Tγδ LGLL identifies a subset with more symptomatic disease: analysis of an international cohort of 137 patients.

Tγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαß LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαß cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2- cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαß cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαß LGLL.

LGL leukemia patients exhibit substantial protective humoral responses following SARS-CoV-2 vaccination.

Large granular lymphocyte leukemia is a rare chronic lymphoproliferative disorder of cytotoxic cells. Other hematological malignancies such as CLL and multiple myeloma have been associated with poor vaccination response and markedly increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mortality rates, specifically in patients who have undergone immunosuppressive therapy. Given the immunosuppressive therapies often used to treat the disease, large granular lymphocytic (LGL) patients may be especially vulnerable to SARS-CoV-2 infection. A questionnaire was sent to all patients in the LGL Leukemia Registry at the University of Virginia (UVA) to obtain information on vaccination status, type of vaccine received, side effects of vaccination, patient treatment status before, during, and after vaccination, antibody testing, history of coronavirus disease 2019 (COVID-19) infection, and presence or absence of booster vaccination. Antibody testing of 27 patients who had quantitative SARS-CoV-2 Spike Protein IgG levels determined by University of Virginia medical laboratories via the Abbott Architect SARS-CoV-2 IgG II assay were collected. The assay was scored as reactive at a threshold of ≥50.0 AU/mL or nonreactive with a threshold of <50.0 AU/mL. LGL patients without treatment as well as patients who held treatment prior to their vaccination have a robust humoral response to SARS-CoV-2 vaccines. Patients who did not hold their immunosuppressive treatments have signifigantly diminished vaccine response compared to those who held their immunosuppressive treatment. Our findings support a dual strategy of pausing immunotherapy during the vaccination window and administration of the SARS-CoV-2 booster to all LGL leukemia patients to maximize protective antibodies.

Allogeneic Bone Marrow Transplant as a Cure for Refractory T-Cell Large Granular Lymphocytic Leukemia in an Adolescent.

T-cell large granular lymphocytic (T-LGL) leukemia is a rare, typically indolent neoplasm with a median age of onset above 60 years. Pathogenesis involves clonal T-cell expansion, and nearly all reported pediatric cases have been associated with concurrent autoimmune disease. Immunosuppressive therapy often mitigates sequelae, but definitive cure is not routinely achieved. Here we present an otherwise healthy 13-year-old with T-LGL leukemia refractory to all standard treatments. Our patient ultimately underwent allogeneic bone marrow transplant (BMT) and is now stable in remission 3 years post-BMT. BMT may offer a viable definitive cure for refractory T-LGL leukemia in very young patients.

CCL22 mutations drive natural killer cell lymphoproliferative disease by deregulating microenvironmental crosstalk.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Genomic landscape of TCRαß and TCRγδ 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 T-cell receptor αß [TCRαß] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 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 < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count 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-γ signaling and decreased phosphatidylinositol 3-kinase-Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

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

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

Bcl-3 inhibits lupus-like phenotypes in BL6/lpr mice.

Bcl-3 is an atypical member of the IκB family that modulates NF-κB activity in nuclei. lpr mice carry the lpr mutation in Fas, resulting in functional loss of this death receptor; they serve as models for lupus erythematosus and autoimmune lymphoproliferation syndrome (ALPS). To explore the biologic roles of Bcl-3 in this disease model, we generated BL6/lpr mice lacking Bcl-3. Unlike lpr mice on an MRL background, BL6/lpr mice present with very mild lupus- or ALPS-like phenotypes. Bcl-3 KO BL6/lpr mice, however, developed severe splenomegaly, dramatically increased numbers of double negative T cells - a hallmark of human lupus, ALPS, and MRL/lpr mice - and exhibited inflammation in multiple organs, despite low levels of autoantibodies, similar to those in BL6/lpr mice. Loss of Bcl-3 specifically in T cells exacerbated select lupus-like phenotypes, specifically organ infiltration. Mechanistically, elevated levels of Tnfα in Bcl-3 KO BL6/lpr mice may promote lupus-like phenotypes, since loss of Tnfα in these mice reversed the pathology due to loss of Bcl-3. Contrary to the inhibitory functions of Bcl-3 revealed here, this regulator has also been shown to promote inflammation in different settings. Our findings highlight the profound, yet highly context-dependent roles of Bcl-3 in the development of inflammation-associated pathology.

Sphingosine kinase-2 is overexpressed in large granular lymphocyte leukaemia and promotes survival through Mcl-1.

Sphingolipid metabolism is increasingly recognised as a therapeutic target in cancer due to its regulation of cell proliferation and apoptosis. The sphingolipid rheostat is proposed to control cell fate through maintaining balance between pro-apoptotic and pro-survival sphingolipids. This balance is regulated by metabolising enzymes involved in sphingolipid production. One such enzyme, sphingosine kinase-2 (SPHK2), produces pro-survival sphingosine 1-phosphate (S1P) by phosphorylation of pro-apoptotic sphingosine. Elevated SPHK2 has been found in multiple cancer types and contributes to cell survival, chemotherapeutic resistance and apoptosis resistance. We have previously shown elevation of S1P in large granular lymphocyte (LGL) leukaemia serum and cells isolated from patients. Here, we examined SPHK2 expression in LGL leukaemia and found SPHK2 mRNA and protein upregulation in a majority of LGL leukaemia patient samples. Knockdown of SPHK2 with siRNA in LGL leukaemia cell lines decreased proliferation. Additionally, the use of ABC294640 or K145, both SPHK2-specific inhibitors, decreased viability of LGL leukaemia cell lines. ABC294640 selectively induced apoptosis in LGL cell lines and freshly isolated LGL leukaemia patient cells compared to normal controls. Mechanistically, SPHK2 inhibition downregulated pro-survival myeloid cell leukaemia-1 (Mcl-1) protein through proteasomal degradation. Targeting of SPHK2 therefore provides a novel therapeutic approach for the treatment of LGL leukaemia.

Advances in the Diagnosis and Treatment of Large Granular Lymphocytic Leukemia.

PURPOSE OF REVIEW: The past decade in LGL leukemia research has seen increased pairing of clinical data with molecular markers, shedding new insights on LGL leukemia pathogenesis and heterogeneity. This review summarizes the current standard of care of LGL leukemia, updates from clinical trials, and our congruent improved understanding of LGL pathogenesis. RECENT FINDINGS: Various clinical reports have identified associations between stem, bone marrow, and solid organ transplants and incidence of LGL leukemia. There is also a potential for underdiagnosis of LGL leukemia within the rheumatoid arthritis patient population, emphasizing our need for continued study. Preliminary results from the BNZ-1 clinical trial, which targets IL-15 along with IL-2 and IL-9 signaling pathways, show some evidence of clinical response. With advances in our understanding of LGL pathogenesis from both the bench and the clinic, exciting avenues for investigations lie ahead for LGL leukemia.