Identification of a miR-146b-FasL axis in the development of neutropenia in T large granular lymphocyte leukemia

T Large Granular Lymphocytes leukemia is characterized by the expansion of several Large Granular Lymphocyte clones, among which a subset of Large Granular Lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNA has not been evaluated in T-Large Granular Lymphocyte Leukemia patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-Large Granular Lymphocyte Leukemia through an altered expression of miRNAs. The expression level of 756 mature miRNAs was assessed on purified T-LGLs by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-Large Granular Lymphocytes. Remarkably, CD8 T-Large Granular Lymphocytes exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of FasL, that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-Large Granular Lymphocytes occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-Large Granular Lymphocytes lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-Large Granular Lymphocyte Leukemia

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

Venetoclax in combination with hypomethylating agents in previously untreated patients with acute myeloid leukemia ineligible for intensive treatment: a real-life multicenter experience

The addition of venetoclax to hypomethylating agents (HMA-V) improved the outcome of patients with newly diagnosed acute myeloid leukemia (AML) ineligible for intensive treatment. The aim of our study was to confirm data reported in literature, in a real-life multicenter experience. We retrospectively evaluated 56 naive AML patients who received HMA-V at 8 different collaborating Hematology Units in the North-East of Italy, from September 2018 to October 2020. Patients received azacitidine or decitabine at standard dose, adding venetoclax starting from cycle 1-3. The median time-to-response was 2 cycles and composite complete remission rate (CCR) was 67.9%. Thirteen out of 38 responders (34.2%) relapsed, with a median response duration of 13.7 months. Transfusion independence (TI) was obtained in 27 (87.0%) and 28 (90.3%) out of 31 patients for red blood cells and platelets, respectively. Median OS was 12.3 months (95% CI, 8.1-16.5), and median PFS was 11.3 months (95% CI, 4.6-17.9). Cytogenetic risk was the only variable impacting on survival, while no differences were observed stratifying patients by age, bone marrow blasts, WHO classification or type of HMA. In conclusion, our real-life multicenter experience indicates that HMA-V treatment allows achieving good response rates in naive AML patients, ineligible for intensive chemotherapy

Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients

Large granular lymphocyte leukemia (LGLL) is a rare and chronic lymphoproliferative disorder characterized by the clonal expansion of LGLs. LGLL patients can be asymptomatic or develop cytopenia, mostly neutropenia. Somatic STAT3 and STAT5b mutations have been recently reported in approximately 40% of patients. The aim of this study is to analyze clinical and biological features of a large cohort of LGLL patients to identify prognostic markers affecting patients' outcome. In 205 LGLL patients, neutropenia (ANC\u2009<\u20091500/mm3) was the main feature (38%), with severe neutropenia (ANC\u2009<\u2009500/mm3) being present in 20.5% of patients. STAT3 mutations were detected in 28.3% patients and were associated with ANC\u2009<\u2009500/mm3 (p\u2009<\u20090.0001), Hb\u2009<\u200990\u2009g/L (p\u2009=\u20090.0079) and treatment requirement (p\u2009<\u20090.0001) while STAT5b mutations were found in 15/152 asymptomatic patients. By age-adjusted univariate analysis, ANC\u2009<\u2009500/mm3 (p\u2009=\u20090.013), Hb\u2009<\u200990\u2009g/L (p\u2009<\u20090.0001), treatment requirement (p\u2009=\u20090.001) and STAT3 mutated status (p\u2009=\u20090.011) were associated to reduced overall survival (OS). By multivariate analysis, STAT3 mutated status (p\u2009=\u20090.0089) and Hb\u2009<\u200990\u2009g/L (p\u2009=\u20090.0011) were independently associated to reduced OS. In conclusion, we identified clinical and biological features associated to reduced OS in LGLL and we demonstrated the adverse impact of STAT3 mutations in patients' survival, suggesting that this biological feature should be regarded as a potential target of therapy

MRD-Based Therapeutic Decisions in Genetically Defined Subsets of Adolescents and Young Adult Philadelphia-Negative ALL

In many clinical studies published over the past 20 years, adolescents and young adults (AYA) with Philadelphia chromosome negative acute lymphoblastic leukemia (Ph− ALL) were considered as a rather homogeneous clinico-prognostic group of patients suitable to receive intensive pediatric-like regimens with an improved outcome compared with the use of traditional adult ALL protocols. The AYA group was defined in most studies by an age range of 18–40 years, with some exceptions (up to 45 years). The experience collected in pediatric ALL with the study of post-induction minimal residual disease (MRD) was rapidly duplicated in AYA ALL, making MRD a widely accepted key factor for risk stratification and risk-oriented therapy with or without allogeneic stem cell transplantation and experimental new drugs for patients with MRD detectable after highly intensive chemotherapy. This combined strategy has resulted in long-term survival rates of AYA patients of 60–80%. The present review examines the evidence for MRD-guided therapies in AYA’s Ph− ALL, provides a critical appraisal of current treatment pitfalls and illustrates the ways of achieving further therapeutic improvement according to the massive knowledge recently generated in the field of ALL biology and MRD/risk/subset-specific therap

Insights into the Molecular Mechanism Accounting for Neutropenia in T-Large Granular Lymphocytes Leukemia

INTRODUCTION: Neutropenia represents the most frequent clinical manifestation in T-Large Granular Lymphocytes Leukemia (T-LGLL) patients. Literature data provided evidence of the involvement of soluble Fas Ligand (sFasL) in this process. Consistently, we confirmed that neutropenic T-LGLL patients were characterized by higher levels of sFasL than non neutropenic patients. We also demonstrated that FasL transcription was mediated by the Signal Transducer and Activator of Transcription (STAT)-3 and we showed that high STAT3 activation correlated with high levels of sFas. However, the mechanism through which STAT3 regulates FasL production still remains elusive. It is well known that FasL expression depends on post-transcriptional events involving ARE-binding proteins, such as Human antigen R (HuR). Important regulators of post-transcriptional modifications are microRNAs (miRNAs), that are small non-coding RNA molecules able to bind target mRNAs, promoting their degradation or blocking protein translation. Among them, miR-146b was identified to be induced by STAT3 in non-transformed cells. This work aims to determine whether miR-146b might regulate STAT3-mediated expression of FasL, thus playing a role in the pathogenesis of neutropenia in T-LGLL patients. METHODS: T-Large Granular Lymphocytes (T-LGLs) were purified by FACSAria cell sorter from PBMCs of untreated T-LGLL patients. High throughput and single miRNA analysis were carried out on purified LGLs by using the TaqMan\uae Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. Transcriptional and protein expression levels were evaluated by Real Time-PCR and Western Blot (WB) assays. RESULTS: By assessing the expression of 756 mature miRNAs on purified patients' T-LGLs, we identified miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC); selected miRNAs were then analyzed for correlation with ANC. Among them, miR-146b expression was the only one correlated with ANC, being down-regulated in neutropenic 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, indicating that miR-146b affected FasL expression at a post-transcriptional level. 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-lymphocytes. 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 the end, we demonstrated the mechanism affecting miR-146b expression in the presence of STAT3 activation, pointing to a role of epigenetic modulation taking place, since the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) can restore STAT3-miR-146b axis. CONCLUSIONS: In this work we suggest a pathogenetic link between STAT3 activation, a defective miR-146b expression and neutropenia development in T-LGLL. Treatment with a demethylating agent may restore STAT3-dependent induction of miR-146b and may represent a new potential therapeutic strategy for the treatment of neutropenia in T-LGLL patients

Multicenter retrospective analysis of clinical outcome of adult patients with mixed-phenotype acute leukemia treated with acute myeloid leukemia-like or acute lymphoblastic leukemia-like chemotherapy and impact of allogeneic stem cell transplantation: a Campus ALL study

: Mixed-phenotype acute leukemia (MPAL) is a rare disease. Treatment is often similar to that of acute lymphoblastic leukemia (ALL), but the outcome in adults and the role of allogeneic stem cell transplantation (AlloSCT) are not well defined. We report on 77 adult patients diagnosed with MPAL over the last 10&nbsp;years and treated with a curative intent. Median age was 49&nbsp;years; 7.6% of cases had a BCR::ABL1 rearrangement. Thirty patients (39%) were treated with an acute myeloid leukemia (AML)-like induction and 47 (61%) with an ALL-like scheme. The complete remission (CR) rate was 67.6% and an ALL-like therapy was associated with a better CR rate (P = 0.048). The median OS was 41.9&nbsp;months; age ≤ 60&nbsp;years was associated with a better OS (67 vs 26&nbsp;months, P = 0.014). An AlloSCT was performed in 50 patients (65%). The 5-year OS of transplanted patients was 54%. The OS post-AlloSCT was better in patients who were minimal residual disease (MRD)-negative prior to transplant (75.8% vs 45.2%, P = 0.06). This study shows that MPAL patients respond better to an ALL-like induction therapy; that consolidation therapy should include, whenever possible, an AlloSCT and that MRD negativity should be a primary endpoint of treatment

Sequential allogeneic transplantation and ruxolitinib maintenance for a synchronous PCM1-JAK2 positive myeloid sarcoma and acute B-lymphoblastic leukemia

The translocation t(8;9)(p22;p24) results in the production of a chimeric PCM1-JAK2 fusion protein leading to the constitutive activation of the Janus Kinase 2 that renders this disease potentially sensitive to ruxolitinib. Here, we report an interesting case of PCM1-JAK2&nbsp;myeloproliferative neoplasm evolving in myeloid sarcoma and B precursor ALL