Granular Lymphocyte Proliferation Research Articles

Incidence, Clinical Features, and Outcomes of Large Granular Lymphocyte Leukemia

Introduction: - Large Granular Lymphocyte (LGL) leukemia is a rare and diverse hematologic disorder characterized by the clonal proliferation of large granular lymphocytes in the peripheral blood and bone marrow. Despite its rarity, population-based studies on LGL leukemia remain limited. This study aims to comprehensively analyze clinical characteristics and outcomes among patients with LGL leukemia. Methods: - We utilized the Surveillance, Epidemiology, and End Results (SEER) database 17 registry to identify patients diagnosed with large granular lymphocytic leukemia between 2000-2020. Data analysis was conducted using SEER*Stat version 8.4.1.2 and STATA. Univariate survival analysis was done using a Kaplan Meir plot with a log-rank test, and the Cox proportion hazard regression model was used for multivariate survival analysis. Results: - We identified a total of 2,589 LGL leukemia cases during the study period. The calculated incidence of LGL leukemia was 0.14 cases per 1,000,000 individuals (range: 0.14 - 0.15). Male patients exhibited a higher incidence rate ratio of 1.19 (95% CI: 1.11 - 1.27, p < 0.01) than females. However, females tended to be diagnosed younger than males (67 vs. 68, p < 0.01). Most cases were observed in the White population (83%) compared to other racial groups. The median age at diagnosis was 67 years (ranging from 7 to 85 years). Patients aged above 70 years showed the highest incidence of LGL leukemia (0.80) in contrast to those aged 45-54 years (0.30) and 55-64 years (0.63). Notably, there was a significant increasing trend in LGL leukemia incidence, with an incidence rate ratio of 13.50 (95% CI: 10.5-17.6, p < 0.001) in the period 2015-2019 compared to 2000-2004. Approximately 32.7% of patients received chemotherapy; the median time from diagnosis to treatment was one month. The survival rates at 12, 36, and 60 months for LGL leukemia patients were 88.9%, 76.5%, and 66.5%, respectively, with a median follow-up of 40 months. Multivariate analysis revealed that females had a more favorable survival outcome than males (HR: 0.78, 95% CI: - 0.64 - 0.97, p: 0.02). Moreover, patients who underwent chemotherapy exhibited better survival than those who did not (HR: 0.72, 95% CI: 0.56 - 0.93, p: 0.01). Elderly patients above 60 years of age had poorer survival rates (HR: 3.1, 95% CI: 2.34 - 4.01, p < 0.01), and no significant difference in outcomes was observed based on the timing of chemotherapy initiation from the time of diagnosis. Conclusion: - This study represents the most extensive and most recent population-based investigation of LGL leukemia. The findings highlight the potential benefits of chemotherapy in improving survival and indicate better outcomes in female patients. Further research is warranted to validate these promising results and gain deeper insights into the management and prognosis of LGL leukemia.

Transdifferentiation, phenotypic infidelity, progression, and transformation in T/NK-cell neoplasms: Report from the 2021 SH/EAHP Workshop.

Sessions 8 and 9 of the 2021 Society for Hematopathology and the European Association for Haematopathology Workshop aimed to collect examples of transdifferentiation, lineage infidelity, progression, and transformation in precursor and mature T/natural killer (NK)-cell neoplasms. Twenty-eight cases were submitted and analyzed, with whole-exome sequencing and genome-wide RNA expression analysis performed in a subset of the cases. In session 8, 7 T-lymphoblastic lymphoma/leukemia cases were received that showed transdifferentiation to clonally related mature myeloid hematopoietic neoplasms, including 6 histiocytic/dendritic cell lineage neoplasms and a mast cell sarcoma. Session 9 included 21 mature T-cell neoplasms that were grouped into 3 themes. The first one addressed phenotypic infidelity in mature T-cell lymphomas (TCLs) and included 8 TCLs expressing aberrant antigens, mimicking classic Hodgkin and non-Hodgkin B-cell lymphomas. The second theme addressed disease progression in TCL and included 5 cutaneous T-cell lymphoproliferative disorders and 2 T-cell large granular lymphocyte proliferations with subsequent progression to systemic TCL. The third theme included 6 patients with TCL with T-follicular helper phenotype, mainly angioimmunoblastic T-cell lymphoma, with concurrent/subsequent clonal hematopoiesis or myeloid neoplasms and/or subsequent/concomitant diffuse large B-cell lymphoma. This cohort of cases allowed us to illustrate, discuss, and review current concepts of transdifferentiation, aberrant antigen expression, and progression in various T/NK-cell neoplasms.

Identification of novel NFKB1 and ICOS frameshift variants in patients with CVID.

Common variable immunodeficiency (CVID) is a 'late-onset' primary immunodeficiency characterized by variable manifestations and genetic heterogeneity. A monogenic cause of CVID has been reported in 10% of patients. In this study, we identified two novel pathogenic variants implicated in monogenic CVID by whole exome sequencing (WES) analysis: a heterozygous nuclear factor κB subunit 1 (NFKB1) p.G686fs mutation and a homozygous inducible T-cell co-stimulator (ICOS) p.L96Sfs mutation. The predicted crystal models indicated premature truncation of the two mutated proteins. Both variants were demonstrated as loss-of-function mutations and were associated with overlapped manifestations of respiratory fungal infection and splenomegaly. We further performed a detailed assessment of immunologic phenotypes and impaired lymphocyte functions in patients. Moreover, we discovered an association between monoclonal T-large granular lymphocyte proliferation and ICOS-deficient CVID for the first time. These observations lead to a new perspective on the underlying genetic heterogeneity of CVID.

TCR γδ Large Granular Lymphocyte Leukemia (LGLL) Displays Features between CD8+ and CD4+ TCR αβ LGLL

BackgroundT-large granular lymphocyte leukemia (T-LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Usually, leukemic cells of this T-cell neoplasia express the αβ T-cell receptor (TCR); only a small subset of cases expresses the γδ TCR denoting the TCRγδ-LGLL. Currently, among the different LGL diseases, TCRγδ-LGLL remains less studied and several clinical and laboratory data already described in TCRαβ-LGLL have not yet been explored in TCRγδ-LGLL. In particular, data are still missing about STAT3 and STAT5b mutations, frequently observed in TCRαβ-LGLL, and about T-LGL clonotypes discovered to be private to TCRαβ-LGLL as compared to healthy controls.AimsThis work was aimed to perform the analysis on: STAT3 and STAT5b mutations, LGL immunophenotype, TCRγ clonotype repertoire and clinical presentations in a group of TCRγδ-LGLL patients. The objective was to reveal common and different features of this rare kind of LGLL with the more frequent TCRαβ-LGLL.MethodsThe cohort of study included 11 patients affected by TCRγδ-LGLL. Sanger sequencing was used for mutational analysis on hot-spot regions of the 2 genes more frequently mutated in LGL disorders, STAT3 and STAT5b . Immunophenotype of LGL clone was defined by flow cytometry analysis. CDR3 repertoire and frequency distribution of TCR gamma gene rearrangements were obtained by Next-Generation Sequencing (NGS).ResultsTCRγδ-LGLL showed a higher occurrence of STAT mutations (82%) as compared to TCRαβ-LGLL (43%). In detail, 9 out of 11 TCRγδ LGLL patients were characterized by STAT3 or STAT5b mutations in a mutually exclusive pattern. Being characterized by both STAT3 and STAT5b mutations, TCRγδ LGLL resembles both CD8+ TCRαβ-LGLL and CD4+ TCRαβ-LGLL, the first being mostly associated to STAT3 mutations and the latter to STAT5b . Similarly, TCRγδ-LGLL showed the same relationship observed between immunophenotype and kind of mutation in TCRαβ-LGLL: TCRγδ-LGLL patients with CD56- LGL immunophenotype were characterized by STAT3 mutations (as in CD8+ TCRαβ-LGLL), while TCRγδ-LGLL patients with CD56+ LGL immunophenotype by STAT5b mutations (as in CD4+ TCRαβ-LGLL). Anyway, in TCRγδ-LGLL no correlation was found between mutations and clinical course as observed in CD8+ TCRαβ-LGLL patients carrying STAT mutations, rather, we observed that patients with γδLGLs positive for Vδ2 showed frequently indolent course, while Vδ1 was associated to a more symptomatic disease (4 out of 5 symptomatic patients were Vδ1+). By NGS we obtained the TCR gamma repertoire of each patient and we revealed that all patients were clonal with the exception of 2 showing a polyclonal pattern. The 2 polyclonal cases were the only 2 patients without STAT mutations. Among the remaining STAT3 mutated patients (n=4), 3 were polyclonal and one biclonal, while STAT5b mutated patients (n=5) were more frequently monoclonal (4/5 monoclonal and 1/5 biclonal). Considering the CDR3 repertoire of the immunodominant clones, as observed in TCRαβ-LGLL, common clonotype sequences were present with low frequency in almost all TCRγδ-LGLL patients. Interestingly, 2 different CDR3 sequences were found shared in different patients at frequency >10% of the total rearrangements. In terms of clonal rearrangements, Vγ3-Jγ1/2, Vγ9-JγP and Vγ8-Jγ1/2 were the combination usages most frequently detected, showing a limited combinatorial diversity of TCR genes similarly to CD4+ and differently from CD8+ TCRαβ-LGLL, this last having larger variability. Analysis about the clonotype repertoire in a 2 years-follow up showed that patients maintain their clonal pattern along the time.ConclusionsOur results highlight common and different characteristics of TCRγδ-LGLL as compared to TCRαβ-LGLL. The data suggest that TCRγδ-LGLL can be considered the intersection of the 2 types of TCRαβ-LGLL, sharing CD8+ or CD4+ T-LGLL mutational and phenotype features. Similarly to TCRαβ-LGLL, also TCRγδ disease showed a decreased diversity of TCR repertoire, with common TCR sequences shared among patients and with limited number of Vγ-Jγ combinations. A distinctive feature of TCRγδ-LGLL is that STAT mutations do not correlate with a symptomatic clinical behavior, rather, Vδ1 expression appears to be indicative for symptomatic disease. DisclosuresGalimberti:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau.

Discovery of Dysimmunity in Large Granular Lymphocytic Leukemia (LGLL) using Bioinformatic Analysis.

Large granular lymphocytic leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by the clonal proliferation of large granular lymphocytes (LGL), classified as T and NK subtypes. Although JAK/STAT pathway gene mutation, such as STAT3/STAT5B, is the dominant driver in the proliferation of LGLL, immune abnormality remains an unsolved puzzle in the pathogenesis. By means of bioinformatic method through the GEO dataset GSE39838, we performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, as well as protein-protein interaction network (PPI) module calculation. As a consequence, differentially expressed genes (DEGs) involved in immune regulation were detected to be related with LGLL, including C1QA, C1QC and CD163 etc. Among all the DEGs, 147 genes were up-regulated, while the number of down-regulated genes was 1,296. In the KEGG pathway of LGLL, infection and immunity were the primary alteration, including tuberculosis and rheumatoid arthritis (RA). However, meticulous experiments are required to validate. To sum up, dysimmunity might be another internal anomaly of LGLL, thus it is a reminder that immune regulation of LGLL should be paid more attention. Moreover, immune microenvironment studies in LGLL covering T, B, and NK cells probably contribute to the molecular pathology, aiming to contribute to the molecular pathology of the LGLL. Additionally, pharmaceutical development directed at immune molecules might be pre-dictive of targeted therapy era in LGLL.

Clinical and laboratory characteristics in patients with myeloid neoplasms complicated with clonal T large granular lymphocyte proliferation

目的探讨髓系肿瘤合并克隆性T大颗粒淋巴细胞（T-LGL）增殖的临床及实验室特征。方法回顾性分析中国医学科学院血液病医院2017年11月至2018年11月收治的5例确诊髓系肿瘤合并克隆性T-LGL增殖患者的临床资料。结果5例患者中位年龄60岁，均存在>6个月的血细胞异常病史。外周血T-LGL绝对计数均<1.0×109/L，LGL免疫表型2例为CD4+CD8−，3例为CD4−CD8+；4例为αβ型T细胞，1例为γδ型T细胞；5例均有克隆性证据；二代测序检测显示1例患者存在STAT3突变，其余4例均为阴性。结论5例髓系肿瘤合并克隆性T-LGL增殖患者起病隐匿，以老年为主，临床以慢性血细胞减少多见。存在克隆性T-LGL，尤其是T-LGL绝对计数<0.5×109/L的患者诊断T大颗粒淋巴细胞白血病需慎重，可能与意义未明的T细胞克隆性疾病部分重叠。STAT3或STAT5b突变检测可能是鉴别两者的主要手段。

Clonal T-cell large granular lymphocyte proliferations in childhood and young adult immune dysregulation conditions.

Proliferation of large granular lymphocytes (LGL) and T-cell LGL (T-LGL) in peripheral blood along with demonstration of clonality are the hallmarks of a heterogeneous group of disorders, including T-LGL leukemia or T-LGL lymphocytosis. They are often associated with neutropenia and responsive to immunosuppression. The true nature of this entity is not well understood. Some cases are reported as reactive phenomena with very limited experience in pediatric population. Hematology/Oncology Flow Cytometry Laboratory database has been reviewed retrospectively. Patients with identifiable distinct CD5-dim T-cell population and positive clonal T-cell receptor rearrangement were included in the analysis. Clinical and laboratory data were then reviewed. Sixteen cases of children and young adults with increased peripheral blood clonal T-LGL population characterized by dim CD5 expression with wide range of underlying immune dysregulation/stimulation disorders were reviewed. Extended follow up with repeat testing suggested the reactive nature of persistent clonal T-LGL proliferations in this group. Our observations indicate that clonal T-LGL proliferations in children and young adults are reactive in nature and some can be persistent with an indolent course with unknown consequentiality. Clonal T-LGL cells could be targeting the most prominent immunogenic stressor(s) involved as a control mechanism.

PF367 MOLECULAR EVIDENCE THAT ATOVAQUONE IS A NEW STAT3‐INHIBITOR COUNTERBALANCING LEUKEMIC CELL PRO‐SURVIVAL IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. The hallmark of leukemic LGLs is a constitutive activation of STAT3 leading to enhanced triggering of many pro‐survival genes. Moreover, 30–40% of patients carry mutations in STAT3 gene bringing to the constitutive activation of the malignant clone.No specific treatment is now available for the patients affected by LGLL and the in‐use therapy is based on low doses of immunosuppressive drugs, i.e. Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA). Anyway, these therapies show limited efficacy, induce many side effects with the Overall Response Rate (ORR) being around 50% and a high rate of relapse in the responding patients.The JAK/STAT signaling pathway, which is central to LGL leukemia pathogenesis, includes several players that provide the basis for new targeted treatments. This offers the rationale for using Atovaquone (ATQ, Mepron™), a recently FDA‐approved anti‐microbial agent characterized by a low adverse effect profile that has been recently demonstrated (Xiang et al., 2016) to work as a powerful STAT3 inhibitor with anti‐cancer efficacy in both animal models and human cell lines.Aims:Our study focuses on in vitro tests to evaluate the effects of ATQ as inhibitor of STAT3 phosphorylation and of LGLs survival, with the goal to develop a targeted approach to LGLL, likely devoid of adverse effects.Methods:Patients’ Peripheral Blood Mononuclear Cells (PBMC) were purified to perform cultures to test serial drug concentrations. Using in vitro cells culture, the inhibition of STAT3 protein phosphorylation and the expression of its target proteins, MCL1 and BCL2, were evaluated by Western Blot assay (WB). In addition, the cytotoxicity and the apoptotic selectivity of ATQ was investigated by Annexin V test; a staining with anti‐CD57, CD56 or CD16 was used to identify leukemic LGLs from PBMCs. Finally, by RT‐PCR, STAT3 activity was analyzed along its transcriptional levels and target genes.Results:Through in vitro experiments we initially tested the best concentration inhibiting cell viability and STAT3 activation on patients’ PBMCs. We observed that ATQ was efficiently able to inhibit STAT3 phosphorylation at a concentration of 25 μM. The results of the apoptosis analysis showed that ATQ has a dose and time dependent cytotoxic effect. The IC50 was reached at a dose of 25 μM after 24 h of culture. ATQ demonstrated a selective cytotoxicity against leukemic LGLs (reaching 3.9 ± 0.7 fold increase of apoptosis vs untreated condition after 48 h of culture) sparing non‐leukemic cells (ATQ treated apoptosis vs untreated in non‐LGL PBMC = 1.18 ± 0.3 fold change). By comparing the in vitro cell effect of ATQ with that of the commonly used therapies, ATQ showed a cytotoxic effect consistent with CTX and CyA, but higher than MTX (p &lt; 0.01). Concerning anti‐apoptotic factors under the STAT3 control, after 6 h ATQ treatment the expression of MCL1 and BCL2 showed only minimal changes, thereby suggesting that different mechanisms of action take place.Summary/Conclusion:These in vitro data indicate that ATQ has a promising action on the inhibition of the JAK/STAT pro‐survival pathway in leukemic LGLs, resulting in a selective cytotoxic effect against LGLs through MCL1‐ and BCL2‐independent mechanisms.

Clonal T-Large Granular Lymphocyte Proliferations in Childhood: Friend or Foe?

Background: The pathophysiology of clonal T-large granular lymphocyte (T-LGL) proliferations is not well understood; the distinction between reactive and malignant entities is not very clear given the fact that acquired STAT3 mutations can be seen with clonal T-LGL proliferations in non-malignant Felty syndrome and with frequent use of immunosuppression for the treatment of T-LGL leukemia. Historically, determination of clonality has been often seen as an indicator of T-LGL leukemia. We reviewed our experience on clonal T-LGL proliferations in children.Material and Methods: T-LGLs were identified by CD3 and dim CD5 staining on flow cytometric analysis and observation of lymphocytes with large granular cytoplasm appearance in peripheral blood. Cases with peripheral blood lymphocyte T-LGL population greater than 10% were reviewed in patients whom had flow cytometry studies performed for different indications. Among them, ones with available T cell receptor (TCR) gene rearrangement studies were selected and 16 cases with clonal TCR rearrangement patterns were included in this study. Fifty-seven peripheral blood flow cytometric tests using 15 surface markers were analyzed in those 16 cases. Patients have been followed for up to 7 years.Results: Sixteen cases of clonal T-LGL proliferation associated with different diseases were included in this analysis. Three patients had chronic graft versus host disease, 2 Evans syndrome, and one case each with the following conditions: common variable immunodeficiency disorder, severe combined immunodeficiency who developed CMV infection following umbilical cord blood transplantation (UCBT) and later acute EBV infection, co-existing Langerhans cell histiocytosis and primary hemophagocytic lymphohistiocytosis, autoimmune hemolytic anemia, chronic idiopathic thrombocytopenic thrombocytopenia, Hodgkin lymphoma, X-linked lymphoproliferative disorder, Rosai Dorfman disease (RDD), acute EBV infection, acute parvovirus B19 infection and paroxysmal nocturnal hemoglobinuria with history of treated severe aplastic anemia. Pathophysiological processes involved in these cases included various combinations of inherited immune deficiency in 4, acquired immune deficiency in 5, lymphoproliferation in 6, alloimmune reaction in 3, autoimmune reaction in 5, infection in 4, inflammation in 3 and malignancy in 1. One patient had three different clonal populations at different times due to maternal engraftment, CMV infection following UCBT and during an acute EBV infection after immune suppression withdrawal, respectively. The patient with RDD developed clonal T-LGL expansion when his disease flared. In some cases, clonal T-LGL proliferations disappeared with the resolution of the underlying pathological process. However, the T-LGL clone persisted and was frequently associated with relative increase over time, if the disease process continued to be active and/or progressed, even despite use of therapeutic approaches often including immunosuppression. Four clonal T-LGL proliferations in 2 patients have resolved, remaining 10 were persistent and have not been reevaluated in 4 cases. None of the cases showed a purely monoclonal pattern. The size of the clonal T-LGL population was variable with a median value of 18.6%, as high as 58.7% of the lymphocyte population. T-LGL population has positively correlated with CD3, CD8, CD57 (p<0.001) and inversely with CD19 (p=0.001) expression. There was also an overall positive correlation between the percent of T-LGL cells and absolute T-LGL count (p<0.001).Discussion: Clonal T-LGL proliferations in childhood is a reactive process reflecting the activity of the underlying disease process, including immunodeficiency, autoimmunity, alloimmunity, infection, inflammation, lymphoproliferation and malignancy. Use of immunosuppression has been associated with relative increase in the clone size despite decrease in peripheral blood absolute lymphocyte count along with relative reduction in T and B lymphocyte compartments in patients with active disease. Presence of clonal T-LGL proliferations in a polyclonal background is not an indicator of malignant process. It is very possible that such proliferations are an effort of the immune system to control the most prominently involved immunogenic stressors. DisclosuresNo relevant conflicts of interest to declare.

T-Cell Large Granular Lymphocytic Leukemia – Case Report

SUMMARY – T-cell large granular lymphocytic leukemia (T-LGLL) is an uncommon but probably underdiagnosed disease caused by clonal proliferation of large granular lymphocytes. Diagnosis is typically based on the high number of morphologically characteristic lymphoid cells and finding of an abnormal immunophenotype by flow cytometry. Because of its relatively indolent clinical behavior, observation is often an appropriate therapy. Here we present a case of a 53-year-old male admitted to the hospital because of abdominal pain. Blood examination revealed mild mycrocitic anemia and multiplied lactate dehydrogenase level. Abdominal ultrasound showed splenomegaly of 16 cm, with no lymphadenopathy. Fine needle aspiration of bone marrow revealed hypocellular marrow with 50% of atypical lymphoid cells. There were 81% of atypical medium sized granular lymphocytes with irregularly shaped nuclei in peripheral blood, so the cytologic diagnosis was lymphoproliferative process. Bone marrow biopsy showed nodular and interstitial proliferation of small, partially atypical T lymphocytic cells positive for CD2, CD3, CD5, CD8, granzyme and TIA, and negative for hairy cell markers, CD10, MUM 1, bcl 1, CD4 and CD56. The finding was consistent with T-LGLL. Due to splenomegaly, the patient was treated with cyclosporine and gradually reduced dose of corticosteroids, leading to regression of splenomegaly and normalization of lactate dehydrogenase level.

Protocolo diagnóstico de las alteraciones leucocitarias en la artritis reumatoide

IntroductionPatients with RA can present a wide variety of hematological disorders. ClassificationWithin leukocyte alterations we can find three groups: leukopenia, leukocytosis and lymphoproliferative disorders. Among the leukopenias, there is the Felty syndrome, which is the most frequent and consists of the triad of neutropenia, splenomegaly and RA; LGL leukemia, which is a proliferation of large granular lymphocytes associated with neutropenia and drug-induced neutropenia, since most of the agents used for treating RA can induce it (methotrexate, cyclophosphamide...). In the presence of leukocytosis, RA can develop neutrophilia in the context of an inflammation or an associated bacterial infection, or eosinophilia, which is related to greater extra-articular involvement. Lymphoproliferative disorders have a higher occurrence in these patients, with an incidence and mortality due to leukemia and lymphoma which doubles the expected values, mainly in those patients with long-term arthritis.

Characterization of Myelodysplastic Syndromes (MDS) with T-Cell Large Granular Lymphocyte Proliferations (LGL)

▪Introduction:MDS include a spectrum of hematopoietic stem cell malignancies characterized by bone marrow failure and dysplastic morphology. LGL is a clonal proliferation of cytotoxic T cells, which manifest as neutropenia, anemia, and thrombocytopenia and is associated with autoimmune disorders. LGL in association with MDS has been previously reported. However, clinicopathological features, prognostic, and predictive factors in those patients diagnosed with both LGL and MDS is not well studied.Methods:We identified patients at Moffitt Cancer Center (MCC) diagnosed with MDS who were previously tested for the presence of LGL clonal populations by peripheral blood flow cytometry at time of first visit. An LGL population was defined by the standard flow cytometry immunophenotype and clonality confirmed by T-cell receptor gamma and beta gene rearrangement.. Next Generation sequencing data was available for 151 patients. Recurrent somatic gene mutations were compared between patients with an LGL clone and those without.Results:Of the 675 patients with MDS tested for LGL in the database, 206 (30.5%) had an LGL clonal population. The mean LGL absolute cell count in the peripheral blood was 335/µL. Table-1 summarizes the baseline characteristics of the two groups.There was no difference in response to azacitidine therapy. Among 50 patients with LGL clone who received azacitidine with available data on response, the rate of hematological improvement or better (HI+) was 38%. The (HI+) was 28% among 105 patients evaluable for response without LGL clone. P .14The median overall survival (OS) was for patients with no LGL clone was 65 months (mo) compared to 46 mo (p .024). The median OS for lower risk MDS patients (low/int-1 by International Prognostic Scoring System [IPSS]) was 68 mo versus 97 mo for those with or without LGL proliferation, respectively (P .005). In higher risk MDS, there was no difference in median OS between those with or without LGL expansion, respectively (20 mo versus 16 mo, p .7). The median OS for patients with very low/ low Revised-Internatinal Prognostic Scoring System (R-IPSS) was 96 mo if LGL proliferation was detected compared to 128 mo if it was not, (p value .016). For intermediate R-IPSS the median OS was 65 mo and 41 mo with or without LGL proliferation (p .16). Finally, for high/very high R-IPSS the median OS was 18 and 16 mo with or without LGL proliferation, (p .84) In cox regression analysis the presence of an LGL clone was independently prognostic for OS after adjusting for age and R-IPSS, Hazard ratio 1.3, p = .05. Somatic gene mutation data were available for 151 patients; there was no statistically significant difference in the distribution of any mutation except IDH-2 (Table-2). The most common somatic mutations observed among patients with LGL clone were SF3B1 19%, TET-2 16%, U2AF1 13%, IDH-2 13%, RUNX-1 13%, and ASXL-1 10%. In patients without an LGL clone the most common somatic mutations were TET-2 26%, ASXL-1 20%, DNMT3A 16%, TP53 13%, SF3B1 12%.Conclusion:An LGL clone is demonstrable in approximately 30% of patients with MDS in association with advancing age. The presence of LGL proliferation was associated with worse OS in lower risk MDS pts. Although the spectrum of somatic gene mutations were similar, the presence of IDH-2 mutation and absence of DNMT3A or TP53 gene mutationscharacterized LGL+ cases. [Display omitted] [Display omitted] DisclosuresRoe:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Seattle Genetics: Speakers Bureau. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy.

T-Cell Large Granular Lymphocyte Proliferation (LGL) in Patients with Myelodysplastic Syndromes (MDS): Not an Innocent Bystander?

T-Cell Large Granular Lymphocyte Proliferation (LGL) in Patients with Myelodysplastic Syndromes (MDS): Not an Innocent Bystander?

Moderate-dose cyclophosphamide in the treatment of relapsed/refractory T-cell large granular lymphocytic leukemia-associated pure red cell aplasia

Background: T-cell large granular lymphocyte leukemia (T-LGLL) is a rare disorder characterized by clonal proliferation of large granular lymphocytes (commonly CD3+/CD8+/CD57+). However, the available data regarding the optimal treatment for relapsed/refractory T-LGLL patients are limited.Methods: We retrospectively reviewed 10 patients treated with immunosuppressive therapy consisting of intravenous moderate-dose cyclophosphamide (MD-CTX) together with oral cyclosporine A for relapsed/refractory T-LGLL in our hospital between July 2006 and March 2013.Results: The overall response rate to MD-CTX was 60% (6/10; hematologic complete remission rate, 50%; hematologic partial remission rate, 10%). The median time to response was 28.5 days (range, 20–118 days). The relapse rate of MD-CTX was 50% (3/6); two of these three patients achieved hematologic complete remission after receiving a second course of MD-CTX. Neutropenia was the major adverse event of the MD-CTX regimen. The median time to neutropenia was 5.5 days (range, 1–10 days) and the median neutropenia duration was 5 days (range, 3–15 days). None of the patients developed severe infection.Conclusions: The MD-CTX regimen appears efficacious and safe in the treatment of relapsed/refractory T-LGLL patients.

T-cell large granular lymphocyte proliferation in myelodysplastic syndromes: Clinicopathological features and prognostic significance

Inflammatory and immune dysregulation are crucial in the initiation and development of myelodysplastic syndromes (MDS). It is noted that clonal T-cell large granular lymphocyte (T-LGL) proliferation associated with MDS is not uncommon. However, clinicopathological features, and prognostic and predictive value of presence of T-LGL proliferation in MDS patients is not very clear. This study compared 35 MDS patients with T-LGL proliferation with 36 MDS patients without T-LGL proliferation and summarized clinicopathologic features, including peripheral blood LGL cell counts, immunophenotype, T cell receptor gene rearrangement, bone marrow hematopoietic status, and adjuvant immunosuppressive therapy. The peripheral blood CD3+/CD57+ cell counts were significantly different (p<0.01) between the two groups. Notably, on examination of the bone marrow, MDS patients with T-LGL proliferation showed more frequent hypocellularity and/or lineage hypoplasia, particularly erythroid hypoplasia. On survival analysis, no overall difference was noted between MDS patients with T-LGL proliferation and those without T-LGL proliferation, and between the patients who received therapy for LGL and those who did not receive adjuvant therapy for LGL in the same risk group. In conclusion, T-LGL proliferation present in MDS patients can be associated with bone marrow hypocellularity and lineage hypoplasia. Although immunosuppressive therapy to eliminate T-LGL cells is potentially beneficial to the MDS patients with associated T-LGL proliferation, there is no overall survival benefit to the patients who received such treatment.

A case of refractory multiple myeloma with proliferation of large granular lymphocytes by lenalidomide treatment and its association with clinical efficacy.

A 72-year-old Japanese male was diagnosed as having monoclonal gammopathy of undetermined significance and was followed up without therapy. Three years later, the patient progressed to symptomatic multiple myeloma. Melphalan + prednisolone was administered as first-line chemotherapy for ~6 years. Since the patient was judged to exhibit refractory multiple myeloma, he subsequently received radiation therapy on the lumbar spine. The patient was enrolled in a clinical trial and received lenalidomide + lowdose dexamethasone (Rd) therapy. The patient achieved very good partial remission following four cycles of Rd. At this time, large granular lymphocytes (LGLs) increased to 25-40% of peripheral blood leukocytes, however, the LGLs were present in the blood (~8%) prior to lenalidomide treatment. By flow cytometry of surface antigens, it was revealed that the LGLs were positive for cluster of differnetiation (CD)2, 7, 8, 16, 56, and 57, and human leukocyte antigen-D related, however, were negative for CD3, 4 and 5, suggesting that these LGLs predominantly exhibited an natural killer (NK) cell phenotype. T-cell receptor β gene rearrangement was not detected by polymerase chain reaction. A 51Cr release assay was performed to investigate whether the NK cells actually possessed activity. A low level of M protein was sustained for ~15 months. This implied the enhancement of immune activation during lenalidomide treatment. The present case study suggested that LGL cells induced by lenalidomide may contribute to long-term restraint of myeloma cells. This immune system component may contribute to disease control.

Long-Term Results and Characteristics of Pleural Effusion in Late Chronic Phase Chronic Myeloid Leukemia Patients at Dasatinib Therapy after Imatinib Failure

Background. The aim of chronic myeloid leukemia (CML) treatment with tyrosine kinase inhibitors (TKI) is not only effectiveness, but also safety. Long treatment duration makes the analysis of most significant complications very important. Pleural effusion (PE) is an important adverse event of dasatinib therapy with largely unclear cause. The optimal management of recurrent PE is unknown and the analysis of its treatment results is actual.Aim. To describe the characteristics of patients with recurrent PE at prolonged dasatinib treatment and to suggest the strategy of their management.Methods. Follow-up data of 23 CML late chronic phase patients at dasatinib therapy after imatinib failure in 2 clinical studies: phase II study comparing dasatinib 140 mg and imatinib 800 mg daily (N = 12) and phase III dasatinib dose-optimizing study in patients with imatinib-resistant or intolerant patients (N = 11). M:F ratio was 7:16. Median age at the beginning of dasatinib - 48 years (26-68), median CML duration - 11 years (4,1-19,2). The reason for TKI change was imatinib resistance: cytogenetic (N = 17) and hematological (N = 6).Results. Median duration of dasatinib treatment - 40 months (10-107); 13 patients (56,3%) are alive, 10 patients (43,5%) have died because of progression of CML. In 19 patients (82,6%) dasatinib treatment was stopped because of: blastic transformation - 6 (26,1%), hematological resistance - 3 (13,1%), cytogenetic resistance - 5 (21,7%), PE - 5 (21,7%). Four patients are still on dasatinib treatment with median duration 8,8 years (8,7-8,9), 3 of them retain complete/major molecular response. The best responses were: complete hematological response in 21 (91,3%), complete cytogenetic response - in 8 (34,8%), major molecular response - in 6 (26,1%) patients. Overall 8-year survival was 55,1%, progression-free survival - 55,4%, event-free survival - 26,1%. PE was observed in 11 (47,8%) patients, in 8 of them (72,7%) - recurrent. In one patient the prolonged PE was associated with fibrosis of adjacent lung and pleura. Median time to PE was 34 (6-83) months. In cases of PE dasatinib was interrupted (mean duration 21 d) and diuretics were started. Six patients (54,5%) also received corticosteroids. Five patients (45,6%) were treated with thoracocentesis. In recurrent PE the dasatinib dosage was decreased. The dasatinib discontinuation in 4 patients with recurrent PE has led to loss of major molecular response in 2 of them; in other 2 it is retained for 6,5 and 1,5 years. Event-free 8-year survival was 36,4% in patients with PE, 16,7% - without it.Discussion. The response rate in patients with PE was not worse, than in those without it. Most often PE begins at 3rd year of treatment; later events were only relapses. Among risk factors of PE 2 patients had arterial hypertension and hypercholesterinemia, 3 patients were > 65 yrs. The significantly high PE on rate (48%) was linked to high initial dasatinib dosage (> 100 mg/d) and bid prescription. We have not observed cases of absolute lymphocytosis due to large granular lymphocytes proliferation. The continuation of treatment generally leads to recurrences of effusion. According to our experience, once arising PE tends to recur. The compensation can be maintained with continuous treatment with diuretics. The prolonged PE may lead to fibrosis of adjacent lung and pleura. Prolonged treatment interruptions and decreased doses can cause treatment failure. The role of corticosteroids is unclear.Conclusion. Our experience in recurrent PE management at dasatinib treatment allow to recommend the usage of alternative TKI in patients with poor treatment response, and discontinuation of treatment in patients with deep molecular response with close monitoring of residual disease by PCR. DisclosuresTurkina:Novartis International AG: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

High Frequency of Thyroid Disorders in Patients Presenting With Neutropenia to an Outpatient Hematology Clinic STROBE-Compliant Article.

Granulopoiesis abnormalities have been described in association with thyroid disorders (TD). However, data regarding systematic evaluation of adult neutropenia and concurrent or prior TD are scarce.To investigate the frequency of TD among patients presenting with neutropenia, and the immunophenotypic and immunologic profile of neutropenic patients with concomitant thyroidopathy.Two hundred eighteen consecutive neutropenic patients were prospectively evaluated in our outpatient Hematology Clinic, with a detailed laboratory screen, including thyroid function tests, antineutrophil antibodies, blood lymphocytes immunophenotyping, and detection of T-cell clonality by PCR.Among 218 patients with neutropenia, 95 (43.6%) had TD, 65 chronic immunologic neutropenia, 20 clonal proliferation of T-large granular lymphocytes (T-LGL), 5 autoimmune disorders, and 33 other diagnoses. TD-patients had an increased frequency of recurrent infections compared with other patients (P = 0.045). The following correlations were found: negative correlation between FT3 and absolute neutrophil count (ANC) (r2 = −0.274, P = 0.007), negative correlation between TPO-Abs/TG-Abs and C4 (r2 = −0.16, P = 0.045; r2 = −0.266, P = 0.001), and CD4+ counts were inversely correlated to T4 and positively to TSH (r2 = −0.274, P = 0.024; r2 = 0.16, P = 0.045). In addition, TD-patients had significantly higher percentages of CD4+ lymphocytes (P = 0.003). Among TD-patients, 23.4% had Hashimoto thyroiditis (HT), 4.1%, Graves disease (GD), 8.2% nontoxic multinodular goiter (NTMG), 5% subclinical hypothyroidism, and 2.8% had undergone total thyroidectomy associated with nodules (TTM). Thirteen TD-patients displayed T-LGL. Patients with autoimmune thyroidopathy had an increased frequency of concomitant autoimmune manifestations (P = 0.03). Significant differences between the different thyroidopathies included: HT-patients had higher percentages of B-lymphocytes, while the opposite was evident for the TTM-subgroup (P = 0.009, 0.02); GD-patients showed an increase of the proportion of NK cells and a decrease in the percentage of TCRγδ+ lymphocytes (P = 0.001, 0.045); and NTMG-patients had significantly higher ANC (P = 0.004) compared to other thyroidopathies. Antineutrophil antibodies were found in 37.2% of TD-patients tested. Anti-TPO titers were significantly higher in patients with positive antineutrophil antibodies (P = 0.04).The frequency of TD among neutropenic patients may be higher than previously reported. The existence of antineutrophil antibodies, as well as the different distribution of lymphocyte subsets among patients with different TD, suggests both humoral and cellular mechanisms in the pathophysiology of thyroid disease-associated neutropenia.

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.