Genomic Landscape of TCR Alpha-Beta and TCR Gamma-Delta T-Large Granular Lymphocyte Leukemia

Large granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 TCRab T-LGL and 12 TCRγδ T-LGL). 76 mutations were observed in three or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (p < 0.01, p < 0.05 respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower ANC values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3 mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon gamma signaling and decreased PI3K-Akt signaling for STAT3 mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

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.

The Value of Flow Cytometry Clonality in Large Granular Lymphocyte Leukemia

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of mature T or NK cells frequently associated with autoimmune disorders and other hematological conditions, such as myelodysplastic syndromes. Immunophenotype of LGL cells is similar to that of effector memory CD8+ T cells with T-cell receptor (TCR) clonality defined by molecular and/or flow cytometric analysis. Vβ usage by flow cytometry can identify clonal TCR rearrangements at the protein level, and is fast, sensitive, and almost always available in every Hematology Center. Moreover, Vβ usage can be associated with immunophenotypic characterization of LGL clone in a multiparametric staining, and clonal kinetics can be easily monitored during treatment and follow-up. Finally, Vβ usage by flow cytometry might identify LGL clones silently underlying other hematological conditions, and routine characterization of Vβ skewing might identify recurrent TCR rearrangements that might trigger aberrant immune responses during hematological or autoimmune conditions.