Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines.

BACKGROUND: Large granular lymphocyte leukemias (LGLLs) are rare lymphoproliferative malignancies caused by clonal expansion of granular lymphocytes. T-cell LGLL and natural killer (NK) cell LGLL are defined based on their cellular origin. Their clinical manifestation and pathophysiology vary depending on the subtype and include, e.g., neutropenia, anemia, recurrent infections, and autoimmunity. A limited number of available patient-derived cell lines are considered valuable tools to study the biology of these malignancies. They differ in the expression of lineage-specific surface markers, but generally contain cytotoxic effector molecules in characteristic granules. METHODS: We investigated the presence and release of lysosome-associated effector proteins in patient-derived LGLL cell lines by flow and imaging cytometry, by Western blotting and by bottom-up proteomics profiling. RESULTS: The tested cell lines did not express FasL (CD178), but did express CD26/DPP4+. Intracellularly, we detected major differences in the abundance and subcellular distribution of granzymes, perforin, and granulysin. Similar differences were seen in enriched lysosome-related effector vesicles (LREVs). The proteomics profiling of enriched EVs from an NK-LGLL line (NKL) and a T-LGLL line (MOTN-1), confirmed individual profiles of effector molecules. CONCLUSION: Our analyses underscore the individual distribution of effector proteins but also open new routes to define the role of intra- and extracellular granules in the disease manifestation or pathology of LGLLs.

A novel NKp80-based strategy for universal identification of normal, reactive and tumor/clonal natural killer-cells in blood.

Purpose: Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes. Methods: A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification. Results: NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56- cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80+ cytotoxic T-cells at the more advanced maturation stages, mostly in adults. Conclusion: Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80- phenotype.

How I diagnose large granular lymphocytic leukemia.

OBJECTIVES: Large granular lymphocytic leukemia (LGLL) represents a rare neoplasm of mature T cells or natural killer (NK) cells, with an indolent clinical course. Diagnosing LGLL can be challenging because of overlapping features with reactive processes and other mimickers. METHODS: By presenting 2 challenging cases, we elucidate the differentiation of LGLL from its mimics and highlight potential diagnostic pitfalls. A comprehensive review of the clinicopathologic features of LGLL was conducted. RESULTS: Large granular lymphocytic leukemia displays a diverse spectrum of clinical presentations, morphologies, flow cytometric immunophenotypes, and molecular profiles. These features are also encountered in reactive conditions, T-cell clones of uncertain significance, and NK cell clones of uncertain significance. CONCLUSIONS: In light of the intricate diagnostic landscape, LGLL workup must encompass clinical, morphologic, immunophenotypic, clonal, and molecular findings. Meeting major and minor diagnostic criteria is imperative for the accurate diagnosis of LGLL.

Genomic landscape of T-large granular lymphocyte leukemia and chronic lymphoproliferative disorder of NK cells: a single institution experience.

LGLL is a rare and chronic lymphoproliferative disorder including T-LGLL and CLPD-NK. Here, we investigated the genomic profiles of LGLL with a focus on STAT3 and STAT5B mutations in a cohort of 49 patients (41 T-LGLL, 8 CLPD-NK). Our study indicated that STAT3 was identified in 38.8% (19/49) of all patients, while STAT5B occurred in only 8.2% (4/49) of patients. We found that STAT3 mutations were associated with lower ANC in T-LGLL patients. The average number of pathogenic/likely pathogenic mutations in STAT3/STAT5B-mutated patients was significantly higher than that in WT patients (1.78 ± 1.17 vs 0.65 ± 1.36, p = 0.0032). Additionally, TET2-only mutated T-LGLL (n = 5) had a significant reduction in platelet values compared with the WT (n = 16) or STAT3-only mutated T-LGLL (n = 12) (p < 0.05). In conclusion, we compared the somatic mutational landscape between STAT3/STAT5B WT and mutated patients and correlate with their distinct clinical characteristics.

[A particular presentation of a T cell large granular lymphocytic leukaemia]. / Une présentation particulière de leucémie à grands lymphocytes granuleux.

T cell prolymphocytic leukemia (T-PLL) is a rare, aggressive neoplasm derived from post-thymic T cells. Patients are typically middle-aged with a slight male predominance who present with a high white blood cell count, hepatosplenomegaly, lymphadenopathy, and other symptoms typically associated with leukemia. Although cutaneous involvement has been reported in up to 30% of cases of T-PLL, to our knowledge, none have presented with a presentation resembling livedoid vasculopathy. In the correct clinical context, an underlying hematolymphoid neoplasm should be included in the differential diagnosis of a patient presenting with livedoid vasculopathy.

Epigenome-wide analysis of T-cell large granular lymphocytic leukemia identifies BCL11B as a potential biomarker.

BACKGROUND: The molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor αß-positive CD8+ memory cytotoxic T cells, is only partly understood. The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples. RESULTS: We identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation. CONCLUSIONS: DNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.

LGL Clonal Expansion and Unexplained Cytopenia: Two Clues Don't Make an Evidence.

Clonal expansions of large granular lymphocytes (LGL) have been reported in a wide spectrum of conditions, with LGL leukemia (LGLL) being the most extreme. However, the boundaries between LGLL and LGL clones are often subtle, and both conditions can be detected in several clinical scenarios, particularly in patients with cytopenias. The intricate overlap of LGL clonal expansion with other disease entities characterized by unexplained cytopenias makes their classification challenging. Indeed, precisely assigning whether cytopenias might be related to inadequate hematopoiesis (i.e., LGL as a marginal finding) rather than immune-mediated mechanisms (i.e., LGLL) is far from being an easy task. As LGL clones acquire different pathogenetic roles and relevance according to their diverse clinical settings, their detection in the landscape of bone marrow failures and myeloid neoplasms has recently raised growing clinical interest. In this regard, the current availability of different diagnostic techniques, including next generation sequencing, shed light on the relationship between LGL clones and cytopenias, paving the way towards a better disease classification for precision medicine treatments. Herein, we discuss the clinical relevance of LGL clones in the diagnostic algorithm to be followed in patients presenting with cytopenias, offering a foundation for rational management approaches.

Inclusion Body Myositis and Neoplasia: A Narrative Review.

Inclusion body myositis (IBM) is an acquired, late-onset inflammatory myopathy, with both inflammatory and degenerative pathogenesis. Although idiopathic inflammatory myopathies may be associated with malignancies, IBM is generally not considered paraneoplastic. Many studies of malignancy in inflammatory myopathies did not include IBM patients. Indeed, IBM is often diagnosed only after around 5 years from onset, while paraneoplastic myositis is generally defined as the co-occurrence of malignancy and myopathy within 1 to 3 years of each other. Nevertheless, a significant association with large granular lymphocyte leukemia has been recently described in IBM, and there are reports of cancer-associated IBM. We review the pathogenic mechanisms supposed to be involved in IBM and outline the common mechanisms in IBM and malignancy, as well as the therapeutic perspectives. The terminally differentiated, CD8+ highly cytotoxic T cells expressing NK features are central in the pathogenesis of IBM and, paradoxically, play a role in some cancers as well. Interferon gamma plays a central role, mostly during the early stages of the disease. The secondary mitochondrial dysfunction, the autophagy and cell cycle dysregulation, and the crosstalk between metabolic and mitogenic pathways could be shared by IBM and cancer. There are intermingled subcellular mechanisms in IBM and neoplasia, and probably their co-existence is underestimated. The link between IBM and cancers deserves further interest, in order to search for efficient therapies in IBM and to improve muscle function, life quality, and survival in both diseases.

Pathogenesis and Treatment of T-Large Granular Lymphocytic Leukemia (T-LGLL) in the Setting of Rheumatic Disease.

A complex relationship exists between rheumatic diseases and cancer. This delicate balance between chronic inflammation and malignant cell transformation in hematologic neoplasms has been observed, but is not well defined. Large Granular Lymphocyte (LGL) leukemia is at the intersection of a clonal lymphoproliferative disease, chronic inflammation, and autoimmunity. The association between rheumatoid arthritis (RA) and the spectrum of Felty's Syndrome is well-known. Other rheumatic disorders have been reported including systemic lupus erythematosus (SLE), Sjogren's Syndrome (SS), vasculitis, Behcet's Disease (BD) and systemic sclerosis. The association between T-LGLL and rheumatic disease pathogenesis has been hypothesized, but has not yet been fully understood. Components of a shared pathogenesis includes chronic antigen stimulation, JAK-STAT pathway activation and overlap of various cytokines. We will summarize current knowledge on the molecular understanding between T-LGLL and rheumatic disease. There are many potential areas of research to help meet this need and lead to development of targeted therapeutic options.

Cytokines in the Pathogenesis of Large Granular Lymphocytic Leukemia.

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

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαß-Large Granular Lymphocytic Leukemia.

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor ß chain (TRBC1) expression for assessing Tαß-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαß cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1- ratios vs. total Tαß cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1- Tαß-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1- Tαß-LGL ranged between 0.36 and 571 cells/µL (3.2-91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/µL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVß families, the CD28- effector-memory and terminal-effector polyclonal Tαß cells ranged between 0 and 25 TRBC1+ or TRBC1- cells/µL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1- cells/µL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαß-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαß cells. However, in the absence of lymphocytosis or in the case of TαßCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαß-LGL-expressing individual TCRVß families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Coexistence of T-Large Granular Lymphocyte Leukemia and Peripheral T Cell Lymphoma-NOS with Indolent Behavior.

T-cell lymphomas and leukemias are highly heterogeneous groups of rare disorders. We report a case of a 68-year-old man patient who developed two different T-cell neoplasms (Large Granular Lymphocyte Leukemia [LGLL] in 2018 and Peripheral T-cell non-Hodgkin lymphoma not otherwise specified [PTCL-NOS] in 2019) with a previous diagnosis of B-cell marginal zone lymphoma in 2010, treated with two lines of chemo-immunotherapy. The coexistence of these different T-cell neoplasms is rarely reported in the literature. Moreover, it is usually described as an LGLL transformation into PTCL-NOS; differently from these examples, herein, the simultaneous conditions appear to be driven by different T-cell clones. Furthermore, the PTCL-NOS had quite unusual behavior, with good disease control without intensive treatment. Because of these features, it could belong to a subgroup of indolent PTCL-NOS, not yet described in the WHO classification of T-cell neoplasms, which could benefit from less aggressive treatment.

Thymoma With Triple Threat: Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and T-Cell Large Granular Lymphocytic Leukemia.

Thymomas are a rare neoplasm of the anterior mediastinum and often associated with paraneoplastic syndromes. Though myasthenia gravis is the most common and well-known, the list of reported paraneoplastic syndromes occurring with thymoma is extensive and ever-growing. Paraneoplastic syndromes can involve nearly every organ system, including hematologic abnormalities affecting any or all cell lines. This can present challenges to the clinician in terms of diagnosis, prognostic impact, and management. We present the case of a previously healthy 41-year-old female who was diagnosed with thymoma and three rare hematologic paraneoplastic syndromes: pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA), and T-cell large granular lymphocytic leukemia (T-LGLL). To the best of our knowledge, there have been only four other reported cases of PRCA and AIHA in a single patient with thymoma, all of which were treated with thymectomy. Upfront surgical resection was not possible in the present case and thus the patient was alternatively treated with corticosteroids and octreotide, which proved successful in resolving the anemia. The authors present this case to share these findings of an alternative treatment strategy for thymoma-associated PRCA and AIHA and to highlight the importance of careful monitoring with routine blood work for these complex patients.

CD8dimCD3+ lymphocytes in fever patients might be biomarkers of active EBV infection and exclusion indicator of T-LGLL.

Background: Massive monoclonal or oligoclonal expansion of CD8+ T cells is a notable feature of primary infections of the Epstein-Barr virus (EBV). However, the clinical significance of this expansion is not clear. Results: An increase in the CD8dimCD3+ lymphocyte subset in patients with active EBV infection was due to caspase-8-dependent apoptosis was found using flow cytometry in this study. The number of these cells was associated with the illness severity. Pan-T-cell antigen and receptor analyses were also compared in patients with active EBV infections and T-cell large granular lymphocytic leukemia to provide additional diagnostic information. Conclusion: The increase in CD8dimCD3+ cells could be a biomarker of active EBV infection and an exclusion indicator of T-cell large granular lymphocytic leukemia with flow cytometric analysis.

Insights Into Genetic Landscape of Large Granular Lymphocyte Leukemia.

Large granular lymphocyte leukemia (LGLL) is a chronic proliferation of clonal cytotoxic lymphocytes, usually presenting with cytopenias and yet lacking a specific therapy. The disease is heterogeneous, including different subsets of patients distinguished by LGL immunophenotype (CD8+ Tαß, CD4+ Tαß, Tγδ, NK) and the clinical course of the disease (indolent/symptomatic/aggressive). Even if the etiology of LGLL remains elusive, evidence is accumulating on the genetic landscape driving and/or sustaining chronic LGL proliferations. The most common gain-of-function mutations identified in LGLL patients are on STAT3 and STAT5b genes, which have been recently recognized as clonal markers and were included in the 2017 WHO classification of the disease. A significant correlation between STAT3 mutations and symptomatic disease has been highlighted. At variance, STAT5b mutations could have a different clinical impact based on the immunophenotype of the mutated clone. In fact, they are regarded as the signature of an aggressive clinical course with a poor prognosis in CD8+ T-LGLL and aggressive NK cell leukemia, while they are devoid of negative prognostic significance in CD4+ T-LGLL and Tγδ LGLL. Knowing the specific distribution of STAT mutations helps identify the discrete mechanisms sustaining LGL proliferations in the corresponding disease subsets. Some patients equipped with wild type STAT genes are characterized by less frequent mutations in different genes, suggesting that other pathogenetic mechanisms are likely to be involved. In this review, we discuss how the LGLL mutational pattern allows a more precise and detailed tumor stratification, suggesting new parameters for better management of the disease and hopefully paving the way for a targeted clinical approach.

Chronic lymphopenia and neutropenia in a dog with large granular lymphocytic leukemia.

T-cell large granular lymphocytic leukemia (T-cell LGLL) is the most common presentation of chronic lymphocytic leukemia (CLL) in dogs. Aleukemic or subleukemic leukemia is a particularly rare variation in both humans and dogs, where bone marrow proliferation is either not or only sparsely translated in the peripheral blood. Neutropenia is a prominent feature in cases of human T-cell LGLL but is normally absent in canine CLL. This report describes a case of a dog presented with an almost 3-year history of asymptomatic neutropenia, lymphopenia, and thrombocytopenia (without anemia). A bone marrow examination, the exclusion of infectious diseases, and clonality testing led to the diagnosis of subleukemic LGLL that responded well to therapy (death occurred 2.5 years later due to an unrelated cause).

T cell large granular lymphocyte leukemia and chronic NK lymphocytosis.

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

Clonal T-cell Large Granular Lymphocytic Disorders Manifesting in Patients with HIV-1 Infection: Case Series and Review of the Literature.

We report five patients with human immunodeficiency virus-1/acquired immunodeficiency syndrome (HIV-1/AIDS) who developed T-cell large granular lymphocytic proliferation (T-LGLP) or leukemia (T-LGLL). None of the patients fulfilled criteria for diagnosis of diffuse infiltrative lymphocyte syndrome (DILS) or HIV-associated CD8+ lymphocytosis syndrome at the time of diagnosis of LGL. The immunophenotype of malignant T-cells was identical in three patients with co-expression of CD3, CD8, CD57, and T-cell receptor (TCR) alpha/beta. Three out of five patients were also diagnosed with clonal disorders of B-cell origin including diffuse large B-cell lymphoma, Burkitt's lymphoma, and monoclonal gammopathy of undetermined significance (MGUS). Two patients developed cytopenias due to T-LGLL prompting initiation of therapy. Our study suggests that chronic viral infection with HIV can contribute to the evolution of T-LGLP. Clinical and laboratory characteristics of T-LGLP associated with HIV-1/AIDS resemble those of immunocompetent patients.

Mixed-phenotype large granular lymphocytic leukemia: a rare subtype in the large granular lymphocytic leukemia spectrum.

Large granular lymphocytic leukemia (LGLL) is a chronic proliferation of cytotoxic lymphocytes in which more than 70% of patients develop cytopenia(s) requiring therapy. LGLL includes T-cell LGLL and chronic lymphoproliferative disorder of natural killer (NK) cells. The neoplastic cells in LGLL usually exhibit a single immunophenotype in a patient, with CD8-positive/αß T-cell type being the most common, followed by NK-cell, γδ T-cell, and CD4-positive/αß T-cell types. We investigated a total of 220 LGLL cases and identified 12 mixed-phenotype LGLLs (5%): 7 cases with coexistent αß T-cell and NK-cell clones and 5 with coexistent αß and γδ T-cell clones. With a median follow-up of 48 months, the clinicopathological characteristics of these patients seemed similar to those of typical LGLL patients. Treatment was instituted in 9 patients, and 5 patients (55%) attained complete hematologic response or partial response. The therapeutic response rate of this cohort is comparable to the reported overall response rate of 40% to 60% in typical LGLL patients. Three patients who did not receive any treatment had progressive or persistent cytopenias. Interestingly, inverted proportions of 2 clones at disease recurrence were identified in 4 patients (36%) and stable clonal proportions in 7 patients (64%). Mixed-phenotype LGLL is rare, and this study underscores the importance of recognizing this rare type of LGLL in patients who may benefit from LGLL treatment.

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.