T-cell Large Granular Lymphocyte Leukemia Patients Research Articles

Pro-inflammatory cells sustain leukemic clonal expansion in T-cell large granular lymphocyte leukemia.

T-cell large granular lymphocyte leukemia (T-LGLL) is a chronic lymphoproliferative disorder characterized by the clonal expansion of T-cell large granular lymphocytes (T-LGL). Immunophenotypic and genotypic features contribute to discriminate symptomatic (CD8+ STAT3-mutated T-LGLL) from clinically indolent patients, this latter group including CD8+ wildtype (wt), CD4+ STAT5B-mutated and wt cases. T-LGL lymphoproliferation is sustained both by somatic gain-offunction mutations (i.e., STAT3 and STAT5B) and by pro-inflammatory cytokines, but little information is available on the activity of T-LGLL non-leukemic cells. In this study, we characterized pro-inflammatory cells in the peripheral blood of T-LGLL patients and analyzed their role in supporting the leukemic growth. In symptomatic patients we found that cell populations not belonging to the leukemic component showed a discrete pro-inflammatory pattern. In particular, CD8+ STAT3-mutated cases showed a skewed Th17/Treg ratio and an abnormal distribution of monocyte populations characterized by increased intermediate and non-classical monocytes. We also demonstrated that monocytes released high levels of interleukin-6 after CCL5 stimulation, a chemokine specifically expressed only by leukemic LGL. Conversely, in asymptomatic cases an altered distribution of monocyte populations was not detected. Moreover, T-LGLL patients' monocytes showed abnormal activation of signaling pathways, further supporting the different pathogenic role of monocytes in patients in discrete clinical settings. Altogether, our data contribute to deepening the knowledge on the different cell subtypes in T-LGLL, focusing particularly on non-leukemic cell populations and thus offering the rationale for new therapeutic strategies.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

Efficacy of Short-Duration Alemtuzumab in T-Cell Large Granular Lymphocytic Leukemia: Potential for a Response-Adapted Strategy

Introduction T-cell large granular lymphocytic leukemia (T-LGL) is a chronic incurable malignancy where once refractory to immunosuppressive therapy, few treatment options and no optimal treatment consensus exists. Alemtuzumab, 10 milligrams (mg) intravenously (IV) administered daily for a total of 10 doses, has been shown to be effective for relapsed or refractory (R/R) T-LGL (Dumitriu, et al. Lancet Haematol 2016); however, it can be associated with severe infusion reactions and infections. Given the palliative nature of T-LGL-directed treatments and the increasing potential for toxicity with cumulative alemtuzumab exposure, we conducted a retrospective analysis to describe the efficacy and safety of abbreviated alemtuzumab regimens in R/R T-LGL patients. Methods We identified patients ≥18 years with R/R T-LGL who were treated with short-duration (defined as ≤ 5 doses) alemtuzumab between 2012 and 2022 from the University of Washington lymphoma database. Patients who received prior alemtuzumab were excluded. The primary endpoint was overall hematologic response rate (ORR). Secondary efficacy endpoints included hematologic response rate at 3 and 6 months, rate of transfusion independence, progression-free and overall survival. Safety endpoints included rate of infusion reactions and infections, specifically Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivation. Results Nine patients meeting criteria were identified (Table 1). Five patients received alemtuzumab 10 mg IV daily for 5 doses and 4 patients received alemtuzumab 10 mg IV weekly for 4 doses. Median age was 65 (range 34-81) years and the median number of prior regimens was 3 (range 1-5). All (100%) patients were refractory to immunosuppressive therapy. Seven (78%), 1 (11%) and 1 (11%) patients had unilineage, bilineage and trilineage cytopenias, respectively. Five patients (56%) had autoimmune comorbidities and five (56%) required periodic transfusions. No patients had myelodysplasia on bone marrow biopsy. Overall, 6 (67%) patients had a hematologic response with 2 patients achieving a partial response (PR) and 4 patients achieving a complete response (CR). ORR at 3 and 6 months was 56% and 44%, respectively. Two (40%) patients achieved transfusion independence. The 3 patients without a hematologic response were retreated with alemtuzumab 10 mg IV daily for 10 doses with 1 subsequently achieving a CR. At a median follow-up time of 7.2 (range 2.3-36.6) months, 3 out of 6 patients who achieved a response had disease relapse; 8 (89%) patients were alive and 1 had died from COVID-19 pneumonia. Among the 5 patients who received alemtuzumab at 10 mg daily for 5 doses, ORR was 40% with 1 patient achieving PR and 1 patient achieving CR. ORR at 3 and 6 months was 40% and 40%, respectively. One (20%) patient had an infusion reaction. Two patients (40%) had subclinical CMV viremia which resolved with valganciclovir. No cases of EBV viremia were noted. Four patients completed alemtuzumab at 10 mg weekly for 4 doses; ORR was 100% with 1 patient achieving PR and 3 achieving CR. Hematologic response rate at 3 and 6 months was 75% and 50%, respectively. Three (75%) patients relapsed at 3, 4 and 14.2 months, respectively; 2 were retreated 4-6 times with alemtuzumab 10 mg IV weekly for 2 to 6 doses each time, with both achieving hematologic responses with each course of treatment. One (25%) patient experienced an infusion reaction. No cases of CMV or EBV viremia were noted. Conclusions Short-duration alemtuzumab is effective and well tolerated in R/R T-LGL with responses observed in 67% overall and no cases of CMV viremia detected in the weekly dosing group. Alemtuzumab given at 10 mg IV weekly for 4 doses or until a response is noted followed by retreatment upon relapse was particularly effective; this abbreviated, response-adapted alemtuzumab strategy should be prospectively explored in this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intense Immunosuppressive Therapy (anti-thymocyte globulin, alemtuzumab) As Salvage Therapy in Refractory T-Cell Large Granular Lymphocytic Leukemia and Pure Red Cell Aplasia

Background T-cell directed immunosuppression (IS: methotrexate, cyclosporine, and cyclophosphamide) is the main treatment modality applied for the therapy of immune single-lineage cytopenias including idiopathic neutropenia and pure red cell aplasia (PRCA). These two conditions are likely T-cell mediated and are often associated with T-cell large granular lymphocytic leukemia (T-LGL). The latter is thought to result from an originally polyclonal process evolving to a clonal proliferation of cytotoxic T-lymphocytes. Generally, response to 1st line therapies is about 30%, whereas rates as high as 80-90% can be achieved with 2nd, and 3rd lines. However, a significant fraction of patients remains refractory to multiple therapies and constitutes a management challenge. These patients remain transfusion dependent and/or have severely depressed neutrophil counts refractory to granulocyte-colony stimulating factor (G-CSF). In this scenario, intense IS with anti-thymocyte globulin (ATG) and alemtuzumab is often considered a salvage option, but no systematic experience exists and only a few controlled clinical trials have been conducted. Particularly, we previously reported an overall response rate (ORR) of 63% (n=23) with low dose alemtuzumab (10 mg sc q week x10) and 2/2 with ATG (40 mg/kg/d x 4 days) in patients with PRCA and/or LGL. Previous experience showed an ORR of 74% in a phase II study of 19 T-LGL patients treated with alemtuzumab, whereas a literature meta-analysis showed an ORR of 33% in 24 patients with PRCA treated with ATG. To better rationalize salvage therapy for refractory immune cytopenias, we explored the effectiveness of these agents at our tertiary referral center. Design and Methods We retrospectively examined treatment outcomes in 296 patients with LGL (113 had neutropenia and 73 anemia), and 78 patients with PRCA (+/- LGL). The study cohort had severe refractory cytopenia despite multiple treatment regimens and received salvage therapy with ATG and/or alemtuzumab. Complete response (CR) was defined as hemoglobin >10 g/dL, and absolute neutrophil count >1.0x109/L. Partial response (PR) was defined not meeting CR criteria. Response to therapy was evaluated while on therapy (alemtuzumab) and 4 months post-therapy (ATG). Overall response rate (ORR) was calculated as the percentage of patients with both CR and PR over the total number of patients who received the drug. The response rate was not available for 4 and 3 cases in the ATG and alemtuzumab cohorts respectively. Results A total of 62 patients who received salvage therapy with ATG (N=23) and alemtuzumab (N=39) for refractory cytopenia associated with LGL (N=38) and without LGL (N=24) were identified. Overall, the median age at disease onset was 58 years (IQR 45-69) with a 1.2 male to female ratio. On average, patients received 4 treatment modalities prior to ATG/alemtuzumab to which they did not respond or became refractory. CR rate (CRR) for patients treated with ATG was 42% (8/19) and ORR 89% (17/19). The CRR and ORR was 22% (8/36) and 81% (29/36), respectively in patients treated with alemtuzumab. No significant difference was observed in terms of ORR in patients treated with alemtuzumab compared to ATG (P = 0.45). In T-LGL cases, the presence of a STAT3 mutation did not impact an ORR to either alemtuzumab (31 vs 45% in STAT3 wild type) or ATG-treated cases (54 vs 38%). There was no significant difference in the LGL and PRCA (+/- LGL) cohorts treated with alemtuzumab (P = 0.89) and ATG (P = 0.13). Overall, no treatment-related death was registered, and toxicities were manageable. Alemtuzumab was held if absolute lymphocyte count was <200/µL. Of note, only one patient had CMV re-activation in our cohort. Conclusion Our data shows that alemtuzumab and ATG represent salvage IS regimens highly effective for the treatment of T-LGL and PRCA refractory to multiple lines of therapy with ORR higher if compared to historical data. This therapy resulted in a durable response in both T-LGL and PRCA (+/- LGL) with a good safety profile. The use of low dose alemtuzumab greatly decreases the complication rate and tolerability.

Identification of novel STAT5B mutations and characterization of TCR\u03b2 signatures in CD4+ T-cell large granular lymphocyte leukemia

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRβ sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.

Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL.Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure)Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively.Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data.To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens.To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response.Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes.Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. [Display omitted] DisclosuresLoughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

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.

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。

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.

Clinical features and treatment outcomes in patients with T-cell large granular lymphocytic leukemia: A single-institution experience

AimLarge granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder associated with failure of hematopoiesis and autoimmune diseases. This study describes the clinical features and treatment responses of 108 patients with T-cell large granular lymphocyte leukemia (T-LGLL). MethodsClinical data were collected from T-LGLL patients treated at an anemia treatment center within the hematology and blood diseases unit of a single hospital from January 2009 to April 2019. ResultsThe majority of patients (78 %) were symptomatic at the time of presentation. Splenomegaly was observed in 41 % of cases, while hepatomegaly and lymphadenopathy were rare (6 % and 7 %, respectively). Cyclosporine (CsA) monotherapy was used as first-line therapy for 16 patients, with an overall response rate (ORR) of 56 %. CsA in combination with steroids was administered in 83 patients, with an ORR of 48 %. Among patients experiencing relapse or resistance to first-line therapy, 10 received antithymocyte globulin (ATG) therapy, with an ORR of 50 %; an additional 9 patients received a modified regimen of high-dose cyclophosphamide (CTX) therapy, yielding an ORR of 78 %. ConclusionsThis study provides new information regarding the clinical features and therapeutic strategies for T-LGLL, which can be used to improve clinical decision making for T-LGLL patients. The data presented here indicate the CsA is an effective option for the treatment of T-LGLL, while modified regimens of high-dose CTX or ATG are safe and effective choices for patients with CsA refractory disease.

Long-Term Experience with Large Granular Lymphocytic Leukemia Evolving after Solid Organ and Hematopoietic Stem Cell Transplantation

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count &gt;500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (&gt;500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P&lt;.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count &lt;1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count &lt;150 x109/L; n=5) and 25% with anemia (hemoglobin &lt;10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

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

Background and Rationale: T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable clonal proliferation of CD8+ memory T-cells that leads to profound neutropenia and anemia with limited treatment options. The primary driver of T-LGLL is overexpression of interleukin-15 (IL-15), a gamma-chain cytokine. Previously, we have demonstrated that mice overexpressing IL-15 develop DNA hypermethylation and chromosomal instability that leads to the spontaneous development of LGLL (Mishra et al. Cancer Cell 2012). Further, the IL-15 promoter is known to be hypermethylated in cutaneous T-cell lymphoma (CTCL), another IL-15 driven malignancy (Mishra et al. Cancer Discovery 2016). In CTCL patients, the counterintuitive increase in IL-15 mRNA was due to hypermethylation of its promoter at the repressor binding sequences in the IL-15 gene. However, the methylation status of the IL-15 promoter in T-LGLL patients remains unknown. Concept: We hypothesize that the IL-15 promoter is hypermethylated in patients with T-LGLL, leading to aberrant overexpression of IL-15 and that this hypermethylation is a critical event in the leukemogenesis of T-LGLL. If true, demethylation of the IL-15 promoter with a resultant decrease in IL-15 transcripts should lead to apoptosis of T-LGLL cells. Hypomethylation of the IL-15 promoter, therefore, may provide a novel therapeutic approach to inhibiting IL-15, the primary driver of T-LGLL. Results: CD3+/CD8+/CD5-/dim T-cells were purified from peripheral blood of LGLL patient (n=3) and normal donor (ND) (n=3) by flow cytometry sorting. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing. With bioinformatics analysis, we determined differential methylation (1-way ANOVA P= 0.0178) and expression (1-way ANOVA P =0.0059). These data sets revealed significant differential hypermethylation of gene promoters in leukemic samples, compared to controls (Figure 1A). Reduced representation bisulfite sequencing that can identify differentially methylated regions at single base-pair resolutions demonstrated an increase in DNA methylation of the IL-15 promoter in patient samples over controls. To determine the functional significance of this finding, we treated the MOTN-1 T-LGLL cell line in vitro with the hypomethylating agent, 5-azacytidine (5-aza) at concentrations of 0.5 uM, 1 uM, 2.5 uM, and 5 uM. At 24 and 48 hours, a marked decrease in the viability of T-LGLL cells was observed, from 100% to 49.50%, p=0.037; particularly at higher concentrations of 5-aza (100% to 27% +11.30%, p=0.0030). Next, we sought to determine whether 5-aza induced hypomethylation of the IL-15 promoter. IL-15 gene expression in MOTN-1 T-LGLL cells treated with 5-aza was measured in comparison to control treated MOTN-1 cells. A marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (Figure 1B, p=0.0001). These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. Finally, to determine whether a decrease in IL-15 alone was the cause of increased apoptosis of T-LGLL cells, 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, IBI-40. Even more profound decrease in cell viability was observed utilizing IBI-15 that targets the binding of IL-15 to its receptor (Figure 1C). Together, these data suggest that hypermethylation of the IL-15 promoter is critical to the pathogenesis of T-LGLL, and that treatment with 5-aza is sufficient to induce hypomethylation of the IL-15 promoter, decrease IL-15 transcription, and induce apoptosis in T-LGLL cells. Conclusions: Hypermethylation of the IL-15 promoter, with subsequent increase in IL-15, is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter hypermethylation by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce apoptosis of T-LGLL cells. These data suggest that 5-aza induced hypomethylation may be a novel method to induce IL-15 inhibition and a potentially efficacious clinical strategy against T-LGLL. Disclosures Brammer: Bioniz Therapeutics, Inc.: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Verastem, Inc: Research Funding. Porcu:Daiichi: Research Funding; BeiGene: Other: Scientific Board, Research Funding; Spectrum: Consultancy; Viracta: Honoraria, Other: Scientific Board, Research Funding; Innate Pharma: Honoraria, Other: Scientific Board, Research Funding; Kyowa: Honoraria, Other: Scientific Board, Research Funding; ADCT: Research Funding; Incyte: Research Funding. OffLabel Disclosure: IBI-15 IBI-40 IL-15 inhibitor

Mutations in the signal transducer and activator of transcription family of genes in cancer.

In recent years, it has become clear that members of the signal transducer and activator of transcription (STAT) family of genes play an important role in cancer. The STAT family consists of seven genes, STAT1‐4,STAT5A, STAT5B and STAT6, that are involved in regulating cellular proliferation, apoptosis, angiogenesis and the immune system response. Constitutive activation of STAT3, via mutational changes, is important in oncogenesis in both solid and hematopoietic cancers. In the case of hematopoietic neoplasms, STAT3 driver mutations have been described in T‐cell large granular lymphocytic (T‐LGL) leukemia and chronic natural killer lymphoproliferative disorders (CLPD‐NK) and are seen in 30%‐40% of T‐LGL leukemia patients. STAT5B is also mutated in T‐LGL leukemia and CLPD‐NK, but in a much smaller proportion. Here we review past and current research on STAT genes in hematopoietic and solid cancers with emphasis on STAT3 and STAT5B and their roles in the pathogenesis of hematopoietic malignancies, particularly T‐LGL leukemia and CLPD‐NK.

Dysregulation of the IFN-γ-STAT1 signaling pathway in a cell line model of large granular lymphocyte leukemia.

T cell large granular lymphocyte leukemia (T-LGLL) is a rare incurable disease that is characterized by defective apoptosis of cytotoxic CD8+ T cells. Chronic activation of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway is a hallmark of T-LGLL. One manifestation is the constitutive phosphorylation of tyrosine 701 of STAT1 (p-STAT1). T-LGLL patients also exhibit elevated serum levels of the STAT1 activator, interferon-γ (IFN-γ), thus contributing to an inflammatory environment. In normal cells, IFN-γ production is tightly controlled through induction of IFN-γ negative regulators. However, in T-LGLL, IFN-γ signaling lacks this negative feedback mechanism as evidenced by excessive IFN-γ production and decreased levels of suppressors of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ. Here we characterize the IFN-γ-STAT1 pathway in TL-1 cells, a cell line model of T-LGLL. TL-1 cells exhibited lower IFN-γ receptor protein and mRNA expression compared to an IFN-γ responsive cell line. Furthermore, IFN-γ treatment did not induce JAK2 or STAT1 activation or transcription of IFN-γ-inducible gene targets. However, IFN-β induced p-STAT1 and subsequent STAT1 gene transcription, demonstrating a specific IFN-γ signaling defect in TL-1 cells. We utilized siRNA targeting of STAT1, STAT3, and STAT5b to probe their role in IL-2-mediated IFN-γ regulation. These studies identified STAT5b as a positive regulator of IFN-γ production. We also characterized the relationship between STAT1, STAT3, and STAT5b proteins. Surprisingly, p-STAT1 was positively correlated with STAT3 levels while STAT5b suppressed the activation of both STAT1 and STAT3. Taken together, these results suggest that the dysregulation of the IFN-γ-STAT1 signaling pathway in TL-1 cells likely results from low levels of the IFN-γ receptor. The resulting inability to induce negative feedback regulators explains the observed elevated IL-2 driven IFN-γ production. Future work will elucidate the best way to target this pathway, with the ultimate goal to find a better therapeutic for T-LGLL.

Divergent roles for antigenic drive in the aetiology of primary versus dasatinib-associated CD8+ TCR-V\u03b2+ expansions

CD8+ T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8+ T-cell receptor (TCR)-Vβ+ populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-Vβ+) and residual (TCR-Vβ−) CD8+ T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8+ TCR-Vβ+ expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8+ TCR-Vβ+ expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8+ TCR-Vβ+ expansions.

T-Cell Large Granular Lymphocytic Leukemia and Coexisting B-Cell Lymphomas: A Study From the Bone Marrow Pathology Group.

T-cell large granular lymphocytic (T-LGL) leukemia is associated with B-cell lymphomas (BCLs), especially small BCLs. We aimed to explore and expand upon its association with BCLs. We retrospectively studied clinicopathologic features of T-LGL leukemia patients with coexisting BCL from January 2001 to December 2016. Among 432 patients with T-LGL leukemia, 22 (5.1%) had an associated B-cell non-Hodgkin lymphoma. Thirteen (59%) patients had large and nine (41%) had small BCL. T-LGL leukemia occurred synchronously with BCL in five, preceded BCL in three, and followed BCL in 14 patients. Anemia was the most common cytopenia (68%). Only one patient had a history of rheumatoid arthritis. To our knowledge, this is the first multicenter study looking at the spectrum and incidence of BCLs in patients with T-LGL leukemia and highlights its association with large BCLs (3% of T-LGL leukemias).

STAT3 mutation and its clinical and histopathologic correlation in T-cell large granular lymphocytic leukemia

Although T-cell large granular lymphocytic leukemia (T-LGLL) is a clinically indolent disorder, patients with moderate to severe cytopenia require therapeutic intervention. The recent discovery of STAT3 mutations has shed light on the genetic basis of T-LGLL pathogenesis. However, the association of STAT3 mutational status with patients' clinical, histopathologic, and other laboratory features has not been thoroughly evaluated in T-LGLL. In this study, STAT3 mutations were identified in 18 of 36 patients with T-LGLL (50%), including Y640F (12/18, 66.7%), N647I (3/18, 16.7%), E638Q (1/18, 5.6%), I659L (1/18, 5.6%), and K657R (1/18, 5.6%). Interestingly, pure red cell aplasia was seen exclusively in T-LGLL patients without STAT3 mutations (6/15 in the wild-type STAT3 group versus 0/13 in the mutant STAT3 group; P = .02); these patients also were the only responders to T-LGLL therapy (mainly cyclophosphamide) in wild-type STAT3 group. Patients harboring STAT3 mutations were more prone to rheumatoid arthritis (4/13 versus 0/15 in the wild-type STAT3 group; P = .04), frequently requiring therapy for neutropenia/neutropenia-associated infections, and demonstrated good therapeutic responses to methotrexate. No significant differences were seen in complete blood count, flow cytometric immunophenotypic features, T-cell receptor γ V-J rearrangement repertoire, and bone marrow biopsy morphology among the STAT3-mutation and wild-type groups other than significantly larger tumor burden in patients with STAT3 mutations. The distinct disease association and therapeutic responses observed in patients with mutant and wild-type STAT3 warrant further investigation to elucidate the underlying mechanisms. They also highlight the importance of identifying STAT3 mutational status in patients with T-LGLL, which may aid in clinical therapeutic choice.

Moderate-dose cyclophosphamide in the treatment of relapsed/refractory T-cell large granular lymphocytic leukemia-associated pure red cell aplasia

Background: T-cell large granular lymphocyte leukemia (T-LGLL) is a rare disorder characterized by clonal proliferation of large granular lymphocytes (commonly CD3+/CD8+/CD57+). However, the available data regarding the optimal treatment for relapsed/refractory T-LGLL patients are limited.Methods: We retrospectively reviewed 10 patients treated with immunosuppressive therapy consisting of intravenous moderate-dose cyclophosphamide (MD-CTX) together with oral cyclosporine A for relapsed/refractory T-LGLL in our hospital between July 2006 and March 2013.Results: The overall response rate to MD-CTX was 60% (6/10; hematologic complete remission rate, 50%; hematologic partial remission rate, 10%). The median time to response was 28.5 days (range, 20–118 days). The relapse rate of MD-CTX was 50% (3/6); two of these three patients achieved hematologic complete remission after receiving a second course of MD-CTX. Neutropenia was the major adverse event of the MD-CTX regimen. The median time to neutropenia was 5.5 days (range, 1–10 days) and the median neutropenia duration was 5 days (range, 3–15 days). None of the patients developed severe infection.Conclusions: The MD-CTX regimen appears efficacious and safe in the treatment of relapsed/refractory T-LGLL patients.

Clinical Relevance of Sensitive and Quantitative STAT3 Mutation Analysis Using Next-Generation Sequencing in T-Cell Large Granular Lymphocytic Leukemia

Diagnosis of T-cell large granular lymphocytic leukemia (T-LGL) is often challenging because clinical and laboratory characteristics are overlapping with nonneoplastic conditions. Recently, mutation in the STAT3 gene has been identified as a recurrent genetic abnormality in T-LGL. STAT3 mutation, therefore, represents a promising marker in T-LGL diagnostics. We developed a new quantitative next-generation sequencing assay that allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of STAT3 mutation analysis as a diagnostic tool in T-LGL. The study included 16 T-LGL patients. A total of 15 mutations, including 2 new mutations (G618R and K658R), were detected in 12 patients (75%), with mutation levels ranging from 2.5% to 45.6% mutation-positive alleles. Next-generation sequencing detected 50% more mutations than Sanger sequencing. Blood samples from 20 healthy blood donors all tested negative, thus demonstrating the specificity of the assay. The results also indicated that mutation levels in blood and bone marrow are not systematically different, and next-generation sequencing-based STAT3 mutation analysis represents a sensitive method for monitoring residual disease as demonstrated in a patient receiving pentostatin. We demonstrate the clinical relevance of next-generation sequencing-based STAT3 mutation analysis, which represents a sensitive and specific diagnostic marker in T-LGL that allows assessment of molecular residual disease, which may improve clinical decision making.

Submicroscopic genomic rearrangements change gene expression in T‐cell large granular lymphocyte leukemia

To better understand the molecular pathogenesis of T-cell large granular lymphocyte leukemia (T-LGL), we decided to search for those genetic alterations in T-LGL patients and MOTN-1 cell line (established from T-LGL patient) that have an impact on gene expression and as a result can influence cell biology. Multicolor fluorescence in situ hybridization (mFISH) analysis of the MOTN-1 cell line was performed as well as paired-end next-generation sequencing (NGS; Illumina HiSeq2000) of this cell line and one T-LGL patient. In addition, chosen 6q region was characterized in three T-LGL patients using high-resolution comparative genomic hybridization (FT-CGH) and LM-PCR. Gene expression was studied by RNA sequencing (RNAseq; SOLID5500). Rearrangements were detected within 1p and 2q in MOTN-1 affecting expression of FGR, ZEB2, and CASP8, and within 6q in MOTN-1 and one T-LGL patient affecting MAP3K5 and IFNGR1. Nineteen genes, among them FOXN3, RIN3, AKT1, PPP2R5C, were overexpressed as a result of an amplification in 14q in one T-LGL patient. Two novel fusion transcripts were identified: CASP8-ERBB4 in MOTN-1 and SBF1-PKHD1L1 in T-LGL patient. This study showed that submicroscopic genomic rearrangements change gene expression in T-LGL. Several genes involved in rearrangements were previously linked to cancer and survival pattern that characterizes T-LGL cells.