEN restrict the oncogenicity of BRAF(V600E) in B-lymphocytes, and therefore that their loss-of-function facilitates BRAF(V600E)-driven HCL ontogeny. Using genetically modified mouse models, we demonstrate that indeed BRAF(V600E)KI together with Trp53KO or pTENKO in B-lymphocytes induces chronic lymphoma with pathological features of human HCL. To further understand the cellular programs essential for HCL ontogeny, we profiled the gene expression of leukemic cells isolated from BRAF(V600E)KI and Trp53KO or pTENKO mice, and found that they had similar but different gene expression signatures that resemble that of M2 or M1 macrophages. In addition, we examined the expression signature of transcription factors/regulators required for germinal center reaction and memory B cell versus plasma cell differentiation in these leukemic cells and found that most transcription factors/regulators essential for these programs were severely inhibited, illustrating why hairy cells are arrested at a transitional stage between activated B cells and memory B cells. Together, our study has uncovered concurrent mutations required for HCL ontogeny, revealed the B cell origin of hairy cells and investigated the molecular basis underlying the unique pathological features of the disease, with important implications for HCL research and treatment.

Flow Cytometry of CD5-Positive Hairy Cell Leukemia.

BACKGROUND AND OBJECTIVE: Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder for which diagnosis is typically straightforward, based on bone marrow morphology and flow cytometry (FC) or immunohistochemistry. Nevertheless, variants present atypical expressions of cell surface markers, as is the case of CD5, for which the differential diagnosis can be more difficult. The aim of the current paper was to describe diagnosis of HCL with atypical CD5 expression, with an emphasis on FC. METHODS: The detailed diagnostic methodology for HCL with atypical CD5 expression is presented, including differential diagnosis from other lymphoproliferative diseases with similar pathologic features, by FC analysis of the bone marrow aspirate. RESULTS: Diagnosis of HCL by means of FC started by gating all events based on side scatter (SSC) versus CD45 and B lymphocytes were selected from the lymphocytes gate as CD45/CD19 positive. The gated cells were positive for CD25, CD11c, CD20, and CD103, while CD10 proved to be dim to negative. Moreover, cells positive for CD3, CD4, and CD8, the three pan-T markers, as well as CD19, showed a bright expression of CD5. The atypical CD5 expression is usually correlated with a negative prognosis and thus chemotherapy with cladribine should be initiated. CONCLUSION: HCL is an indolent chronic lymphoproliferative disorder and diagnosis is usually straightforward. However, atypical expression of CD5 renders its differential diagnosis more difficult, but FC is a useful tool that allows an optimal classification of the disease and allows initiation of timely satisfactory therapy.

Hairy cell leukemia: a specific 17-gene expression signature points to new targets for therapy.

BACKGROUND: Hairy cell leukemia (HCL) is a rare chronic B cell malignancy, characterized by infiltration of bone marrow, blood and spleen by typical "hairy cells" that bear the BRAFV600E mutation. However, in addition to the intrinsic activation of the MAP kinase pathway as a consequence of the BRAFV600E mutation, the potential participation of other signaling pathways to the pathophysiology of the disease remains unclear as the precise origin of the malignant hairy B cells. MATERIALS AND METHODS: Using mRNA gene expression profiling based on the Nanostring technology and the analysis of 290 genes with crucial roles in B cell lymphomas, we defined a 17 gene expression signature specific for HCL. RESULTS: Separate analysis of samples from classical and variant forms of hairy cell leukemia showed almost similar mRNA expression profiles apart from overexpression in vHCL of the immune checkpoints CD274 and PDCD1LG2 and underexpression of FAS. Our results point to a post-germinal memory B cell origin and in some samples to the activation of the non-canonical NF-κB pathway. CONCLUSIONS: This study provides a better understanding of the pathogenesis of HCL and describes new and potential targets for treatment approaches and guidance for studies in the molecular mechanisms of HCL.

Hairy Cell Leukemia-Japanese Variant: Report of a Patient and Literature Review.

Hairy cell leukemia-Japanese variant (HCL-jv) shares some features with, but differs in other features from, HCL variant. Recently, it has been pointed out that HCL-jv and splenic diffuse red pulp small B-cell lymphoma (SDRPL) possibly constitute the same disease. We report a patient with HCL-jv, in which the neoplastic cells in the resected spleen were positive for CD11c, CD103, tartrate-resistant acid phosphatase (by immunohistochemistry), and weakly positive for cyclin D3. They were negative for CD25, CD123, annexin A1, and BRAF V600E-derived protein. Meta-analysis of HCL-jv cases in the literature showed considerable variation in the expression of HCL-related molecules. Although the clinical features and pattern of splenic involvement of HCL-jv are similar to those of SDRPL, some cytomorphological and phenotypical differences can be pointed out. To confirm whether the weak expression of cyclin D3 in our case suggests a spectrum from HCL-jv to SDRPL or one of the characteristics of HCL-jv, further studies on a large number of cases are necessary.

A Review on Splenic Diffuse Red Pulp Small B-Cell Lymphoma.

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is a rare disease, representing <1% of all non-Hodgkin lymphomas (NHL). The most common clinical manifestations include splenomegaly, lymphocytosis, and hemocytopenia. A diagnosis of SDRPL can be challenging, as it shares multiple clinical and laboratory features with splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), and HCL variant (HCL-v). Obtaining splenic tissue remains the gold standard for diagnosis. In the cases where splenic tissue is not available, diagnosis can be established by a review of peripheral blood and bone marrow studies. SDRPL is characterized by a diffuse involvement of the splenic red pulp by monomorphous small-to-medium sized mature B lymphocytes effacing the white pulp. The characteristic immunophenotype is positive for CD20, DBA.44 (20 to 90%), and IgG, and typically negative for CD5, CD10, CD23, cyclin D1, CD43, annexin A1, CD11c, CD25, CD123, and CD138. The Ki-67 proliferative index is characteristically low. Cyclin D3 is expressed in the majority of SDRPL in contrast with other types of small B-cell lymphomas, thus facilitating the recognition of this disease. There is no standard treatment regimen for SDRPL. Initial treatment options include splenectomy, rituximab monotherapy, or a combination of both. Chemoimmunotherapy should be considered in patients with advanced disease at baseline or progression.

Dissection of the bone marrow microenvironment in hairy cell leukaemia identifies prognostic tumour and immune related biomarkers.

Hairy cell leukaemia (HCL) is a rare CD20+ B cell malignancy characterised by rare "hairy" B cells and extensive bone marrow (BM) infiltration. Frontline treatment with the purine analogue cladribine (CDA) results in a highly variable response duration. We hypothesised that analysis of the BM tumour microenvironment would identify prognostic biomarkers of response to CDA. HCL BM immunology pre and post CDA treatment and healthy controls were analysed using Digital Spatial Profiling to assess the expression of 57 proteins using an immunology panel. A bioinformatics pipeline was developed to accommodate the more complex experimental design of a spatially resolved study. Treatment with CDA was associated with the reduction in expression of HCL tumour markers (CD20, CD11c) and increased expression of myeloid markers (CD14, CD68, CD66b, ARG1). Expression of HLA-DR, STING, CTLA4, VISTA, OX40L were dysregulated pre- and post-CDA. Duration of response to treatment was associated with greater reduction in tumour burden and infiltration by CD8 T cells into the BM post-CDA. This is the first study to provide a high multiplex analysis of HCL BM microenvironment demonstrating significant immune dysregulation and identify biomarkers of response to CDA. With validation in future studies, prospective application of these biomarkers could allow early identification and increased monitoring in patients at increased relapse risk post CDA.

Hairy Cell Leukaemia: From Hairy Beginnings to a BRAF New World.

For a disease initially described in 1958 as a leukaemic reticulo-endotheliosis associated with poor outcomes, we have come a long way in our understanding of Hairy cell leukaemia. The vast majority of patients diagnosed with this rare, often diagnostically challenging, leukaemia can now expect a lifespan that is similar to the general population. This article covers some of the highlights from the last 6 decades that have led to our current understanding of this fascinating leukaemia - from elucidation of its B-cell origin to discovery of the almost universal occurrence of the BRAF V600E mutation; from the initial successes reported with splenectomy to the more recent development of targeted therapies such as Vemurafenib and Moxetumomab Pasudotox. It also pays tribute to some of the outstanding research in this field focusing particularly on the significant contributions made by the clinical and scientific community in the UK.

Usefulness of Dual Immunohistochemistry Staining in Detection of Hairy Cell Leukemia in Bone Marrow.

OBJECTIVES: We evaluated efficacy of two dual immunohistochemistry (IHC) staining assays in assessing hairy cell leukemia (HCL) involvement in core biopsies and compared the results with concurrently collected flow cytometric data. METHODS: Overall, 148 patients with HCL (123 male, 25 female; mean age: 59.8 years; range: 25-81 years) had multiparameter flow cytometry performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5, and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/tartrate-resistant alkaline phosphatase (TRAP) dual IHC stains. RESULTS: Overall sensitivity of dual IHC stains was 81.4%, positive predictive value was 100%, and negative predictive value was 81.7%. All IHC-positive cases concurred with flow cytometry data, even when HCL burden was extremely low in the flow cytometry specimens (as low as 0.02% of all lymphoid cells). CONCLUSIONS: Dual IHC stain is a sensitive tool in detecting HCL, even in cases with minimal disease involvement.

New treatment options in hairy cell leukemia with focus on BRAF inhibitors.

Hairy cell leukemia (HCL) responds initially very well to chemotherapy with purine analogues. However, up to 50% of patients relapse, often multiple times, and become progressively less sensitive to these myelotoxic and immune-suppressive drugs. At progression, viable therapeutic strategies include addition of rituximab to purine analogues, and treatment with the anti-CD22 immunotoxin moxetumomab pasudotox, which has been recently approved by the FDA in HCL patients after at least two prior therapies. Identification of the BRAF-V600E kinase mutation as the genetic cause of HCL has opened the way, in the relapsed/refractory experimental setting, to targeted and non-myelotoxic effective strategies that are based on inhibition of BRAF with vemurafenib, co-inhibition of BRAF and its target MEK with dabrafenib and trametinib, and BRAF inhibition with vemurafenib combined with anti-CD20 immunotherapy. In particular, vemurafenib plus rituximab is emerging as a short, safe, chemotherapy-free regimen able to induce deep complete remissions in most HCL patients refractory to, or relapsed multiple times, after chemo(immuno)therapy.

Moxetumomab pasudotox: A first-in-class treatment for hairy cell leukemia.

Hairy cell leukemia is a rare indolent B-cell lymphoid malignancy. Durable remission can be obtained with purine analogues, but relapse is inevitable, and effective treatment options may be limited. Moxetumomab pasudotox is a recombinant CD22-targeting immunotoxin that has recently been approved by the United States Food and Drug Administration for the treatment of relapsed or refractory hairy cell leukemia. Approval was based on a pivotal phase III study in this unique patient population. Rationale for use, clinical trial data, and current treatment recommendations are detailed. Common adverse effects are reviewed, and management strategies for select adverse effects are suggested. Implications for contemporary practitioners are also provided, as use of this novel agent is likely to increase as follow-up studies are reported.

BRAF in the cross-hairs.

INTRODUCTION: Hairy cell leukemia (HCL) is a rare, chronic B-cell lymphoproliferative disorder characterized by distinctive morphologic features and an indolent clinical course. The discovery of a recurrent activating mutation in BRAF (BRAF V600E) as a disease-defining genetic event in HCL has substantial diagnostic and therapeutic implications. Areas covered: Herein the authors review the role of BRAF V600E and RAF-MEK-ERK signaling in the pathogenesis of HCL, anecdotal clinical reports of BRAF inhibitor monotherapy in management of relapsed or refractory HCL, larger phase 2 trials investigating efficacy of BRAF inhibitor therapy for HCL, adverse effects commonly associated with BRAF inhibitor therapy, including cutaneous toxicity, and mechanisms of therapeutic resistance. Expert opinion: Ongoing and planned studies will help to optimize the use of BRAF inhibitor therapy for HCL by determining the efficacy of BRAF inhibition in combination with other antigen targeted or molecularly targeted therapies, and more broadly, to determine how hematologists can best utilize and sequence emerging diagnostic and therapeutic modalities in the care of patients with newly diagnosed and relapsed or refractory HCL.

Identification of the BRAF V600E mutation in Japanese patients with hairy cell leukemia and related diseases using a quenching probe method.

Hairy cell leukemia (HCL) is a rare B-cell lymphoid malignancy that is difficult to distinguish from other morphological variants. The frequency of HCL has not been determined accurately in Japan. Recent studies revealed that the BRAF V600E mutation is the causal genetic event in HCL. We assessed the BRAF mutation in Japanese patients with HCL and related diseases using the quenching probe (QP) method, a single-nucleotide polymorphism detection system, and evaluated the incidence rate of HCL among Japanese patients with chronic lymphocytic leukemia, and related diseases. We identified 18 cases (33.3%) harboring the BRAF mutation among 54 patients diagnosed with, or suspected of having HCL. Of BRAF V600E-positive patients, 7 were only detected using the QP method, not by direct sequencing, whereas 11 were positive using both tests. In a larger cohort of Japanese patients diagnosed with chronic lymphoid leukemia or related diseases, the frequency of HCL was 4%. Patients with the BRAF V600E mutation had a significantly higher frequency of neutropenia, thrombocytopenia, and elevated soluble interleukin-2 receptor and common B-cell surface markers than patients without the mutation. Our results confirm that BRAF V600E-positive HCL is a relatively rare disorder in the Japanese leukemia patient population.

Immunohistochemical Detection of Hairy Cell Leukemia in Paraffin Sections: The Role of Pax5 and CD103 Double Staining to Improve Specificity and Sensitivity.

CONTEXT: - In hematopathology practice, abnormal expression of CD103 on B cells is detected by flow cytometry in hairy cell leukemia (HCL) and, in combination with other phenotypic and morphologic findings, provides diagnostic specificity and sensitivity. Immunostaining on paraffin sections makes it possible to perform immunophenotyping in situ. However, normal bone marrow contains CD103-positive cells, which are not B cells, making it difficult to be certain about low-level involvement by HCL. OBJECTIVE: - To develop dual immunostaining for confirmation that CD103 is expressed in B cells, which may be highly desirable in assessing low-level involvement by HCL. DESIGN: - We developed a dual immunostaining approach using a B-cell marker, Pax5, expressed in the nucleus, in combination with a membrane marker, CD103. RESULTS: - We analyzed 6 HCLs, 7 marginal zone lymphomas, 12 lymphoplasmacytic lymphomas, 7 follicular lymphomas, 5 chronic lymphocytic leukemias, 5 mantle cell lymphomas, 1 multiple myeloma (lymphocytic variant), and 3 bone marrows not involved by any B-cell neoplasm. Our dual staining approach confirmed that only the neoplastic cells of HCL were positive for both CD103 and Pax5. CONCLUSIONS: - This dual-staining method is particularly helpful in cases with low-level involvement by HCL and can be used for determining minimal residual disease after treatment.

CD27-positive hairy cell leukemia-Japanese variant.

We report a very rare case of a 45-year-old Japanese male patient with hairy cell leukemia-Japanese variant (HCL-JV) expressing CD27. The patient showed a high number of abnormal peripheral lymphocytes, thrombocytopenia, and severe splenomegaly but no lymphadenopathy. Histology of the resected spleen showed small-sized lymphoma cells diffusely infiltrating the red pulp without follicle formation. By immunohistochemistry, lymphoma cells were negative for CD3, CD5, CD8, CD10, CD34, cyclin-D1, and annexin A1 but positive for CD20 and BCL2. BRAF V600E mutation was not observed. Bone marrow aspirate showed preserved normal hematopoietic cells with invasion of lymphoma cells in an interstitial pattern without obvious nodules. The cells had abundant pale cytoplasm and round nuclei with inconspicuous nucleoli. After natural drying, the cells had unevenly distributed microvilli. Flow cytometric analysis demonstrated positivity for CD11a, CD11c, CD19, CD20, CD22, CD27, surface IgG, and λ but not for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD21, CD23, CD25, CD30, CD34, CD38, CD43, CD56, CD57, CD103, IgD, IgM, and κ. Monoclonal expansion of B cells was confirmed by an immunoglobulin heavy chain (IgH) rearrangement band as demonstrated by Southern blot hybridization. The lymphoma cells had unevenly distributed long, large, and broad-based microvilli, which resembled splenic diffuse red pulp small B cell lymphoma (SDRPL) cells. CD27 expression is extremely rare in HCL-JV, but the young age of the patient and high peripheral WBC counts were similar to HCL-JV, which suggests, in this case, an intermediate disease between SDRPL and HCL-JV.

Potential role of AKT/mTOR signalling proteins in hairy cell leukaemia: association with BRAF/ERK activation and clinical outcome.

The potential role of AKT/mTOR signalling proteins and its association with the Raf-MEK-ERK pathway was investigated in hairy cell leukaemia (HCL). BRAFV600E expression and activated forms of AKT, mTOR, ERK1/2, p70S6k and 4E-BP1 were immunohistochemically assessed in 77 BM biopsies of HCL patients and correlated with clinicopathological and BM microvascular characteristics, as well as with c-Caspase-3 levels in hairy cells. Additionally, we tested rapamycin treatment response of BONNA-12 wild-type cells or transfected with BRAFV600E. Most HCL cases expressed p-p70S6K and p-4E-BP1 but not p-mTOR, being accompanied by p-ERK1/2 and p-AKT. AKT/mTOR activation was evident in BONNA-12 cells irrespective of the presence of BRAFV600E mutation and was implicated in cell proliferation enhancement. In multivariate analysis p-AKT/p-mTOR/p-4E-BP1 overexpression was an adverse prognostic factor for time to next treatment conferring earlier relapse. When p-AKT, p-mTOR and p-4E-BP1 were examined separately only p-4E-BP1 remained significant. Our findings indicate that in HCL, critical proteins up- and downstream of mTOR are activated. Moreover, the strong associations with Raf-MEK-ERK signalling imply a possible biologic interaction between these pathways. Most importantly, expression of p-4E-BP1 alone or combined with p-AKT and p-mTOR is of prognostic value in patients with HCL.