Large Granular Lymphocyte Leukemia Research Articles

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.

Coexpression of natural killer cell antigens by T-cell large granular lymphocytes in hydroa vacciniforme lymphoproliferative disorder and the involvement of Vδ1 + epithelial-type γδT cells

Hydroa vacciniforme lymphoproliferative disorder (HV-LPD) is a cutaneous variant of chronic active Epstein–Barr virus disease. We examined the coexpression of T- and natural killer (NK)-cell antigens in five patients with classic HV (cHV) and five with systemic HV (sHV). T-cell receptor (TCR) repertoire analysis was performed with high‑throughput sequencing. All five cHV patients had increased γδT cells (> 5%), whereas five sHV patients showed γδT- and αβT-cell dominance in two patients each, and a mixture of abnormal γδT and αβT cells in one. Circulating CD3 + T cells expressed CD16/CD56 at 7.8–42.3% and 1.1–9.7% in sHV and cHV, respectively. The percentage of CD16/CD56 + T cells was higher in the large granular lymphocyte or atypical T-cell fractions in sHV, but no TCR Vα24 invariant chain characteristic of NKT cells was detected. Considerable numbers of CD3 + cells expressing CD56 were observed in sHV skin infiltrates. Of the circulating γδT cells tested, TCR Vδ1 + cells characteristic of the epithelial type of γδT cells were dominant in two sHV cases. Thus, atypical αβT and γδT cells in HV-LPD can express NK-cell antigens, such as CD16 and CD56, and Vδ1 + epithelial-type γδT cells are a major cell type in some HV-LPD cases.

T-Cell Large Granular Lymphocytic Leukaemia in a South African Patient Living with HIV

T-Cell Large Granular Lymphocytic Leukaemia in a South African Patient Living with HIV

Progressive multifocal leukoencephalopathy (PML) treated with pembrolizumab in a patient with mosaic STAT3 gain-of-function mutation

Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal disease of the central nervous system caused by JC virus. It occurs exclusively in immunocompromised hosts and is strongly associated with HIV infection and iatrogenic immunosuppression. Monogenic defects underlying the pathogenesis and reactivation of JC virus are not well understood, although some reports of primary immunodeficiencies (including four cases of STAT1 gain-of-function) have been previously described.A 77 year old male with history of prostate cancer (in remission; treated with radiation and hormonal therapy >10 years ago) presented with dysarthria, ataxia, left-sided weakness, diagnosed with CSF and biopsy-confirmed PML. He underwent an extensive workup upon which no other chronic infectious or oncologic processes were identified. He has never received any form of immunomodulation (including chemotherapy) in the past. Workup revealed low CD4 (347/mm3) and CD8 (282/mm3) T cell counts with normal proportions of naive vs. memory populations. Mitogen stimulation demonstrated poor CD8T cell proliferation. Targeted sequencing revealed a heterozygous mosaic mutation in the DNA-binding domain of STAT3 (c.1229 A > G p.H410R, 14% in 96 reads). This variant had been previously identified in two individuals with large granular lymphocyte (LGL) leukemia and validated as a gain-of-function via downstream signaling assays.Our patient demonstrated a favorable response to PD-1 blockade with pembrolizumab (2 mg/kg) and was discharged to a rehabilitation facility. His MRI brain showed no further progression of cerebellar white matter lesions following three doses of pembrolizumab.To our knowledge, this is the first case to implicate that STAT3 gain-of-function may confer a predisposition to JC virus reactivation and/or control in the pathogenesis of PML. Furthermore, we propose that this patient’s response to pembrolizumab may be partially attributed to STAT3-mediated regulation of the PD-1/PD-L1 axis. Further studies are currently underway to characterize this patient’s mosaic variant in flow cytometry-sorted subsets (including CD8T cells). Additional studies may include measurement of PD-1/PD-L1 expression and basal vs. induced STAT3 phosphorylation in sorted cell populations.

T-large granular lymphocytic leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is characterized by clonal expansion of cytotoxic T cells resulting in cytopenia. The proliferation of clonal LGLs is caused by prolonged antigenic stimulation, which leads to apoptotic dysregulation owing mainly to the constitutive activation of survival pathways, notably the JAK/STAT pathway. Understanding how leukemic T-LGL persists can aid in the development of future immunosuppressive therapies. In this review, we summarize the diagnosis and current standard of therapy for T-LGL leukemia, as well as recent advances in clinical trials.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.

Distributed Pinning Set Stabilization of Large-Scale Boolean Networks

In this article, we design the distributed pinning controllers to globally stabilize a Boolean network (BN), especially a sparsely connected large-scale one, toward a preassigned subset of states through the node-to-node message exchange. Given an appointed set of states, system nodes are partitioned into two disjoint parts, whose states are, respectively, fixed or arbitrary with respect to the given state set. With such node division, three parts of pinned nodes are selected and the state feedback controllers are accordingly designed such that the resulting BN satisfies all three conditions: the information of the arbitrary-state nodes cannot be passed to the others, the subgraph of network structure induced by the fixed-state nodes is acyclic, and the fixed states of these nodes are compatible with the preassigned state set. If the network structure of controlling BN is acyclic, the stabilizing time is revealed to be no more than the diameter of the resulting subgraph plus one. Based on this, we further design the pinning controllers with the constraint of stabilizing time. Noting that the overall procedure runs in an exponentially increasing time complexity with respect to the largest number of functional variables in the dynamics of pinned nodes, the sparsely connected large-scale BNs can be well addressed in a reasonable amount of time. Finally, we demonstrate the applications of our theoretical results in a T-cell large granular lymphocyte (T-LGL) survival signal network with 29 nodes and a T-cell receptor signaling network with 90 nodes.

Retrospective Study of T Cell Leukaemia (Large Granular Lymphocyte Variant) in Dogs Associated with Suspected Immune-Mediated Cytopaenia(s) in the Absence of Peripheral Lymphocytosis.

Canine chronic large granular lymphocyte (LGL) leukaemia is commonly characterised by moderate to marked lymphocytosis but not neutropaenia. In humans, LGL leukaemia is often associated with autoimmune disorders, including immune-mediated cytopaenias (mainly neutropaenia). This presentation is rare in dogs. The aim of this retrospective study was to describe the clinical characteristics, treatments, and outcomes of dogs with chronic LGL leukaemia with suspected immune-mediated cytopaenia. Six dogs with a median age of 4.5 years (range 2-8 years) were included in the study. The most common presenting signs were pyrexia and lethargy. All dogs had severe neutropaenia (median neutrophil count 0.07 × 109/L), three had thrombocytopaenia (median platelet count 66 × 109/L), and one had anaemia (HCT 0.32 L/L). In all dogs, bone marrow cytology revealed infiltration of granular T lymphocytes; PARR analysis confirmed clonality in four, and bone marrow flow cytometry identified CD3+ CD8+ neoplastic cells in two cases. All patients received systemic chemotherapy, and the cytopaenias resolved after 1-19 weeks. Two dogs were euthanised 133 and 322 days after diagnosis, two were lost to follow-up after 224 and 357 days, and two were alive at 546 and 721 days. A subset of LGL leukaemia in dogs is associated with immune-mediated cytopaenia and has a unique clinical presentation.

Prevalence of STAT3 mutations in patients with rheumatoid arthritis-associated T-cell large granular lymphocytic leukaemia and Felty syndrome.

Neutropenia is a key presentation of Felty syndrome (FS) and rheumatoid arthritis (RA)-associated T-cell large granular lymphocytic (T-LGL) leukaemia. Clonal rearrangement of T-cell receptor (TCR) gene supports the diagnosis of T-LGL leukaemia but not FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) gene are highly specific for T-LGL leukaemia, but their prevalence in FS remains poorly clarified. The study included 100 patients with RA and unexplained neutropenia. TCR rearrangements were examined in blood (100 cases), bone marrow (47 cases), and spleen (12 cases) using the BIOMED-2 protocol. Patients were stratified into RA-associated T-LGL leukaemia cohort if a clonal TCR rearrangement was identified in any of the tested patient samples, and into FS cohort in other cases. Mutations in the STAT3 were examined using next-generation sequencing (NGS) technology in blood (100 cases), bone marrow (37 cases), and spleen (7 cases). STAT3 mutations were identified in 71% (49/69) patients with RA-associated T-LGL leukaemia and in 10% (3/31) patients with FS (p=4.7×10-8). Three samples from the RA-associated T-LGL leukaemia cohort and 5 samples from the FS cohort had STAT3 mutations in the absence of clonal TCR rearrangement. The results suggest that STAT3 mutations are significantly less common in FS than in RA-associated T-LGL leukaemia. Moreover, NGS can detect clones undetectable by fragment analysis. We speculate that in patients with RA and neutropenia, the detection of STAT3 mutations can point to T-LGL leukaemia even in the absence of clonal TCR rearrangement.

SBNO2 is a critical mediator of STAT3-driven hematological malignancies

AbstractGain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.

Multi-organ carcinogenicity by inhalation exposure to 2-Bromopropane in rats.

The purpose of this study was to investigate the carcinogenicity of 2-bromopropane (2-BP) in rats. Male and female F344 rats were exposed by whole body inhalation to 2-BP vapor at concentrations of 0, 67, 200, and 600 ppm for 6h/day, 5 days/week for 2 years. All rats of both sexes exposed to 600 ppm died or became moribund within 85 weeks. Death/moribunditywas caused by 2-BP induced tumors. In males, significantly increased tumors were malignant Zymbal's gland tumors; sebaceous adenoma and basal cell carcinoma of the skin/appendage; adenocarcinoma of the small/large intestine; follicular cell adenoma of the thyroid; fibroma of the subcutis, and malignant lymphoma of the lymph node. In addition, an increased trend in tumor incidence was found in the preputial gland, lung, forestomach, pancreas islet, brain, and spleen. In females, significantly increased tumors were adenocarcinoma and fibroadenoma of the mammary gland, squamous cell papilloma of the vagina, and large granular lymphocytic leukemia of the spleen. In addition, an increased trend in tumor incidence was found in Zymbal's gland, the clitoral gland, skin, large intestine, pancreas islet, uterus, and subcutis. Particularly, malignant Zymbal's gland tumors were induced even in males exposed to the lowest concentration, 67 ppm. Two-year inhalation exposure to 2-BP resulted in multi-organ carcinogenicity in rats. Based on sufficient evidence of carcinogenicity in this study, 2-BP has the potential to be a human carcinogen.

Large granular lymphocytic leukemia and its association with immune dysregulation

Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disease of cytotoxic T cells or NK cells with LGL morphology and frequently complicated cytopenia and/or different autoimmune diseases, which often require medical interventions, although LGL leukemia itself is seldom lethal. Immunologic dysregulations in LGL leukemia contribute to the development of complications, for example, neutropenia with the involvement of Fas ligand system and, in pure red cell aplasia, which is a common complication among the patients of East Asian origin, impairing erythroid developments by cytotoxic T cells. Rheumatoid arthritis (RA) is the most prevalent nonhematological consequence, and Felty syndrome, a rare form of RA, and T-LGL leukemia have a lot in common. When patients have LGL leukemia-associated complications, immunosuppressive medication is a mainstay of treatment. Characteristic mutational features in STAT3, STAT5B, CCL22, and other genes in specific subtypes of LGL leukemia have been detected, that would be associated with immunologically mediated molecular pathogenesis in LGL leukemia, and these new findings may help in creating optimal diagnostic approaches or novel therapies for LGL leukemia.

Carcinogenicity and chronic toxicity of butyl methacrylate in rats and mice by a two-year inhalation study.

We conducted a two-year inhalation study of butyl methacrylate using F344/DuCrlCrlj rats and B6D2F1/Crl mice. Rats were exposed to 0, 30, 125 and 500 ppm (v/v) and mice were exposed to 0, 8, 30 and 125 ppm (v/v) using whole-body inhalation chambers. Non-neoplastic lesions developed in the nasal cavities of both rats and mice, but neoplastic lesions were not found. There was also a positive trend in the incidence of large granular lymphocytic (LGL) leukemia in the spleen of male rats. No changes were observed in female rats. Overall, there is some evidence of carcinogenicity in male rats, but there is no evidence of carcinogenicity in female rats. In male mice, there was a positive trend by Peto's test in the incidence of hepatocellular adenomas, and the incidence of hepatocellular adenomas and hepatocellular carcinomas combined was significantly increased compared to the controls by Fisher's exact test in the 30 ppm exposed male group. In female mice, the incidence of hemangiosarcoma in all organs combined showed a positive trend by Peto's test. Therefore, there is some evidence of carcinogenicity in male mice, and there is equivocal evidence of carcinogenicity in female mice.

Gamma Heavy Chain Disease Associated with T-Cell Large Granular Lymphocyte Lymphoproliferative Disorder: Case Report and Literature Review.

Heavy chain diseases are rare B-cell neoplasms consisting of the production of a monoclonal immunoglobulin composed of the only heavy chain without corresponding light chains. It is a rare adult disease that may involve several sites with a variable clinical course. It manifests itself on a large spectrum from indolent to rapidly progressive. We present a case of heavy chain disease and concomitant T- cell large granular lymphoproliferative disorder, an association described in only six cases before.

Prevalence and significance of large granular lymphocytes in patients with immune thrombocytopenia

The association between T-cell large granular lymphocytes (T-LGL) and ITP is uncertain. The aims of this study were to determine the prevalence of T-LGL in patients with ITP and to describe its association with ITP disease severity. We analyzed flow cytometry results for T-LGL (using a threshold of 0.3 x109 or greater cells/L) or positive T-cell receptor clonality in patients with ITP and nonimmune thrombocytopenia. Descriptive statistics were used to characterize the association between T-LGL and ITP, response to ITP treatments (rituximab and splenectomy) and response to T-LGL treatment. Among ITP patients, 14.3% (13/91) had evidence of a T-LGL population compared to 10.3% (3/29) of patients with non-immune thrombocytopenia. ITP patients with T-LGL had lower nadir platelet counts (2 vs. 47 × 109/L) and received more ITP treatments (median 6 vs. 3) than ITP patients without T-LGL. Response to rituximab was observed in 14.3% (1/7) of ITP patients with T-LGL and 54.5% (6/11) without T-LGL. Response to splenectomy was observed in 25% (2/8) with T-LGL and 56.2% (9/16) without T-LGL. Four patients with ITP and T-LGL received treatment for T-LGL with methotrexate; none had an improvement in platelet count levels. T-LGL may appear in patients with ITP, and the meaning of this finding remains unclear; however, for some patients, the presence of abnormal T-LGL may indicate a more severe form of ITP that tends to be less responsive to therapy. In this cohort, treatment of T-LGL with methotrexate did not improve platelet counts in the few patients who were treated.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation

The association between cancer and autoimmune disease is unexplained, exemplified by Tcell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ Tcell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ Tcell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subsetalso expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ Tcells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ Tcell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Presence of Activating STAT3 Mutations in the CD8+ T Cells of Rheumatoid Arthritis Patients

Rheumatoid Arthritis (RA) is a systemic autoimmune disorder afflicting around 1% of the global population. It is treated with a variety of immunosuppressants typically progressing from first line therapies such as low dose methotrexate (MTX) or other disease modifying anti-rheumatic drugs (DMARDs) to second line treatments such as tumor necrosis factor (TNF) inhibitors or other biologics designed to target pro-inflammatory cytokines. Several factors are used to anticipate and measure disease severity including Disease Activity Measures (DAM) and positivity of rheumatoid factor (RF), antibodies to cyclic citrullinated protein (CCP), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Presence of anti-CCP antibody is the most specific of these markers of RA, and its presence may be associated with more severe disease. Large granular lymphocyte (LGL) leukemia is a rare hematologic malignancy characterized by clonal proliferation of natural killer or CD8+ T cells, somatic activating mutations in the signaling transducer and activator of transcription 3 (STAT3) gene, and clinical features including neutropenia and anemia. Interestingly, LGL leukemia is strongly associated with RA, with as high as 36% of patients exhibiting concomitant RA. There is significant overlap between the diseases. Both RA alone and in combination with LGL leukemia exhibit cytotoxic T cell expansions, HLA-DR4 enrichment, female bias, and RA-associated autoantibodies. The dominant molecular hallmark of LGL leukemia is somatic activating STAT3 mutation. Most patients exhibit increased activation of STAT3 through phosphorylation, and 50% or more patients have a mutation in STAT3, the most common being missense mutations, Y640F and D661Y. Of note, LGL leukemia patients with one or more STAT3 mutations are more likely to suffer from concomitant RA. Based on these observations, we hypothesized that STAT3 mutations would be enriched in RA patients compared to the general population and that the presence of mutations may correlate with specific clinical characteristics of RA. In this study, blood samples and demographic data were simultaneously collected from 96 RA patients at the UVA Rheumatology Clinic. CD8+ cells were isolated from blood samples and DNA was extracted and analyzed via droplet digital PCR to detect Y640F, D661Y, and S614R_G618R STAT3 mutations. Figure A shows that roughly 3% (3/96) of the RA samples had a STAT3 mutation in greater than 5% of the DNA analyzed. Additionally, an even larger proportion of RA patients had either Y640F or D661Y mutations at a rate of between 1:100 and 1:1000 cells (10/96 Y640F, 14/96 D661Y). These samples were classified as mutated for the purposes of statistical analysis. Of the 9 healthy control samples analyzed, only one exhibited a mutation of this size (1/9 Y640F, 0/9 D661Y). Correlation with clinical parameters showed similar disease manifestations between WT and STAT3 mutant samples. However, CCP differed between mutational types, with the Y640F group exhibiting higher positivity than the D661Y group (Figure B). Taken together, the increased presence of STAT3 activating mutations in CD8+ cells isolated from RA blood samples as well as the difference in CCP positivity between D661Y and Y640F mutational groups merits further investigation of these pathways in RA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Mechanistic Relationship of T-Cell Large Granular LymphocyticLeukemia (TLGL) andmonoclonal Gammopathy of Undetermined Significance(MGUS)

Introduction: The incidental finding of asymptomatic clonal expansion of plasma cells (MGUS), and T cells (TCUS) is not uncommon and its prevalence increases with age. While the propensity of such conditions to progress to multiple myeloma (MM) and TLGL, respectively is not fully elucidated, we observed in our clinical practice an intertwine between TLGL and MGUS, suggesting a possible mechanistic relationship. Per definition, MGUS should be asymptomatic and without cytopenia, the presence of which would upstage this condition. Thus, most often cytopenias in patients (pts) with MGUS and TLGL coexistence have been primarily attributed to the autoimmune sequelae of the T cell clonopathy. However, TLGL can also be asymptomatic in 20-50% of cases. In such a scenario the identification of the culprit etiology of cytopenia is crucial for the selection of proper therapies. Moreover, understanding the relationship between MGUS and TLGL may provide important lessons regarding the nature of the latter. One could stipulate that TLGL cells are the closest natural equivalent to the chimeric antigen receptor T cells (CART) and naturally arise from the immune trigger provided by clonal plasma cells. The initially polyclonal CTL responses may subsequently evolve to a clonal process, in particular upon acquisition of STAT3 mutations, ultimately controlling the MGUS clone. Alternatively, both humoral and CTL responses are triggered by the same offending agent and its persistence leads to the selection of dominant MGUS and TLGL clones as markers of an overshooting immune response. Methods/Results: We accrued a cohort of 280 pts with TLGL and explored both deep clinical and genomic phenotypes on 171 informative pts for whom complete data was available. Overall, pts had a median age of 67 years (IQR 58-73) with almost 1.09 M:F ratio. Among them, 23% had a concomitant plasma cell dyscrasia, with the majority being MGUS (19%), whereas only 2% and 1% of pts presented with Waldenstrom's macroglobulinemia/lymphoplasmacytic lymphoma (WM-LPL) and MM, respectively. Of note, no MGUS was found in TLGL pts <50 years, suggesting other pathogenetic drivers in younger cases. Overall, TLGL/MGUS had IgG (63%), IgM (25%) and IgA (9%) monoclonal protein phenotypes. In addition, polyclonal hypergammaglobulinemia was present in another 12% of pts. Whereas the overall expected MM evolution is 1-2% per year in typical MGUS, at a median follow up time of 12.5 years (8-17) only 1 MGUS/TLGL case progressed to MM as opposed to the estimated 3-4 pts. Furthermore, while the median overall survival (OS) of pts with MGUS has been reported as 8 years in historical cohorts, in our TLGL/MGUS cases reached 19 years. Looking at clinical characteristics, 13.5% of pts were asymptomatic, 61% had anemia, 53% neutropenia, and 34.5% thrombocytopenia. Anemia (76% vs 58%, p=.04), and bilineage cytopenia (38% vs 22%, p=.04) were more common in pts with co-associated MGUS. Gender, age distribution and disease-specific features such as TLGL counts (median 1785 vs 1680/µL), presence of STAT3 mutations (26% vs 33%) and co-occurring rheumatoid arthritis (15% vs 14%) did not differ between the two groups. Overall response rate (ORR) to T cell-directed therapy achieved 87% in pts with TLGL vs 64% in pts with concomitant MGUS (p=.0017). Indeed, because of the higher refractoriness rate in the latter group (median line of therapies 4 vs 3 in non-MGUS cases), 6 of these immunosuppression-refractory pts received bortezomib-dexamethasone resulting in a 67% ORR with a median time-to-next-treatment of 6.5 months (3-11). These observations argue against TLGL being the culprit of cytopenia in all TLGL/MGUS, suggesting that the monoclonal protein may have a specificity against myeloid or erythroid precursors. Conclusion: Our study shed light on the intertwining relation of the TLGL/MGUS clinical dyad. Rather than a coincidental finding in older pts, such clinical presentation may represent the first paradigmatic example of natural CART cells, analogous to those currently used in MM clinical practice. Finally, the etiology of cytopenias in TLGL need to be cautiously investigated, not automatically assuming a relation to TLGL. To further dissect such complicated dynamics, we are investigating a large cohort of MGUS pts for the presence of TCUS/TLGL and assessing the specificity of polyclonal and monoclonal proteins in MGUS pts with cytopenia using 2 million 12-mers peptide arrays.

DR-01, a Non-Fucosylated Anti-CD94 Antibody, Depletes Leukemic Cells in Ex Vivo and In Vivo Models of Large Granular Lymphocyte Leukemia

Large granular lymphocyte leukemia (LGLL) is a rare leukemia more frequently occurring in the elderly population with three major subtypes: T-LGLL, chronic lymphoproliferative disorder of NK cells (CLPD-NK) and aggressive NK cell leukemia (ANKL). Over 50% of patients develop complications including severe neutropenia, thrombocytopenia and anemia, thus predisposing patients to opportunistic infections. In addition, treatments are limited to standard immunosuppressive therapy such as methotrexate, cyclosporine or cyclophosphamide, with complete response rates of only 50% or less for each agent. Thus, novel and effective therapies for LGLL patients are critically needed. Dren Bio is developing DR-01, a non-ligand blocking, non-fucosylated monoclonal antibody targeting CD94. CD94 is a type II transmembrane receptor identified on mature, cytotoxic lymphocytes such as healthy NK and terminally differentiated CD8 T cells. In the context of disease, CD94 is known to be upregulated on LGLL cells. DR-01 functions through depletion of target cells via antibody-mediated cellular cytotoxicity (ADCC) by means of fratricide, a method in which the same cell type induces ADCC on each other. Through characterization of DR-01 using various ELISA and cell-based assays, DR-01 was determined to bind with nanomolar affinity to CD16a (EC50: 0.04µg/mL) and to CD94 on healthy human donor NK cells, cynomolgus monkey NK cells and LGLL cells (EC50: 0.5-1nM). DR-01 induces potent, dose-dependent depletion of CD94+ cells in ex vivo ADCC assays (IC50: 1ng/mL) of peripheral blood mononuclear cells (PBMCs) from healthy and diseased individuals. Furthermore, DR-01 has been shown to be well tolerated in a 4-week dose-range finding and Good Laboratory Practice (GLP) study in cynomolgus monkeys from 5-100mg/kg on a weekly dosing regimen. A 12-color flow cytometry panel was applied to a cohort of patient LGLL PBMCs to profile CD94 expression and other cytotoxic markers associated with the disease, including CD16, CD57 and functional binding partners of CD94 such as NKG2A and NKG2C. CD94 was identified on 97% (37/38) of patient samples screened, ranging from 400 to 725,000 receptors per cell. All patient leukemic cells expressed variable levels of CD16 and CD57. NKG2A and NKG2C were present in over 5% of total leukemic cells in 37% (14/38) and 21% (8/38) of patient samples, respectively. In addition, DR-01 was able to deplete LGL leukemic cells with high potency and in a dose-dependent manner in a functional ADCC assay (IC50:1-10ng/mL). The mechanism of fratricide was demonstrated through degranulation of the leukemic population via expression of CD107a. Cytokine profiling results are pending. To demonstrate in vivo efficacy of DR-01 in a disease model of LGLL, humanized NSG-Tg(hIL-15) mice engrafted with patient LGLL splenocytes were utilized. Optimal splenocyte engraftment (5-20% human CD45+ cells in blood) was achieved within 3 weeks post-inoculation with 10 million PBMCs per pre-sublethally irradiated mouse. The engrafted leukemic population partially preserved the CD94 expression profile from the patient leukemic cells and was successfully depleted (>90%) in blood, bone marrow, spleen and liver 1-week post-treatment with a single dose of DR-01 at 5mg/kg. Overall, DR-01 is a non-fucosylated anti-CD94 antibody with high affinity and potent leukemic cell depleting ability ex vivo and in vivo. The favorable toxicity profile of DR-01 in cynomolgus monkeys precede clinical studies in LGLL patients. Additional profiling and evaluation of DR-01 in LGLL patient samples ex vivo as part of a pre-phase I study is ongoing. A phase I/II trial has been initiated to assess the safety and efficacy of DR-01 in previously treated LGLL patients.

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