Case Report: Large Granular Lymphocyte Leukemia (LGLL)—A Case Series of Challenging Presentations

Large granular lymphocyte leukemia (LGLL) represents a rare group of diseases with considerable difficulties in their correct diagnostic workup and therapy. The major challenges lie in their distinction from reactive (including autoimmune) lymphoproliferations. Moreover, monoclonal LGL proliferative diseases are in fact a heterogeneous group of disorders, as recognized by the three subtypes in the current WHO classification. It distinguishes two chronic forms (the focus of this case series), namely T-LGLL and chronic lymphoproliferative disorders of Natural Killer cells (CLPD-NK) as well as aggressive NK-cell leukemia. In the clinical routine, the variable presentations and phenotypes of T-LGLL and CLPD-NK are underappreciated. The relevant differential diagnoses range from benign reactive T-cell expansions to other mature T-cell leukemias to highly aggressive γδ-lymphomas. T-LGLL or CLPD-NK patients suffer from a wide variety of symptoms often including, but not limited to, cytopenias or classical autoimmune phenomena. They receive treatments ranging from mere supportive measures (e.g. antibiotics, growth factors, transfusions) over strategies of immunosuppression up to anti-leukemic therapies. The diagnostic pitfalls range from recognition of the subtle T-cell proliferation, repeated establishment of monoclonality, assignment to a descript immunophenotypic pattern, and interpretations of molecular aberrancies. Here, we report a series of selected cases to represent the spectrum of LGLL. The purpose is to raise awareness among the scientifically or practically interested readers of the wide variety of clinical, immunological, and phenotypic features of the various forms of LGLL, e.g. of T-cell type, including its γδ forms or those of NK-lineage. We highlight the characteristics and courses of four unique cases from two academic centers, including those from a prospective nationwide LGLL registry. Each case of this instructive catalogue serves to transport a key message from the areas of (chronic inflammatory) contexts in which LGLL can arise as well as from the fields of differential diagnostics and of various treatment options. Implications for optimization in these areas are discussed.

Coexistence of T-Large Granular Lymphocyte Leukemia and Peripheral T Cell Lymphoma-NOS with indolent behaviour

T-cell lymphomas and leukemias are highly heterogeneous groups of rare disorders. We report a case of a 68-year-old man patient who develops 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 literature and, moreover, 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 a quite unusual behaviour, with a 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 of less aggressive treatment.

Impact of large granular lymphocyte leukemia on blood DNA methylation and epigenetic clock modeling in Fischer 344 rats

Age-dependent differences in methylation at specific cytosine-guanosine sites (CpGs) have been used in “epigenetic clock” formulas to predict age. Deviations of epigenetic age from chronological age are informative of health status and are associated with adverse health outcomes, including mortality. In most cases, epigenetic clocks are performed on methylation from DNA extracted from circulating blood cells. However, the effect of neoplastic cells in the circulation on estimation and interpretation of epigenetic clocks is not well understood. Here, we explored this using Fischer 344 (F344) rats, a strain that often develops large granular lymphocyte leukemia (LGL). We found clear histological markers of LGL pathology in the spleens and livers of 27 out of 61 rats aged 17-27 months. We assessed DNA methylation by reduced representation bisulfite sequencing with coverage of 3 million cytosine residues. Although LGL broadly increased DNA methylation variability, it did not change epigenetic aging. Despite this, inclusion of rats with LGL in clock training sets significantly altered predictor selection probability at 83 of 121 commonly utilized CpGs. Furthermore, models trained on rat samples that included individuals with LGL had greater absolute age error than those trained exclusively on LGL-free rats (39% increase; p<0.0001). We conclude that the epigenetic signals for aging and LGL are distinct, such that LGL assessment is not necessary for valid measures of epigenetic age in F344 rats. The precision and architecture of constructed epigenetic clock formulas, however, can be influenced by the presence of neoplastic hematopoietic cells in training set populations.

Neutropenia and Large Granular Lymphocyte Leukemia: From Pathogenesis to Therapeutic Options

Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL or NK cells. Chronic isolated neutropenia represents the clinical hallmark of the disease, being present in up to 80% of cases. New advances were made in the biological characterization of neutropenia in these patients, in particular STAT3 mutations and a discrete immunophenotype are now recognized as relevant features. Nevertheless, the etiology of LGLL-related neutropenia is not completely elucidated and several mechanisms, including humoral abnormalities, bone marrow infiltration/substitution and cell-mediated cytotoxicity might cooperate to its pathogenesis. As a consequence of the multifactorial nature of LGLL-related neutropenia, a targeted therapeutic approach for neutropenic patients has not been developed yet; moreover, specific guidelines based on prospective trials are still lacking, thus making the treatment of this disorder a complex and challenging task. Immunosuppressive therapy represents the current, although poorly effective, therapeutic strategy. The recent identification of a STAT3-mediated miR-146b down-regulation in neutropenic T-LGLL patients emphasized the pathogenetic role of STAT3 activation in neutropenia development. Accordingly, JAK/STAT3 axis inhibition and miR-146b restoration might represent tempting strategies and should be prospectively evaluated for the treatment of neutropenic LGLL patients.

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.

AB0183 T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA IN PATIENTS WITH FELTY’S SYNDROME

Background:Felty’s syndrome (FS) is a rare subtype of seropositive rheumatoid arthritis (RA) and is characterized by neutropenia and splenomegaly. Some researchers suggest that FS and T-cell large granular leukemia (T-LGLL) may have common pathogenetic relationships [1].Objectives:to characterize the clinical and laboratory manifestations of FS, to evaluate the frequency of T-LGLL and Sjogren’s syndrome (SS) in this group of patients with RA, neutropenia and splenomegaly (pts).Methods:We observed 18 pts with ACCP-positive (100%) and RF-positive (94.5%) RA diagnosed according to ACR 2010 criteria, who also had neutropenia and hepatosplenomegaly. All 18 pts underwent T-LGLL diagnostics using blood smears and phenotyping of peripheral lymphocytes for the presence of granular lymphocytes, determination of T-cell clonality using the rearrangement of the γ-chain of the T-cell receptor of lymphocytes by PCR, histological/immunohistochemical study of bone marrow biopsy specimens for the presence of LGL invasion studies, as well as the study of 4 spleens after splenectomy. SS was diagnosed in 8 out of 18 pts (44.5%) according ACR 2016 criteria.Results:Twelve (66.6%) of 18 pts with RA, neutropenia and splenomegaly were diagnosed with T-LGLL, the patients were divided in 2 groups: FS (6 pts) and RA+T-LGL (12 pts). Pts with FS debuted with arthritis of small hand joints, extremely rarely with extra-articular manifestations, mainly at a young age (36.5±3.9 years), and developed neutropenia after 10 years of RA. Pts with T-LGLL debuted at a younger age (39,5±4,5 and 51,5 + 7,8 years, respectively), had a longer course of RA before the development of neutropenia (14.3±3.3 and 5±1.5 years, respectively, p=0.03), and more often had extra-articular manifestations at the onset of the disease. RA activity did not differ between groups and in most cases was characterized by a mild course of articular syndrome. Though the course of RA+T-LGLL group was characterized by low (50%) and moderate (33%) DAS28-CRP activity and active synovitis in only 41.5% of pts, severe joint deformities (stage III and IV) developed in 58.5% of pts. Pts with T-LGLL showed a higher incidence of hepatomegaly (75% and 16.5%, respectively, p=0.02) and more severe neutropenia (p=0.02). The development of severe leukopenia (<1x109) and massive hepatosplenomegaly was observed only in pts with T-LGLL, which required splenectomy in 4 cases. SS was more often detected in the FS group than in the RA+T-LGLL group (83.5% and 25%, respectively, p = 0.02).Conclusion:Clinical and laboratory manifestations of FS and T-LGLL are extremely close, therefore, pts who are diagnosed with FS should be examined to exclude T-LGLL.References:[1]Liu, Xin, and Thomas P Loughran Jr. “The spectrum of large granular lymphocyte leukemia and Felty’s syndrome.” Current opinion in hematology vol. 18,4 (2011): 254-9. doi:10.1097/MOH.0b013e32834760fb.Disclosure of Interests:None declared