Large 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.

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.

T Cell Receptor (α, β, γ) Gene Rearrangements and Expression in Normal and Leukemic Large Granular Lymphocytes/Natural Killer Cells

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

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.

PS1261 CLINICAL CHARACTERISTICS AND PROGNOSTIC FACTORS OF 33 PATIENTS WITH SEZARY SYNDROME AT MOFFITT CANCER CENTER: A SINGLE‐INSTITUTION COHORT

Background:Sezary syndrome (SS) is a rare aggressive leukemia variant of cutaneous T‐cell lymphoma (CTCL) characterized by erythroderma and Sezary cells in peripheral blood and lymphoadenopathy. It has a poor prognosis with reported median overall survival ranging from 2–4 years. Limited studies exist to describe the SS cohorts in modern era of biological therapy.Aims:To analyze clinical and laboratory characteristics such as age, lesion characteristics, hematological parameters to determine their impact on survival in patients with SS.Methods:A retrospective chart review was conducted to identify patients with SS seen at Moffitt Cancer Center, Tampa, FL from 2016 to 2018. Kaplan Meier estimates were calculated for overall survival. Log rank test and cox proportional hazard model were used to evaluate categorical and continuous factors associated with overall survival respectively.Results:In total, 33 patients were identified with a clinicopathologic diagnosis of SS. 76% were white, 21% African American and 3% Hispanic; 67% were male and 33% female (2:1 ratio). Mean age was 67.3 years (range: 43–89). 49% had primary SS and 51% had a previous diagnosis of mycosis fungoides (MF). The average time to progression to SS was 6.4 years. The majority presented with advanced stage (III/IV). Seven patients were known to be deceased with a median time to death of 2.4 years. Other 26 patients had a median follow up time of 3.8 years. OS was 68.5% and 42.8% at 1 and 5 years, respectively. Prior diagnosis of MF indicated a poor prognosis (p = 0.005). Age, race, the presence of erythroderma, absolute Sezary cell count, LDH level, CD4/CD8 ratio, ferritin level, and lymph node involvement at the time of diagnosis did not correlate with OS. There was a trend to favorable survival in the group of patients &lt;60 years old at diagnosis and with the presence of large granular lymphocytes (LGL) proliferations.Sezary cells of the immunophenotype of CD3+CD4+CD7‐CD26‐ were present in 78% patients. TCR gene was clonally rearranged in 30/31 tested patients. 28 (90.3%) was TCRβ+TCRγ+; 1 TCRβ+TCRγ‐; 1 TCRβ+TCRγ‐. Median Sezary cell count is 1.81x109 cells/L.Foundation Medicine Hematology gene panel was done in selected patients, which tested 437 genes that are known to be somatically altered in human hematologic malignancies, sarcomas and pediatric cancers and that unambiguous driver of oncogenesis based on current knowledge. 21 mutations were detected in 4 patients: CCND3, MKI67, CDKN2A, EP300, MAPK1, BCOR, RHOA in cell cycle regulation and cell proliferation; TET2, ASXL1, DNMT in DNA methylation; STED2, ARID2 in DNA repair; CBL, DTX1, TNFAIP3 in protein Ubiquination; TP63 (P53 mediated apoptosis), TNFAIP3 (NFkB signaling apoptosis), MPAK1 in Cell Apoptosis; CD 58, BTK and CIITA in immune surveillance; STAT3 in JAK/STAT pathway; TP63 and DTX1 in Notch signaling pathway. GRIN2A, involved in NMDA receptor signaling, was detected with unclear role in oncogenesis. GRIN2A and CDKN2A were detected in 2/4 patients. TET2, DNMT3A, TNFAIP3 were previously reported in CTCL. CCND3, CDKN2A, MAP1, ASXL1, EP300, STAT3 were reported in other T‐cell malignancies.Summary/Conclusion:Our study confirmed overall poor prognosis of SS with OS of 68.5% and 42.8% at 1 and 5 years despite advances in therapy. The preexisting diagnosis of MF was a poor prognostic factor. Younger age at diagnosis and the presence of LGL proliferations are potentially favorable prognostic factors. However, due to limited sample sizes, the results need to be interpreted with caution and verified with a larger cohort.image

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.

Distinctive clinical characteristics and favorable outcomes in patients with large granular lymphocytosis after allo‐HCT: 12‐year follow‐up data

An increase in large granular lymphocytes (LGL) is frequently seen in patients following allogeneic hematopoietic cell transplantation (allo-HCT) and it has been associated with better outcomes in some reports. We assessed 826 consecutive patients at our institution with over 12years of follow-up for the occurrence of LGL lymphocytosis after allo-HCT. The 3-year cumulative incidence of LGL lymphocytosis was 14.5% with a median duration of over 3.5years. The development of LGL lymphocytosis was strongly correlated with CMV viremia and GVHD. The clinical course of patients with LGL lymphocytosis after allo-HCT was indolent, with the majority of these patients not displaying any clinical signs or symptoms related to the LGL proliferation. LGL lymphocytosis was associated with better outcomes, including higher overall survival (OS 86.6% vs 44.7% at 3years), lower non-relapse mortality (NRM 5.5% vs 30.4% at 3years), and lower risk of relapse (8.9% vs 22.9% at 3years). A time-dependent multivariable analysis confirmed the favorable impact of LGL lymphocytosis on OS and NRM, but not on the risk of relapse. In multivariable analysis, a longer duration of LGL lymphocytosis was associated with better OS and NRM. Improved immunomodulatory properties of these cells, regulating GVHD and infections, may explain the observed favorable outcomes of patients who developed LGL lymphocytosis following allo-HCT.

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.

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.