Large Granular Lymphocyte Proliferation Research Articles

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.

Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells.

The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.

CD4 positive/CD8 negative/CD56 positive T cell large granular lymphocyte proliferations; clonal disorders of uncertain significance

Clonal expansions of CD4 positive T large granular lymphocytes with or without co-expression of CD8 are extremely uncommon. These typically display an indolent clinical course and are said to be associated with other solid malignancies. We present the clinicopathologic features and immunophenotypic analyses of two rare cases of CD4 positive T large granular lymphocyte proliferation with expression of natural killer cell associated antigen CD56. Both had high positive anti cytomegalovirus (CMV) IgG titres and persistent lymphocytosis with a relatively benign course at 15 and 13 months of follow up, respectively. Case 1 A 58 year old Indian man presented with fever and cough of one week duration. On examination, he was febrile with crackles in the right lower zone. Hematological investigations revealed mild neutropenia and a monotypic CD3 positive/CD4 positive/CD8 negative/CD56 positive/T cell receptor alpha-beta positive large granular lymphocyte population representing 50% of blood and bone marrow lymphocytes. Clonality was demonstrated using a T cell receptor gamma polymerase chain reaction. A chest roentgenogram showed features suggestive of bronchopneumonia. Patient was treated with broad spectrum antibiotics and antipyretics with improvement. At follow up of 15 months, he continued to have persistent large granular lymphocytosis. Case 2 A 38 year old Indian woman presented with generalized weakness and malaise of two months duration. On examination, she was afebrile with no lympadenopathy or hepatosplenomegaly. A routine hemogram showed absolute lymphocytosis. Flow cytometric evaluation was performed on peripheral blood affirmed the cells to be CD4 positive T large granular lymphocytes, with similar findings to the first case. At follow up of 13 months, patient was otherwise asymptomatic but continued to have persistent CD4 positive large granular lymphocytosis. Flow cytometric immunophenotyping remains an essential tool for the diagnosis of T cell proliferations and helped to clinch the diagnosis in these cases. The aim of this case series is to present clinical and immunophenotypic features of these rare CD4 positive T large granular lymphocyte proliferations that fulfil the WHO defining criteria, however are better regarded as clonal expansions rather than as leukemias in view of their indolent and non progressive nature.

Are T-LGL Leukemia and NK-Chronic Lymphoproliferative Disorder really two distinct diseases?

Mature Large Granular lymphocytes (LGL) disorders include a spectrum of conditions, ranging from polyclonal to clonal indolent and/or overt leukemic LGL proliferations. Most cases are represented by clonal expansions of TCRα/β+ LGL displaying a CD8+ phenotype with expression of cytotoxic T-cell antigens (CD57, CD16, TIA-1, perforin and granzyme B). Proliferations of CD3-CD16+ NK cells with a restricted patter of NK receptors are less common, usually comprising 15% of the cases. Main features are cytopenias, splenomegaly and autoimmune phenomena. Morphology, immunophenotyping and molecular analyses are crucial to establish a correct diagnosis of disease. According to the 2008 WHO classification, two separate entities account for the majority of cases, T-LGL leukemia and Chronic Lymphoproliferative Disease of NK cell (this latter still provisional). Although these disorders are characterized by the expansion of different cells types i.e. T and NK cells, with specific genetic features and abnormalities, compelling evidence supports the hypothesis that a common pathogenic mechanism would be involved in both disorders. As a matter of fact, a foreign antigen driven clonal selection is considered the initial step in the mechanism ultimately leading to generation of both conditions. In this chapter we will discuss recent advances on the pathogenesis of chronic T and NK disorders of granular lymphocytes, challenging the current WHO classification on the opportunity to separate T and NK disorders, which are likely to represent two sides of the same coin.

Lack of common TCRA and TCRB clonotypes in CD8+/TCRαβ+ T-cell large granular lymphocyte leukemia: a review on the role of antigenic selection in the immunopathogenesis of CD8+ T-LGL

Clonal CD8+/T-cell receptor (TCR)αβ+ T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vβ and TCR-Vα clonotypes in a cohort of 26 CD8+/TCRαβ+ T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRβ (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8+/TCRαβ+ T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4+/TCRαβ+ T-LGL and TCRγδ+ T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8+/TCRαβ+ T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.

LGL Disorders: From An Inflammatory-Mediated To a Self-Maintaining Proliferation

T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients' LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated.With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients' plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs >60%: 9.1 ± 2.7 pg/ml; p <0.05). Considering that IL-6 is a mediator of inflammation, we suggest that these low burden disease patients (LGLs <60%) are mostly characterized by an active inflammatory background. By Real Time-PCR, we observed that IL-15 mediated IL-6 expression in patients PBMCs (IL-6 increased 4.7 fold after IL-15) while inhibited IL-6Rα in leukemic LGLs (by 2-fold down), indicating that IL-15 favors IL-6 trans-signalling. By western blotting analysis we showed that both IL-6 and IL-15 were able to activate STAT3 and ERK, then sustaining LGL survival. Interestingly, in the patients showing STAT3 mutations (20% of patients) we demonstrated a significantly lower level of plasma IL-6 (8.9 pg/ml). This feature was found to be associated to a high proliferative disease, with more than 60% circulating LGLs.These results suggest that an initial step along the development of disease characterized by a low lymphocytosis (LGLs <60% of total PBMCs) is mostly sustained by extrinsic factors contributing to the relevant inflammatory background. A subsequent stage is characterized by a high lymphocytosis (LGLs >60% of total PBMCs) in which LGL disease goes on independently from exogenous stimuli, likely becoming self-maintaining due to the contribution of the emerging STAT3 mutations. It is suggested that these phases can represent two sequential steps in the progression of disease. Disclosures:No relevant conflicts of interest to declare.

The Association Of Dasatinib-Induced Lymphocytosis With Treatment Outcome In Patients With Chronic Myeloid Leukemia (CML)

The Association Of Dasatinib-Induced Lymphocytosis With Treatment Outcome In Patients With Chronic Myeloid Leukemia (CML)

Molecular evidence for repertoire skewing of T large granular lymphocyte proliferation after allogeneic hematopoietic SCT: report of two cases

Molecular evidence for repertoire skewing of T large granular lymphocyte proliferation after allogeneic hematopoietic SCT: report of two cases

Investigation of theBRAFV600E Mutation by Pyrosequencing in Lymphoproliferative Disorders

The presence of the BRAF c.1799T>A V600E mutation was recently described in cases of hairy cell leukemia (HCL) but not in other common lymphomas. However, many uncommon subtypes of lymphoma have not been studied. We designed a BRAF pyrosequencing assay specific for the V600E mutation, which has a sensitivity of 5% and is applicable to paraffin-embedded tissue. DNA was sequenced in 9 cases of HCL; 6 cases of variant HCL; 10 cases each of nodal marginal zone lymphoma (NMZL), extranodal marginal zone lymphoma (ENMZL), posttransplantation lymphoproliferative disorder (PTLD), and large granular lymphocyte (LGL) proliferations; 11 cases of peripheral T-cell lymphoma (PTCL); and 12 cases of anaplastic large cell lymphoma (ALCL). All (100%) cases of HCL were positive for BRAF mutations. No mutations were identified in variant HCL, NMZL, ENMZL, PTLD, PTCL, ALCL, or LGL proliferations. Among lymphoproliferative disorders, BRAF mutations are restricted to HCL.

Late Onset Neutropenia Develops Selectively in Only a Subset of Patients with T Large Granular Lymphocyte Proliferation After Rituximab Treatment for Lymphoma,

Abstract 3675Emerging evidence suggests that B cells can actively modulate T cell immune responses by presenting antigen, providing co-stimulation and secreting cytokines. This has prompted investigation whether B cell depletion by monoclonal antibodies, including Rituximab, can alter the subset composition, activation or function of T cells. Rituximab-associated late onset neutropenia (R-LON) is increasingly recognized as a long-term adverse event of Rituximab. Although the etiology of R-LON is not fully elucidated, the syndrome seems to be multifactorial and likely caused by immune-mediated mechanisms. We have previously shown that at least a proportion of R-LON may develop in a setting of expanded cytotoxic T cell populations in peripheral blood (PB) with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Here, we extend our observations regarding PB lymphocyte subset composition in a cohort of 107 Rituximab-treated patients with available results from PB flow cytometry analysis performed at roughly similar intervals after the initial Rituximab administration. The present cohort included 107 patients, aged 16–83 (median 60), who received Rituximab for the treatment of chronic lymphocytic leukemia (CLL) (29), diffuse large B cell lymhoma (DLBCL) (20), marginal zone lymphomas (15), follicular lymhoma (FL) (15), mantle cell lymphoma (MCL) (8) and auto-immune cytopenias (20). Overall, we found: (i) increased (>1.0×109/l) CD8+ cells in 45/107 (42%) cases; (ii) CD4+/CD8+ cell ratios <0.7 in 56/107 (52%) cases; and, (iii) T-LGLs >20% in 66/107 (63%) cases. Within this cohort, 33 cases (group A) developed R-LON, whereas the remainder (group B, n=74) did not develop this syndrome over a comparable observation period. Importantly, no patient with autoimmune cytopenia developed R-LON. R-LON was significantly more frequent in patients with lymphoma subtypes treated with intensive chemotherapy (CLL, DLBCL, MCL), as well as patients who underwent autologous transplantation (p\\q0.001 for all comparisons). No significant differences were noted between groups A or B regarding PB lymphocyte subset composition. We next evaluated the findings from the histopathological study of bone marrow biopsy samples, available in 17 Group A and 19 Group B cases, all with a diagnosis of lymphoma. The morphological and immunohistochemical examination revealed a series of features common in both groups, summarized as follows: (i) mild-to-moderate small lymphocytic infiltration by CD20-CD79a-CD3+CD45RO+CD43+ (CD3>CD45RO) cells, predominantly nodular and/or interstitial (non intrasinusoidal); (ii) pronounced hyperplasia of the erythroid and megakaryocytic series with prominent dyserythropoiesis and dysmegakaryopoiesis, respectively, including abnormal paratrabecular localization, suggestive of myelodysplasia (MDS); and (iii) remarkable shift-to-the-left of the granulocytic series, often with abnormal localization of immature progenitors (ALIP), always with \\q2% CD34+ cells. A proportion of cases showed hyperplasia of the granulocytic series. However, a major difference between the two Groups concerned hypoplasia of the granulocytic series, which was noted almost exclusively in group A. We conclude that lymphoma patients treated with Rituximab often develop cytotoxic T cell expansions than can have a variable impact on hematopoiesis, with R-LON perhaps representing the end of a spectrum of T-LGL-mediated autoimmune myelopathy/myelodyplasia. The selective development of R-LON in only a proportion of cases with expanded cytotoxic T cells associated with prominent hypoplasia of the granulocytic series and MDS-like changes of the hematopoietic marrow post Rituximab raises several questions regarding the underlying (genetically determined?) immunopathogenetic mechanisms. Disclosures:No relevant conflicts of interest to declare.

Immunoprofile of Patients with Chronic Myeloid Leukemia Treated with Imatinib, Nilotinib or Dasatinib,

Abstract 3764Recent reports showed that dasatinib induces significant immunostimulation with clonal expansion of large granular lymphocytes (LGL) which, in chronic myeloid leukemia (CML), is related to both better prognosis and to autoimmune-like side effects. It is speculated that lower levels of circulating T-regulatory cells play a partial role in LGL proliferation in patients receiving dasatinib.The immunoprofile was studied using flow cytometry to evaluate lymphocyte subsets and NK-cell reactivity in the peripheral blood of 61 patients in the chronic phase of CML during treatment with a tyrosine kinase inhibitor (TKI) (Median age: 58 years; imatinib 36, nilotinib 9, dasatinib 16). Furthermore, we measured plasma levels of 27 types of cytokines or chemokines in 58 patients in the chronic phase of CML so that a comprehensive comparison could be made of the differences in immunoprofile among the patients receiving these three TKIs.There were no significant differences between the three TKI-treated groups in terms of CD4/8 ratios or the number of T-cells (CD3+CD8+ or CD4+) and NK cells (CD3-CD56+). However, the number of NK-LGL (CD56+CD57+) and T-LGL (CD3+CD57+) increased significantly in the group that received dasatinib. Furthermore, dasatinib significantly enhanced NK-cell reactivity as compared to imatinib and nilotinib. In contrast nilotinib significantly suppressed NK-cell reactivity (E/T ratio =10:1: Median (interquartile range), 8.7% (5.0–16.2), 5.2% (4.8–11.4), 20.8% (13.4–33.3), for imatinib, nilotinib and dasatinib, respectively). In addition, the number of regulatory T-cells (CD4+CD25int-hiCD127low) was similar among the three groups (Median (interquartile range), 36/mm3 (27–53), 48/mm3 (34–60), 39/mm3 (26–53), for imatinib, nilotinib and dasatinib, respectively).Furthermore, in the analysis of cytokines and chemokines, plasma levels of IL-8, IP-10, and MCP-1 were significantly elevated while the level of PDGF-bb was significantly decreased in all three groups compared to those of healthy control. Plasma levels of IL-1 beta, IFN-gamma, and FGF-basic were significantly decreased in only the dasatinib group compared to those of control (P=.02, P=.04, P=.03, respectively). In addition, plasma levels of GM-CSF were significantly elevated in the imatinib and dasatinib groups (Median (interquartile range), 6.1 pg/ml (2.7–11.7) and 7.9 pg/ml (4.5–8.2), P=.02, and P=.03, respectively) but not in the nilotinib group (P=.34) when compared to those of control. In the multiple regression analysis that evaluated the relationship between NK-reactivity and cytokines or chemokines in the patients receiving dasatinib, only plasma levels of GM-CSF were significantly associated with NK-reactivity (P=.03).Notably, in our data, dasatinib and nilotinib exerted opposite effects on NK-cell reactivity, expansion of LGL, and showed different cytokine and chemokine profiles. Based on our results, the activation of NK-cell reactivity induced by dasatinib might be caused by a mechanism other than a decrease in the number of regulatory T-cells. Additionally, in an unphysiological immunological status mediated by dasatinib, GM-CSF might make some contribution to NK-cell reactivity. Disclosures:Nakamae:BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Hino:BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Evaluation of Therapy in Symptomatic Patients with Lymphoproliferative Disorders of Large Granular Lymphocytes in Italy

Abstract 4607 Background.Chronic proliferations of large granular lymphocyte (LGL) are characterized by the expansion of cytotoxic T or NK cells. Relevant clinical features include neutropenia and anemia, either symptomatic or asymptomatic. The immunological and biological features of patients'LGL and the frequent association with autoimmune conditions represent the rationale for the immunosuppressive therapy frequently used in these conditions. However, no common criteria for starting therapy have been defined nor standard therapy has been established. We herein present data on 30 symptomatic patients treated in different Italian Centers, in the context of a collaborative study sponsored by the GIMEMA group. Methods.Diagnosis of lymphoproliferative disorder of LGL was made according to published criteria (Blood 1997, 1:89) and WHO classification 2008. Twentyseven patients were diagnosed as LGL leukemia and 3 as NK chronic lymphoproliferative disorder (NK-CLPD). Patients required treatment for symptomatic neutropenia, more frequently represented by recurrent bacterial infections (23/30), transfusion dependent anemia (4/30), other reasons (5/30; n: 2 pulmonary hypertension, n:2 aggressive disease and n:1 neurologic involvement). Treatments.First line therapy consisted of methotrexate (MTX) at 7,5 mg/m2/week in 13/30 patients; cyclosporin A (CyA, (2-3 mg/Kg/die) in 10/30 patients; cyclophosphamide (CY, 50–100mg/die) in 5/30 patients; (immuno)-chemotherapy (Campath-fludarabine, CVP, CHOP-like regimens) in 2 patients characterized by aggressive clinical presentation of disease with B symptoms. Steroid was generally given for short period (1 mg/kg p.o. prednisone) and then tapered off in all but 2 patients. Growth factors (G-CSF and EPO) were also used for short period of times. Before considering failure, each treatment was continued for at least 4 months. Response criteria were defined according to Lamy and Loughran (Blood 2011, 10:117).Second line therapy with MTX, CyA and CY was used in 2, 4 and 3 patients, respectively. Results.Time to response to MTX therapy ranged from 1 to 12 months. The overall response rate were 60% (9/15), with 3 CR and 6 PR. The duration of response ranged from 5 to 48 months. The 5 patients failing MTX as first line therapy were treated with CY (n:2, 1 CR, 1 PD), with CyA (n:2, 2 PR) and 1 rapidly progressed and died.Time to response to CyA ranged from 2 to 12 months. The overall response rate were 50% (7/14) with 2 CR and 5 PR. The duration of response ranged from 1 year to 10 year. The 5 patients failing first line CyA therapy were treated with MTX (n:1, with PR), with CY (n:2, with failure, but one case was rescued using the combination of CyA/CY), 1 patient with chemotherapy with failure; for 1 patient the follow up is too short.Time to response to CY ranged from 3 to 9 months. The overall response rate were 37.5% (3/8):2 CR, 1 PR. The duration of response ranged from 5 months to 5 years. The 3 patients failing CY at first line were treated with bendamustine (n:1), with alemtuzumab (n:1), with MTX (n:1) with failure in each casesTwo patients with very aggressive presentation were treated with chemotherapy: 1 case rapidly progressed and died; 1 case, who failed CT, was treated first with CyA and then with MTX with failure. This patient underwent allotransplant. Conclusions.Immunosuppressive therapy with MTX or CyA or CY represents the common initial approach to patients with symptomatic LGL proliferation. Patient failing one therapy can be efficiently rescued by one of the other immunosuppressive drugs. Although any conclusive evaluation is difficult due to the low number of patients, our data are likely to indicate that the duration of response after treatment with CY and CyA is longer than the response obtained with MTX. The aggressive initial presentation of disease correlates with a very dismal prognosis. A prospective randomized clinical trial comparing these three different clinical approaches is going to start in Italy. Disclosures:No relevant conflicts of interest to declare.

Advances in diagnosis and treatment of large granular lymphocyte syndrome

Large granular lymphocyte (LGL) syndrome comprises a clonal spectrum of T-cell and natural killer (NK)-cell LGL lymphoproliferative disorders associated with neutropenia. This review presents advances in diagnosis and therapy of LGL syndrome. Due to the lack of a single unique genetic or phenotypic feature and clinicopathological overlap between reactive and neoplastic entities, accurate LGL syndrome diagnosis should be based on the combination of morphologic, immunophenotypic, and molecular studies as well as clinical features. For diagnosis and monitoring of LGL proliferations, it is essential to perform flow cytometric blood and/or bone marrow analysis using a panel of monoclonal antibodies to conventional and novel T-cell and NK-cell antigens such as NK-cell receptors and T-cell receptor β-chain variable region families together with TCR gene rearrangement studies. Treatment of symptomatic cytopenias in patients with indolent LGL leukemia is still based on immunosuppressive therapy. Treatment with purine analogs and alemtuzumab may be considered as an alternative option. Progress in understanding the pathogenetic mechanisms of these entities, especially resistance of clonal LGLs to apoptosis, due to constitutive activation of survival signaling pathways, has its impact on identification of potential molecular therapeutic targets.

Interleukin-15 Is a Proto-Oncogene In Acute Large Granular Lymphocyte Leukemia

AbstractAbstract 704Interleukin (IL)-15 is critical for the differentiation, proliferation, activation and survival of large granular lymphocytes (LGL). Malignant blasts from patients with acute LGL leukemia (LGLL) can express membrane bound IL-15 and often require IL-15 or IL-2 to survive and expand in vitro, suggesting a pivotal role of IL-15 in the genesis of LGLL in vivo. Indeed, 30% of mice engineered to over express IL-15 develop LGLL (J Exp Med 193:219-231, 2001), suggesting that IL-15 is a proto-oncogene. The present study examined the mechanism by which this may occur in mouse and in man. We observed ~2.5-fold increased levels of DNA methyltransferase 3b (Dnmt3b) in IL-15tg mice with LGLL compared to wild type (Wt) splenocytes (2.6 ± 0.6 -fold higher, N = 3 each, P =.03) and a ~2-fold increased levels of global DNA methylation (GDM) compared to Wt splenocytes (% global DNA levels measured by mass spec as % 5mC/(5mC+2dC): 3.6 ± 0.11%, N = 4 for IL-15tg LGLL; 1.5 ± 0.08%, N = 4 for Wt, P <.0001). Remarkably, samples from three LGLL patients exhibited significantly increased levels of DNMT3B when compared to their normal donor counterparts, ranging from 38-fold to 5448-fold (P <0.008), and a significant increase in GDM (4.4% ± 0.01 N = 3 for LGLL patients; 4.2 ± 0.01% N = 3 for normal donors, P =.008). Dnmt3b is negatively regulated by miR-29b, which is under the regulation of Myc (Nature Genetics 40:43-50, 2008). Blasts from both IL-15tg LGLL mice and LGLL patients had a significant decrease in miR-29b expression when compared to their Wt LGL and normal donor counterparts respectively (2-fold lower in mouse, P <.02; 11 to 230-fold lower in human samples, N = 3 each, P <.0009). This was accompanied by increased expression of Myc in LGLL samples (mouse LGLL: Wt LGL = 10.4 ± 1.3-fold higher, N = 3 each, P < 0.005; human LGLL: normal donor LGL = 7.7 ± 1.6 -fold higher, N = 4, P <.0015). Since IL-15 signaling appears important in development of LGLL we investigated the oncogenic effect of IL-15 by culturing sorted mouse Wt (NK1.1+) LGLs in medium supplemented with 10% FCS and 100 ng/ml IL-15. Within 12 hours of culture, these IL-15–stimulated Wt LGLs displayed 2.0 ± 0.5-fold lower levels of miR-29b due to increased binding of a repressor complex that included Myc in addition to NFkBp65 and Hdac1 at the miR-29b promoter (Fig. 1A). By day 30 of Wt LGL culture in IL-15, we observed a 2.6 ± 0.6-fold mRNA increase in Dnmt3b (N=3, P=.03) and a 10.4 ± 1.3-fold increase in Myc (N = 3, P < .005) compared to fresh Wt LGL, also confirmed at the protein level (Fig. 1B, green color). Compared to fresh Wt LGLs, day 30 IL-15-stimulated Wt LGLs exhibited a significant increase in GDM measured by mass spectrometry (%GDM: 2.5 ± 0.3 in stimulated Wt LGLs, N = 4, vs 1.5 ± 0.08 in non-stimulated Wt LGLs, N = 4, P <.01). Over several months, the IL-15-stimulated Wt LGLs continued to expand and accumulate centrosome aberrations and aneuploidy, with over expression of Aurora kinase B when compared to fresh Wt LGLs (170.5 ± 58.23 -fold higher, N = 3, P = .02). SCID mice injected with these transformed cells died of LGLL in the absence of exogenous IL-15. Thus, chronic in vitro exposure of Wt LGLs to excessive IL-15 results in leukemic transformation via a process that likely includes decreased miR-29b, increased Dnmt3b, global DNA hypermethylation, centrosome aberrations and chromosomal instability. To address the in vivo role of DNMT3B in IL-15-mediated leukemogenesis, we created DNMT3Btg mice under a vav-1 promoter. DNMT3Btg mice had normal survival at 2 years, while IL-15/DNMT3Btg mice showed a significantly lower latency and higher incidence of LGLL compared with IL-15tg mice (median survival: 20.7 wk in IL-15/DNMT3Btg vs 31.1 wk in IL-15tg, P <.0001; Fig. 2). Thirty-one of 42 (73%) IL-15/DNMT3Btg mice died of LGLL compared to 8 of 38 (21%) IL-15tg mice. Leukemic blasts from LGLL mice demonstrated excessive expression of Aurora kinase B over Wt splenocytes (257.7 ± 39.4 -fold higher, N = 3 each, P = .01) and aneuploidy. Collectively, we provide evidence that IL-15 is a proto-oncogene: excessive expression of this lymphocytotropic cytokine results in activation of a repressor complex that decreases expression of miR-29b, induces Dnmt3b expression, global DNA hypermethylation, aberrant centrosome formation, aneuploidy and leukemic transformation. We provide evidence for these mechanisms in both in vitro and in vivo mouse models as well as in primary LGLL patient samples. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Transient monoclonal CD3+ T large granular lymphocyte proliferation in a case of mantle cell lymphoma with Rituximab‐associated late onset neutropenia

Transient monoclonal CD3+ T large granular lymphocyte proliferation in a case of mantle cell lymphoma with Rituximab‐associated late onset neutropenia

Hypertension artérielle pulmonaire et hémopathies malignes

Many recent publications have evidenced a statistical link between pulmonary arterial hypertension and myeloproliferative disorders, once excluded secondary causes of pulmonary arterial hypertension (more particularly, portal hypertension or thromboembolic events). Nonetheless, the frequency of such an association is quite variable, from 10 to 50 % and depends both on the population studied and on the type of myeloproliferative disorder. The most robust association is observed with essential thrombocythemia. Pulmonary arterial hypertension has also been described in association with lymphoma, acute leukemia, myeloma or large granular lymphocyte proliferation. The absence of large series call for a prospective study of pulmonary arterial hypertension in malignant hematologic disorders to firmly establish the exact frequency of such an association, to define more precisely its physiopathology in order to elaborate more specific diagnostic criteria together with guidelines for both treatment and follow-up.

The Spectrum of T-cell and Natural Killer – Large Granular Lymphocytic Proliferations

Large granular lymphocyte (LGL) disorders comprise a spectrum of conditions that result from the expansion of a T cell or natural killer (NK) cell with a distinct morphology. The World Health Organization (WHO) considers T-cell LGL leukaemia as a distinct entity and chronic lymphoproliferative disorders (CLD) of NK cells as a provisional category. Patients are asymptomatic or manifest cytopenia, splenomegaly and autoimmune phenomena. Morphology, immunophenotype and molecular analysis are important diagnostic investigations. Most T-cell LGL leukaemias are T-cell receptor (TCR)-〈β+ LGL expansions displaying a CD8+ CD4- CD57+ or rarely a CD4+CD8±dim phenotype; a minority are clonal expansions of TCRγδ+LGL. NK LGL proliferations are CD3-, TCR-, CD16+, CD56+ with variable expression of T-cell non-specific markers. Expression of cytotoxic antigens T1A-1, granzymes and perforins are seen in both diseases. The pathogenesis is unknown. It is postulated that these disorders result from an expansion of a terminal memory-activated cytotoxic lymphocyte or an NK cell due to persistent antigenic stimulation with failure to undergo activation-induced cell death as a consequence of an impaired apoptotic pathway. Gene profiling has shown dysregulation of genes involved in apoptosis. The course is chronic, and transformation or aggressive disease is rare. Treatment strategies comprise immunomodulatory agents, purine analogues and antibody therapy.

Prevalence of Large Granular Lymphocyte Proliferation in Chronic Myeloid Leukemia (CML) Patients Treated with Dasatinib.

Abstract 1111Poster Board I-133Dasatinib is a potent inhibitor of the BCR-ABL tyrosine kinase which is effective in the treatment of imatinib refractory CML. While hematologic toxicities of neutropenia and thrombocytopenia are well known, large granular lymphocytosis has been reported in only a small number of patients without prior allogeneic stem cell transplant treated with dasatinib for CML. During routine follow up of leukocyte counts in 15 consecutive patients (age range 27-77 years) treated with dasatinib, 4 patients (2 chronic-phase, 1 accelerated phase with clonal cytogenetic progression, 1 blast-phase) developed a lymphocytosis (> 3800/mm3). Peripheral blood smear and peripheral blood flow cytometry revealed a population of large granular lymphocytes (LGLs) expressing CD3, CD8, CD57, and variable expression of CD56. Lymphocytosis was first noted between 1 and 9 months after initiation of dasatinib and has persisted in 3 of the patients with a median follow up of 33 months from the onset of lymphocytosis. Peak absolute lymphocyte count ranged from 5000/mm3 to 6900/mm3 and approximately 40 to 60% of the lymphocytes were LGLs by flow cytometry with the remainder being predominantly T lymphocytes. These 4 patients with LGL lymphocytosis have all have major molecular responses and the patient with blast-phase has remained in a complete cytogenetic remission with a major molecular response 44 months after initiation of dasatinib. The 11 other patients (6 chronic-phase, 2 accelerated-phase, 3 blast-phase) treated with dasatinib for CML have not developed lymphocytosis. These patients have been followed for a median of 25 months (range 3-50 months) although some were treated with dasatinib for a relatively short period of time because of poor response of their advanced CML. Review of the peripheral blood smears from 3 of the 6 chronic phase patients without lymphocytosis who remain on dasatinib treatment did not reveal any LGLs. All of these 6 patients have had complete, sustained cytogenetic responses. A persistent pleural effusion developed in the blast phase patient with lymphocytosis approximately 12 months after lymphocytosis developed; no significant side effects were noted in the other 3 patients although one remains thrombocytopenic. Pleural effusions developed in 2 of the 6 patients without lymphocytosis who remain on dasatinib treatment. Previous reports have suggested an increased incidence of “inflammatory” type side effects such as pleural effusions and pneumonitis in patients with dasatinib related LGL proliferation, although the small number of patients in this series precludes analysis of this association. In summary, LGL proliferation was detected in a minimum of 27% of dasatinib recipients and may be associated with a beneficial response. While the mechanism of LGL proliferation has not been fully explained, it has been suggested that dasatinib mediated inhibition of immunoregulatory kinases such as Src is permissive of LGL proliferation. Further evaluation of the frequency and clinical impact of this phenomenon in the large clinical trials of patients treated with dasatinib is warranted. DisclosuresSchiffer:Bristol Myers: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

P083 A father and his son presenting with cytopenias associated with CD3 T large granular lymphocyte proliferation

P083 A father and his son presenting with cytopenias associated with CD3 T large granular lymphocyte proliferation

Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy

IntroductionThe purpose of this study was to analyze the data of patients with T-cell large granular lymphocyte (T-LGL) lymphocytosis associated with inflammatory arthropathy or with no arthritis symptoms.MethodsClinical, serological as well as histopathological, immuhistochemical, and flow cytometric evaluations of blood/bone marrow of 21 patients with T-LGL lymphocytosis were performed. The bone marrow samples were also investigated for T-cell receptor (TCR) and immunoglobulin (IG) gene rearrangements by polymerase chain reaction with heteroduplex analysis.ResultsNeutropenia was observed in 21 patients, splenomegaly in 10, autoimmune diseases such as rheumatoid arthritis (RA) in 9, unclassified arthritis resembling RA in 2, and autoimmune thyroiditis in 5 patients. T-LGL leukemia was recognized in 19 cases. Features of Felty syndrome were observed in all RA patients, representing a spectrum of T-LGL proliferations from reactive polyclonal through transitional between reactive and monoclonal to T-LGL leukemia. Bone marrow trephines from T-LGL leukemia patients showed interstitial clusters and intrasinusoidal linear infiltrations of CD3+/CD8+/CD57+/granzyme B+ lymphocytes, reactive lymphoid nodules, and decreased or normal granulocyte precursor count with left-shifted maturation. In three-color flow cytometry (FCM), T-LGL leukemia cells demonstrated CD2, CD3, and CD8 expression as well as a combination of CD16, CD56, or CD57. Abnormalities of other T-cell antigen expressions (especially CD5, CD7, and CD43) were also detected. In patients with polyclonal T-LGL lymphocytosis, T cells were dispersed in the bone marrow and the expression of pan-T-cell antigens in FCM was normal. Molecular studies revealed TCRB and TCRG gene rearrangements in 13 patients and TCRB, TCRG, and TCRD in 4 patients. The most frequently rearranged regions of variable genes were Vβ-Jβ1, Jβ2 and Vγ If Vγ10-Jγ. Moreover, in 4 patients, additional rearrangements of IG kappa and lambda variable genes of B cells were also observed.ConclusionRA and neutropenia patients represented a continuous spectrum of T-LGL proliferations, although monoclonal expansions were most frequently observed. The histopathological pattern and immunophenotype of bone marrow infiltration as well as molecular characteristics were similar in T-LGL leukemia patients with and without arthritis.