Expansion Of Large Granular Lymphocytes Research Articles

Expansion of large granular lymphocytes after autologous hematopoietic stem cell transplantation

Expansion of large granular lymphocytes (LGLs) is sometimes observed in allogeneic hematopoietic stem cell transplantation (HSCT) recipients, and is reported to be associated with a favorable transplant outcome. LGLs are also observed after autologous HSCT, but their clinical implications have not been well investigated. We retrospectively reviewed peripheral blood smears of consecutive autologous HSCT recipients. LGL lymphocytosis was defined as the observation of LGLs in the peripheral blood (> 20% white blood cells) in at least two consecutive blood tests. We evaluated the clinical impact of LGL lymphocytosis on autologous HSCT recipients. LGL lymphocytosis was observed in 18 of 197 patients (9.1%) who received autologous HSCT, at a median of 49 days after transplantation, with a median duration of 120.5 days. Incidence of cytomegalovirus reactivation was significantly higher in patients with LGL lymphocytosis than those without (16.7% vs. 3.3%, p = 0.038). No significant difference in survival rates was observed between groups (3 year OS 90.9% vs. 90.5%, p = 0.793 for lymphoma; 100 vs. 92.4%, p = 0.328 for myeloma). LGL lymphocytosis was observed in almost 10% of autologous HSCT recipients. In contrast to allogeneic HSCT, the duration of LGL was shorter and no significant improvement in survival was observed.

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.

POS1342 DEPLETION OF KLRG1+ T CELLS IN A FIRST-IN-HUMAN CLINICAL TRIAL OF ABC008 IN INCLUSION BODY MYOSITIS (IBM)

BackgroundInclusion body myositis (IBM), a relentlessly progressive autoimmune skeletal muscle disease, has no effective available pharmacological therapy. A prominent feature of IBM on microscopy is highly differentiated effector CD8+ cytotoxic T (Tc) cells invading non-necrotic myofibers [1]. These Tc cells, known to be relatively resistant to apoptosis, express markers including killer cell lectin-like receptor G1 (KLRG1) [2]. ABC008, a first-in-class humanized, afucosylated monoclonal antibody (mAb) specific for KLRG1, selectively depletes these highly differentiated Tc cells while sparing other blood cell populations, e.g., naïve, central memory, and regulatory T cells and B cells. ABC008 has been designed to treat diseases mediated by these Tc cells, including IBM and T-cell large granular lymphocytic leukemia (T-LGLL). IBM and rheumatoid arthritis overlap clinically with T-LGLL and share similar expansions of large granular lymphocytes (LGLs), which also express KLRG1. We report here our preliminary data from our ongoing trial of ABC008 in IBM (NCT04659031).ObjectivesEvaluate the safety, pharmacodynamics (PD), and pharmacokinetics (PK) of ABC008 administered subcutaneously (SC) in adults with IBM.MethodsIn this first-in-human, open-label, single ascending dose trial with 3 + 3 design evaluating ABC008 SC, eligible participants must have clinicopathologically defined, clinically defined, or probable IBM according to the European Neuromuscular Centre 2011 research diagnostic criteria [3] and an IBM Functional Rating Scale (IBMFRS) score ≤38. Four dose cohorts are planned: ABC008 0.1, 0.5, 2.0, and 5.0 mg/kg. PD, PK, safety, and disease severity assessments are performed pre-dose (Day 0) and during the 6-month follow-up period.ResultsFive of the 6 (83.3%) participants were male with baseline mean age = 65.7 years, mean IBM disease duration = 6.8 years, and mean IBMFRS score = 27.5 (Table 1). Each received a single dose of ABC008 SC: Cohorts 1 (C1) and 2 (C2) received 0.1 and 0.5 mg/kg and have completed 168 and 56 days of follow-up, respectively.Table 1.Baseline DemographicsBaseline CharacteristicsABC008 SC Treatment GroupsCohort 10.1 mg/kg SC (n=3)Cohort 20.5 mg/kg SC (n=3)ABC008 Overall (N=6)Age (years), mean ± SD64.0 ± 11.3667.3 ± 6.6665.7 ± 8.52Male sex, n (%)3 (100)2 (66.7)5 (83.3)Caucasian3 (100)3 (100)6 (100)Body Mass Index (kg/m2)28.5 ± 3.5828.3 ± 4.2528.4 ± 3.52Disease Duration (years), mean ± SD9.7 ± 5.973.9 ± 4.486.8 ± 5.70IBMFRS score, mean ± SD30.0 ± 4.0825.0 ± 6.1627.5 ± 5.80Abbreviations: IBMFRS, Inclusion Body Myositis Functional Rating Scale; n or N, number; SC, subcutaneous; SD, standard deviation.Maximum depletion of CD8+KLRG1+ cells for C1 and C2 ranged from 46-96% and 98-99%, respectively (Figure 1A), with depletion evident by the next assessment on Day 1. Recovery in C1 began at Day 84 with Day 168 depletion at 29-71%. Other hematologic parameters generally were stable (e.g., T regulatory and B cells). CD8+CD57+ LGLs, mostly KLRG1+, were also depleted (Figure 1B). Preliminary PK showed that ABC008 SC displays a long absorption phase and slow clearance properties typical of mAb therapies. No severe adverse events (AEs) or discontinuations due to AEs have been reported. One unrelated serious AE of fall with muscle tear in a C1 participant with a prior history of falls occurred.Figure 1.Key pharmacodynamic parameters in Study ABC008-IBM-101. Baseline and change (A) of KLRG1+CD8+ T cells and (B) CD8+CD57+ large granular lymphocytes (LGLs). C1 and C2 are Cohorts 1 and 2; P1, P2, and P3 are Participants 1, 2, and 3.ConclusionIn study participants with IBM, single SC doses of 0.1 and 0.5 mg/kg of ABC008 resulted in the depletion of CD8+KLRG1+ cells and CD8+CD57+ LGLs with clear evidence of a dose response for KLRG1+ T cell depletion and no apparent safety signals. Based on these results, a study evaluating ABC008 for the treatment of T-LGLL is planned.

Large Granular Lymphocytic Leukemia: Current State of Diagnosis, Pathogenesis and Treatment.

This manuscript aims at updating the knowledge on the clinico-biological characteristics, pathogenesis, and the diagnostic challenges of T-LGLL and CLPD-NK disorders and reviews the advances in the management and treatment of these patients. It has been shown that clonal large granular lymphocyte (LGL) expansions arise from chronic antigenic stimulation, leading to resistance to apoptosis. All the above findings have facilitated the diagnosis of LGLL and provided insights in the pathogenesis of the disease. At present, there is no standard first-line therapy for the disease. Immunosuppressive agents are the treatment routinely used in clinical practice. However, these agents have a limited capacity to eradicate the LGL clone and induce long-lasting remission. Advances in the knowledge of pathogenesis have made it possible to explore new therapeutic targets with promising results. Since LGLL is a rare disease, international efforts are needed to carry on prospective clinical trials with new potentially active drugs that could include a large number of patients.

Chronic Lymphocytic Leukemia, T-Cell Large Granular Lymphocytic Leukemia in a Pediatric Patient without Underlying Condition

BackgroundLarge granular lymphocytic (LGL) leukemia is a rare hematological malignancy in children. The two types of LGL leukemia that have been described are T-cell and Natural Killer cell leukemia. It is most commonly diagnosed in older adults, average age of 60-year-old. About 20 cases of LGL leukemia have been reported in children and young adults. All the patients in the reported cases had immune dysregulation conditions, such as chronic graft versus host disease, common variable immunodeficiency disorder, Crohn's disease and autoimmune hemolytic anemia. Here we report a case of T-cell LGL leukemia in a 11-year-old boy without underlying condition who presented with chronic neutropenia associated with gingival hypertrophy, recurrent skin abscesses, aphthous ulcers, clubbing of nails and low bone density.MethodsMulti-institution collaboration and literature review.Case Description11-year-old male with two years history of episodic gum bleeding with gingival hypertrophy, skin abscesses, aphthous ulcers, chronic neutropenia and lymphocytosis presented to our clinic for further evaluation. Initial workup demonstrated moderate to severe neutropenia (absolute neutrophil count between 400/ul to 800/ul) with low segmented neutrophils of 2-4% and high lymphocytes of more than 80%, but normal white blood cell count, hemoglobin for age and platelet count. Peripheral blood smear showed several variant lymphocytes with cytoplasmic blebs and no immature cells present. Expansion of T-cell large granular lymphocytes were detected in peripheral blood by flow cytometry. Due to new symptom of lower back pain, a lumbar Magnetic Resonance Imaging was performed. Results showed low bone density with mild compression deformity of L1 and abnormal heterogeneous marrow signal with heterogeneous contrast enhancement. The abnormal bone marrow signal promoted the investigation of bone marrow aspiration and biopsy. Flow cytometry detected forty-five percent of lymphocytes with immuno-phenotype of CD3+, CD8+, CD57+, CD16+, CD7+ and CD5-. The morphology of minimal cytoplasm and mature chromatin along with immunophenotype were consistent with clonal T-cell large granular lymphocytic proliferation/leukemia. Further cytogenetic tests showed TCR gamma and beta genes rearrangement, STAT3 N647I mutation with normal male karyotype. A peripheral blood congenital neutropenia panel, which included a total of 18 genes, found a heterozygous mutation c 279 G>A in the Gata2 gene; a variant of uncertain significance. Next generation sequencing showed somatic mutations of TRGV10, TRGV8 TRGJ1, TNFAIP3 and STAT3. However, there was no germline mutations detected in sample from skin biopsy. Comprehensive evaluation by immunology, rheumatology and gastroenterology failed to detect any underlying conditions.ConclusionDue to rarity of LGL leukemia in pediatrics, standard of care guidelines are currently unavailable. Extrapolated from limited literature, two management options are considered: watch and wait approach versus early initiation of immunosuppressant chemotherapy. Improved diagnostics can aide management strategies in this patient population. DisclosuresNo relevant conflicts of interest to declare.

Large Granular Lymphocyte Expansion in Myeloid Diseases and Bone Marrow Failure Syndromes: Whoever Seeks Finds.

Large granular lymphocytes (LGL) are lymphoid cells characterized by either a T-cell or a natural killer phenotype whose expansion may be reactive to toxic, infectious, and neoplastic conditions, or result from clonal selection. Recently, the higher attention to LGL clones led to their detection in many clinical conditions including myeloid neoplasms and bone marrow failures. In these contexts, it is still unclear whether LGL cells actively contribute to anti-stem cell autoimmunity or are only a reaction to dysplastic/leukemic myelopoiesis. Moreover, some evidence exists about a common clonal origin of LGL and myeloid clones, including the detection of STAT3 mutations, typical of LGL, in myeloid precursors from myelodysplastic patients. In this article we reviewed available literature regarding the association of LGL clones with myeloid neoplasms (myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemias) and bone marrow failures (aplastic anemia and pure red cell aplasia, PRCA) focusing on evidence of pathogenic, clinical, and prognostic relevance. It emerged that LGL clones may be found in up to one third of patients, particularly those with PRCA, and are associated with a more cytopenic phenotype and good response to immunosuppression. Pathogenically, LGL clones seem to expand after myeloid therapies, whilst immunosuppression leading to LGL depletion may favor leukemic escape and thus requires caution.

Therapeutic Efficacy of Mcl-1 Antagonism in Large Granular Lymphocyte Leukemia

Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit. DisclosuresGru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Clonal expansion of large granular lymphocytes in patients with spondyloarthritis and psoriatic arthritis treated with TNFα inhibitors.

To determine the prevalence of clonal T-large granular lymphocyte (T-LGL) cells in patients with spondyloarthritis (SpA) and psoriatic arthritis (PsA) and to define possible risk factors for this condition. We present a cross-sectional analysis with retrospective and prospective aspects. 115 SpA patients, 48 PsA patients and 51 controls were recruited between December 28, 2017 and January 23, 2019. Flow cytometry (FACS) was performed to screen for aberrant T-LGL cells. Molecular analysis was then employed to confirm the diagnosis in patients with suggestive FACS findings. Patients with clonal T-LGL populations were followed prospectively by FACS analysis. Electronic patient files were retrospectively analyzed to determine risk factors. Median age was 49years for SpA, 55.5years for PsA, and 54years for controls. Median disease duration of SpA and PsA was 15years and 11years, respectively. 79.8% of patients had received biologics at some point, 75.5% had ever received tumor necrosis factor (TNF) inhibitors. 59.5% were treated with TNF inhibitors at the time of study inclusion. We identified clonal T-LGL expansions in 13 individuals equaling a prevalence of 6% (13/214). T-LGL patients were taking TNF inhibitors more frequently at the time of study inclusion (p = 0.022) and were more likely to have ever been treated with TNF inhibition (p = 0.046). Clonal T-LGL expansions can be detected in patients with SpA, PsA and also in healthy controls. Confirming earlier results, exposure to TNFα-blocking agents appears to increase the risk of developing clonal expansions of T-LGL cells.

Nilotinib treatment induced large granular lymphocyte expansion and maintenance of longitudinal remission in a Philadelphia chromosome-positive acute lymphoblastic leukemia

It is well known that the second-generation tyrosine kinase inhibitor dasatinib evokes an immunological reaction as an off-target effect and induces large granular lymphocytes (LGLs) expansion in 30% of patients. However, LGLs expansion in nilotinib-treated patients is rare. We report the case of a 65-year-old patient with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) who showed LGLs expansion during nilotinib treatment. The patient achieved complete remission (CR) after multi-agent chemotherapy combined with dasatinib treatment. However, ALL relapsed in the central nervous system and bone marrow when treatment was interrupted due to interstitial pneumonia. Nilotinib treatment was subsequently started and the patient achieved second CR. Marked peripheral blood lymphocytosis emerged after the start of nilotinib treatment. CD8 + CD57 + cytotoxic T cells were predominantly expanded and showed strong cytocidal activity against K562 Ph-positive leukemia cells. These results suggest that similar to dasatinib, nilotinib can induce LGLs expansion, possibly contributing to long-term remission in patients with Ph-ALL.

What is new in mature T-cell leukemias?

Mature T-cell and NK-cell leukemias represent a clinically heterogeneous group of diseases, ranging from indolent expansions of large granular lymphocytes, to aggressive diseases that are associated with a fulminant clinical course. Recent advances in genomic methodologies have massively increased the understanding of the pathogenesis of this group of diseases. While the entities are genetically heterogeneous, JAK-STAT pathway activation appears to be important across these disorders. The identification of constitutively activated pathways and the emergence of novel targeted pharmaceutical agents raise the expectation that more effective therapies will be identified for these disorders in the coming years.

Prognostic Value of Lymphopenia and Lymphocytosis after Peripheral Blood Haplo-Identical Stem Cell Transplantation

Introduction Chronic expansion of large granular lymphocytes (LGL) has been reported after classical matched allogeneic hematopoietic stem cell transplantation (SCT) with bone marrow, peripheral (PB) or cord blood (CB) as source of graft. This proliferation is indolent and carries a favorable prognosis. Little is known of the incidence and features of LGL expansions in haplo-SCT with post-transplant cyclophosphamide (PTCy), where the impact of the duration of lymphopenia is also ill-documented. Patients &amp; methods This study included 85 adult patients (pts) who received a haplo-SCT between 11/2013 and 03/2019 for hematological diseases. The 3 conditioning regimen used were i) Baltimore (n=28, 11/2013-05/2017) i.e. fludarabine 30 mg/m²/day (d), d -6 to -2, cyclophosphamide 14.5 mg/kg d -6, low dose total body irradiation (LDTBI) 2 Grays d-1, ii) Clo-Baltimore (n=28, 03/2014-04/2017), i.e. Clofarabine 30 mg/m²/d, d -6 to -2, cyclophosphamide 14.5 mg/kg d -6, LDTBI 2 Grays d -1, iii) CloB2A1 (n=29, 05/2017-03/2019) with Clofarabine 30 mg/m²/d,d -6 to -2, busulfan 3,4 mg/kg d -3 and -2, ATG 2,5 mg/kg d-1. All pts received mobilized PB as SC source on d 0 and PTCY 50 mg/kg/d on d +3 and +4 with cyclosporine and mycophenolate mofetyl as graft versus host disease (GVHD) prophylaxis. All pts provided informed consent for data collection. The duration of lymphopenia (&lt;1.5x109/L) as well as occurrence, duration and immunophenotype of LGL expansion (&gt;4x109/L) were recorded. The patients with primary graft failure (n=6) or dead before 3 months (mo) post-SCT were excluded (n=7). Engraftment was monitored by qPCR on PB cells and sorted CD3+ T-cells. TCR-γ/β gene rearrangements of CD3+ collected during sustained lymphocytosis were assessed with the Biomed-2 PCR method (n=7). Data were analyzed considering viral reactivation episodes (CMV, EBV, HHV6 or BK virus), acute or chronic GVHD, relapse and survival. Results The study included 72 adults treated with haplo-SCT (43 males, median age: 59 yo (24-71)) with a median follow-up of 31 mo for alive patients. Most pts had a myeloid disease (64%) and 57% were in complete remission at the time of haplo-SCT. The median duration of lymphopenia was 6 mo (1-49), significantly shorter in pts with a CloB2A1 conditioning (151 d vs. Baltimore 293 d vs. Clo-Baltimore 387 d, p=0.003) or with CMV reactivation (138 d vs 361 d, p&lt;0.0001). Brisk LGL expansion was characterized morphologically in 10 pts (14%), of donor origin in the 9 pts tested. It occurred at a median of 5 mo (2-8), whatever the GVHD prophylaxis. These pts had a shorter duration of lymphopenia (4 vs 10 mo, p=0.0002). The median duration of LGL expansion was 6 mo (0.1-22) with a median lymphocyte count of 5.8x109/L (4.3-19.4). Immunophenotyping disclosed expansions of NK-cells (n=2), CD8+ CD4- T-cells (n=6) or CD4- CD8- TCR gd T-cells (n=2). They were oligoclonal (n=4) or monoclonal (n=3). A recipient CMV+ status was strongly associated with the onset of LGL expansion (89% vs 20%, p=0.0001), and with CMV reactivation (35% vs 4%, p=0.001) but not with that of other viruses. Grade 2-4 acute and chronic GVHD were not correlated with LGL expansion. ROC curve analyses identified that pts with more than 216 d of lymphopenia (AUC=0.83, p&lt;0.001) had a better 2y disease-free survival (DFS) (77% vs 38%, p=0.0008) and 2y overall survival (OS) (81% vs 45%, p =0.0006) (Fig. 1). LGL expansion was associated with a significantly lower incidence of relapse (10% vs 50%, p=0.03), better 2y-DFS (86% vs. 51%, p=0.05) and a trend towards a better 2y-OS (86% vs. 54%, p=0.1) (Fig. 2). Only 1 of these pts has relapsed and died of transplant-related mortality. Neither the recipient's CMV status nor CMV reactivation influenced DFS or OS. Multivariate analysis showed that the disease risk index score (Armand 2014), lymphopenia (&gt;216 days) and LGL expansion, but not age (&gt; 60yo), were independently associated with a better DFS and OS (p&lt;0.0001). Conclusion A shorter duration of lymphopenia after haplo-SCT confers unexpectedly shorter survivals, suggesting the expansion of non-allo-reactive T-cells with a reduced graft versus leukemia effect. LGL expansion (14%) is not a rare event after haplo-SCT, mainly involves CD8+ T-cells, occurs preferably in CMV+ recipients or in pts with CMV reactivation. It is associated with a favorable outcome, similar to that observed in matched and CB SCT. Disclosures Peterlin: AbbVie Inc: Consultancy; Jazz Pharma: Consultancy; Daiichi-Sankyo: Consultancy; Astellas: Consultancy. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Chevallier:Incyte: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria.

Prevalence and Characteristics of Persistent Clonal T Cell Large Granular Lymphocyte Expansions in Rheumatoid Arthritis: A Comprehensive Analysis of 529 Patients.

Up to one-third of patients with T cell large granular lymphocyte (T-LGL) leukemia display symptoms of rheumatoid arthritis (RA). In Crohn's disease and psoriasis, treatment with tumor necrosis factor (TNF) inhibitors is associated with hepatosplenic γδ T cell lymphoma and with clonal expansion of γδ T cells, respectively. This study was undertaken to determine the prevalence of clonal T-LGL cells in patients with RA and define risk factors for this rare hematologic malignancy. A total of 529 RA patients were recruited between November 2013 and August 2015. Eight-color flow cytometry (fluorescence-activated cell sorting [FACS]) was performed to screen for aberrant T cell populations of LGLs. Molecular analysis of the T cell receptor was used to confirm the diagnosis in patients with suggestive FACS findings. Electronic patient files were used to determine risk factors. Patients with clonal populations were monitored prospectively for up to 4 years. The median patient age was 61 years, and 74% were female. The median duration of RA was 12 years. The median Disease Activity Score in 28 joints was 2.8, and 69.9% of patients had ever been treated with biologic disease-modifying antirheumatic drugs. We identified clonal T-LGL expansions in 19 patients, equaling a prevalence of 3.6%. The T-LGL cell clone was constant over time in most patients and was significantly associated with the duration of the exposure to TNF-blocking agents (P = 0.01). No other risk factors could be detected. RA patients with long-term exposure to TNF-blocking agents were at a greater risk of developing clonal expansions of LGLs. This finding may prompt clinicians to refrain from using these substances in RA patients with known T cell aberrations.

A case of Alemtuzumab-induced neutropenia in multiple sclerosis in association with the expansion of large granular lymphocytes

BackgroundAlemtuzumab has been demonstrated to reduce the risks of relapse and accumulation of sustained disability in Multiple Sclerosis (MS) patients compared to β-interferon. It acts against CD52, leading primarily to lymphopenia. Recent data have shown that mild neutropenia is observed in 16% of treated MS-patients whereas severe neutropenia occurred in 0.6%.Case presentationHerein, we present the case of a 34-year-old woman with relapsing-remitting MS, with a history of treatment with glatiramer acetate and natalizumab, who subsequently received Alemtuzumab (12 mg / 24 h × 5 days). 70-days after the last Alemtuzumab administration, the patient displayed neutropenia (500 neutrophils/μL) with virtual absence of B-cells (0.6% of total lymphocytes), low values of CD4-T-cells (6.6%) and predominance of CD8-T-cells (48%) and NK-cells (47%); while large granular lymphocytes (LGL) predominated in the blood-smear examination. Due to prolonged neutropenia (5-days) the patient was placed on low-dose corticosteroids leading to sustained remission.ConclusionThis is the first case of a patient with relapsing-remitting MS with neutropenia two months post-Alemtuzumab, with simultaneous presence of LGL cells in the blood and a robust therapeutic response to prednisolone. We recommend testing with a complete blood count every 15 days in the first 3 months after the 1st Alemtuzumab administration and searching for large granular lymphocytes cell expansion on microscopic examination of the peripheral blood if neutropenia develops.

Pure Red Cell Aplasia Due to T-Cell Large Granular Lymphocytic Leukemia After Hematopoietic Stem Cell Transplantation

Abstract Although reactive polytypic large granular lymphocyte (LGL) expansions after hematopoietic stem cell transplant (HSCT) due to viral infection are common, T-cell large granular lymphocytic leukemia (T-LGLL) is extremely rare and usually clinically indolent and requires no treatment in the post-HSCT setting. In contrast, here we present a case of post-HSCT donor-derived T-LGLL that led to pure red cell aplasia with severe patient morbidity. After diagnosis, the patient received immunosuppressive treatment with a dramatic response and has been transfusion independent for more than a year. To our knowledge, this is the first case of a post-HSCT T-LGLL with a documented significant adverse clinical impact requiring and responding to therapeutic intervention. It underscores the importance of recognizing such a rare entity in posttransplant patients.

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

Large granular lymphocyte (LGL) leukemia has been recognized in the World Health Organization classifications among mature T cell and natural killer cell neoplasms and is divided into three categories. Chronic T cell leukemia and natural killer cell lymphocytosis can be considered as a similar spectrum of an indolent disease characterized by cytopenias and autoimmune conditions. The last category, aggressive natural killer cell LGL leukemia is very rare, related to Epstein-Barr virus, and seen mainly in young Asian people. Clonal LGL expansion arises from chronic antigenic stimulation sustained by interleukin-15 and platelet-derived growth factor cytokine signal. Those leukemic cells are resistant to apoptosis, mainly because of constitutive activation of survival pathways including Jak/Stat, MapK, Pi3k-Akt, RasRaf-1, MEK1/ERK, sphingolipid, and NFκB. Stat3 constitutive activation is the hallmark of this lymphoproliferative disorder. Socs3 is downregulated, but no mutation could be found to explain this status. However, several somatic mutations, including Stat3, Stat5b, and tumor necrosis factor alpha-induced protein 3, have been demonstrated recently in LGL leukemia; they are identified in half of patients and cannot explain by themselves LGL leukemogenesis. Recurrent infections as a result of chronic neutropenia, anemia, and autoimmune disorders are the main complications related to LGL leukemia. Despite an indolent presentation, 10% of patients die, mainly because of infectious complications. Current treatments are based on immunosuppressive therapies. A better mechanistic understanding of LGL leukemia will allow future consideration of a personalized therapeutic approach perhaps based on Jak/Stat inhibitors, which may offer better results than current immunosuppressive therapy.

Frequency of secondary malignancies (SM) in patients with large granular lymphocytic leukemia (LGLL): A single institutional experience.

7542 Background: Large granular lymphocyte (LGL) disorders represent a spectrum of aberrant T-cell or natural killer cell lymphocytic proliferations. LGLL is classically associated with autoimmune conditions and bone marrow (BM) failure disorders. SM has been reported in association with LGLL in about 10%. The aim of this study is to evaluate the impact of SM on the clinical course of LGLL. Methods: This is a retrospective study of LGLL patients (pts) evaluated at Moffitt Cancer Center between January 1995 and May 2016. The diagnostic clinico-pathological criteria consisted of LGL count &gt; 0.5 k/μL with T-cell receptor gene rearrangement. Lower absolute number of clonal circulating LGLs with characteristic immunophenotype associated with BM involvement, cytopenias, splenomegaly and/or associated symptoms were also diagnostic. Pts with myelodysplastic syndrome were excluded. Survival analysis was performed using the Kaplan-Meier method with log-rank test. Chi-square and T-test were used to analyze association among various variables. Significant P-value was considered &lt; 0.05. Results: Out of 668 screened pts with LGL expansions in peripheral blood, 261 met criteria for LGLL. SM were present in 44% (116/261) of LGLL pts, of which 38% were hematological and 80% arose prior to onset of LGLL. Most common solid SM included skin cancer (14%), prostate cancer (12%), and breast cancer (12%), while most common hematological SM consisted of non-Hodgkin lymphoma (17%) and chronic leukemia (14%). 5-year overall survival (OS) for all LGLL pts was 75% and 10-year OS 63%. There was a statistically significant difference in 5-year OS between LGLL pts with SM compared to without (p = 0.049), but no difference between both groups in median OS or 10-year OS. Pts diagnosed with SM prior to LGLL had worse 5-year OS (p = 0.031) and 10-year OS (p = 0.05) compared to all other LGLL pts. Conclusions: This study showed that the frequency of SM is higher than previously described, especially with onset prior to diagnosis of LGLL. Even though median age of LGLL is around 60 years, it appears that age itself cannot explain this phenomenon. Our results suggest that having a SM is a poor prognostic factor in LGLL patients.

Multifactorial neutropenia in a patient with acute promyelocytic leukemia and associated large granular lymphocyte expansion: A case report.

Neutropenia in the setting of acute hematological malignancies may impact disease prognosis, thus affecting therapy dose intensity. This is often due to chemotherapy-induced aplasia as well as to the disease itself. However, chronic neutropenia deserves further investigation, as the management of reversible concomitant causes may avoid treatment delay. The present study describes a case of an acute promyelocytic leukemia patient with chronic severe neutropenia of multifactorial origin, including acute leukemia itself, chemotherapy, autoimmune activation with anti-platelets and anti-neutrophil antibodies positivity, and the rare association of large granular lymphocyte (LGL) expansion. As neutropenia may challenge the diagnosis and treatment of acute malignancies, clinicians and hematopathologists must discuss the differential diagnosis in order to avoid misdiagnosing and undertreating concomitant diseases. In particular, LGL chronic expansion and autoimmunity should be considered.

Late Graft Rejection in Association With T-Large Granular Lymphocyte Expansion of Recipient Origin After Human Leukocyte Antigen–Haploidentical Stem Cell Transplantation: A Case Report

Large granular lymphocyte (LGL) expansion occasionally occurs after allogeneic stem cell transplantation (allo-SCT), and is thought to be a good prognostic sign that is associated with a lower relapse rate. However, there have been no reports of late graft failure (LGF) due to graft rejection in association with oligoclonal LGL expansion. We herein report a case of LGF associated with the transient expansion of recipient-derived T-LGL after allo-SCT. A 65-year-old man underwent peripheral blood stem cell transplantation (PBSCT) from his human leukocyte antigen (HLA)-haploidentical son for the treatment of acute myeloid leukemia, which had evolved from a myelodysplastic syndrome (MDS). Neutrophil engraftment occurred on day 20. A chimerism analysis on day 29 showed both granulocytes and mononuclear cells in the peripheral blood to be completely of donor origin. However, his neutrophil count gradually decreased and a chimerism analysis on day 61 showed that 84% of the patient's T cells were of recipient origin while the granulocytes were 100% donor-derived. His LGLs rapidly increased to 4.01× 109/L on day 113 and decreased thereafter. The percentage of donor cells in his granulocytes gradually decreased, and the patient's leukocytes were completely replaced by recipient cells on day177. The clinical course suggests that the expansion of recipient-derived T-LGLs after allo-SCT can be a sign of graft rejection. Early intervention may be needed if the LGL expansion is recipient-derived.

Association of inclusion body myositis with T cell large granular lymphocytic leukaemia.

SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present on follow-up testing in 15 patients a median of 350 days later. T cell aberrant loss of CD5 or gain of expression of CD16 and CD94 were common (19/42, 45%). In comparison, 2/15 (14%) age-matched patients with dermatomyositis, polymyositis, or necrotizing myopathy, and 0/20 (0%) age-matched healthy subjects had large granular lymphocyte expansions, with none of these patients having T cell aberrant expression of CD5, CD16 or CD94. Reduced blood CD4/CD8 ratio, increased blood CD8 count, and lymphocytosis were additional biomarkers highly correlated with flow cytometry-measured large granular lymphocyte expansions. Cross-sectional data suggested more aggressive disease in patients with such expansions than without. Muscle immunohistochemistry demonstrated invasion of large granular lymphocytes into muscle in 15/15 inclusion body myositis patients but in only 1/28 patients with dermatomyositis or polymyositis. The extent of CD8+ and CD57+ cells in inclusion body myositis muscle correlated with the size of blood large granular lymphocyte populations. Myofibre-invading cells expressed CD57, a marker of persistent T cell exposure to antigen and T cell aggressiveness. In many patients with inclusion body myositis, the autoimmune T cell expansion has evolved into a neoplastic-like or overtly neoplastic disorder, perhaps contributing to its relative refractoriness to immune-directed therapies previously reported.

Expansion of T or B Lymphocytes after Unrelated Cord Blood (UCB) Allogeneic Stem Cell Transplantation in Adults Correlates with CMV Reactivation and Is Associated with a Better Outcome

Introduction: Peripheral lymphocytosis encountered after myeloablative (MAC) or reduced-intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT) is an ill-defined feature. Most reports in the literature deal with large granular lymphocytes (LGL) expansions and only seldom of B-cell increases (Bellucci, Blood, 2002). With an incidence of 3 to 18%, LGL proliferations occur generally late after allo-SCT with a median onset of 9 to 16 months. Such expansions can be polyclonal, oligoclonal or monoclonal, arising from either CD3+ T-cells or CD3- NK cells or both. LGL expansion has been frequently linked to CMV reactivation, indolent clinical course and a usually favorable outcome. Most available data were mainly described in the setting of allo-SCT using bone marrow (BM) or peripheral blood (PBSC) as stem cell source. Here, we report data regarding the incidence and features of lymphocyte expansions after unrelated cord blood (UCB) transplantation.Patients and Methods: Ninety-nine UCB allo-SCT performed in adults between October 2005 and October 2014 were considered for the purpose of this study. Most patients received double CB units (n=94) and a RIC regimen (n=89), for various hematological diseases. Whenever detected, we collected the date of onset and termination of peripheral blood lymphocyte expansions (4x109/L) among the 86 UCB-SCT patients alive at 3 months post-transplant. LGL expansion was defined as sustained LGL above 0.5x109/L and/or >40% of LGL in peripheral blood (Zambello, Haematologica, 1998). Concomitant immunophenotypic results, allowed to discriminate expansions of cytotoxic T-cells (CD3+CD8+CD56+), NK-cells (CD3-CD16+/CD56+) and B-cells (CD19+). LGL expansion data were also analyzed with respect to viral reactivation episodes, acute or chronic graft vs host disease, relapse and survival.Results: Lymphocytosis was observed in 21 cases (24%; 10 females and 11 males; median age: 58 y., range: 32-69). Most patients had a myeloid-lineage disease (67%) and were in complete remission at time of UCB-SCT (76%). The median onset of lymphocyte expansion after UCB-SCT was 12.6 months (range, 1.4-49). The median initial lymphocyte count was 4.76x109/L at time of expansion diagnosis. The median duration of expansion was 12 months (range: 1-52). Twenty patients could be further analyzed phenotypically, showing 8 CD8+ T, 1 NK and 1 T-NK LGL expansions. Interestingly, 7 cases of polyclonal B-lymphocytes expansions were also documented while 3 patients presented both T CD8+ and B expansions. Of note, B-cell expansions were CD5+. For 6 patients with T-cell expansion, concomitant DNA from CD3+ sorted cells is available to test clonality.Lymphocyte expansion were from donor origin for 12/14 tested patients. Acute and chronic GVHD developed respectively in 31% and 68% of lymphocytosis patients, and in 57 and 45% of the 65 patients without lymphocyte expansion (P=NS). Comparing these two groups for viral reactivations, the rates were 86% and 76% for HHV-6 (P=NS) and 23% and 39% for EBV (P=NS) respectively. CMV reactivation was significantly more frequent in the group of lymphocytosis patients (76% vs. 29%, P=0.0001). Interestingly, CMV reactivation was significantly higher in the 10 patients of the T or NK group compared to the 7 patients with B cell expansion (100% vs 57%, P=0.05).At time of analysis, 1 patient had relapsed and 4 had died, the causes of death being disease in 1 case and transplant-related mortality in 3. These events were significantly lower than in the group of patients without lymphocytosis (p=0.003 for relapses and p=0.04 for death). Two-year disease-free survival (Fig A) and overall survival (Fig B) were significantly different at respectively 85% vs. 55% (p=0.01) and 85% vs. 63%. (p=0.03).Conclusion: Lymphocyte expansion, at 24%, is not a rare event in adults receiving UCB allo-SCT. These expansions involve equally the T or B-lineages. The latter are often CD5+ suggesting a proliferation of innate B1 cells from the UCB. Lymphocyte expansions are significantly associated with previous reactivation of CMV, but not HHV-6 or EBV. Because these cells were of donor origin, it can be postulated that they represent primo-activation upon encounter with CMV. Finally, both types of lymphocyte expansions are associated with a significant favorable outcome, suggesting a possibly bystander anti-GVL effect. [Display omitted] DisclosuresMoreau:Celgene, Janssen, Takeda, Novartis, Amgen: Membership on an entity's Board of Directors or advisory committees.