Cell Large Granular Lymphocyte Leukemia Research Articles

Abstract 3201: Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies

Abstract The histone deacetylase inhibitors (HDACi) have long been regarded as an important class of drugs for treating T-cell lymphoid malignancies (TCL). Romidepsin (Romi), a bicyclic peptide, was approved for patients with relapsed or refractory cutaneous (CTCL) and peripheral T-cell lymphomas (PTCL). Unfortunately, the approval of Romi for relapsed/refractory PTCL was withdrawn due to a negative Phase 4 post-marketing requirement. Given its potent activity against Class I HDACs, Romi is considered one of the most effective HDAC inhibitors studied and used in patients with TCL. Herein, we report on a first-in-class nanoparticle of Romi (NanoRomi) with a superior safety and efficacy profile compared to the free drug in preclinical models of PTCL and large granular lymphocyte leukemia (LGLL). We developed a first-in-class NanoRomi using our proprietary amphiphilic di-block copolymer-based nanochemistry platform. NanoRomi was characterized in cell viability, flow cytometry, and western blotting (WB) assays to determine the concentration-effect relationships with regard to the induction of apoptosis and histone acetylation in cell lines representative of TCL and LGLL, as well as primary LGLL patient PBMC samples. In addition, we performed extensive in vivo modeling using murine xenografts of CTCL, including repeated dose toxicity, biodistribution, efficacy, and survival studies. Cytotoxicity assays demonstrated superior/comparable activity of NanoRomi (IC50: 1.1-2.3 nM) compared to free Romi (IC50: 4.8-10.6 nM) across all PTCL and LGLL cell lines studied, while in primary LGLL samples, NanoRomi exhibited superior cytotoxicity (2-fold) compared to Romi. WB analysis showed that NanoRomi induced apoptosis and increased acetylation of histone H3 and H4 in a time and concentration-dependent manner in cell lines representing CTCL, ALK+-ALCL, and LGLL. Repeat dose toxicity studies in CTCL xenografts confirmed that the overall tolerability of NanoRomi was substantially better than Romi. Tracking studies with a fluorescent dye (DiO) in the NanoRomi demonstrated preferential localization of NanoRomi in tumors of mice compared to the normal organs after 6 hours of drug administration. NanoRomi exhibited superior efficacy in the murine CTCL xenograft models, including a significant improvement in overall survival compared to Romi. Collectively, these data show that NanoRomi exhibits markedly superior pharmacokinetics, tolerability, and efficacy in models of TCL. We anticipate that the improved activity and tolerability of NanoRomi relate to the altered pharmacologic profile and superior bioavailability of the drug, which optimizes the effects of the drug for its influence on epigenetic biology. This unique platform offers the prospect of improving the tolerability and efficacy of epigenetic treatment strategies in these challenging diseases. Citation Format: Ipsita Pal, Anuradha Illendula, Andrea Joyner, Tess M. Deddens, Deepthi P. Damera, Todd E. Fox, Marya E. Dunlap-Brown, Kallesh D. Jayappa, Thomas P. Loughran, David J. Feith, Owen A. O'Connor. Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3201.

Effective treatment with the selective cytokine inhibitor BNZ-1 reveals the cytokine dependency of T-LGL leukemia

AbstractT-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes that can result in severe neutropenia, anemia, and bone marrow failure. Strong evidence from patients and mouse models demonstrate the critical role of interleukin-15 (IL-15) in T-LGLL pathogenesis. BNZ-1 is a pegylated peptide that selectively inhibits the binding of IL-15 and other γc cytokines to their cellular receptor complex, which has demonstrated efficacy in ex vivo T-LGLL cells and transgenic mice in preclinical studies. We conducted a phase 1/2 trial of BNZ-1 in patients with T-LGLL who had hematocytopenias (anemia or neutropenia) and required therapy. Clinical responses were assessed using hematologic parameters (improvement in hematocytopenias) based on response criteria from the Eastern Cooperative Oncology Group 5998 T-LGLL trial. BNZ-1 demonstrated clinical partial responses in 20% of patients with T-LGLL with minimal toxicity and the maximum tolerated dose was not reached. Furthermore, T-LGL leukemic cells showed significantly increased apoptosis in response to BNZ-1 treatment as early as day 2, including in clinical nonresponders, with changes that remained statistically different from baseline throughout treatment (P < .005). We report first-in-human proof that T-LGL leukemic cells are dependent on IL-15 and that intervention with IL-15 inhibition with BNZ-1 in patients with T-LGLL shows therapeutic effects, which carries important implications for the understanding of the pathogenesis of this disease. This trial was registered at www.clinicaltrials.gov as #NCT03239392.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.

DR-01, a Non-Fucosylated Anti-CD94 Antibody, Depletes Leukemic Cells in Ex Vivo and In Vivo Models of Large Granular Lymphocyte Leukemia

Large granular lymphocyte leukemia (LGLL) is a rare leukemia more frequently occurring in the elderly population with three major subtypes: T-LGLL, chronic lymphoproliferative disorder of NK cells (CLPD-NK) and aggressive NK cell leukemia (ANKL). Over 50% of patients develop complications including severe neutropenia, thrombocytopenia and anemia, thus predisposing patients to opportunistic infections. In addition, treatments are limited to standard immunosuppressive therapy such as methotrexate, cyclosporine or cyclophosphamide, with complete response rates of only 50% or less for each agent. Thus, novel and effective therapies for LGLL patients are critically needed. Dren Bio is developing DR-01, a non-ligand blocking, non-fucosylated monoclonal antibody targeting CD94. CD94 is a type II transmembrane receptor identified on mature, cytotoxic lymphocytes such as healthy NK and terminally differentiated CD8 T cells. In the context of disease, CD94 is known to be upregulated on LGLL cells. DR-01 functions through depletion of target cells via antibody-mediated cellular cytotoxicity (ADCC) by means of fratricide, a method in which the same cell type induces ADCC on each other. Through characterization of DR-01 using various ELISA and cell-based assays, DR-01 was determined to bind with nanomolar affinity to CD16a (EC50: 0.04µg/mL) and to CD94 on healthy human donor NK cells, cynomolgus monkey NK cells and LGLL cells (EC50: 0.5-1nM). DR-01 induces potent, dose-dependent depletion of CD94+ cells in ex vivo ADCC assays (IC50: 1ng/mL) of peripheral blood mononuclear cells (PBMCs) from healthy and diseased individuals. Furthermore, DR-01 has been shown to be well tolerated in a 4-week dose-range finding and Good Laboratory Practice (GLP) study in cynomolgus monkeys from 5-100mg/kg on a weekly dosing regimen. A 12-color flow cytometry panel was applied to a cohort of patient LGLL PBMCs to profile CD94 expression and other cytotoxic markers associated with the disease, including CD16, CD57 and functional binding partners of CD94 such as NKG2A and NKG2C. CD94 was identified on 97% (37/38) of patient samples screened, ranging from 400 to 725,000 receptors per cell. All patient leukemic cells expressed variable levels of CD16 and CD57. NKG2A and NKG2C were present in over 5% of total leukemic cells in 37% (14/38) and 21% (8/38) of patient samples, respectively. In addition, DR-01 was able to deplete LGL leukemic cells with high potency and in a dose-dependent manner in a functional ADCC assay (IC50:1-10ng/mL). The mechanism of fratricide was demonstrated through degranulation of the leukemic population via expression of CD107a. Cytokine profiling results are pending. To demonstrate in vivo efficacy of DR-01 in a disease model of LGLL, humanized NSG-Tg(hIL-15) mice engrafted with patient LGLL splenocytes were utilized. Optimal splenocyte engraftment (5-20% human CD45+ cells in blood) was achieved within 3 weeks post-inoculation with 10 million PBMCs per pre-sublethally irradiated mouse. The engrafted leukemic population partially preserved the CD94 expression profile from the patient leukemic cells and was successfully depleted (>90%) in blood, bone marrow, spleen and liver 1-week post-treatment with a single dose of DR-01 at 5mg/kg. Overall, DR-01 is a non-fucosylated anti-CD94 antibody with high affinity and potent leukemic cell depleting ability ex vivo and in vivo. The favorable toxicity profile of DR-01 in cynomolgus monkeys precede clinical studies in LGLL patients. Additional profiling and evaluation of DR-01 in LGLL patient samples ex vivo as part of a pre-phase I study is ongoing. A phase I/II trial has been initiated to assess the safety and efficacy of DR-01 in previously treated LGLL patients.

Safety of mRNA COVID-19 Vaccines in Patients with Inborn Errors of Immunity: an Italian Multicentric Study

PurposeLittle is known about vaccine safety in inborn errors of immunity (IEI) patients during the current vaccination campaign for COVID-19. To better investigate the reactogenicity and adverse event profile after two, three, and four doses of mRNA vaccines, we conducted an observational, multicentric study on 342 PID patients from four Italian Referral Centres.MethodsWe conducted a survey on self-reported adverse reactions in IEI patients who received mRNA vaccine by administering a questionnaire after each dose.ResultsOver the whole study period, none of the patients needed hospitalization or had hypersensitivity reactions, including anaphylaxis and delayed injection site reaction. After two vaccination doses, 35.4% of patients showed only local reactogenicity-related symptoms (RrS), 44.4% reported both systemic and local RrS, and 5% reported only systemic RrS. In more than 60% of cases, local or systemic RrS were mild. After the first and second booster doses, patients showed fewer adverse events (AEs) than after the first vaccination course. Patients aged 50 years and older reported adverse events and RrS less frequently. Among AEs requiring treatment, one common variable immune deficiency patient affected by T cell large granular lymphocytic leukemia developed neutropenia and one patient had Bell’s paralysis perhaps during herpes zoster reactivation.ConclusionAlthough our follow-up period is relatively short, the safety data we reported are reassuring. This data would help to contrast the vaccine hesitancy often manifested by patients with IEI and to better inform their healthcare providers.

The spectrum of somatic mutations in large granular lymphocyte leukemia, rheumatoid arthritis, and Felty's syndrome

T cell large granular lymphocyte leukemia (T-LGLL) is an interesting case at the intersection of autoimmunity and cancer. In T-LGLL, T cells with somatic pathogenic mutations (mainly in STAT3) are linked to rheumatoid arthritis (RA) and neutropenia. A rare subtype of RA, Felty's syndrome, exhibits overlapping clinical features and comparable frequencies of activating STAT3 mutations in T cells as T-LGLL, which hints at a potential T-LGLL–Felty's syndrome–RA axis. Somatic mutations could shed light on the unexplained pathologies of these disorders. However, the causality of somatic mutations–do somatic mutations in immune cells cause inflammation, or does prolonged inflammation predispose to mutagenesis–remains unanswered. This review will focus on the recent advances in understanding somatic mutations in T-LGLL and related autoimmune conditions as a master regulatory network that sustains lymphoproliferation and inflammation.

Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia

T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCRαβ-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell–cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFNγ. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype.

T cell clonal expansion and STAT3 mutations: a characteristic feature of acquired chronic T cell-mediated pure red cell aplasia

Acquired chronic pure red cell aplasia (PRCA) develops idiopathically or in association with other medical conditions, including T cell large granular lymphocytic leukemia (T-LGLL) and thymoma. T cell dysregulation is considered a cardinal pathogenesis of PRCA, but genetic-phenotypic associations in T cell abnormalities are largely unclear. We evaluated an extended cohort of 90 patients with acquired PRCA, including 26 with idiopathic, 36 with T-LGLL-associated and 15 with thymoma-associated PRCA, for their T cell immuno-phenotypes, clonalities and STAT3 mutations. TCR repertoire skewing of CD8+ T cells was detected in 37.5% of idiopathic, 66.7% of T-LGLL-associated and 25% of thymoma-associated PRCA patients, and restriction to Vβ1 was most prominent (41%). Clonalities of TCRβ or γ chain and STAT3 mutational status were statistically associated (P = 0.0398), and they were detected in all three subtypes. The overall response rate to cyclosporin A was 73.9%, without significant difference by subtypes nor STAT3 mutational status. The T cell dysregulations, such as TCR repertoire skewing with predominant Vβ1 usage, clonality and STAT3 mutations, were frequently found across the subtypes, and the close associations between them suggest that these T cell derangements reflect a common pathophysiological mechanism among these PRCA subtypes.

Cytokines in the Pathogenesis of Large Granular Lymphocytic Leukemia

Large granular lymphocytic leukemia (LGLL) is a lymphoproliferative disorder of older adults characterized by the clonal expansion of cytotoxic T/natural killer cells due to constitutive pro-survival signaling. In recent years, it has become clear that cytokines and their receptors are aberrantly expressed in LGLL cells. The exact initiation process of LGLL is unknown, although several cytokine-driven mechanisms have emerged. Elevated levels of several cytokines, including interleukin-15 (IL-15) and platelet-derived growth factor (PDGF), have been described in LGLL patients. Evidence from humans and animal models has shown that cytokines may also contribute to the co-occurrence of a wide range of autoimmune diseases seen in patients with LGLL. The goal of this review is to provide a comprehensive analysis of the link between cytokines and pro-survival signaling in LGLL and to discuss the various strategies and research approaches that are being utilized to study this link. This review will also highlight the importance of cytokine-targeted therapeutics in the treatment of LGLL.

Clinical and pathophysiological features of acquired pure red cell aplasia: based on the concept of T-cell dysregulations

Acquired pure red cell aplasia (PRCA) develops in a variety of contexts and thus, should be regarded as a syndrome. The three major subtypes of acquired PRCA are idiopathic PRCA, T cell large granular lymphocytic leukemia (T-LGLL)-associated PRCA, and thymoma-associated PRCA. Although the Japanese National Research Group on Idiopathic Bone Marrow Failure Syndromes of the Ministry of Health, Labor and Welfare of Japan has made significant contributions to our understanding of PRCA, details of its clinical characteristics, and pathophysiological mechanisms remain largely unknown. A recent epidemiological analysis using the JSH Hematologic Disease Registry revealed that approximately 100 new cases with acquired PRCA were diagnosed annually in Japan, which was higher than previously thought to be. The median age of the patients was 73 years. A prospective observational study on chronic PRCA (PRCA2016) is currently ongoing, and it may provide new clinical insights into acquired PRCA. Dysregulation of T cells has been shown to play a central role in PRCA. We studied T cell clonalities and STAT3 mutations in 90 PRCA patients and discovered that clonal T cell expansions were frequently recognized and closely associated with STAT3 mutations in the three major types of PRCA.

Therapeutic Efficacy of Mcl-1 Antagonism in Large Granular Lymphocyte Leukemia

Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit. DisclosuresGru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Case Report of Aggressive T-Cell Large Granular Lymphocytic Leukemia

Large granular lymphocyte (LGL) leukemia is a spectrum of rare lymphoproliferative disorders. Two entities are reported: indolent T-cell LGL leukemia and aggressive NK cell leukemia. However, a review of the literature identified cases of aggressive T-cell LGL. Considering this unusual and rare clinical presentation, we are reporting a case of aggressive T-cell LGL leukemia.A 53-yo man presented in May 2014 with a one year history of constitutional symptoms, splenomegaly, mild lymphocytosis and a pancytopenia (hemoglobin 105 g/L, neutrophils 0,5 x 109 /L and platelets 80 x 109 /L). He had similar symptoms at the age of 24 that resolved without further investigation. Bone marrow evaluation revealed a massive infiltration by small to medium sized lymphocytes with irregular nucleus CD3 +, CD4 -, CD7 -, CD8 +, CD25 -, CD33 -, CD34 -, CD56 -, CD57 -, TCRαβ+. HTLV-1 serology was negative. His pancytopenia did not improve with 3 cycles of CHOP followed by 4 cycles of ESHAP. In January 2015, indolent T LGL was finally retained. The patient was observed up to July 2015 where methotrexate was begun because of frequent infections and symptomatic splenomegaly. A splenectomy was performed in January 2016 due to a toxicity to methotrexate and persistent symptoms from splenomegaly. Pathology report showed a splenic and lymph node infiltration by mature T CD8+ lymphocytes supporting the T LGL diagnosis. Post splenectomy, cytopenia improved and progressed in June 2016 with a lymphocytosis (82 x 109/L), new lung infiltrates and acute renal insufficiency. The lung biopsy revealed an infiltration by the T-cell LGL and renal imaging suggested infiltration by this T-cell LGL as well. Based on the weak expression of CD20+ on the splenic pathology, rituximab was attempted in August 2016. The patient rapidly progressed fatally with systemic involvement of T-cell LGL (skin, lungs, liver, kidney, facial cranial nerves, conus medullaris and bone marrow).This case therefore depicts a very aggressive case of T-cell LGL with an atypical clinical presentation. In contrast to the indolent variant and as seen in the few similar cases retrieved in literature, the clinical presentation of the aggressive T-cell LGL leukemia is characterized by constitutional symptoms, rapidly progressive hepatosplenomegaly, cytopenia with/without organic infiltration and an important resistance to treatment. Moreover, concerning the pathological findings, the medullary involvement of our patient was massive without real images of intra-sinusal lymphocytosis and the T-cell of this T-cell LGL were atypical because of their small-medium size with slightly irregular nuclei and the lack of expression of CD56 and CD57. However, the few studies on aggressive T-cell LGL leukemia retrieved similar findings. Even though there is no consensus on the optimal therapeutic management of this disease, we believe that the treatments given to our patient were consistent with literature and did not add an additional mortality risk.To conclude, LGL cell leukemia represent a spectrum of indolent and aggressive diseases, whereby an indolent form can evolve into an aggressive form. Aggressive T-cell LGL leukemia are characterized by a multisystem disease, an atypical immunophenotype (CD56-, CD57-) and are associated with an uncertainty regarding therapeutics. [Display omitted] DisclosuresFleury:Gilead: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

STAT3 activation in large granular lymphocyte leukemia is associated with cytokine signaling and DNA hypermethylation

Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NFκB levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3Y640F and STAT3G618R mutants compared to KAI3 NK cells overexpressing STAT3WT. Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments.

Altered Spectrum of Lymphoid Neoplasms in a Single-Center Cohort of Common Variable Immunodeficiency with Immune Dysregulation

PurposeCommon variable immune deficiency (CVID) confers an increased risk of lymphoid neoplasms, but reports describing the precise WHO specification of the lymphoma subtypes and their immunological environment are lacking. We therefore classified lymphomas—occurring in a cohort of 21 adult CVID patients during a 17-year period at our center—according to the 2016 WHO classification and characterized the local and systemic immunological contextResultsThe median time between the onset of CVID and lymphoma was 14 years. Patients showed a high prevalence of preceding immune dysregulation: lymphadenopathy (n = 13, 62%), splenomegaly (n = 18, 86%), autoimmune cytopenia (n = 14, 67%), and gastrointestinal involvement (n = 15, 71%). The entities comprised extranodal marginal zone lymphoma (n = 6), diffuse large B cell lymphoma (n = 7), plasmablastic lymphoma (n = 1), classic Hodgkin lymphoma (n = 4, including three cases with germline CTLA4 mutations), T cell large granular lymphocytic leukemia (n = 2), and peripheral T cell lymphoma, not otherwise specified (n = 1), but no follicular lymphoma. An Epstein-Barr virus association was documented in eight of 16 investigated lymphomas. High expression of PDL1 by tumor cells in five and of PDL1 and PD1 by tumor-infiltrating macrophages and T cells in 12 of 12 investigated lymphomas suggested a tolerogenic immunological tumor environment.ConclusionIn summary, a diverse combination of specific factors like genetic background, chronic immune activation, viral trigger, and impaired immune surveillance contributes to the observed spectrum of lymphomas in CVID. In the future, targeted therapies, e.g., PD1/PDL1 inhibitors in CVID associated lymphomas with a tolerogenic environment may improve therapy outcome.

Thymoma-associated multiorgan autoimmunity with cutaneous only presentation: A case report.

Thymoma-associated multiorgan autoimmunity disease can be seen in patients with thymomas and presents with features of graft versus host disease. Here, we report a case of a 52-year-old woman with a complex medical history including malignant thymoma, myasthenia gravis, Good’s syndrome, and T cell large granular lymphocytic leukemia who presented with a diffuse pruritic rash ultimately found to be compatible with a cutaneous presentation of thymoma-associated multiorgan autoimmunity disease. The eruption heralded the recurrence of the malignant thymoma and the rash was ultimately found to be resistant to a multitude of therapies except for oral corticosteroids.

Chronic urticaria versus dermatomyositis in a case of T- cell large granular lymphocytic leukemia

Aim: The reported case involved a complicated diagnostic path, not only because of the usual difficulties specific to chronic urticaria but, also because the trigger was a T-cell large granular lymphocytic leukemia (T-LGLL), a rare type of leukemia with an indolent course whose etiology is still not well known. This leukemia is also known for its propensity to cause autoimmune diseases. The aim of this study was to identify whether the muscle damage was caused by dermatomyositis or by T-LGLL. Methods: After elevated muscle enzyme levels had been discovered magnetic resonance imaging (MRI) revealed muscle damage. Consequently, a muscle biopsy was performed in a targeted manner. In addition to muscle biopsy, transmission electron microscopy and anti-CN1A antibody testing were performed. Results: MRI of the lower limbs and pelvic girdle indicated moderate fibroadipose substitution in many muscles, moderate edema in others. No involvement of the shoulder girdle and upper limbs. Histological examination of the muscle fibers showed an “inflammatory myopathy with isolated phagocytotic fibers.” Inclusion-body myositis, which is known to be associated with chronic T-LGLL was excluded. On the same biopsy transmission electron microscopy confirmed inflammatory myopathy and anti-CN1A antibodies were positive. DNA extracted from the muscle of the micro-rearrangement for the y-chain of the TCR identified on the DNA extracted from peripheral blood was positive. Conclusions: Conclusions: Chronic urticaria was an indication of immunoproliferative disease. Myositis was the pathology due to T-LGLL, and dermatomyositis was excluded.

Unusual Case of Hepatic Sinusoidal Obstruction Syndrome Induced By Low Dose Oral Cyclophosphamide

Hepatic sinusoidal obstruction syndrome (SOS) has been reported in patients receiving high dose intravenous (IV) cyclophosphamide especially as a part of preparative regimen for hematopoietic stem cell transplantation. Hepatic SOS from low dose oral cyclophosphamide has not been reported. Here we present a rare case of hepatic SOS induced by low dose oral cyclophosphamide. A 78-year-old male with history of coronary artery disease status post coronary artery bypass graft (CABG) was initially seen in hematology clinic for evaluation of normocytic anemia. During workup, bone marrow biopsy revealed NK cell large granular lymphocytic leukemia (LGL). He was then started on low dose oral cyclophosphamide 50 mg daily. After 2 months, his dose was reduced to 25 mg daily due to severe neutropenia. He then tolerated cyclophosphamide well and his hemoglobin (Hb) improved from his baseline 8 gm/dl to 12 gm/dl in 4 months. After 6 months of treatment, he was admitted to the hospital for abdominal distension, ascites and 15 pounds weight gain over 3 weeks. Labs most prominent on admission were Hb of 12.5 gm/dL , WBC 5.3 x 103/µL, Platelets 197 x 103/µL, total bilirubin 1.6 mg/dL, aspartate aminotransferase (AST) 89 U/L , alanine aminotransferase (ALT) 35 U/L and alkaline phosphatase (ALP) 283 U/L. Workup of ascites included abdominal paracentesis which revealed serum ascites albumin gradient(SAAG) &amp;lt;1.1 and fluid cytology was negative for carcinoma. Ultrasound of abdomen showed diffuse parenchymal heterogeneity and increased echogenicity of liver with patent hepatic vessels with normal direction of blood flow. Echocardiogram revealed preserved left ventricular systolic function with estimated left ventricular ejection fraction of 60%. He then underwent transjugular liver biopsy which revealed chronic lymphoproliferative disorder of NK cells, prominent centrilobular sinusoidal dilatation and congestion, lobular disarray, cholestasis and sinusoidal fibrosis suggestive of veno-occlusive disease secondary to drug/toxin induced liver injury. The Naranjo adverse drug reaction probability scale indicated a probable relationship between hepatic SOS and cyclophosphamide. Cyclophosphamide was held. Bone marrow biopsy showed the patient was in remission. His liver enzymes are improving and hematologic counts continue to be stable while on observation. High dose of IV cyclophosphamide given as myeloablative therapy in combination of total body irradiation or busulfan (or other agents) in preparation for hematopoietic stem cell transplantation can induce hepatic SOS, which can be severe leading to acute liver failure and death. The diagnosis is usually based on clinical features of tender hepatomegaly, weight gain, ascites and jaundice. Liver biopsy is usually diagnostic. Mechanism of injury is related to direct toxic effects of cyclophosphamide on sinusoidal cells in the liver, causing their necrosis and release into the sinusoids, obstruction and obliteration of hepatic veins. This case demonstrates that hepatic SOS can also be caused by low dose oral cyclophosphamide. Given this rare occurrence, it is prudent for the clinicians to keep an open mind and consider hepatic SOS as a potential side effect even with oral cyclophosphamide. Disclosures Yacoub: Novartis: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Roche: Other: Support of parent study and funding of editorial support.

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&gt;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.

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.