Variant Form Of Hairy Cell Leukemia Research Articles

New Generation Sequencing of Targeted Genes in Hairy Cell Leukemia Highlighted Mutations Associated to BRAFV600E anda New Recurrent Altered Gene, KDM6A, in Hairy Cell Leukemia Variant Patients

Introduction: BRAFV600E detected in more than 80% of Hairy cell leukemia (HCL) cases, was described as a driver mutation but additional genetic abnormalities seem necessary for the progression of the disease. Within HCL harboring BRAFWT, the differential diagnosis with the variant form of HCL (HCL-v) is complex.Sequencing of targeted genes by next generation sequencing (NGS) has proved its interest for classification and prognosis of lymphoid neoplasms and opens the lead to new perspectives for personalized medicine.Methods : We selected a panel of 21 relevant genes (defined as Trichopanel) based on literature review of whole exome sequencing studies. At diagnosis, we analyzed 20 HCL and 4 HCL-v samples and at relapse 2 HCL and 3 HCL-v. HCL diagnosis was based on hairy cell morphology with the co-expression of CD103, CD123, CD25 and CD11c, and HCL-v on hairy cell morphology with a prominent nucleolus and the lack of CD25 and CD123 expression. DNA was extracted from peripheral blood mononuclear cells isolated from blood (17 samples), bone marrow aspirates (10 samples) or biopsies (spleen: 1 and cutaneous nodule: 1 case). NGS was performed on the Ion Torrent PGMTM device according to the manufacturer' recommendations. The Trichopanel covered 71,020 bases using 712 amplicons. Analyzed genes belong to different gene ontology families: MAPK pathway (BRAF, MAP2K1, DUSP2, MAPK15), epigenetic regulation (ARID1A, ARID1B, EZH2, KDM6A, CREBBP), cell cycle/apoptosis (TP53, CDKN1B, XPO1), homing (KLF2, CXCR4), NOTCH pathway (NOTH1, NOTCH2), NF-kB pathway (MYD88), inflammation (ANXA1), splicing (U2AF1), differentiation (BCOR) and extracellular transport (ABCA8). Data analysis was performed with the Torrent suiteTM software, then variant analysis was performed using an in-house bioinformatics pipeline detailed as previously described1. The ratio of variant allele frequency (rVAF) was pondered according to the % of tumor cells estimated by Flow Cytometry.Results : The Trichopanel was informative for 96% (23/24) of patients and revealed single nucleotide variants (SNVs) in BRAF (18 pts), KLF2 (4 pts), MAP2K1 (3 pts), KDM6A (2 pts), CDKN1B (2 pts), ARID1A (2 pts), CREBBP (2 pts) NOTCH1 (1 pts) and ARID1B (1 pts). BRAFV600E was found in 90% (18/20) of HCL patients and was not detected in HCL-v patients. In one BRAFWT HCL patient, we found a mutation on MAP2K1. In the last case, we found no SNV in the genes sequenced by the Trichopanel. In HCL BRAFV600E patients, other mutations were found in 39% (7/18) of cases: KLF2 (3 pts), CDKN1B (2 pts), NOTCH1 (1 pt), ARID1B (1 pt) and CREBBP (1 pt). All 4 HCL-v patients had SNVs in epigenetic regulation genes e.g.: KDM6A (2 pts), CREBBP (1 pt) or ARID1A (1 pt). KDM6A SNVs were either frameshift deletions or splicing variations with a loss of exon (confirmed by RNA sequencing). We also analyzed at diagnosis and relapse serial samples from 5 patients (2 HCL and 3 HCL-v). In one HCL, patient we observed at diagnosis and at first relapse the same mutational pattern and in the second case, we observed the presence of 2 news sub-clonal mutations (BCORE1430X and XPO1E571K). In the 3 HCL-v patients (2 treated and 1 untreated), no new mutation was observed but variations of the rVAF were identified in 2 cases.Discussion : In addition to BRAFV600E mutations, one third of HCL patients had mutations with a rVAF close to BRAFV600E rVAF. KLF2, which is mutated in 3 patients, is known to play a key role for B-cell homing in lymph nodes and inhibition of NF-kB pathway. KLF2275N, KLF2275T, KFL2T271I variants found in those patients have been previously described in splenic zone marginal lymphoma (SMZL) as inhibitory mutations, possibly suggesting a genetic similarity between both diseases2. We also describe new mutations targeting KDM6A, a lysine demethylase protein, in HCL-v disease. Stop-gain KDM6A mutations have been already described in 2 HCL patients with atypical clinical features3,4. Those mutations and the 2 mutations found in our series leads to the loss of the highly-conserved C-terminal region (included Jumomji and Zinc binding domains) which is essential for its demethylase activity. Loss of KDM6A activity may sensibilise tumor cells to demethylating agents such as 5-azacytidine or EZH2 inhibitors5.Conclusion : the Trichopanel is a useful tool for the diagnosis, relapse and prognosis of HCL and HCL-v. [Display omitted] DisclosuresTroussard:ABBVIE: Honoraria; JANSSEN: Honoraria; GILEAD: Honoraria; ROCHE: Honoraria. Jardin:Roche: Honoraria; Janssen: Honoraria; celgen: Honoraria.

A Single-center Experience in Splenic Diffuse Red Pulp Lymphoma Diagnosis.

The World Health Organization 2008 classification highlighted a new nosology-splenic diffuse red pulp lymphoma (SDRPL) with clinical and laboratory features similar to both splenic marginal zone lymphoma and hairy cell leukemia (HCL) and variant form of HCL. Experience of hematologists on the diagnosis and differential diagnosis of SDRPL is extremely limited. The aim of our report was to characterize the clinical and immunomorphologic features of SDRPL on our own observations. During 2013-2014, in National Research Center for Hematology, 87 spleen specimens removed from various B-cell lymphomas were analyzed. In four (4.6%) cases, the diagnosis SDRPL was made based on morphologic, immunohistochemical, immunophenotypic, molecular examination of spleen biopsies, blood and bone marrow samples. In all cases of SDRPL were observed significant splenomegaly, lymphocytosis from 56% to 94% (in two cases with leukocytosis 55.000 and 75.000 109/l). The circulating "villous" lymphocytes phenotype was CD20+ (bright), CD11c+/±, CD103 (weakly)+/±, LAIR-1+, CD25-, CD5-, CD10-, and CD23-. Mutation BRAFV600E was not detected. Bone marrow with minor lymphoid CD20+, CD25-, Annexin1-, Cyclin D1- cell infiltration. The average weight of the spleen was 3900 g (1450-9500 g), and morphologically, there was revealed lymphoid infiltration of red pulp with phenotype CD20+, DBA.44+, CD25-, Annexin1-, Cyclin D1-, CD103-, CD123-, CD27-, focal SD11c± and TRAP±. Now patients are observed in remission: two patients after splenectomy, two after splenectomy and cladribine+rituximab chemotherapy. SRDPL-a rare lymphoma that is suspected in the cases with significant splenomegaly and lymphocytosis with villous lymphocytes forms that have only a part of the classic markers HCL, with minor bone marrow infiltration. The standard diagnosis and treatment is splenectomy. Differential diagnosis of SMZL and HCL has clear criteria, but criteria of differentiation with variant HCL are still unknown.

Chronic B-cell lymphoproliferative disorders with hairy cells

The standardized blood smear examination is the first step in the diagnosis of a B-cell chronic lymphoproliferative disorder and can guide further investigations. In the laboratory, the identification of hairy cells on blood smear is a matter of daily practice. Hairy cell proliferations represent heterogeneous entities and their respective diagnoses can be difficult. If hairy cell leukemia (HCL) and splenic marginal zone lymphoma (SMZL) represent separate entities, the variant form of HCL (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL) remain provisional entities in the 2008 WHO classification. We discuss the main clinical and biological characteristics of these four entities and appropriate means to characterize, identify and distinguish from each other; standardized blood smear examination, multiparameter flow cytometry analysis, analysis of the repertoire of immunoglobulins heavy chains genes and their mutational status (mutated or unmutated profile), molecular analyses: BRAF gene V600E mutation in HCL and MAP2K1 gene mutations in HCLv. We also discuss the main therapeutic aspects with emphasis on the new targeted drugs that enter into force in the therapeutic arsenal.

Recommendations of the SFH (French Society of Haematology) for the diagnosis, treatment and follow-up of hairy cell leukaemia.

Hairy cell leukaemia (HCL) is a rare haematological malignancy, with approximately 175 new incident cases in France. Diagnosis is based on a careful examination of the blood smear and immunophenotyping of the tumour cells, with a panel of four markers being used specifically to screen for hairy cells (CD11c, CD25, CD103 and CD123). In 2011, the V600E mutation of the BRAF gene in exon 15 was identified in HCL; being present in HCL, it is absent in the variant form of HCL (HCL-v) and in splenic red pulp lymphoma (SRPL), two entities related to HCL. The management of patients with HCL has changed in recent years. A poorer response to purine nucleoside analogues (PNAs) is observed in patients with more marked leukocytosis, bulky splenomegaly, an unmutated immunoglobulin variable heavy chain (IgVH) gene profile, use of VH4–34 or with TP53 mutations. We present the recommendations of a group of 11 experts belonging to a number of French hospitals. This group met in November 2013 to examine the criteria for managing patients with HCL. The ideas and proposals of the group are based on a critical analysis of the recommendations already published in the literature and on an analysis of the practices of clinical haematology departments with experience in managing these patients. The first-line treatment uses purine analogues: cladribine or pentostatin. The role of BRAF inhibitors, whether or not combined with MEK inhibitors, is discussed. The panel of French experts proposed recommendations to manage patients with HCL, which can be used in a daily practice.

Leucémie à tricholeucocytes

Summary Hairy cell leukemia (HCL) accounts for 2% of all haematologic cancers. The diagnosis is based on the identification of hairy cells in the peripheral blood, bone marrow and/orspleen. Hairy cells are B-cells, which express CD11c, CD25, CD103, DBA-44 and CD123 without expressing CD5. No specific genetic abnormality was described in HCL and it is necessary to distinguish HCL from all other B-cell chronic lymphoproliferative disorders, specially splenic lymphoma with villous lymphocytes (SLVL) and the variant form of HCL (HCL-V) because specific treatments are required. All the immunologic and molecular data suggest that hairy cell is a postgerminal center memory B-cell. Purine analogs, 2’-deoxycoformycin (DCF) or 2-chloro-deoxyadenosine are very active and used as first line treatment. DCF changed the natural history of HCL: however the risk of cytopenia, immunosuppression and second cancer must be evaluated carefully. Eradication of minimal residual disease (MRD) in HCL using monoclonal antibodies (rituximab) after therapy with nucleoside analogs is feasible.

Phase II study of cladribine (2CDA) followed by rituximab for eradication of minimal residual disease (MRD) in hairy cell leukemia (HCL)

6620 Background: HCL is an indolent lymphoproliferative malignancy characterized by infiltration of the bone marrow, liver, and spleen by neoplastic B-cells with cytoplasmic hair-like projections. Despite the success of the nucleoside analogs 2CDA and pentostatin in achieving long-term remissions in HCL, some patients (pts) continue to relapse and the relapse-free survival curve does not appear to achieve a plateau. Rituximab has been used effectively to treat pts with relapsed HCL. We investigated whether the prolonged administration of rituximab one month after therapy with 2CDA can effectively eradicate MRD in pts with newly diagnosed HCL or those who have relapsed after one prior therapy. Methods: 2CDA 5.6 mg/m2 is administered over 2 hours daily for 5 days. One month later, rituximab 375 mg/m2 is administered weekly for 8 weeks. MRD is assessed by flow cytometry as well as by IgH polymerase chain reaction (PCR) assay using framework-1, -2 and -3 primer sets. MRD is evaluated at one month following 2CDA and after completion of rituximab. Results: To date, ten pts have been enrolled onto the study. Eight pts had received no prior therapy, one pt had relapsed after prior 2CDA therapy 9 years ago and one pt had received chlorambucil only. Two pts had the variant form of HCL. Six pts have completed all therapy, 3 are receiving rituximab, and 1 pt has completed 2CDA only. So far, six (100%) of the evaluable pts have achieved a complete remission (CR) with no evidence of MRD by flow in 6 and by PCR in 3. PCR remained positive in 1 pt and was not evaluable in 2 pts. Eight of 9 pts with evaluable flow studies one month after completing 2CDA remained positive; 1 pt was negative after 2CDA only and 6 became negative after completing rituximab. Five of 6 pts with evaluable PCR studies one month after completing 2CDA remained positive; 1 pt was negative after 2CDA only and 3 became negative after rituximab. Conclusions: We conclude that assessment and eradication of MRD by flow and PCR in HCL is feasible. Longer follow-up is necessary to determine whether MRD status at completion of therapy can predict relapse. This may allow design of regimens that may further improve long-term disease-free survival in HCL. No significant financial relationships to disclose.

Remission in Hairy Cell Leukemia-Variant Following Splenic Radiotherapy Alone

The therapeutic approach to hairy cell leukemia (HCL) is in some instances still debated. A variant form of HCL (HCL-V) characterized by high white cell count, splenomegaly, hypercellular and aspirable bone marrow has been described; differential diagnosis often arises with some other B-cell disorders which also show circulating hairy or villous lymphocytes. Conventional treatment for HCL is often less effective in HCL-V. In this report we describe a case with features consistent with HCL-variant treated with splenic radiotherapy. We not only obtained an hematological response but also the near total disappearance of bone marrow infiltration, compatible with a clinical complete remission. Clinical and biological implications of this phenomenon are discussed on the basis of this unexpected therapeutic result, obtained with splenic radiotherapy alone.

Fludarabine therapy in hairy cell leukemia

This study evaluated the efficacy of fludarabine, a new adenine nucleoside analogue, in typical and variant forms of hairy cell leukemia (HCL). Two patients with HCL and one patient with a variant form of HCL (HCL-variant) with resistant or progressive disease with prior treatments were studied. Fludarabine (30 mg/m2) was administered intravenously over 30 minutes daily for 5 days every month. Two patients (one with HCL and one with HCL-variant) achieved partial responses; the third patient had a minor response. This is the first report of encouraging activity of fludarabine in typical and variant forms of HCL. Further experience with fludarabine in these disorders is indicated.

Treatment of Hairy Cell Leukaemia with 2'-Deoxycoformycin.

The adenosine deaminase inhibitor 2'-deoxycoformycin (DCF) has been used to treat 40 patients with hairy cell leukaemia (HCL) who have been followed up for a minimum of 6 months. 21 patients had previously undergone splenectomy. 33 had received treatment with alpha-interferon (IFN), half of whom had relapsed and the remainder had either been partially treated due to intolerance or had an incomplete response. Deoxycoformycin was administered by slow intravenous injection at a dose of 4 mg/m(2) weekly for 4 consecutive weeks and then 2-weekly for 4 doses followed by 4-weekly injections until a complete remission was achieved. No maintenance therapy was given. The overall response rate was 97yi, with 82% complete remission (CR) and 15% partial remission (PR). CR have lasted from 3+ to 30+ months (median 12 +) with no relapses recorded so far. Only one evaluable patient, suffering from a variant form of HCL, failed to respond to DCF, whilst two other HCL-variant cases who had been resistant to alpha-IFN have achieved a PR and are still on treatment. DCF was generally well tolerated, the major toxicity being related to cytopenia and associated infection. Four patients developed severe infections, one of whom died of septicaemia before it was possible to evaluate the response to DCF. A group of 12 patients who had received alpha-IFN immediately prior to treatment with DCF and had obtained clinical and haematological benefit had fewer infections with DCF than the group who had either not received IFN immediately before or who had failed to respond to it. In view of the infections associated with DCF, we would now recommend initial therapy for HCL patients with alpha-IFN for 2-4 months to obtain clinical and haematological improvement, followed by DCF given 2-weekly, to eradicate residual disease. This approach may achieve a higher proportion of sustained CR with a short treatment time and minimal toxicity.