Pathogenesis of Neutropenia in large granular lymphocyte leukemia: immunophenotypic and molecular characterization of neutropenic patients

Large Granular Lymphocytes (LGLs) leukemia (LGLL) is a chronic lymphoproliferation of clonal cytotoxic LGLs, which can be divided in two different subsets, based on Cluster of Differentiation (CD)-3 expression: CD3- Natural Killer (NK) cells and CD3+/CD8+/CD4- or CD3+/CD8-/dim/CD4+ T-LGLs. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways. Among these, the most relevant is the JAK-STAT axis, whose constitutive activation is in part explained by somatic activating mutations in STAT3 and STAT5b. Neutropenia is the most frequent clinical manifestation, but its pathogenesis is still unknown, although high levels of soluble Fas Ligand (sFasL) were detected in serum of LGLL patients and supposed to trigger neutrophil’s death. This work aimed i) to describe the distinctive biological features of T-LGLs or NK cells of neutropenic LGLL patients; ii) to investigate the role of microRNAs in the regulation of sFasL expression in leukemic LGLs. LGLs were purified by FACSAriaIII cell sorter from peripheral blood mononuclear cells (PBMCs) of untreated LGLL patients and their immunophenotype was evaluated by Flow Cytometry. The screening of STATs mutations was performed by Sanger sequencing. PBMCs of patients were cultured in vitro and STAT3 transcriptional activity were inhibited or stimulated with Stattic or IL-6, respectively. Transcriptional and protein expression levels were evaluated by Real Time-PCR, Western Blot (WB) assays and ELISA test.High throughput and single miRNA analysis were carried out on purified T-LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. We showed that leukemic T-LGLs of neutropenic patients were characterized by CD3+/CD8+/CD16+/CD56- phenotype, higher STAT3 activations, higher incidence of STAT3 mutations and higher levels of sFasL, compared to T-LGLs of non-neutropenic patients. We also demonstrated that FasL transcription was mediated by STAT3 activation in T-LGLL patients. The characterization of neutropenic CLPD-NK patients, instead, showed that they are characterized by CD56-/dim/CD16high/CD57- cytotoxic NK cells expansion. However, we observed a heterogeneous level of STAT3 activation and a heterogeneous expression of FasL in this subset of patients. To investigate whether STAT3 could play its pathogenetic role in T-LGLs through an altered expression of microRNAs, we studied miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC). We showed that miR-146b expression, found down-regulated in neutropenic patients, was correlated with the ANC of T-LGLL patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes10. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non-neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients. In this work we identify distinctive features of neutropenic LGLL patients, that could acquire relevance to correctly address the management of each patient, and we identify a miR-146b-FasL axis involved in neutropenia development in leukemic T-LGLs

STAT3 mutation impacts biological and clinical features of T-LGL leukemia

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features.Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8\ub1) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France).Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8\ub1 T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia

A high definition picture of somatic mutations in chronic lymphoproliferative disorder of natural killer cells

The molecular pathogenesis of chronic lymphoproliferative disorder of natural killer (NK) cells (CLPD-NK) is poorly understood. Following the screening of 57 CLPD-NK patients, only five presented STAT3 mutations. WES profiling of 13 cases negative for STAT3/STAT5B mutations uncovered an average of 18 clonal, population rare and deleterious somatic variants per patient. The mutational landscape of CLPD-NK showed that most patients carry a heavy mutational burden, with major and subclonal deleterious mutations co-existing in the leukemic clone. Somatic mutations hit genes wired to cancer proliferation, survival, and migration pathways, in the first place Ras/MAPK, PI3K-AKT, in addition to JAK/STAT (PIK3R1 and PTK2). We confirmed variants with putative driver role of MAP10, MPZL1, RPS6KA1, SETD1B, TAOK2, TMEM127, and TNFRSF1A genes, and of genes linked to viral infections (DDX3X and RSF1) and DNA repair (PAXIP1). A truncating mutation of the epigenetic regulator TET2 and a variant likely abrogating PIK3R1-negative regulatory activity were validated. This study significantly furthered the view of the genes and pathways involved in CLPD-NK, indicated similarities with aggressive diseases of NK cells and detected mutated genes targetable by approved drugs, being a step forward to personalized precision medicine for CLPD-NK patients.Peer reviewe

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Pathogenesis of Neutropenia in large granular lymphocyte leukemia: immunophenotypic and molecular characterization of neutropenic patients

Large Granular Lymphocytes (LGLs) leukemia (LGLL) is a chronic lymphoproliferation of clonal cytotoxic LGLs, which can be divided in two different subsets, based on Cluster of Differentiation (CD)-3 expression: CD3- Natural Killer (NK) cells and CD3+/CD8+/CD4- or CD3+/CD8-/dim/CD4+ T-LGLs. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways. Among these, the most relevant is the JAK-STAT axis, whose constitutive activation is in part explained by somatic activating mutations in STAT3 and STAT5b. Neutropenia is the most frequent clinical manifestation, but its pathogenesis is still unknown, although high levels of soluble Fas Ligand (sFasL) were detected in serum of LGLL patients and supposed to trigger neutrophil’s death. This work aimed i) to describe the distinctive biological features of T-LGLs or NK cells of neutropenic LGLL patients; ii) to investigate the role of microRNAs in the regulation of sFasL expression in leukemic LGLs. LGLs were purified by FACSAriaIII cell sorter from peripheral blood mononuclear cells (PBMCs) of untreated LGLL patients and their immunophenotype was evaluated by Flow Cytometry. The screening of STATs mutations was performed by Sanger sequencing. PBMCs of patients were cultured in vitro and STAT3 transcriptional activity were inhibited or stimulated with Stattic or IL-6, respectively. Transcriptional and protein expression levels were evaluated by Real Time-PCR, Western Blot (WB) assays and ELISA test.High throughput and single miRNA analysis were carried out on purified T-LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. We showed that leukemic T-LGLs of neutropenic patients were characterized by CD3+/CD8+/CD16+/CD56- phenotype, higher STAT3 activations, higher incidence of STAT3 mutations and higher levels of sFasL, compared to T-LGLs of non-neutropenic patients. We also demonstrated that FasL transcription was mediated by STAT3 activation in T-LGLL patients. The characterization of neutropenic CLPD-NK patients, instead, showed that they are characterized by CD56-/dim/CD16high/CD57- cytotoxic NK cells expansion. However, we observed a heterogeneous level of STAT3 activation and a heterogeneous expression of FasL in this subset of patients. To investigate whether STAT3 could play its pathogenetic role in T-LGLs through an altered expression of microRNAs, we studied miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC). We showed that miR-146b expression, found down-regulated in neutropenic patients, was correlated with the ANC of T-LGLL patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes10. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non-neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients. In this work we identify distinctive features of neutropenic LGLL patients, that could acquire relevance to correctly address the management of each patient, and we identify a miR-146b-FasL axis involved in neutropenia development in leukemic T-LGLs.Large Granular Lymphocytes (LGLs) leukemia (LGLL) is a chronic lymphoproliferation of clonal cytotoxic LGLs, which can be divided in two different subsets, based on Cluster of Differentiation (CD)-3 expression: CD3- Natural Killer (NK) cells and CD3+/CD8+/CD4- or CD3+/CD8-/dim/CD4+ T-LGLs. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways. Among these, the most relevant is the JAK-STAT axis, whose constitutive activation is in part explained by somatic activating mutations in STAT3 and STAT5b. Neutropenia is the most frequent clinical manifestation, but its pathogenesis is still unknown, although high levels of soluble Fas Ligand (sFasL) were detected in serum of LGLL patients and supposed to trigger neutrophil’s death. This work aimed i) to describe the distinctive biological features of T-LGLs or NK cells of neutropenic LGLL patients; ii) to investigate the role of microRNAs in the regulation of sFasL expression in leukemic LGLs. LGLs were purified by FACSAriaIII cell sorter from peripheral blood mononuclear cells (PBMCs) of untreated LGLL patients and their immunophenotype was evaluated by Flow Cytometry. The screening of STATs mutations was performed by Sanger sequencing. PBMCs of patients were cultured in vitro and STAT3 transcriptional activity were inhibited or stimulated with Stattic or IL-6, respectively. Transcriptional and protein expression levels were evaluated by Real Time-PCR, Western Blot (WB) assays and ELISA test.High throughput and single miRNA analysis were carried out on purified T-LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. We showed that leukemic T-LGLs of neutropenic patients were characterized by CD3+/CD8+/CD16+/CD56- phenotype, higher STAT3 activations, higher incidence of STAT3 mutations and higher levels of sFasL, compared to T-LGLs of non-neutropenic patients. We also demonstrated that FasL transcription was mediated by STAT3 activation in T-LGLL patients. The characterization of neutropenic CLPD-NK patients, instead, showed that they are characterized by CD56-/dim/CD16high/CD57- cytotoxic NK cells expansion. However, we observed a heterogeneous level of STAT3 activation and a heterogeneous expression of FasL in this subset of patients. To investigate whether STAT3 could play its pathogenetic role in T-LGLs through an altered expression of microRNAs, we studied miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC). We showed that miR-146b expression, found down-regulated in neutropenic patients, was correlated with the ANC of T-LGLL patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes10. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non-neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients. In this work we identify distinctive features of neutropenic LGLL patients, that could acquire relevance to correctly address the management of each patient, and we identify a miR-146b-FasL axis involved in neutropenia development in leukemic T-LGLs

Neutropenia and Large Granular Lymphocyte Leukemia: From Pathogenesis to Therapeutic Options

Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL or NK cells. Chronic isolated neutropenia represents the clinical hallmark of the disease, being present in up to 80% of cases. New advances were made in the biological characterization of neutropenia in these patients, in particular STAT3 mutations and a discrete immunophenotype are now recognized as relevant features. Nevertheless, the etiology of LGLL-related neutropenia is not completely elucidated and several mechanisms, including humoral abnormalities, bone marrow infiltration/substitution and cell-mediated cytotoxicity might cooperate to its pathogenesis. As a consequence of the multifactorial nature of LGLL-related neutropenia, a targeted therapeutic approach for neutropenic patients has not been developed yet; moreover, specific guidelines based on prospective trials are still lacking, thus making the treatment of this disorder a complex and challenging task. Immunosuppressive therapy represents the current, although poorly effective, therapeutic strategy. The recent identification of a STAT3-mediated miR-146b down-regulation in neutropenic T-LGLL patients emphasized the pathogenetic role of STAT3 activation in neutropenia development. Accordingly, JAK/STAT3 axis inhibition and miR-146b restoration might represent tempting strategies and should be prospectively evaluated for the treatment of neutropenic LGLL patients

All that glitters is not LGL Leukemia

LGL disorders are rare hematological neoplasias with remarkable phenotypic, genotypic and clinical heterogeneity. Despite these constraints, many achievements have been recently accomplished in understanding the aberrant pathways involved in the LGL leukemogenesis. In particular, compelling evidence implicates STAT signaling as a crucial player of the abnormal cell survival. As interest increases in mapping hematological malignancies by molecular genetics, the relevance of STAT gene mutations in LGL disorders has emerged thanks to their association with discrete clinical features. STAT3 and STAT5b mutations are recognized as the most common gain-of-function genetic lesions up to now identified in T-LGL leukemia (T-LGLL) and are actually regarded as the hallmark of this disorder, also contributing to further refine its subclassification. However, from a clinical perspective, the relationships between T-LGLL and other borderline and overlapping conditions, including reactive cell expansions, clonal hematopoiesis of indeterminate potential (CHIP) and unrelated clonopathies are not fully established, sometimes making the diagnosis of T cell malignancy challenging. In this review specifically focused on the topic of clonality of T-LGL disorders we will discuss the rationale of the appropriate steps to aid in distinguishing LGLL from its mimics, also attempting to provide new clues to stimulate further investigations designed to move this field forward

Not all LGL leukemias are created equal

: Large Granular Lymphocyte (LGL) Leukemia is a rare, heterogeneous even more that once thought, chronic lymphoproliferative disorder characterized by the clonal expansion of T- or NK-LGLs that requires appropriate immunophenotypic and molecular characterization. As in many other hematological conditions, genomic features are taking research efforts one step further and are also becoming instrumental in refining discrete subsets of LGL disorders. In particular, STAT3 and STAT5B mutations may be harbored in leukemic cells and their presence has been linked to diagnosis of LGL disorders. On clinical grounds, a correlation has been established in CD8+ T-LGLL patients between STAT3 mutations and clinical features, in particular neutropenia that favors the onset of severe infections. Revisiting biological aspects, clinical features as well as current and predictable emerging treatments of these disorders, we will herein discuss why appropriate dissection of different disease variants is needed to better manage patients with LGL disorders

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition

Identification of a STAT3-miRNA Axis in T-LGL Leukemia

Introduction T large granular lymphocytes leukemia (T-LGLL) is a rare disease characterized by the abnormal expansion of T-large granular lymphocytes (T-LGLs) in the peripheral blood. The etiology of this disease is still largely unknown. LGL proliferation is maintained through an impairment of the apoptotic machinery due to the activation of many survival signals. Among these, JAK/STAT signaling represents one of the most important deregulated pathways in T-LGLL. In particular, leukemic LGLs are equipped with STAT3 constitutively over-expressed and over-activated. Moreover, in 30-40% of patients, STAT3 has been demonstrated carrying hot-spot mutations, likely resulting in STAT3 activation. Although STAT3 is an inducer of transcription of a large number of oncogenes, its relationship with microRNAs (miRNAs) has not yet been extensively evaluated in T-LGLL patients. As a matter of fact, several miRNAs contribute to normal hematopoietic processes and many miRNAs act both as tumor suppressors and oncogenes in the pathology of hematological malignancies, including acute and chronic leukemias and lymphomas, where they contribute to lymphomagenesis acting in various cellular functions, such as the regulation of cell survival and proliferation. Aims We investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. A high throughput quantitative and qualitative analysis of the miRNA expression profile in leukemic LGLs compared to healthy controls was performed with the aim to investigate whether STAT3 activation and/or mutation were correlated to some miRNAs in leukemic LGLs. Methods Six patients (3 characterized by STAT3 mutations and 3 with wild type STAT3) and three healthy controls were enrolled in a pilot study. STAT3 mRNA expression and protein activation levels were analyzed by Real Time-PCR and Western Blot, respectively. The expression level of 756 mature miRNAs was assessed by using a TaqMan-based Low Density Array on purified LGLs. Experimental data were analyzed by ViiA7 RUO software and the relative miRNA expression values were calculated using U6 as endogenous control. miRNA array data underwent hierarchical cluster analysis (HCL) by using MEV. miRNAs with a 2 or 0.5 fold change and p value < 0.05 in samples as compared to controls were considered as differentially expressed. Results of this pilot study were validated on additional 12 T-LGLL and 3 healthy controls subjects. Results Two clusters were identified by HCL analysis: cluster A included healthy controls and LGL patients characterized by comparably low levels of STAT3 activation (S3low) and absence of STAT3 mutations. Cluster B included four patients characterized by high levels of STAT3 activation (S3high). Remarkably, three out of four LGL patients in cluster B shared STAT3 mutation. Comparative analysis of the miRNAs expressed identified 33 miRNAs upregulated and 9 miRNAs downregulated in S3high as compared to S3low. Interestingly, the level of expression of these selected miRNAs correlated with the level of STAT3 expression/activation in LGL. Among these, three miRNAs, miR-484, miR-501 and miR-1249, have been validated and confirmed in the validation cohort. Conclusions These data firstly describe the miRNA pattern in T-LGLL, providing evidence that a series of miRNAs are correlated with relevant key factors in T-LGLL pathogenesis, including STAT3 activation/expression and mutations. Our results suggest the hypothesis that STAT3 could mediate its role through some defined miRNAs