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Gregorio Barila - One of the best experts on this subject based on the ideXlab platform.

  • stat3 mutations impact on overall survival in large granular lymphocyte leukemia a single center experience of 205 patients
    Leukemia, 2020
    Co-Authors: Gregorio Barila, Vanessa Rebecca Gasparini, Giulia Calabretto, Antonella Teramo, Cristina Vicenzetto, Laura Pavan, Matteo Leoncin, Susanna Vedovato, Anna Chiara Frigo, Monica Facco
    Abstract:

    Large granular lymphocyte leukemia (LGLL) is a rare and Chronic Lymphoproliferative Disorder characterized by the clonal expansion of LGLs. LGLL patients can be asymptomatic or develop cytopenia, mostly neutropenia. Somatic STAT3 and STAT5b mutations have been recently reported in approximately 40% of patients. The aim of this study is to analyze clinical and biological features of a large cohort of LGLL patients to identify prognostic markers affecting patients’ outcome. In 205 LGLL patients, neutropenia (ANC < 1500/mm3) was the main feature (38%), with severe neutropenia (ANC < 500/mm3) being present in 20.5% of patients. STAT3 mutations were detected in 28.3% patients and were associated with ANC < 500/mm3 (p < 0.0001), Hb < 90 g/L (p = 0.0079) and treatment requirement (p < 0.0001) while STAT5b mutations were found in 15/152 asymptomatic patients. By age-adjusted univariate analysis, ANC < 500/mm3 (p = 0.013), Hb < 90 g/L (p < 0.0001), treatment requirement (p = 0.001) and STAT3 mutated status (p = 0.011) were associated to reduced overall survival (OS). By multivariate analysis, STAT3 mutated status (p = 0.0089) and Hb < 90 g/L (p = 0.0011) were independently associated to reduced OS. In conclusion, we identified clinical and biological features associated to reduced OS in LGLL and we demonstrated the adverse impact of STAT3 mutations in patients’ survival, suggesting that this biological feature should be regarded as a potential target of therapy.

  • t cell large granular lymphocyte leukemia and Chronic nk lymphocytosis
    Best Practice & Research Clinical Haematology, 2019
    Co-Authors: Gregorio Barila, Vanessa Rebecca Gasparini, Giulia Calabretto, Antonella Teramo, Cristina Vicenzetto, Gianpietro Semenzato, Renato Zambello
    Abstract:

    Abstract Large Granular Lymphocyte Leukemia (LGLL) is a rare Chronic Lymphoproliferative Disorder characterized by the clonal expansion of Large Granular Lymphocytes (LGLs). Among LGLL, the 2016 WHO classification recognizes two different entities, i.e. T-LGLL and the provisional entity Chronic Lymphoproliferative Disorder of NK cells (CLPD-NK). In both subtypes neutropenia represents the hallmark of the disease and is frequently regarded as the leading reason to start treatment. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways, the most relevant being the JAK-STAT axis, whose constitutive activation is partly explained by somatic mutations in STAT3 and STAT5b. In addiction, in the last few years, a relationship between STAT3 mutations/activation and the development of neutropenia was found. Given that backbone treatment relying on immunosuppressive agents is generally unsatisfactory, novel agents targeting the JAK/STAT pathway can represent a turning point in LGLL treatment.

Raajit K Rampal - One of the best experts on this subject based on the ideXlab platform.

  • hematopoietic stem cell origin of brafv600e mutations in hairy cell leukemia
    Science Translational Medicine, 2014
    Co-Authors: Stephen S Chung, Jae H Park, Eunhee Kim, Young Rock Chung, Piro Lito, Julie Teruyafeldstein, Wendy Beguelin, Sebastien Monette, Cihangir Duy, Raajit K Rampal
    Abstract:

    Hairy cell leukemia (HCL) is a Chronic Lymphoproliferative Disorder characterized by somatic BRAFV600E mutations. The malignant cell in HCL has immunophenotypic features of a mature B cell, but no normal counterpart along the continuum of developing B lymphocytes has been delineated as the cell of origin. We find that the BRAFV600E mutation is present in hematopoietic stem cells (HSCs) in HCL patients, and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies. Quantitative sequencing analysis revealed a mean BRAFV600E-mutant allele frequency of 4.97% in HSCs from HCL patients. Moreover, transplantation of BRAFV600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAFV600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. Consistent with the human genetic data, expression of BRafV600E in murine HSPCs resulted in a lethal hematopoietic Disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B lineage cells—all classic features of human HCL. In contrast, restricting expression of BRafV600E to the mature B cell compartment did not result in disease. Treatment of HCL patients with vemurafenib, an inhibitor of mutated BRAF, resulted in normalization of HSPC frequencies and increased myeloid and erythroid output from HSPCs. These findings link the pathogenesis of HCL to somatic mutations that arise in HSPCs and further suggest that Chronic lymphoid malignancies may be initiated by aberrant HSCs.

  • brafv600e mutations occur in the hematopoietic stem cell compartment in hairy cell leukemia
    Blood, 2013
    Co-Authors: Jae H Park, Eunhee Kim, Young Rock Chung, Piro Lito, Julie Teruya Feldstein, Raajit K Rampal, Leon Telis, Minal Patel, Neal Rosen, Christopher Y Park
    Abstract:

    Hairy cell leukemia (HCL) is a Chronic Lymphoproliferative Disorder recently found to be characterized by somatic BRAF V600E mutations. The malignant cell in HCL exhibits features consistent with a mature B-lymphocyte, including cell-surface expression of the pan-B-cell marker CD19 and monotypic surface immunoglobulins with clonal rearrangements of immunoglobulin heavy and light chains. Despite possessing these stereotypic features, the cell of origin of HCL has been long debated, and no cell type along the continuum of developing B-lymphocytes has been definitively identified as the normal counterpart of HCL cells. We hypothesized that HCL may originate from immature hematopoietic cells, and therefore investigated the hematopoietic-stem/progenitor cell (HSPC) compartment in HCL patients. We found that HCL patients exhibited a significantly increased frequency of immunophenotypically defined long-term hematopoietic stem cells (LT-HSCs; lineage-negative (Lin-neg) CD34+CD38-CD90+CD45RA- cells), pro-B cells (Lin-neg CD10+ cells), and CD34-CD38+ CD10+CD19+ hematogones, as well as a decreased frequency of granulocyte-macrophage progenitor cells (Lin-neg CD34+CD38+CD45RA+CD123+) relative to age-matched normal controls. Sequencing of cDNA from highly pure FACS-sorted cell populations from the bone marrow of HCL patients revealed the presence of the BRAF V600E allele in LT-HSCs and in pro-B cells ( [Figure][1] ). Transplantation of LT-HSCs from the pretreatment bone marrow of HCL patients into NOD/SCID/IL2r-gnull mice resulted in stable human grafts characterized by an expanded B-progenitor population and development of a clonal population of hCD19+hCD103+hCD25+ B cells characteristic of HCL 6 months after transplantation. Together, these data suggest that HCL arises from HSCs that then differentiate into committed B-cells which ultimately give rise to the characteristic clonal B-cell proliferation of HCL. ![Figure][2] Given the human HSC genetic and functional cell data, we conditionally expressed BRaf V600E from its endogenous locus at different stages of hematopoiesis, including in HSPCs and committed B cells. Mice with conditional expression of BRaf V600E in Mx1 Cre+ BRaf V600E knock-in mice died of a lethal hematopoietic malignancy characterized by features of human HCL including splenomegaly, anemia, thrombocytopenia, increased circulating sCD25, and increased clonogenic capacity of B-lineage cells (evidenced by infinite serial replating in the presence of IL-7) ( [Figure][1] ). This Disorder was transplantable into lethally-irradiated recipient mice. In contrast, mice with expression of BRaf V600E restricted to the B-cell lineage with Cd19 Cre manifested no overt malignant phenotype up to one year of age. Stimulation of these mice with alloantigen through injections of sheep red blood cells resulted in germinal center B-cell hyperplasia, but still did not result in development of a clonal B-cell proliferation. Recent case reports have noted that refractory HCL patients respond to mutant BRAF inhibition with vemurafenib. We investigated the effect of vemurafenib on HSPCs and hematopoiesis in patients treated on a phase II study of the mutant BRAF inhibitor vemurafenib for relapsed/refractory HCL as well as in our in vivo murine models. Flow cytometric analysis of bone marrow cells from vemurafenib treated HCL patients revealed normalization of HSPC frequencies within three months of starting therapy, concomitant with an improvement in peripheral blood counts. Consistent with this, evaluation of the in vitro clonogenic capacity of sorted LT-HSC's from the bone marrow of HCL patients revealed a significant increase in myeloid/erythroid colony formation in HCL patients treated for 3 months with vemurafenib compared to their pretreatment marrows. Likewise, treatment of wildtype mice transplanted with Mx1 Cre+ BRaf V600E mutant bone marrow cells revealed improvement in anemia and hepatosplenomegaly with in vivo therapy. Overall, these findings link the pathogenesis of HCL to a specific somatic genetic abnormality present in HSCs and provide evidence that mature B-cell malignancies can initiate in the HSC compartment. Moreover, these data suggest that the use of therapies targeting MAP kinase signaling in HCL may lead to durable remissions not only by eliminating the mature leukemic cells but also through targeted inhibition of signaling and survival in HCL initiating cells. Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes

Thomas P Loughran - One of the best experts on this subject based on the ideXlab platform.

  • fty720 fingolimod targets the sphingolipid pathway and induces autophagy related apoptosis in human natural killer large granular lymphocyte leukemia
    Blood, 2015
    Co-Authors: Francis R Leblanc, David J Feith, Hong-gang Wang, Thomas P Loughran
    Abstract:

    Introduction: Natural killer large granular lymphocytic leukemia (NK-LGL) and Chronic Lymphoproliferative Disorder of NK cells (CLPD-NK) are rare Disorders of cytotoxic CD3-/CD56+ natural killer cells. Aggressive NK-LGL leukemia patients present with a malignant clinical course and a fatal outcome with median survival time of two months from diagnosis. Aggressive NK-LGL is refractory to conventional chemotherapy and pathogenetic mechanisms remain undefined. The 'sphingolipid rheostat' has been identified as a key player in determining cell fate. The balance between pro-apoptotic ceramide and pro-survival sphingosine-1-phosphate (S1P) in this rheostat is determined by the expression of acid ceramidase (AC) which converts ceramide to sphingosine, and sphingosine kinase-1 and -2 (SPHK1 and SPHK2), which convert sphingosine to S1P. The S1P pathway is involved in cancer pathogenesis, cell survival, and resistance to chemotherapy and radiation, therefore it is considered a potential target for anti-cancer therapy. S1P signals both extracellularly through a family of five related GPCRs; the S1P receptors (S1PR1-5) and intracellularly through various incompletely defined mechanisms. FTY720 is a sphingolipid modulator that targets the S1P system and has potent immunosuppressive and anti-cancer properties. We have previously shown that FTY720 shows therapeutic efficacy in a rat model of NK-LGL leukemia, but its mechanism-of-action in inducing cell death is incompletely defined. The aim of this study is to explore these molecular mechanisms and further characterize the actions of FTY720 in NK-LGL leukemia. Results: We identified that the key enzymes in S1P biosynthesis, SPHK1 and AC, and the S1P receptor S1PR5 were all expressed in LGL cell lines and primary PBMCs from NK-LGL patients. Increased mRNA levels of AC, SPHK1 and S1PR5 were detected in NK-LGL samples (n=8) compared to PBMCs from healthy donors (n=8) (p < 0.05). These results were verified by immunoblot in NK-LGL samples (n=6) versus PBMCs from healthy donors (n=3) (p < 0.05). In vitro treatment of human and rat LGL cell lines (NKL, RNK16 and TL-1) resulted in time- and dose-dependent decreases in cell proliferation and increased apoptosis (IC50 5.0 - 12.5 μM). Interestingly, treatment of the LGL cell lines with the phosphorylated form of FTY720, FTY720-P, which acts antagonistically at the S1PRs, resulted in minimal effects on proliferation and apoptosis. This suggests that FTY720-induced cell death is independent of its effects on S1PRs. However, treatment of LGL cell lines with FTY720 did target multiple sphingolipid pathway members. We observed dose-dependent decreases in AC and S1PR5 protein expression. Interestingly, the combination of FTY720 with LCL204, an AC inhibitor, resulted in additive effects on cell proliferation and apoptosis in the NKL cells. Further characterization of cell death mechanisms demonstrated involvement of autophagy-related apoptosis. We observed increased PARP and caspase-3 cleavage after FTY720 treatment, which occurred after increased expression of LC3A/B and the conversion of LC3-I to LC3-II, suggesting autophagy is being induced prior to apoptosis induction. We identified the mTOR pathway as a target of FTY720. The mTOR pathway negatively regulates autophagy and its inhibition can induce autophagy. FTY720 treatment suppressed mTOR signaling in LGL cell lines as demonstrated by decreased phosphorylation of mTOR, p70S6K and S6 Kinase. These effects preceded the autophagy-related changes in LC3A/B. Furthermore, PARP and caspase-3 cleavage could be blocked by pre-treatment with 3-methyladenine and BafilomycinA1, both inhibitors of autophagy. Interestingly, the effects of FTY720 in LGL cell lines could also be blocked by pre-treatment with autophagy inhibitors, further suggesting that autophagy is required for the FTY720-induced effects. Importantly, these key findings were also reproduced in PBMCs from NK-LGL patients (n=3). Conclusions : Taken together, our findings demonstrate that FTY720 targets multiple sphingolipid pathway members in NK-LGL leukemia and that FTY720-induced cell death is autophagy-dependent. The down-stream targeting of mTOR by FTY720, combined with mTOR's prominent role in inhibiting autophagy, suggests that co-treatment with specific mTOR inhibitors in combination with FTY720 may be a promising and novel therapeutic strategy in LGL leukemia. Disclosures No relevant conflicts of interest to declare.

  • pathogenesis of neutropenia in large granular lymphocyte leukemia and felty syndrome
    Blood Reviews, 2006
    Co-Authors: Eric Burks, Thomas P Loughran
    Abstract:

    T-cell large granular lymphocyte leukemia (TLGL) is an atypical Chronic Lymphoproliferative Disorder derived from cytotoxic T-cells (CTL). Unlike most forms of leukemia, the pattern of bone marrow infiltration in TLGL may be subtle and the cytopenias are often lineage specific, with neutropenia dominating. Both granulocytic survival and proliferation defects are observed and are mediated by humoral and cell-mediated mechanisms respectively. Splenic production of immune complexes induces a neutrophil survival defect, where as Fas expression by leukemic CTL results in a marrow based proliferation defect. These humoral and cell-mediated pathways induce granulocytic apoptosis through independent intracellular mechanisms which are not mutually exclusive and may be observed concurrently in individual patients with either TLGL or FS. A variety of therapeutic interventions have been utilized in the management of TLGL and Felty syndrome, including methotrexate, cyclosporine A, cyclophosphamide, glucocorticoids, myeloid colony stimulating factors and splenectomy. Their efficacy and mechanisms of action are reviewed.

Luigi Petrucci - One of the best experts on this subject based on the ideXlab platform.

Renato Zambello - One of the best experts on this subject based on the ideXlab platform.

  • t cell large granular lymphocyte leukemia and Chronic nk lymphocytosis
    Best Practice & Research Clinical Haematology, 2019
    Co-Authors: Gregorio Barila, Vanessa Rebecca Gasparini, Giulia Calabretto, Antonella Teramo, Cristina Vicenzetto, Gianpietro Semenzato, Renato Zambello
    Abstract:

    Abstract Large Granular Lymphocyte Leukemia (LGLL) is a rare Chronic Lymphoproliferative Disorder characterized by the clonal expansion of Large Granular Lymphocytes (LGLs). Among LGLL, the 2016 WHO classification recognizes two different entities, i.e. T-LGLL and the provisional entity Chronic Lymphoproliferative Disorder of NK cells (CLPD-NK). In both subtypes neutropenia represents the hallmark of the disease and is frequently regarded as the leading reason to start treatment. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways, the most relevant being the JAK-STAT axis, whose constitutive activation is partly explained by somatic mutations in STAT3 and STAT5b. In addiction, in the last few years, a relationship between STAT3 mutations/activation and the development of neutropenia was found. Given that backbone treatment relying on immunosuppressive agents is generally unsatisfactory, novel agents targeting the JAK/STAT pathway can represent a turning point in LGLL treatment.