Large Granular Lymphocyte Leukemia Research Articles

Blockade of IL-2 and IL-15 Gamma Chain Receptor Signaling Decreases Leukemic Cell Viability in T-Cell Large Granular Lymphocyte Leukemia and Adult T-Cell Leukemia

IntroductionInterleukin-15 (IL-15) and IL-2 are important cytokines that drive the progression of many T-cell malignancies such as T-cell Large Granular Lymphocyte Leukemia (T-LGLL) and HTLV-1 derived Adult T-cell Leukemia (ATL).T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with a constitutively active JAK/STAT pathway controlled by cytokines including IL-15 and IL-2. A computational systems approach identified IL-15 as a master regulator of T-LGLL survival and patients have increased IL-15/IL-15Ra serum levels. Cytokine signaling leads to increased phosphorylation and activation of STAT proteins. T-LGLL cells exhibit STAT3 or STAT5 mutational activation in some cases and constitutive activation in all patients. T-LGLL treatments are not selective or curative, and the commonly utilized immunosuppressive agents are not effective in all patients.ATL is an aggressive T-cell disorder that also exhibits aberrant IL-2 and IL-15 cytokine signaling. Tax, identified as an essential oncogene in the disease, is associated with increased cytokine and cytokine receptor expression. ATL patients have high IL-2Ra gene expression and PBMCs from chronic/smoldering ATL patients can depend on autocrine production of cytokines for proliferation. Patients with ATL have poor prognosis and current cytokine-targeting therapies have not proven to be efficacious.The gc receptor is shared by the gc-family cytokines including IL-2, -4, -7, -9, -15 and -21. BNZ-1 (formerly known as BNZ132-1-40) is a pegylated peptide designed to specifically bind the gc receptor to block IL-2, IL-15 and, to a lesser extent, IL-9 cytokine signaling. Because these cytokines are vital to the survival of T cells in T-LGLL and ATL, these diseases represent ideal models to test the hypothesis that treatment of leukemic cells with BNZ-1 will result in reduced proliferation and viability.MethodsThese studies utilized a human T-LGL cell line (TL1), PBMCs from T-LGLL patients and ATL patients, and human IL-15 transgenic NSG mice injected with the ED40515(+) ATL cell line expressing luciferase. In vitro assays were performed after an overnight cytokine withdrawal and 20-minute pretreatment with BNZ-1 prior to addition of cytokine. Leukemic mice were divided into 3 treatment groups; BNZ-1 (40 mg/kg i.v. 2x per week for 4 weeks), JAK1/2 inhibitor Ruxolitinib (Ruxo) (50 mg/kg/day s.c. by pump for 28 days) and PBS control. Tumor burden was measured through bioluminescence imaging (Xenogen IVIS).ResultsOur results demonstrate that in vitro treatment of TL1 cells with BNZ-1 inhibits IL-2 and IL-15 mediated expansion and viability. BNZ-1 impaired the growth of these cytokine-dependent cells to the level observed in cells without exogenous cytokine supplementation. In addition, BNZ-1 was able to drastically block IL-15 and IL-2 induced phosphorylation events, specifically STAT1, STAT3, STAT5, ERK and Akt. The compound blocked IL-15 mediated viability and signaling more effectively than that mediated by IL-2.The BNZ-1 mediated decreased viability of PBMCs from T-LGLL patients was observed in the presence of either IL-2 or IL-15 stimulation and regardless of STAT3 mutational status. A potent reduction of IL-2 and IL-15 mediated STAT1, STAT3, STAT5 and ERK phosphorylation was also observed in T-LGLL PBMCs with BNZ-1 treatment. Proliferation of PBMCs from ATL patients was inhibited by 58 to 80 percent by the addition of either the antiIL-2Ra antibody daclizumab or and the combination treatment of daclizumab, anti-IL-15 and anti-IL-9. BNZ-1 at 10 µM inhibitedproliferation by 60 to 80 percent.Leukemic burden in the ATL tumor bearing mice was reduced by both BNZ-1 and Ruxo when compared to controls. The BNZ-1 treatment group showed comparable efficacy to that observed in the Ruxo group. Furthermore, BNZ-1 therapy was associated with lower levels of IL-2Ra, which reflects reduced tumor burden at 4 and 7 weeks when compared to that observed with the PBS group.In summary, BNZ-1 shows promise as a potential therapy for T-LGLL and ATL. Results from this study support its advancement in clinical trials in the treatment of T-LGLL and ATL patients. DisclosuresTagaya:Bioniz Therapeutics: Employment. Azimi:Bioniz Therapeutics: Employment.

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.

Acquired Somatic Mutations in T Cells in Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndromes

Background: Vigorous cell division increases the probability of acquiring mutations during DNA replication. Such may occur in T cells when they recognize their cognate antigen and proliferate. Conventionally, acquired somatic mutations have been associated with the development of malignancy. In T cells, somatic mutations may also lead to autoimmunity, as in large granular lymphocyte (LGL) leukemia with STAT3 mutations. Somatic STAT3 mutations have also been detected in patients with immune-mediated bone marrow failure (BMF) disorders without concomitant LGL lymphoproliferation. These BMF disorders, aplastic anemia (AA) and hypoplastic myelodysplastic syndromes (hMDS), are responsive to immunosuppressive therapy, indicating autoimmune nature of diseases. In AA and hMDS, cytotoxic T cell receptor (TCR) repertoire is skewed and clonal autoreactive cytotoxic T cells are thought to cause hematopoietic stem cell destruction. Because previous findings suggest that these immune-mediated BMF disorders and LGL leukemia share common features, we investigated whether also in AA and hMDS cytotoxic T cells accumulate acquired somatic mutations in genes that are important for cell survival and immune regulation.Methods: We sorted CD8+ and CD4+ T cell fractions from blood and BM samples of patients diagnosed with AA (n=24, median age 60) or hMDS (n=13, median age 64). TCR repertoire was analyzed with TCRB CDR3 deep sequencing or TCR Vb-antibody based flow cytometry, and all selected patients had at least one cytotoxic CD8+ T cell clone >7%. CD4+ and CD8+ T cells were then sequenced with our custom gene panel covering 2000 essential genes of innate and adaptive immunity with mean sequencing depth of 270x. Results were compared to healthy controls (n=9, median age 48). For each subject, somatic mutations in CD8+ cells were called with VarScan2 using CD4+ cells as a “germline” control and vice versa. To reduce possible false positives, all detected mutations were evaluated visually in Integrative Genomics Viewers by two independent researchers in a blinded manner, with agreed and precise criteria to distinguish real and false mutations. Concordant results were validated with amplicon sequencing. The enrichment of cell specific somatic mutations in biological pathways among AA and MDS patients was compared to healthy controls.Results: Somatic mutations in CD4+ and CD8+ T cells were detected both in patients (29/35, 83%) and controls (7/9, 78%). The number of found somatic mutations was associated with the TCR clonality index in cytotoxic CD8+ T cells (r=0.76, p<0.0001). Although single mutations were frequently found in healthy controls' T cells, the number of mutations per subject was significantly higher in patients' CD8+ T cells compared to controls (mean[range] of found somatic mutations per subject for each group: AA CD4 0.9[0-6] and CD8 3.5[0-10]; MDS CD4 0.7[0-2] and CD8 2.3[0-8]; controls CD4 1.1[0-3] and CD8 0.9[0-3]). The number of mutations did not correlate with age. In cases from which both blood and BM samples were available, mutations were detected in T cells derived from both compartments. Most of the genes that had somatic mutations in healthy controls had also mutations in AA and MDS patients (e.g. DNMT3A, TLR2). Although mutations were mostly distinct in individual patients, they accumulated in important immune regulatory pathways. When compared to healthy controls, AA and hMDS patients had more mutations in JAK-STAT signaling (SOCS7, STAT3, PTPN2), MAPK signaling (KRAS, PTPN5, TP53), other cancer-related pathways and in pathways related to viral carcinogenesis and HTLV-I infection. Notably, most of the BMF patients' T-cell mutations were in other genes than STAT3 or genes related to clonal hematopoiesis.Conclusions: Our findings confirm that a subset of immune-mediated BMF disorders may have similar pathogenesis to LGL leukemia highlighting the importance of JAK-STAT signaling pathway in immunoregulation and autoimmunity. Acquired somatic mutations in inflammatory cells leading to dysregulation of immune system may be the common denominator in the pathogenesis of autoimmune disorders and hematologic malignancies. Deep molecular depth analysis and functional studies on found somatic mutations can further illuminate the pathogenesis of immune-mediated BMF syndromes and other autoimmune disorders. DisclosuresMaciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees. Mustjoki:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.

High Frequency of Subclonal Myeloid Mutations Can be Found in Large Granular Lymphocyte Leukemia

Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferation of cytotoxic T-lymphocytes often associated with immune cytopenias. Somatic mutations in STAT3 were found in a proportion of LGL and provide a good diagnostic marker for positive cases but a large proportion of LGL cases is negative. More polyclonal CTL reactions directed against hematopoietic stem and progenitor cells operate in aplastic anemia but have been also postulated as a pathogenetic factor leading to cytopenias in some cases of myelodysplastic syndromes (MDS). Increased CTL activation and LGL can be occasionally seen in MDS and previously, concomitant LGL and MDS have been described. According to one theory behind LGL pathogenesis, proliferation is driven by a more or less specific tumor surveillance reaction. Moreover, asymptomatic clonal hematopoiesis, T cell clonopathy of unclear significance as well as LGL show an overlapping demographic.By applying a rational screening algorithm for LGL in MDS (to identify treatable causes of immune cytopenia) we have identified patients with concomitant MDS and T-LGL and determined their somatic molecular profile. We have hypothesized that some of the T-LGL cases may be associated with the presence of clonal somatic mutations seen in MDS, consistent with subclinical MDS or clonal hematopoiesis of indeterminate potential (CHIP). We stipulated that LGL may indeed correspond to more or less successful tumor surveillance reaction and thus myeloid mutations would be more commonly seen in LGL than expect based on age. Consequently, according to the reverse principle we have screened patients with LGL for the presence of clonal myeloid mutations.We have studied 225 LGL patients; among them by traditional pathomorphologic criteria we have identified 9 (3%) patients with concomitant MDS; 1/9 had STAT3D661V, 8/9 patients had chromosomal abnormalities, including del(20q) (n=1), del(5q) (n=2), -X (n=1), -7 (n=3) and complex karyotype in 1 patient. When “pure” LGL patients were assessed using deep NGS we found STAT3 mutations in 27% of our LGL cohort including most common D661V/Y followed by Y640F but also less frequent hits such as N647I, S614R, K658*/H/N mainly mapped in the SH2 domain (584-674) of the protein. Conversely, the analysis of 472 MDS did not identify any STAT3 mutations.A subset of LGL patients without evidence for the morphologic or cytogenetic presence of MDS (n=66; excluding the cases with concomitant MDS) were then selected for targeted NGS with a panel containing 65 genes recurrently found in myeloid neoplasms (MDS panel). Excluding STAT3 mutations, hits in myeloid cells were found in 36/66 (55%) of patients with 12 myeloid mutations in STAT3MT and 24 mutations in STAT3WT. For those with clonal events the average number of mutations was 2.3 and average VAF was 17-57%. The most common mutated genes included: KMT2D (6/66; 9%), KDM6A/B (5/66; 8%), TET2 and (3/66; 5% each), and ASXL1 (2/66; 3%) SF3B1, CUX1, GNAS, IDH1, NPM1 among others (e.g., ATR, BARD1, BRIP1, CDKN2A, SETD2; N=17 ) .In conclusion, somatic mutations in common “myeloid” genes can be found in patients with LGL without morphologic presence of MDS. The much higher than expected rate detection in LGL (median age of 63 yrs.) when compared to the elderly patients with clonal hematopoiesis (NEJM 2014, Jaiswal et.al) may lead to a stipulation that the presence of aberrant myeloid clones may be sensed by LGL and thus some of LGL occurring in older patients may reflect an abortive tumor surveillance reaction. Targeted gene sequencing has helped identify a spectrum of MDS-related subclonal myeloid mutations in LGL and thereby further our understanding of the pathogenesis of LGL. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

TCR γδ Large Granular Lymphocyte Leukemia (LGLL) Displays Features between CD8+ and CD4+ TCR αβ LGLL

BackgroundT-large granular lymphocyte leukemia (T-LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Usually, leukemic cells of this T-cell neoplasia express the αβ T-cell receptor (TCR); only a small subset of cases expresses the γδ TCR denoting the TCRγδ-LGLL. Currently, among the different LGL diseases, TCRγδ-LGLL remains less studied and several clinical and laboratory data already described in TCRαβ-LGLL have not yet been explored in TCRγδ-LGLL. In particular, data are still missing about STAT3 and STAT5b mutations, frequently observed in TCRαβ-LGLL, and about T-LGL clonotypes discovered to be private to TCRαβ-LGLL as compared to healthy controls.AimsThis work was aimed to perform the analysis on: STAT3 and STAT5b mutations, LGL immunophenotype, TCRγ clonotype repertoire and clinical presentations in a group of TCRγδ-LGLL patients. The objective was to reveal common and different features of this rare kind of LGLL with the more frequent TCRαβ-LGLL.MethodsThe cohort of study included 11 patients affected by TCRγδ-LGLL. Sanger sequencing was used for mutational analysis on hot-spot regions of the 2 genes more frequently mutated in LGL disorders, STAT3 and STAT5b . Immunophenotype of LGL clone was defined by flow cytometry analysis. CDR3 repertoire and frequency distribution of TCR gamma gene rearrangements were obtained by Next-Generation Sequencing (NGS).ResultsTCRγδ-LGLL showed a higher occurrence of STAT mutations (82%) as compared to TCRαβ-LGLL (43%). In detail, 9 out of 11 TCRγδ LGLL patients were characterized by STAT3 or STAT5b mutations in a mutually exclusive pattern. Being characterized by both STAT3 and STAT5b mutations, TCRγδ LGLL resembles both CD8+ TCRαβ-LGLL and CD4+ TCRαβ-LGLL, the first being mostly associated to STAT3 mutations and the latter to STAT5b . Similarly, TCRγδ-LGLL showed the same relationship observed between immunophenotype and kind of mutation in TCRαβ-LGLL: TCRγδ-LGLL patients with CD56- LGL immunophenotype were characterized by STAT3 mutations (as in CD8+ TCRαβ-LGLL), while TCRγδ-LGLL patients with CD56+ LGL immunophenotype by STAT5b mutations (as in CD4+ TCRαβ-LGLL). Anyway, in TCRγδ-LGLL no correlation was found between mutations and clinical course as observed in CD8+ TCRαβ-LGLL patients carrying STAT mutations, rather, we observed that patients with γδLGLs positive for Vδ2 showed frequently indolent course, while Vδ1 was associated to a more symptomatic disease (4 out of 5 symptomatic patients were Vδ1+). By NGS we obtained the TCR gamma repertoire of each patient and we revealed that all patients were clonal with the exception of 2 showing a polyclonal pattern. The 2 polyclonal cases were the only 2 patients without STAT mutations. Among the remaining STAT3 mutated patients (n=4), 3 were polyclonal and one biclonal, while STAT5b mutated patients (n=5) were more frequently monoclonal (4/5 monoclonal and 1/5 biclonal). Considering the CDR3 repertoire of the immunodominant clones, as observed in TCRαβ-LGLL, common clonotype sequences were present with low frequency in almost all TCRγδ-LGLL patients. Interestingly, 2 different CDR3 sequences were found shared in different patients at frequency >10% of the total rearrangements. In terms of clonal rearrangements, Vγ3-Jγ1/2, Vγ9-JγP and Vγ8-Jγ1/2 were the combination usages most frequently detected, showing a limited combinatorial diversity of TCR genes similarly to CD4+ and differently from CD8+ TCRαβ-LGLL, this last having larger variability. Analysis about the clonotype repertoire in a 2 years-follow up showed that patients maintain their clonal pattern along the time.ConclusionsOur results highlight common and different characteristics of TCRγδ-LGLL as compared to TCRαβ-LGLL. The data suggest that TCRγδ-LGLL can be considered the intersection of the 2 types of TCRαβ-LGLL, sharing CD8+ or CD4+ T-LGLL mutational and phenotype features. Similarly to TCRαβ-LGLL, also TCRγδ disease showed a decreased diversity of TCR repertoire, with common TCR sequences shared among patients and with limited number of Vγ-Jγ combinations. A distinctive feature of TCRγδ-LGLL is that STAT mutations do not correlate with a symptomatic clinical behavior, rather, Vδ1 expression appears to be indicative for symptomatic disease. DisclosuresGalimberti:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau.

A Single Institutional Experience of 49 Patients with Large Granular Lymphocytic Leukemia: Clinical and Immunophenotypic Characteristics and the Significance of Immune Therapy

T-cell large granular lymphocytic leukemia (LGL) is a rare type of T-cell lymphomas characterized by proliferation of clonal LGL (in the amount exceeding 0.5 x 109/L) that generally have CD3+, CD8+, CD16+, CD57+, CD5+ and/or CD7+, TCRαβ+, TIA1+, granzyme B+ phenotype, as well as by chronic benign clinical course (median overall survival is >10 years). So far, first-line therapy for the disease (if it is clinically indicated, i.e. in case of anemia - hemoglobin concentration <100 g/L, thrombocytopenia <50х109/L, neutropenia <0.5х109/L, splenomegaly, recurrent infections, autoimmune disorders, B-symptoms) has not been established.The goal of the study: to evaluate clinical and immunophenotyping characteristics, hematological parameters and the results of initiating therapy in a large patient cohort.Forty-nine patients with verified T-LGL diagnosis were enrolled in the study based on the results of immunophenotyping and molecular genetic testing.Results: Our study showed that T-cell large granular lymphocytic leukemia was more often diagnosed in females (M:F=1:1.3) with the median age of 52 years. The results of the clinical data analysis were as follows: cytopenia affecting two lineages was observed in 51% of the patients (33% - anemia; 18% - thrombocytopenia); histologically confirmed bone marrow infiltration was revealed in 24% of patients; splenomegaly - in 31% of patients. Specific febrile fever, night sweats, weight loss (B-symptoms), frequently recurrent infectious complications were registered in 31% of patients. Diagnostically confirmed autoimmune disorders (rheumatoid arthritis etc.) were revealed only in 8% of cases.The immunophenotyping features of the tumor cells were as follows: CD3+ (in 59% of patients), CD8>CD4 (55%), CD4>CD8 (8%), TCRab+ (51%), TCRgd+ (35%), CD16CD56+ (45%), while double-negative profile (CD4-CD8) was revealed in 8% of cases.25 of 49 patients (51%) did not have indications for treatment initiation and were followed up. In other patients who required therapy initiation, the distribution of treatment options was as follows: low-dose methotrexate was used in 21% (80% demonstrated progressive disease/no response to therapy), cyclophosphamide (100 mg/day) - in 33% (CR was achieved in 50% of patients), cyclosporine A - in 8% (disease progression in all cases) cases, respectively; and 54% of patients received immune therapy with interferon alfa-containing drugs (CR and PR were achieved in 42% and 24% of patients, respectively). Median follow-up time in patients who received first-line therapy was 16.5 months.Conclusions: our retrospective study conducted in a large patient cohort showed that the disease was most often prevalent clinical signs being B-symptoms, recurrent infectious complications, anemia and anemia-related syndrome. 50% of patients did not require the initiation of specific therapy. It is the first study evaluating the use of immune therapy with interferon-alfa-containing drugs as a first-line therapy for the disease, which has demonstrated good and promising results (general response was achieved in 66% of patients). DisclosuresNo relevant conflicts of interest to declare.

Measurement of PD-1 Expression on Lymphoma Cells By Flow Cytometry Is a Useful Tool in Diagnosing and Monitoring Minimal Residual Disease in Angioimmunoblastic T-Cell Lymphoma

Introduction: Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most common types of peripheral T-cell lymphoma (PTCL), representing about 15-20% of cases of PTCL. Diagnosis of AITL is often challenging and a definitive diagnosis requires a use of ancillary testing including broad panels of immunohistochemistry and molecular studies. We evaluated the utility of evaluation of Programmed-Death-1 (PD-1) expression on lymphoma cells by flow cytometry as a diagnostic tool as well as monitoring minimal residual disease (MRD) of AITL.Methods: The study group was composed of 81 patients who were diagnosed with PTCL at Memorial Sloan Kettering Cancer Center between May 2015 and July 2017. This includes 36 patients with AITL, 6 with ALK-negative anaplastic large cell lymphoma (ALCL, ALK-), 2 with ALK-positive anaplastic large cell lymphoma (ALCL, ALK+), 9 with adult T-cell leukemia/lymphoma (ATLL), 11 with peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), 6 with nodal involvement by mycosis fungoides (MF), 5 with T-cell large granular lymphocytic leukemia (T-LGL), and 6 with T-cell prolymphocytic leukemia (T-PLL). The expression of PD-1 (CD279) on lymphoma cells was evaluated by 10-color flow cytometry using BV605-conjugated anti-CD279 antibody, and mean fluorescence intensity (MFI) was evaluated. Immunohistochemistry for PD-1, CD10, BCL6, and CXCR13 were also performed on tissue samples of AITL. Flow cytometry for variable regions of T-cell receptor beta chain (Vbeta) and genetic analysis for T-cell receptor gene rearrangement were performed on sorted abnormal T-cell population by flow cytometry in selected cases for the evaluation of clonality. Peripheral blood sample was used for the monitoring of MRD.Results: All AITL cases with tissue biopsy showed PD-1 immunoreactivity by immunohistochemistry (IHC), and PD-1 expression by flow cytometry showed excellent correlation with IHC. The abnormal CD4-positive PD-1 bright T-cell population detected by flow cytometry in AITL cases was confirmed to be clonal by restricted Vbeta fragment expression and molecular genetic analysis (Fig 1). On tissue samples, expression of other T-follicular helper cell markers was not observed by IHC in 7 cases (25%), and additional 3 cases (11%) presented with concomitant lymphoid neoplasm, which obscured morphologic and immunohistochemical evaluation for AITL. The latter 3 cases would be misdiagnosed by morphologic assessment alone. However, flow cytometry reliably detected AITL in all 10 challenging cases. Overall flow cytometry including PD-1 expression had 100% sensitivity in detection of AITL. The expression of PD-1 was significantly higher in AITL than that of other PTCL (CD279-BV605 MFI; Mean ± SEM; 6990.0 ± 934.9 vs. 547.3 ± 155.5; p<0.0001) (Fig 2A). PD-1 MFI differentiated AITL from other PTCL with high sensitivity and specificity (AUC 0.978; Fig 2B). The significant increase in PD-1 expression in AITL was a consistent finding with peripheral blood specimen. In 54 patients who had circulating lymphoma cells in peripheral blood, PD-1 expression was significantly higher in AITL than that of other PTCL (CD279-BV605 MFI; Mean ± SEM; 3079.0 ± 508.8 vs. 341.8 ± 54.17; p<0.0001: AUC 0.986) (Fig 2C, 2D). In 12 AITL patients who had tissue sample with concomitant bone marrow and peripheral blood specimens, flow cytometry detected lymphoma cells in bone marrow and peripheral blood in all patients. Among them, 9 patients were followed for monitoring of MRD. MRD in peripheral blood was not detected in 4 patients after therapy (Median 4.5 months; Range; 2-6 months); 2 patients received stem cell transplant, and 2 patients received chemotherapy only, and all 4 patients were free of disease at the time of last follow up (Median 18.5 months; Range 17-26 months). 5 patients kept having MRD after therapy (Median 13 months; Range: 3-21 months), and one of 5 patients presented with new lesion of AITL 12 months after the initial diagnosis.Conclusions: Flow cytometric immunophenotyping with anti-PD-1 antibody reliably detects neoplastic population in AITL cases, and PD-1 MFI differentiates AITL from other PTCL with high sensitivity and specificity. Bright PD-1 expression alone is a reliable indicator of TFH immunophenotype by flow cytometry. Measurement of PD-1 expression by flow cytometry is a useful tool both in diagnosing and monitoring MRD of AITL. [Display omitted] DisclosuresMoskowitz:ADC Therapeutics: Research Funding; Seattle Genetics: Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy, Research Funding; Takeda: Honoraria; Incyte: Research Funding. Horwitz:Seattle Genetics: Consultancy, Research Funding; Millenium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; ADCT Therapeutics: Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; HUYA: Consultancy; BMS: Consultancy; Mundipharma: Consultancy; Aileron Therapeutics: Research Funding. Dogan:Celgene: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peer Review Institute: Consultancy.

Identification of Adverse Risk Factors for Survival in Acquired Pure Red Cell Aplasia Receiving Immunosuppressive Therapy By the Japan PRCA2004/2006 Study and Introduction to the New Cohort Study PRCA2016

Pure red cell aplasia (PRCA) is characterized by normocytic normochromic anemia associated with reticulocytopenia and erythroid hypoplasia in otherwise normal bone marrow. The acquired chronic PRCA may present as a primary hematological disorder in the absence of any other diseases or develop associated with certain conditions such as thymoma, lymphoproliferative disorders, infections, collagen vascular diseases, or after exposure to a wide variety of drugs. Idiopathic PRCA and secondary PRCA not responding to the treatment of the underlying diseases are generally treated by immunosuppressive therapy and the most patients require long-term treatment. Glucocorticoids, cyclosporine and cyclophosphamide are commonly used for initial treatment as monotherapy or combination therapy and majority of the patients respond to immunosuppression. However, most patients treated with these medications relapse during tapering of the treatment. Because of the extreme rarity of this disease, the long-term outcome of chronic PRCA following immunosuppression remains unclear and the adverse risk factors for survival have to be elucidated.In order to identify the adverse risk factors for survival in acquired chronic PRCA following immunosuppression and improve the prognosis, we have conducted nationwide surveys for adult acquired chronic PRCA in Japan. We conducted the first study for adult chronic PRCA in japan in 2004 and 2006 that had been diagnosed between 1990 and 2006 across 109 institutions, and collected the data on a total of 185 patients (PRCA2004/2006 study). We have also started another cohort study in 2016 in collaboration with the Japanese Society of Hematology (JSH) (PRCA2016 study) that was approved by the institutional review board and the ethical committee of the JSH.In PRCA2004/2006 study 73 patients (39%) were classified as having idiopathic PRCA and 112 patients (61%) as having secondary PRCA, including 42 thymoma- and 14 large granular lymphocyte leukemia (LGL)-associated PRCA. The details on idiopathic, thymoma-associated and LGL-leukemia-associated PRCA have been previously reported (Sawada K, et al. Haematologica 2007;92:1021; Hirokawa M, et al. Haematologica 2008;93:27; Fujishima N, et al. Haematologica 2008;93:1555). The last questionnaires for the patients with these three subtypes of PRCA were sent to the institutions in 2012 for collecting the data on relapse, subsequent treatment and efficacy and outcome.The PRCA2004/2006 study revealed that the median overall survival (OS) has not yet been reached in idiopathic PRCA following immunosuppression and the estimated median OS times in thymoma-associated and LGL leukemia-associated PRCA were 142 and 148 months, respectively. Twenty-two deaths were reported and the major causes of death were infection and organ failure (Hirokawa M, et al. Brit J Haematol 2015;169:879). Poor response to induction therapy and relapse of anemia were demonstrated to be associated with death and this was also confirmed by the Mantel-Byar analysis (p<0.001) (Fig. 1). The efficacy of re-induction therapy for relapsing PRCA was inferior to the initial inductive therapy (p<0.001) (Fig. 2).With the results of this study, we have proposed the clinical guidelines for management of PRCA in 2011 and we conducted another cohort study in 2016 in collaboration with the Japanese Society of Hematology (JSH) (PRCA2016 study). The primary endpoint of this study is the OS in chronic PRCA following immunosuppression and the principal aim is to learn whether or not the prognosis would be improved. Between 2006 and 2014, 554 PRCA patients were registered in the JSH hematological disorder registry and we asked the physicians to participate in this study (Fig. 3). The potential size of patient cohort is 181, including 116 idiopathic and 65 secondary PRCA and these patients will be followed up during the next ten years. The PRCA2016 cohort study is expected to reveal the unbiased results regarding the long-term survival and quality of life in PRCA following immunosuppression. [Display omitted] DisclosuresFujisawa:Bristol myers Squibb: Honoraria; Beckman Coulter: Honoraria; Eisai: Honoraria; Janssen Pharmaceutical: Honoraria; Novartis: Honoraria; Astellas: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Otsuka Pharmaceutical: Honoraria, Research Funding; SHIONOGI CO, LTD: Honoraria, Research Funding; Shire plc: Honoraria; Alexion Pharmaceutical: Honoraria; Pfizer: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; MSD: Research Funding. Yonemura:Alexion Pharma GK: Honoraria, Research Funding. Kurokawa:Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding.

Clinical Utility of Flow Cytometry Immunophenotyping and Hematological Profile in Chronic Lymphoproliferative Disorders

Background: Chronic lymphoproliferative disorders (CLPD) are a group of lymphatic malignancies characterized by proliferation of neoplastic mature lymphocytes, and may be sourced B, T and natural killer cells (NK). Flow cytometric (FC) immunophenotyping is a routine practice in the differential diagnosis of these tumors, contributing to greater accuracy and new therapeutic perspectives. Methods: Patients (n = 500) with CLPD were assessed by FC. In addition, we gathered patient information such as age, sex and hematologic data. Results: The results showed that 86.6% of cases were B-cell and 13.4% T/NK CLPD. B-cell CLPD distribution was as follows: 265 B-cell chronic lymphocytic leukemia, 17 B-cell prolymphocytic leukemia, 11 hairy cell leukemia, 3 follicular lymphoma in leukemic phase, 3 splenic lymphoma with villous lymphocytes, 12 mantle cell lymphoma, one Waldenstrong macroglobulinemia, 47 multiple myeloma, 12 plasma cell leukemia, 9 Burkitt lymphoma and 37 B-cell CLPD with non-specific immunoclassification. The T/NK CLPD classifications were as follows: 11 large granular lymphocyte leukemia, 14 T-cell prolymphocytic leukemia, 10 adult T-cell leukemia lymphoma, 8 Sézary syndrome and 24 T-cell peripheral lymphoma. Conclusion: The data suggest that immunophenotyping is an important method in diagnosis, monitoring and prognosis when determining the pathological mechanisms of CLPD evolution. DisclosuresNo relevant conflicts of interest to declare.

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.

Clinical characteristics of T-cell large granular lymphoblastic leukemia with STAT3 gene mutation

目的探讨携带STAT3基因突变的T细胞大颗粒淋巴细胞白血病（T-LGLL）患者的临床特征，为此类患者的临床管理提供参考。方法回顾性分析2009至2019年就诊于江苏省人民医院的T-LGLL患者的临床资料，比较STAT3突变患者与未突变患者的基线临床数据、治疗反应及生存结局。结果共纳入80例患者，STAT3未突变组66例，STAT3突变组14例（17.5％），其中Y640F突变发生频率最高（42.9％）。STAT3突变组与STAT3未突变组相比，HGB减低（67.5 g/L对82.5 g/L，P＝0.018），中性粒细胞计数减少（0.665×109/L对1.465×109/L，P<0.001），乳酸脱氢酶升高（229 U/L对198 U/L，P＝0.041），铁蛋白升高（402.5 g/L对236.0 g/L，P＝0.029），TCR Vβ亚家族表达率升高（89.2％对65.4％，P＝0.014），具备治疗指征患者比例升高（100％对74％，P＝0.033）。STAT3突变组与未突变组一线免疫抑制治疗的完全缓解率分别为38.5％和32.7％，差异无统计学意义（P＝0.748）。STAT3突变组与未突变组一线免疫抑制治疗的总有效率分别为69.2％和69.4％，差异无统计学意义（P＝1.000）。中位随访63（2～121）个月，两组总生存时间（均未达到）的差异无统计学意义（P＝0.170）。结论STAT3基因突变的T-LGLL患者可能有更高的肿瘤负荷和治疗需求，一线应用免疫抑制剂疗效良好。STAT3基因突变对T-LGLL患者预后的意义尚需进一步验证。

CD94 expression patterns in reactive and neoplastic T-cell and NK-cell proliferations

Lymphomas and leukemias of T-cell and NK-cell lineages are highly heterogeneous disorders and lack effective therapeutic strategies. Targeted therapies including anti-CD94 agents are currently under clinical investigation, but studies of CD94 expression on mature T/NK-cell neoplasms are limited. In this study, we investigated the landscape of CD94 protein expression in 15 patients with reactive T/NK-cell proliferations and 124 patients with various T/NK cell neoplasms. CD94 expression was detected at a high level in reactive NK-cells, with a lower level of expression in a subset of reactive CD8 + T-cells; reactive CD4 + T-cells were negative for CD94 expression. All NK-cell neoplasms surveyed had high-level CD94 expression, which was significantly higher than that in T cell neoplasms (p = 0.0174). In neoplastic T-cell proliferations, CD94 expression was positive in all 10 hepatosplenic T-cell lymphoma cases tested, with a high mean fluorescence intensity. Fifty-six percent of T-cell large granular lymphocytic leukemia cases were positive for CD94 expression in a subset of neoplastic cells. All T-cell prolymphocytic leukemia and 97 % of peripheral T-cell lymphoma cases showed no CD94 expression. Our findings demonstrate a broad range of CD94 expression among T/NK-cell neoplasms, in some at levels that suggest therapeutic vulnerability to CD94-targeted therapies.

Clinical and Pathologic Spectrum of DDX41-Mutated Hematolymphoid Neoplasms.

This study seeks to further characterize the clinicopathologic spectrum of DDX41-mutated hematolymphoid malignancies. We identified DDX41 mutations from a cohort of known or suspected hematologic disorders and reviewed the corresponding clinical, genetic, phenotypic, and morphologic findings. DDX41 mutations were identified in 20 (1.4%) of 1,371 cases, including 8 cases of acute myeloid leukemia (AML), 5 cases of myelodysplastic syndrome (MDS), 2 cases of therapy-related MDS/AML, 1 case of primary myelofibrosis, 1 case of chronic myeloid leukemia, 1 case of clonal cytopenia of uncertain significance (CCUS), 1 case of T-cell large granular lymphocytic leukemia (T-LGL), and 1 case of multiple myeloma. DDX41-mutated neoplasms were morphologically heterogeneous with a median cellularity of 20% (range, 10%-100%). Megakaryocyte dysplasia occurred in 7 (35%) of 20 cases and trilineage dysplasia in 1 (5%). Frequently comutated genes include a second, somatic DDX41 mutation (8/19, 42%) followed by mutations in TET2 (20%), DNMT3A (20%), ASXL1 (20%), and CUX1 (20%). Karyotypes were noncomplex in 17 (89%) of 19. This report extends the spectrum of DDX41-mutated disorders to include CCUS, T-LGL, and plasma cell disorders. The morphologic features are heterogeneous and nonspecific, highlighting the importance of DDX41 testing during routine workup of hematolymphoid neoplasms.

Large granular lymphocytic leukemia - A retrospective study of 319 cases.

Large granular lymphocytic leukemia (LGLL) is a rare hematological malignancy that arises from cytotoxic T lymphocytes (T-LGLL) in 85% of cases and natural killer (NK) cells in the rest. A significant knowledge gap exists regarding the pathogenesis, treatment choices, and prognostic factors of LGLL. We report a cohort of 319 consecutive LGLL patients who presented to our cancer center between 2001 and 2020. A total of 295 patients with T-LGLL and 24 with chronic NK-cell lymphoproliferative disorder (CLPD-NK) were identified. The median age was 65 years (range, 17-90 years). Eighty-three patients (26.0%) had autoimmune diseases. A total of 119 patients (37.3%) had coexisting malignancies, 66 (20.7%) had solid tumors, and 59 (18.5%) had hematological malignancies. Most coexisting malignancies were diagnosed before the diagnosis of LGLL. Treatment was needed for 57% of patients. Methotrexate (MTX), cyclophosphamide (Cy), and cyclosporine A (CSA) were most used and had similar response rates between 61.5%-74.4%. Cy produced more complete responses (32.3%) compared to MTX and CSA (15.7% and 23.1%, respectively). Thrombocytopenia, splenomegaly, and female gender (after controlling for autoimmune diseases) were associated with decreased response rates to MTX, CSA, or Cy. Autoimmune diseases were associated with increased response rates. Thrombocytopenia was an independent risk factor for worse survival.

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.

Eastern Equine Encephalitis in a Patient with Chronic Neutropenia Secondary to T-Cell Large Granular Lymphocytic Leukemia (1403)

Eastern Equine Encephalitis in a Patient with Chronic Neutropenia Secondary to T-Cell Large Granular Lymphocytic Leukemia (1403)

Frequent somatic TET2 mutations in chronic NK-LGL leukemia with distinct patterns of cytopenias

AbstractChronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher's exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch's t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Inherited bone marrow failure syndromes. An overview

Inherited bone marrow failure syndromes are a diverse set of genetic disorders characterized by insufficient blood cell production leading to cytopenias/pancytopenia. Bone marrow failure can be restricted to one or two blood cell lineages, with symptoms specific to the particular cell lineage or it can affect all cell lineages, causing clinical symptoms similar to aplastic anemia. Inherited bone marrow failure syndromes are genetically heterogeneous diseases resulting from germline mutations that affect key cellular pathways namely ribosomal biogenesis, telomerase biology, DNA repair, and structural proteins. Common Inherited bone marrow failure syndromes are Diamond-Blackfan anemia, Fanconi anemia, Dyskeratosis Congenita, and Shwachman-Diamond syndrome. These different syndromes have variable prognoses and risks of developing hematological or solid malignancies. Thus the accurate diagnosis of these diseases differentiating it from other Inherited bone marrow failure syndromes and other causes of bone marrow failure is of utmost importance for management and surveillance of long-term squeal of the disease. Other causes of BMF can be acquired. The most common forms of Bone marrow failure can occur from chemicals, radiation, drugs, viral infections, immune disorders, myelodysplastic syndrome, paroxysmal nocturnal hemoglobinuria, or large granular lymphocytic leukemia. Moreover, these Inherited bone marrow failure syndromes are often heritable, affecting other family members, thus requiring insightful genetic counseling. This review discusses the frequent Inherited bone marrow failure syndromes along with their differential diagnosis.

Clinical Utility of Targeted Next-Generation Sequencing in the Evaluation of Low-Grade Lymphoproliferative Disorders.

We investigated the usefulness of a custom-designed 31-gene next-generation sequencing (NGS) panel implemented on a routine basis for the evaluation of low-grade lymphoproliferative disorders (LPDs). In total, 147 blood, bone marrow, and tissue specimens were sequenced, including 81% B-cell, 15% T-cell, and 3% natural killer (NK)-cell neoplasms. Of the cases, 92 (63%) of 147 displayed at least one pathogenic variant while 41 (28%) of 147 had two or more. Low mutation rates were noted in monoclonal B-cell lymphocytoses and samples with small T- and NK-cell clones of uncertain significance. Pathogenic molecular variants were described in specific disorders and classified according to their diagnostic, prognostic, and potential therapeutic value. Diagnostically, in addition to confirming the diagnosis of 15 of 15 lymphoplasmacytic lymphomas, 10 of 12 T large granular lymphocytic leukemias, and 2 of 2 hairy cell leukemias (HCLs), the panel helped resolve the diagnosis of 10 (62.5%) of 16 challenging cases lacking a specified diagnosis based on standard morphology, phenotype, and genetic analysis. Overall, implementation of this targeted lymphoid NGS panel as part of regular hematopathology practice was found to be a beneficial adjunct in the evaluation of low-grade LPDs.

79696 Reversible DNA Hypermethylation of the Interleukin-15 Promoter Induces IL-15 Expression, Drives the Pathogenesis of T-Cell Large Granular Lymphocytic Leukemia (T-LGLL) and Provides a Therapeutic Approach Using 5-Azacitidine

ABSTRACT IMPACT: This work describes, for the first time, the methylome in patients with T-LGLL, focusing on the IL-15 promoter, and clearly demonstrates that 5-azacytidine decreases IL-15 production leading to T-LGLL cell death. These results form the basis a translational clinical trial in T-LGLL that will begin accrual in 2021 OBJECTIVES/GOALS: T-LGLL is an incurable leukemia with few treatment options driven by overexpression of IL-15. Our objective is to characterize the methylation status of the IL-15 promoter in T-LGLL patients and evaluate the potential use of 5-azacytidine (5-aza) in a translational trial by studying the effect of 5-aza in vitro on IL-15 levels, and the IL-15 promoter. METHODS/STUDY POPULATION: We sorted T-LGLL patient (n=3) and normal donor (ND) samples (n=3) for CD3+/CD8+/CD5-/dim for T-LGLL immunophenotype. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing and determined differential methylation and gene expression using 1-way ANOVA analysis. To determine the functional significance of differential methylation, we evaluated MOTN-1 T-LGLL cell viability in vitro with 5-aza at increasing concentrations. Next, we evaluated IL-15 gene expression in MOTN-1 cells treated with 5-Aza versus MOTN-1 with control using western immunoblot. Finally, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control. RESULTS/ANTICIPATED RESULTS: There was significant differential methylation (P= 0.0178) and expression (P =0.0059) in T-LGLL patients vs ND. These data revealed significant differential hypermethylation of gene promoters, including an increase in DNA methylation of the IL-15 promoter in T-LGLL cells vs ND. In MOTN-1 cells treated in vitro with 5-Aza at 24 and 48 hours, a dose-dependent decrease in the viability of T-LGLL cells was observed, from 100% to 49.5%, p=0.037. Further, a marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (p=0.0001). Finally, a decrease in cell viability was observed utilizing the IL-15 inhibitor IBI-15 vs control. These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Hypermethylation of the IL-15 promoter and subsequent increase in IL-15 is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce T-LGLL cell death. Based on these results, a phase I trial will be conducted using CC-486 (oral 5-Aza) in T-LGLL.