Large Granular Lymphocyte Leukemia Research Articles

Overexpression and Targeted Activation of the Protein Phosphatases SHP-1 Abrogates Survival Pathways in Large Granular Lymphocyte Leukemia (LGLL)

Introduction: Aberrant phosphorylation-mediated signaling is a well-established condition involved in the onset and progression of virtually all types of cancer. That not only depends on the over-activation of protein kinases, but also on the lack of the proper counterbalance of protein phosphatases, because of their decreased expression or activity. In turn, this is likely to be due to genetic and epigenetic alterations, or interactions with cellular inhibitors and inhibitory post-translational modifications, respectively. In Large Granular Lymphocyte Leukemia (LGLL), a rare lymphoproliferative disorder characterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, CLPD-NK), constitutive activation of phosphorylation-regulated survival pathways, especially the JAK2/STAT3 axis, but also PI3K/Akt and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since such pathways are modulated by the tyrosine phosphatase SHP-1 under normal conditions, it is conceivable that putative alterations of this phosphatase, and consequent inability to disrupt hyperactive pathways, play a role in LGLL pathogenesis. In this study, we assessed the ability of small molecules inducing the expression and stimulating the activity of SHP-1 to abrogate the survival pathways in LGL by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were collected from untreated patients with LGLL (both T-LGLL and CLPD-NK). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were further separated from PBMCs by using immunomagnetic beads (Miltenyi Biotec). LGLs were incubated either with acetyl-11-keto-β-boswellic acid (AKBA), a pentacyclic triterpene capable of inducing SHP-1 expression, or SC-78, a regorafenib-like derivative specifically stimulating SHP-1 activity, or both at increasing concentrations and discrete time points. After such treatments, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the abundance of SHP-1 and the phosphatase activity thereof. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both AKBA and SC-78 proved effective at eliciting a significant level of apoptosis in the low micromolar range, exhibiting an additive effect when used in combination. Moreover, both compounds activated SHP-1 activity, as demonstrated by in-vitro phosphatase assays. As to the phosphorylation-dependent signals affected by SHP-1 activation, we observed a marked decrease in the phosphorylation status of STAT3, resulting in the downregulation of the STAT3-induced expression of downstream target genes (Mcl-1, survivin, Cyclin D and S1P5), as well as the dephosphorylation of Akt and p65, which confirmed the role of SHP-1 as a functional antagonist of survival pathways in LGLs. Conclusion: Taken together, our results support the newly emerging evidence that the targeted activation of SHP-1, which is already recognized as a tumor suppressor in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening new options for the treatment of LGLL. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

STAT3 Mutations and Anemia with Pure Red Cell Aplasia Are the Distinctive Features of T Cell Receptor Gamma Delta Type Large Granular Lymphocyte Leukemia

Large granular lymphocytic leukemia (LGLL) is a heterogenous group of disorders. The clinical presentations of LGLL are highly variable; it may present with neutropenia or anemia, complicated with recurrent infections, autoimmune diseases such as rheumatoid arthritis, and neurological symptoms, requiring therapeutic interventions. It may present only with indolent laboratory changes without any symptoms. The cytological phenotypes of LGLL also vary and they commonly include CD8+TCR αβ-type, CD4+TCR αβ-type, and NK cell-type. NK cell-type LGLL is designated as chronic lymphoproliferative disorder of NK cells (CLPD-NK). TCR γδ-type LGLL (γδT-LGLL) is even rarer and it often confers cytopenia. The genetic backgrounds of LGLL with CD8+TCRαβ-type, CD4+TCRαβ-type and NK cell-type have been elucidated with the recent sequencing technologies. JAK/STAT signaling genes and chromatin-modifying genes were frequently mutated in the non-leukemic counterpart of the lymphoid malignancies with γδ TCR immunophenotypes, such as hepatosplenic T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma and primary cutaneous gamma delta T-cell lymphoma. However, the genetic basis of γδT-LGLL and the genetic-phenotypic associations remain unclear. The aim of this study is to identify clinical and genetic characteristics of γδT-LGLL in comparison with other LGL leukemias. In total, 56 LGLL patients were recruited; six patients with γδT-LGLL, 25 CD8+ TCRαβ type, 8 CD4+TCR αβ type and 17 CLPD-NK, in this study. All cases were negative for EBV. The median age of patients with γδT-LGLL was 48 years old and five of them were male. The median LGL counts and hemoglobin level were 1.81 ×109/L (range; 0.60-4.42) and 5.8 g/dL (range; 3.0-11.2), respectively. All patients with γδT-LGLL had anemia and/or neutropenia, although no patients were thrombocytopenic. 4 patients presented with pure red cell aplasia. Common immunophenotypes of γδT cells were CD2+ CD3+ CD4-CD7+. 3 patients were CD8+. CD16, CD56 and CD57 were positive in 3, 0, and 1 patient, respectively. γδT cells of all patients showed a monoclonal pattern of TCRγ gene rearrangements with BioMed 2 protocol. We performed amplicon sequencing of entire coding regions of STAT3, STAT5B and TNFAIP3 genes and allele-specific PCR for several hot spot regions of STAT3 and STAT5B genes using extracted DNA from mononuclear cells of peripheral blood. Mutations in STAT3 and TNFAIP3 were identified in 100% (6/6) and 17% (1/6), respectively. Among the10 STAT3 mutations identified, 5 were located in the SH-2 domain, while other 3 were in coiled-coiled domain and the other 2 were in the DNA binding domain. The median variant allele frequencies of STAT3 was 2.0% (0.7-22.6) and STAT3 mutations were restricted to sorted γδT cells in all 3 patients examined. No patient was positive for STAT5B mutations, including N642H and Y665F with allele specific PCRs. One of 5 patients who were treated with cyclosporin A was refractory. Two patients were treated with cyclophosphamide, showing clinical improvements. The patients with γδT-LGLL were younger than those with other types of LGLL (P=0.048) and anemia and STAT3 mutations were significantly more frequent in γδT-LGLL (P value; 0.003 and 0.02, respectively). PRCA was a consistent cause of anemia in γδT-LGLL. Low frequency of STAT5B mutations among γδT-LGLL patients was in contrast to other aggressive γδT cell lymphoma in which STAT5B frequently mutated. The clinical characteristics of γδT-LGLL patients may be defined by younger age and the anemia due to pure red cell aplasia, and the frequent STAT3 mutations might be a novel molecular marker for γδT-LGLL. Disclosures Yamane: Pfizer: Research Funding; Celgene: Honoraria; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Astellas Pharma: Research Funding; Eli Lily and Company: Research Funding. Ishida:Eli Lily and Company: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Celgene: Honoraria.

CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Aberrant SYK Expression As a Potential Therapeutic Target in T/NK Cell Neoplasms

Background: During T cell development spleen tyrosine kinase (SYK) is highly expressed at pre-TCR signaling stages, and gradually becomes undetectable at the mature stage. We have previously reported aberrant expression of SYK in peripheral T-cell lymphoma (PTCL). Additionally, ITK-SYK fusion protein detected in a subset of PTCL, mimics a constitutively active TCR signal and drives oncogenesis in mouse models of PTCL. Multiple SYK inhibitors are currently under active investigation in clinical trials for patients with B cell lymphoma and acute myeloid leukemia, and show promising results. Multiparameter flow cytometry can quantitate protein levels at single cell resolution in immunophenotypically defined neoplastic populations. In this study, we employ multiparameter flow cytometry to quantitate the SYK expression in T and NK cell malignancies and to guide the clinical trial design and as potential tool to evaluate the therapeutic responses. Methods: Patients with T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), T-cell large granular lymphocyte leukemia (T-LGL), HTLV-1+ adult T-cell leukemia/lymphoma (ATLL), T-cell prolymphocytic leukemia (T-PLL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), mixed phenotype acute leukemia (MPAL) and natural killer cell large granular lymphocyte leukemia (NK-LGL) were included. Abnormal samples included the peripheral blood (46 cases), bone marrow aspirates (10 cases) or tissue (9 cases) from 65 patients diagnosed with T or NK cell malignancies, including AITL (8 cases), ATLL (5 cases), ALCL (2 cases), CTCL (25 cases), PTCL-NOS (5 cases), T-PLL (7 cases), T-LGL (7 cases), NK-LGL (1 case), MEITL (1 case), MAPL (3 cases) and T-ALL (1 case). Normal controls included peripheral blood from healthy donors (15 cases), or patient derived- peripheral blood (6 cases), bone marrow aspirates (13 cases) and lymph node (16 cases) with diagnostic normal immunophenotype. Results: SYK was highly expressed in all the B and most NK cells from peripheral blood, bone marrow and lymph node of normal controls and there was no difference in the percentage of positive cells or intensity of staining in different specimen types. In normal T cell subsets SYK was expressed in a small proportion of T cells and at very low level. Only 3.9% (95% CI: 3.3% - 6.3%) of the CD4+ T cells from peripheral blood, 2.6% (1.5% - 6.6%) from bone marrow, and 1.9% (1.4% - 5.8%) from lymph node were positive for SYK. Similar to CD4+ T cells, SYK was expressed in 5.4% (95% CI: 4.3% - 7.8%) of the CD8+ T cells from peripheral blood. These data suggest SYK expression is nearly undetectable in the normal mature T cells which are part of adaptive immunity. In contrast, lymphoid cells of innate immunity such as NK cells express high levels of SYK. Similar to normal T-cell subsets, the neoplastic T-cells of most PTCL believed to originate from cells of adaptive immunity (AITL, ATLL, ALCL, CTCL, PTCL-NOS and T-PLL) showed low levels of SYK expression (median: 5.8%; 95% CI: 5.8% -10.4%) with rare exceptions (Figure 1). In contrast, the T and NK cell neoplasms of innate immunity origin (NK-LGL, MEITL) and immature precursor T-cell origin (MPAL and T-ALL) showed high levels of SYK expression (median 91.1%, 95% CI as 97.1% - 99.6%). SYK expression in T-LGL was more modest and variable (median 43.6%, 95% CI as 43.6% - 78.0% (Figure 1). Conclusion: SYK is highly expressed in the neoplastic lymphoid cells of innate immunity such as NK-LGL, MEITL and T-LGL cases, and immature hematopoietic neoplasms with T-cell differentiation. The expression of SYK in neoplastic cells from patients with mature T cell lymphoma of adaptive immunity remains low. Given the significant physiological role of SYK in innate immunity, SYK expression in NK/T-cell neoplasms is likely to have an oncogenic role, suggesting that SYK might be a good therapy target for these tumors. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Dogan:Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy.

T-Cell Mediated Cytopenias in Patients with Myelodysplastic Syndromes Respond to Intravenous Immunoglobulin

Background: Patients with Myelodysplastic Syndrome (MDS) with cytopenias have limited treatment options to improve blood counts apart from immune-modulatory drugs (IMIDS), hypomethylating agents (HMA), cytokines and transfusional support. Immune dysregulation is common in MDS and occurs in up to 35% of pts (LR Silverman, Cancer Med, 2017). Expanded T-cell clonal populations in the peripheral blood and bone marrow in the presence of MDS may play a role in aggravating MDS related cytopenias, increasing the need for transfusions or exacerbating symptoms. Intravenous immunoglobulin (IVIG) may interact with Fc receptors on T cells or have suppressive effects on the concurrent large granular lymphocytic leukemia (LGL) clone. Targeted therapy with IVIG could be a novel strategy to mitigate the cytopenias in MDS and other bone marrow failure pts. Methods: We reviewed in a single-center series 27 consecutive pts who had either MDS, aplastic anemia (AA), or a myeloproliferative neoplasm (MPN) along with an LGL clone seen on TCR-rearrangement studies and/or peripheral blood flow cytometry in the blood and/or bone marrow. Of the 27 pts, 22 had MDS, 2 had MPN, and 1 each with AA, LGL, or Multiple Myeloma. IVIG was initially used as a single agent administered at a dose of 500mg/kg/day over 4 hours once weekly x4 weeks. After "induction", administration was changed to every other week, and then in responding pts, the interval was extended to every 3-4 weeks. In pts with a stable response, IVIG was discontinued and pts observed. After the initial 4 pts, pts were treated concurrently with prednisone 20 mg/d with a taper over 4 weeks, except in pts in which prednisone was a relative contra-indication. Overall hematologic improvement was determined by MDS IWG 2006 criteria, while hemolytic response was determined to be either a complete normalization (CR) or a partial normalization (PR) if there was >50% improvement in deviation from normal LDH, reticulocytes, indirect bilirubin, or haptoglobin values from baseline. Results: All pts received IVIG administered at 500mg/kg, once weekly, to be spaced out maximally as tolerated. 74% (20/27) of pts received steroids. Baseline characteristics of the pt population: median age of 70 years (range 39-87), representing 21 males and 6 females. Of the pts with MDS evaluable for risk stratification by IPSS-R criteria [n=18], 3 were very low, 5 low, 6 intermediate, 2 high, and 2 very high risk categories. By WHO criteria, the greatest representation was 11/20 RCMD, followed by 3/20 with RAEB2. Baseline cytogenetics [n=21] included 3 pts with very poor, 2 poor, 3 intermediate, 11 good, and 2 very good risk profiles. Pts had a range of 0-6 mutations, most common were SETBP1, ASXL1, TP53 (n=4) and SRSF2, U2AF1, PDGFRB (n=3). 24/27 pts had positive TCR beta and 13/27 had positive TCR gamma gene rearrangements. ORR for any cytopenia was 77.8% (21/27), and for any hemolytic response was 76.2% (16/21). ORR for individual cell lines included: 59.2% (16/27) erythroid, 48.1% (13/27) neutrophil, and 33.3% (9/27) platelet responses. For hemolytic responses, CR/PR rate were measured by LDH (29.6%/11.1%), bilirubin (22.2%/14.8%), retic (33.3%/14.8%), and haptoglobin (22.2%/11.1%). For the 14 MDS pts with very low, low, and intermediate IPSS-R risk, ORR was: 64.3% (9/14) any cytopenia, 35.7% (5/14) erythroid, 50.0% (7/14) neutrophil, 14.3% (2/14) platelets. Combined CR+PR rates for hemolysis in the lower risk categories were 42.9% LDH, 21.4% bili, 42.9% retic, 28.6% haptoglobin responses. For the 5 non-MDS pts, ORR for any cytopenia was 100%, and 60% for any hemolytic response. There were no reported significant adverse events with IVIG administration. Conclusions: IVIG can be used as a first line treatment for pts with autoimmune cytopenias associated with LGL clones and MDS (along with other hematological malignancies). Treatment with IVIG yields an ORR of 77.8% in the entire cohort and 64.3% in pts with lower risk MDS, with a reduction in hemolysis as well. This report expands on a previous analysis and demonstrates further evidence of efficacy for the use of IVIG in treating cytopenias exacerbated by LGL T-cell clones. IVIG therapy can permit a delay in the start of an HMA or IMID treatment, lowering transfusion burden, and increasing ANC to help prevent infectious complications. Further evaluation in a larger cohort of pts is warranted. Studies exploring the mechanism of action are underway. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Novel Drugs and Clinical Trial-Based Treatments Prolong Survival of Peripheral T-Cell Lymphomas (PTCL) Patients: Single Institution Retrospective Analysis

Introduction Peripheral T-cell lymphomas (PTCL) are a group of rare and heterogeneous diseases associated with unfavorable prognoses. Conventional chemotherapy has been the standard of care, but is considered marginally effective. This outcome has been attributed to the fact that most PTCL treatments are derived from the management of aggressive B-cell lymphomas. While relapse is common, subsequent line therapies are heterogeneous, and can include many lines of non-specific chemotherapy. Recently approved novel agents, such as pralatrexate and histone deacetylase (HDAC) inhibitors, and clinical trials however, tend to either not be used or used too late in the natural history of the disease. Herein, we compared the efficacy of chemotherapy and novel therapies, in first line, second line and third line, to identify factors that might influence the outcome of PTCL patients in a single institutional cohort. Methods A search of the electronic medical record and clinical trial databases from January 1, 1994 to May 31, 2019 was performed to identify patients with PTCL. Only patients 18 years and older with pathology-confirmed PTCL and complete clinical data were included. Due to substantially different disease characteristics, behavior, and treatment, we excluded patients with T/NK large granular lymphocytic leukemia and primary cutaneous T-cell lymphoma, with the exception of transformed mycosis fungoides. Overall survival (OS) was calculated from the time of diagnosis to death from any cause. Kaplan-Meier curves were generated and compared based on the log-rank test. Cox proportional hazard models were used to investigate the association by adjusting for the type of treatments and the lines of therapy. Statistical analyses were performed with SAS version 9.4. Results There were 213 patients (1.2 males:1 female) included in the analysis with a median age of diagnosis of 56.9 years (range 18.1-90.3). Front-line therapies typically consisted of chemotherapy, including CHOP, CHOEP, and EPOCH. The use of clinical trials and novel agents increased as patients progressed through multiple lines of therapy. Figure 1A showed that patients who achieved a CR with first line chemotherapy had a better overall survival than patients who did not (median 8.9 years vs 0.83 years, p<0.0001), with trends suggesting improvement in survival with novel combinations and single agents. Although limited by small numbers, patients with primary refractory or relapsed disease were generally given additional lines of chemotherapy, and the percentage of patients who achieved CR trended down more drastically (42% first line in Figure 1A, 16% second line in Figure 1D, 21% third line in Figure 1G) than patients who received novel combinations (50% first line in Figure 1B, 23% second line in Figure 1E, 30% third line in Figure 1H) or single agents (27% first line in Figure 1C, 40% second line in Figure 1F, 20% third line in Figure 1I). There were no significant differences in the frequency of radiotherapy or autologous stem cell transplantation over the study period. Our data demonstrate that exposure to novel agents (p=0.002) in Figure 2A and novel combinations (p=0.003) in Figure 2B, especially histone deacetylase inhibitors (HDACI, p=0.045) in Figure 2D, during the treatment course of PTCL patients, was associated with prolonged overall survival. Pralatrexate had a trend toward improving outcomes in the first ten years as shown in Figure 2C, but this was not statistically significant. Conclusions Clinical trials and novel therapies improve outcomes and should be considered earlier in the therapy of patients with PTCL. Patients who obtain CR early have the best overall survival. Our findings suggest that patients with R/R PTCL who fail chemotherapy should be enrolled in clinical trials or be given novel approved agents rather than repeated rounds of non-specific cytotoxic therapy. Disclosures Marchi: Spectrum Pharmaceuticals, Verastem Oncology: Research Funding. O'Connor:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADCT Therapeutics, Affimed, Agensys, Merck, Seattle Genetics, Spectrum, Trillium, and Verastem Oncology.: Research Funding; TG Therapeutics: Other: Travel Support, Research Funding. OffLabel Disclosure: Drugs that are approved for the treatment of patients with relapsed and refractory peripheral T-cell lymphoma, such as pralatrexate (a novel antifolate) and histone deacetylase inhibitors, were used in the front line setting when patients were not candidates for chemotherapy or enrolled in clinical trials. Outcomes are also described for non-FDA approved drugs used as monotherapy or in combination in clinical trials as well as FDA approved drugs for other indications used in clinical trials are described though not explicitly listed.

Final Results of a Phase II Biomarker-Driven Study of Ruxolitinib in Relapsed and Refractory T-Cell Lymphoma

Final Results of a Phase II Biomarker-Driven Study of Ruxolitinib in Relapsed and Refractory T-Cell Lymphoma

Severe Chronic Neutropenia in the Large Granular Lymphocyte Syndrome: Outcomes in Response to Granulocyte Colony Stimulating Factor (G-CSF) and Immunosuppressive Therapies

The large granular lymphocyte (LGL) syndrome and large granular lymphocytic leukemia (LGLL) are rare hematological disorders. Through the Severe Chronic Neutropenia International Registry (SCNIR) we have followed the clinical course and treatment responses of 27 LGL patients presenting with severe chronic neutropenia without evidence of leukemia, rheumatoid arthritis or autoimmune diseases. This report focuses on their treatment responses to G-CSF and outcomes with and without other immunosuppressive therapies. Methods: Patients with severe chronic neutropenia and a diagnosis of LGL syndrome (at least 3 ANC values < 0.5 x 109/L over a 3 month period) were enrollment in the SCNIR if they do not have concomitant systemic autoimmune disease, myeloid or lymphoid malignancy or drug-induced neutropenia. Findings: The 27 LGL patients (8 males, median age 53; 19 females, median age 57) had chronic neutropenia for approximately 4 years (range 0.4 - 25.6 years) before enrollment. Diagnoses were primarily based on FACS analysis (12 bone marrow [50%]; 11 blood and bone marrow [46%]; 1 blood only [4%]). None of these patients presented with lymphocytosis, i.e., ALC > 5.0 x 109/L. Before enrollment, 7 patients had anti-neutrophil antibody tests (3 positive, 4 negative), 4 patients had antinuclear antibody tests (2 positive, 2 negative), and ANCA was positive for the one patient tested. After enrollment, additional results were: 1 patient had a negative anti-neutrophil antibody, 8 patients had antinuclear antibody tests (2 positive, 6 negative), and ANCA was positive in two additional patients. The diagnosis of LGL was based on increased LGL cells in the blood or bone marrow, most frequently increased CD 3+, CD 8+, CD 57+ cells. Increases in lymphocytes in the marrow, often in clusters were a common finding; lymphocytes in the marrow ranged from 6-79%. The range of CD 57+ in blood 11-19%, bone marrow 19-61%; CD 56+/NK cells in blood 0.06-14%, bone marrow <1-76.8%; increased CD 8+/CD 3+ in blood 1.5-90%, bone marrow <1-91%. Ten patients have gene rearrangement studies, 7 prior to enrollment (3 negative, 4 positive) and 3 patients had these studies after enrollment, all positive. Because most patients had neutropenia, fevers and infections we evaluated the effectiveness and safety of G-CSF therapy. Treatments were G-CSF alone (13), G-CSF with prednisone (5), G-CSF with methotrexate and prednisone (4), G-CSF with methotrexate alone (2), G-CSF with cyclosporine (1), G-CSF with cyclosporine and prednisone (1), and G-CSF with prednisone, methotrexate and cyclosporine (1). Prior to G-CSF treatment the median ANC was 0.54 x 109/L, mean 1.48 +/- 0.35 SEM. The ALC median was 1.67 x 109/L, mean 2.74 +/- 0.74. The G-CSF dosing median was 1.44 mcg/kg/day, mean 2.27 +/- 0.31. All responded to G-CSF alone or in combination therapy to achieve a mean ANC greater than 1.0 x 109/L. After G-CSF treatment the median ANC was 2.23 x 109/L, mean 3.78 +/- 0.40. The ALC median was 2.24 x 109/L, mean 3.17 +/- 0.48. Time period for SCNIR observation median years is 9.6 years; mean 10.0 +/- 1.2, range 0.4 to 23.1 years. There was a total 270 years of observation in the SCNIR and 307 years of G-CSF administration in this group of 27 patients. The increase in neutrophils was consistently associated with fewer episodes of fever, infections and antibiotic use. One of 27 patients had episodic thrombocytopenia (<50 x 109/L) before G-CSF and continued while on G-CSF. Seven of 26 patients reported episodic thrombocytopenia after starting G-CSF. There were no report episodes of significant bleeding. Fourteen of 27 are now living, ages 17 to 72, and all have continued on treatment. Over the 24 year observation period, 5 patients developed Hodgkin disease (1) or non-Hodgkin lymphoma (5); and 5/6 died. Five other LGL patients died of sepsis or pneumonia. Other deaths were attributed to a T-cell lymphoproliferative disorder (1), chronic pancreatitis (1) and lung cancer (1). There were no myeloid malignancies. Conclusions: Our long term observations suggests that the combination of low dose G-CSF plus methotrexate or cyclosporine appears to be more effective than either drug alone, titrated each to minimal effective doses, and continued indefinitely. There was no increased frequency of opportunistic infections. The expected course is a long period of normal ANCs without other autoimmune diseases. Evolution to lymphoid, not myeloid, malignancies is the greatest concern. Disclosures Dale: Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding; Athelas: Equity Ownership; Prolong: Consultancy; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Amgen: Consultancy, Research Funding; Sanofi Aventis: Consultancy, Honoraria; Beheringer/Ingelheim: Consultancy. Newburger:TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.

Prospective, Multicentric Phase II Randomized Trial Comparing the Efficacy of Methotrexate or Cyclophosphamide in Large Granular Lymphocytic Leukemia: A French National Study. Report on the Interim Analysis

Introduction Large granular lymphocyte (LGL) leukemia is characterized by a clonal expansion of CD3+ cytotoxic T or CD3- NK cells. Prominent clinical features include neutropenia, anemia and autoimmune-associated diseases such as rheumatoid arthritis (RA). Although the disease is usually chronic and indolent, some patients may be symptomatic and require treatment. No standard therapy has been established due to the absence of prospective clinical trials. So far, low dose methotrexate, oral cyclophosphamide, and cyclosporine represent the 3 main options for initial therapy. In 2014, we launched a prospective clinical trial comparing methotrexate to cyclophosphamide in previously-untreated patients with LGL leukemia in need of treatment. Patients and methods The study was designed as a multicentric, national, open label, randomized, controlled trial on two parallel groups, comparing methotrexate and cyclophosphamide. Patients were included if they had at least one of the following indications of treatment: isolated severe neutropenia (ANC <0.5x109/L) or neutropenia (ANC <1.5x109/L) with infections, anemia requiring transfusions or symptomatic anemia, associated complications such as systemic diseases or auto-immune diseases resistant to steroids and/or immunomodulating agents (colchicin, disulone, hydrochloroquine). They were randomly assigned to receive either methotrexate (10 mg/m²/w) or cyclophosphamide (100 mg/d) for 4 months. Responders at M4 continued with the same treatment until M12 (cyclophosphamide was then delivered at 50 mg/d). Non-responders at M4 were randomly assigned to receive either cyclosporine (3 mg/kg/d) or the drug which had not been administered at the first randomization (methotrexate or cyclophosphamide). Response was assessed using previously published criteria (Lamy T, Blood 123:1182, 2014). Complete response (CR) was defined as a normalization of clinical exam (disappearance of splenomegaly or associated autoimmune symptoms) and a complete normalization of blood counts. Partial response (PR) was defined as an improvement in blood counts which did not meet criteria for complete remission (e.g., ANC >0.5x109/L or decrease of transfusion requirements). Treatment failure was defined as no response or any response which did not meet the above-mentioned criteria within four months after the beginning of treatment. To stop the trial as soon as sufficient information was collected, a sequential analysis was planned each time 20 patients were included and evaluated using the triangular test (Sébille V, Bellissant E. Fundam Clin Pharmacol. 2003;17(5):505-16). The primary endpoint was the hematological CR rate evaluated at M4 (binary endpoint). Secondary endpoints were overall response rate (ORR) at M4, M8 and M12, time to relapse. For non-responders at M4, cyclosporine was compared to the treatment which had not been administered during the first phase. Results From Nov 2013 to July 2019, 99 patients met inclusion criteria among which 96 were randomized. The baseline characteristics of these patients are shown in Table1. STAT3 mutation was observed 52% of cases. After the 4th sequential analysis performed on the first 80 patients evaluable for response at M4, the sample path remained in the continuation region of the triangular test. Thus, the trial has to be continued. At M4, 13 patients were in CR (16.3%) and 29 patients were in PR (36.3%), ORR was 52.6%, 36 patients were considered as refractory and underwent a second randomization: 18 patients received cyclosporine and 17 received methotrexate or cyclophosphamide. Conclusions This first prospective randomized clinical trial in LGL leukemia shows that the CR after first line therapy using either methotrexate or cyclophosphamide is relatively low (< 20%). Recruitment is still ongoing to assess if there is a difference in terms of response between the two drugs. Predictive biomarkers of response will be presented at the meeting. Regarding a 52% of incidence of Stat3 mutation (higher than that previously published), Jak/Stat targeted therapy should be prospectively evaluated in this disease. Disclosures Houot: Bristol Myers Squibb: Honoraria; Merck Sharp Dohme: Honoraria. Gyan:Pfizer: Honoraria. Feugier:janssen: Honoraria, Research Funding, Speakers Bureau; abbvie: Honoraria, Research Funding, Speakers Bureau; gilead: Honoraria, Research Funding, Speakers Bureau; roche: Honoraria, Research Funding, Speakers Bureau.

Pathophysiologic Mechanisms And Management Of Large Granular Lymphocytic Leukemia Associated Pure Red Cell Aplasia.

Large granular lymphocytic leukemia (LGLL) is a chronic clonal lymphoproliferative disease of mature T or NK cells, and produces a variety of hematological abnormalities. Pure red cell aplasia (PRCA) is a rare haematological disease and is one of the most common complications of LGLL. LGLL-associated PRCA may represent a relatively indolent type and may be more common than reported, but its natural history and clinical course have not been well described. The ethnic origin of the patients is an important consideration in determining the relationship between PRCA and LGLL. Guidelines and progresses for management of LGLL-associated PRCA rely on accumulation of empirical experiences, integrative analyses of several cases and clinical trials. The purpose of this review is to evaluate occurrence, possible mechanisms, diagnosis, clinical features, treatments and outcomes of LGLL-associated PRCA.

PP-55 - Behçet’s disease and T-cell large granular lymphocytic leukemia: two case reports

PP-55 - Behçet’s disease and T-cell large granular lymphocytic leukemia: two case reports

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4− phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

The Road Less Travelled, from Large Granular Lymphocytic Leukemia to Myelodysplastic Syndrome

Evolution of MDS and other myeloid clonal aberrations in LGLL.

Recurrent anti-GBM disease with T-cell large granular lymphocytic leukemia

Rationale:Anti-glomerular basement membrane disease (anti-GBM disease) is a rare small vessel vasculitis caused by autoantibodies directed against the glomerular and alveolar basement membranes. Anti-GBM disease is usually a monophasic illness and relapse is rare after effective treatment. This article reports a case of coexistence of recurrent anti-GBM disease and T-cell large granular lymphocytic (T-LGL) leukemia.Patient concerns:A 37-year-old man presented with hematuria, edema, and acute kidney injury for 2 months.Diagnosis:Anti-GBM disease was diagnosed by renal biopsy, in which crescentic glomerulonephritis was observed with light microscopy, strong linear immunofluorescent staining for immunoglobulin G on the GBM and positive serum anti-GBM antibody. Given this diagnosis, the patient was treated with plasmapheresis, steroids, and cyclophosphamide for 4 months. The anti-GBM antibody titer was maintained to negative level but the patient remained dialysis-dependent. One year later, the patient suffered with a relapse of anti-GBM disease, after an extensive examination, he was further diagnosed T-LGL leukemia by accident.Interventions:The patient received cyclosporine A therapy for T-LGL leukemia.Outcomes:After treatment with cyclosporine A, serum anti-GBM antibody became undetectable. During the 16 months follow-up, anti-GBM titer remained normal and abnormal T-lymphocytes in the bone marrow and peripheral blood were also decreased.Lessons:T-LGL leukemia is an indolent lymphoproliferative disorder that represents a monoclonal expansion of cytotoxic T cells, which has been reported to be accompanied by some autoimmune diseases. This is the first report of coincidence of T-LGL leukemia and anti-GBM disease, and suggests there are some relationships between these 2 diseases. Clinical physicians should exclude hematological tumors when faced with recurrent anti-GBM disease.

CD4+/CD8- T cell Large Granular Lymphocytic Leukemia: A rare Entity

Large granular lymphocytic leukemia (LGL) is a well-recognized disorder of mature T-cells or NK cells. T-cell LGL leukemia (T-LGL) is characteristically a disorder of mature CD3+/CD8+ cytotoxic T-cells. Rare variants include CD 3+/CD4+/CD8-cases. To the best of our knowledge, 11 such cases (4 cases by Lima et al.[1] in 2003, 4 cases by Olteanu et al. in 2010,[2] 2 cases by Mutreja et al.[3] in 2014, and 1 case by Rabade et al.[4] in 2014) of T-LGL showing CD3+/CD4+/CD8-immunophenotype has been published in literature so far. There is a paucity of literature explaining the monoclonal expansion of CD3+/CD4+ T-LGL.[1] Unlike CD8+ T-LGL, CD4+ T-LGL does not show cytopenia, autoimmune phenotypes,[1],[5] or splenomegaly. However, CD4+ T-LGL is frequently associated with nonhematological malignancies.[1] Here, we report a case presenting with CD3+/CD4+/CD8-immunophenotype. Such immunophenotypic variant form of T-LGL cases should have a close clinical follow-up as they are prone to develop either simultaneously or months and years after, secondary hematological or nonhematological malignancies.[1]

Retrovirus insertion site analysis of LGL leukemia patient genomes

BackgroundLarge granular lymphocyte (LGL) leukemia is an uncommon cancer characterized by sustained clonal proliferation of LGL cells. Antibodies reactive to retroviruses have been documented in the serum of patients with LGL leukemia. Culture or molecular approaches have to date not been successful in identifying a retrovirus.MethodsBecause a retrovirus must integrate into the genome of an infected cell, we focused our efforts on detecting a novel retrovirus integration site in the clonally expanded LGL cells. We present a new computational tool that uses long-insert mate pair sequence data to search the genome of LGL leukemia cells for retrovirus integration sites. We also utilize recently published methods to interrogate the status of polymorphic human endogenous retrovirus type K (HERV-K) provirus in patient genomes.ResultsOur data show that there are no new retrovirus insertions in LGL genomes of LGL leukemia patients. However, our insertion call tool did detect four HERV-K provirus integration sites that are polymorphic in the human population but absent from the human reference genome, hg19. To determine if the prevalence of these or other polymorphic proviral HERV-Ks differed between LGL leukemia patients and the general population, we used a recently developed tool that reports sites in the human genome occupied by a known proviral HERV-K. We report that there are significant differences in the number of polymorphic HERV-Ks in the genomes of LGL leukemia patients of European origin compared to individuals with European ancestry in the 1000 genomes (KGP) data.ConclusionsOur study confirms that the clonal expansion of LGL cells in LGL leukemia is not driven by the integration of a new infectious or endogenous retrovirus, although we do not rule out that these cells are responding to retroviral antigens produced in other cell types. However, our computational analyses revealed that the genomes of LGL leukemia patients carry a higher burden of polymorphic HERV-K proviruses compare to individuals from KGP of European ancestry. Our research emphasizes the merits of comprehensive genomic assessment of HERV-K in cancer samples and suggests that further analyses to determine contributions of HERV-K to LGL leukemia are warranted.

Indications and Outcomes of Splenectomy for Hematological Disorders.

Background and AimSplenectomy is a frequent component of the diagnosis and treatment of hematological disorders. The aim of this study was to define the indications and outcomes of splenectomy for benign and malign hematological disorders.Materials and MethodsOne hundred and two patients with hematological disease who had splenectomy at Hacettepe University Hospital between the years of 2010 and 2018 were evaluated.ResultsA total of one hundred and two patients were included in this study. The median age was 52 (20-82) years at the time of splenectomy. Most of the patients were female (57.9%). The median follow up time was 11.0 (0.03-87.9) months after splenectomy. Splenectomy was performed to diagnose thirty patients (29.4%). Seventy-two patients underwent splenectomy for the treatment of hematological disease (70.6%). Twenty-seven patients (90%) were diagnosed with various lymphomas. Two patients (6.7%) were diagnosed with hairy cell leukemia and one patient (3.3%) was diagnosed with large granular lymphocytic leukemia.ConclusionIn conclusion, an improvement in medical therapy, especially with monoclonal antibodies, the indications and outcomes of splenectomy for hematologic disorders have changed extremely in last years. Nevertheless, splenectomy has an important role for diagnosis and treatment of benign and malign hematological disorders.

PF479 CLINICAL AND BIOLOGICAL PREDICTORS OF OUTCOME IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA: A SINGLE CENTER EXPERIENCE OF 205 PATIENTS

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare chronic lymphoproliferative disorder characterized by the expansion of LGLs. Among LGLL, WHO 2016 classification recognizes a CD3+ T‐LGLL and a CD3‐ Chronic Lymphoproliferative disorder of Natural Killer cells (CLPD‐NK), accounting for 85% and 15% of cases respectively. Moreover, according to the T cell receptor rearrangement, the Tαβ and Tγδ subsets can be identified. From a clinical point of view, LGLL is a heterogeneous disease with some patients being asymptomatic and other developing cytopenias, mostly neutropenia. The aetiology is still unknown but a constitutive activation of JAK/STAT pathway is involved in LGL proliferation, supported by the evidence of somatic STAT3 and STAT5b mutations in approximately 40% of patients. We previously demonstrated that in T‐LGLL STAT3 mutations are associated to symptomatic disease.Aims:The aim of this study is to analyse clinical and biological features of a large cohort of LGLL patients to identify prognostic markers affecting patients’ outcome.Methods:From 1992 to 2018, clinical and biological data of 205 patients affected by LGLL have been collected. STAT3 exon 21 mutation analysis and STAT5b exon 16–18 mutation analysis was performed by Sanger sequencing.Results:In our cohort, median age at diagnosis was 58 years. By phenotype, 129/205 (62.9%) patients were affected by Tαβ LGLL, 23/205 (11.2%) by Tγδ LGLL, and 36/205 (17.6%) by CLPD‐NK. Moreover, 17 patients (8.3%) were characterized by a bi‐phenotypical variant, identified by a concomitant T/NK or Tαβ/Tγδ clone. According to CD4 and CD8 expression, Tαβ LGLL was further classified in CD4‐/CD8+ LGLL (CD8+ T‐LGLL, 84/205, 41%) and CD4+/CD8dim/neg LGLL (CD4+ T‐LGLL, 45/205, 21.9%).Neutropenia (Absolute Neutrophil's Count, ANC<1,500/mm3) was the most relevant feature (78/205, 38%), severe neutropenia (ANC<500/mm3) being present in 20.5% of patients. Anemia (Hb<120 g/L, 27/205, 13.2%), severe anemia (Hb<90 g/L, 20/205, 9.8%) and thrombocytopenia (PLTs <100.000/mm3, 12/205, 4.8%) were less recurring. 44 patients (21.5%) were affected by concomitant autoimmune/inflammatory disease, mostly autoimmune hemolytic anemia and rheumatoid arthritis.DNA samples of 166 and 152 patients were available for STAT3 and STAT5b mutations analysis, respectively. STAT3 mutations were detected in 47 patients (28.3%), with higher frequency in CD8+ Tαβ LGLL towards Tγδ LGLL (60% vs 25%, p = 0.0232) and CLPD‐NK (60% vs 5.8%, p < 0.0001). At variance, STAT5b mutations were found in 15 patients (9.8%), mostly in CD4+ Tαβ LGLL (12/35 cases available, 34.3%) and in 3 cases were Tγδ LGLL. Interestingly, STAT3 and STAT5b mutations were mutually exclusive in CD8+ and CD4+ Tαβ LGLL.With a median follow up of 8 years, median overall survival (OS) of our cohort was not reached. Major features associated to reduced OS were ANC<500/mm3 (267 months vs not reached, p = 0.0297), severe anemia (144 months vs not reached, p < 0.0001), treatment requirement (214 vs not reached, p = 0.0011) and presence of STAT3 mutations (267 months vs not reached, p = 0.0113) (Figure 1, Panel A to D). No significant survival differences were found between STAT5b mutated and wild‐type patients (not reached vs 267 months, p = 0.3514).Summary/Conclusion:Our results confirmed the remarkable biological and clinical heterogeneity of LGLL. We identified clinical and biological features associated to reduced OS in LGLL patients and, for the first time, we demonstrated the dismal impact of STAT3 mutations in patients’ survival, suggesting that this marker should be regarded as a potential target of therapy.image

PB1871 ONCOGENIC MIRNAS CONTROL PROLIFERATION AND APOPTOSIS IN TCRαβ+ CD8+ LARGE GRANULAR LYMPHOCYTE (LGL) LEUKEMIA CELLS

Background:TCRαβ+ CD8+ large granular lymphocyte (LGL) leukemia is a rare heterogeneous hematological disorder that has a chronic disease course and mostly affects the elderly. LGL leukemia is estimated to account for 2‐5% of all chronic lymphoproliferative disorders and most commonly TCRαβ+ CD8+ T cells are affected. Although the precise disease etiology is largely unknown and disease course is heterogeneous, it is generally accepted that LGL cells arise through chronic antigenic stimulation and inflammation and thrive as a consequence of dysregulation of proliferation and apoptosis pathways. One level of dysregulation of these pathways could involve miRNAs. miRNAs are a class of endogenous noncoding RNAs (19‐25 nt) that are involved in regulation of gene expression. miRNAs primarily act at the post‐transcriptional level and are involved in various cellular processes such as proliferation and apoptosis. Moreover, dysregulated expression of miRNAs that hold tumor suppressor or oncogenic capacity has been associated with the pathogenesis of human cancers and leukemias. Here, we explored the miRNAome of TCRαβ+ CD8+ LGL leukemic cells by performing small noncoding RNA sequencing of sorted TCRαβ+ CD8+ LGL cells (n = 7) and sorted age‐matched healthy control TCRαβ+ CD8+ TEMRA effector T cells (n = 6). Furthermore, we evaluated whether differentially expressed miRNAs could potentially drive the dysregulation of proliferation and apoptosis pathways in LGL leukemia.Aims:To determine the miRNAome of TCRαβ+ CD8+ LGL cells and to elucidate whether the aberrant expression of miRNAs drives the dysregulation of proliferation and apoptosis pathways in LGL cells.Methods:Purified LGL leukemia cells and healthy control cell subsets were sorted with FACS Aria III. Cells were sorted directly in Trizol reagent and RNA was isolated according to manufacturer's protocol. Libraries were prepared with cleantag small RNA library prep kit and SMART‐seq v4 ultra low input RNA kit for sequencing according to manufacturer's protocol.Results:Using next generation sequencing, we determined differential expression of a subset of miRNAs between LGL leukemia cells and healthy control T cell subsets. Principle component analysis of miRNA expression profiles clearly showed LGL leukemia cells clustering away from healthy control T cells, with more than 100 miRNAs significantly upregulated or downregulated between patient cells and control cells. In silico pathway analysis of top differentially expressed miRNAs indicated that these miRNAs could be involved in cell cycle regulation through repression of cyclin dependent kinases CDKs, the NF‐κB pathway and potentially drive apoptosis resistance through inhibition of the p53 and BCL2 pathways. Currently we are exploiting the RNAome by RNA sequencing to validate the involvement of these pathways in LGL leukemia. By integrating both the miRNA and RNA datasets the association of top candidates in these pathways will be further investigated.Summary/Conclusion:Collectively our data implicate miRNAs as potential drivers in the maintenance of established LGL leukemic cells. Likewise, in silico prediction tools imply that long term survival of these cells is a result of miRNAs that enhance proliferation and inhibit apoptosis.

PF367 MOLECULAR EVIDENCE THAT ATOVAQUONE IS A NEW STAT3‐INHIBITOR COUNTERBALANCING LEUKEMIC CELL PRO‐SURVIVAL IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. The hallmark of leukemic LGLs is a constitutive activation of STAT3 leading to enhanced triggering of many pro‐survival genes. Moreover, 30–40% of patients carry mutations in STAT3 gene bringing to the constitutive activation of the malignant clone.No specific treatment is now available for the patients affected by LGLL and the in‐use therapy is based on low doses of immunosuppressive drugs, i.e. Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA). Anyway, these therapies show limited efficacy, induce many side effects with the Overall Response Rate (ORR) being around 50% and a high rate of relapse in the responding patients.The JAK/STAT signaling pathway, which is central to LGL leukemia pathogenesis, includes several players that provide the basis for new targeted treatments. This offers the rationale for using Atovaquone (ATQ, Mepron™), a recently FDA‐approved anti‐microbial agent characterized by a low adverse effect profile that has been recently demonstrated (Xiang et al., 2016) to work as a powerful STAT3 inhibitor with anti‐cancer efficacy in both animal models and human cell lines.Aims:Our study focuses on in vitro tests to evaluate the effects of ATQ as inhibitor of STAT3 phosphorylation and of LGLs survival, with the goal to develop a targeted approach to LGLL, likely devoid of adverse effects.Methods:Patients’ Peripheral Blood Mononuclear Cells (PBMC) were purified to perform cultures to test serial drug concentrations. Using in vitro cells culture, the inhibition of STAT3 protein phosphorylation and the expression of its target proteins, MCL1 and BCL2, were evaluated by Western Blot assay (WB). In addition, the cytotoxicity and the apoptotic selectivity of ATQ was investigated by Annexin V test; a staining with anti‐CD57, CD56 or CD16 was used to identify leukemic LGLs from PBMCs. Finally, by RT‐PCR, STAT3 activity was analyzed along its transcriptional levels and target genes.Results:Through in vitro experiments we initially tested the best concentration inhibiting cell viability and STAT3 activation on patients’ PBMCs. We observed that ATQ was efficiently able to inhibit STAT3 phosphorylation at a concentration of 25 μM. The results of the apoptosis analysis showed that ATQ has a dose and time dependent cytotoxic effect. The IC50 was reached at a dose of 25 μM after 24 h of culture. ATQ demonstrated a selective cytotoxicity against leukemic LGLs (reaching 3.9 ± 0.7 fold increase of apoptosis vs untreated condition after 48 h of culture) sparing non‐leukemic cells (ATQ treated apoptosis vs untreated in non‐LGL PBMC = 1.18 ± 0.3 fold change). By comparing the in vitro cell effect of ATQ with that of the commonly used therapies, ATQ showed a cytotoxic effect consistent with CTX and CyA, but higher than MTX (p < 0.01). Concerning anti‐apoptotic factors under the STAT3 control, after 6 h ATQ treatment the expression of MCL1 and BCL2 showed only minimal changes, thereby suggesting that different mechanisms of action take place.Summary/Conclusion:These in vitro data indicate that ATQ has a promising action on the inhibition of the JAK/STAT pro‐survival pathway in leukemic LGLs, resulting in a selective cytotoxic effect against LGLs through MCL1‐ and BCL2‐independent mechanisms.