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

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

Identification of novel STAT5B mutations and characterization of TCR beta signatures in CD4+T-cell large granular lymphocyte leukemia

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCR beta sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.Peer reviewe

Dissection of the microenvironment role in T-large granular lymphocite leukemia

T Large granular lymphocytes leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of T-LGL. According to the expression on the leukemic clone of CD8- (CD8+ T-LGLL) and CD4-related determinants (CD4+ T-LGLL) two main groups of patients are identified. Somatic mutations are involved in T-LGLL pathogenesis; they have been identified in genes that are central for the survival of the leukemic clone and are differently distributed among the patients. STAT3 mutations characterize CD8+ T-LGLL cases with a discrete immunophenotype (i.e. CD16+/CD56-) and correlate with neutropenia, which represents the most relevant clinical manifestation in T-LGLL. On the other hand, STAT5b mutations can be observed in CD4+ patients, usually characterized by an indolent disease. The leukemic clone survival is mediated by several deregulated pathways that are likely under the control of external stimuli. Indeed, several pro-inflammatory cytokines are increased in patients’ plasma, including CCL5 and IL-6. This latter cytokine has been demonstrated to sustain LGL survival via STAT3 activation. The role of soluble factors have been described in the pathogenesis of T-LGLL, but no data are up to now available on the cellular compartment of the microenvironment. For this reason we studied the involvement of tumour microenvironment (TME) cells and we found that: a) monocytes are central for leukemic LGL survival; b) leukemic cells belonging to CD8+ and CD4+ T-LGLL patients take advantage from different TME-mediated survival mechanisms. In CD8+ patients, particularly in cases with severe neutropenia, monocytes were altered in their population distribution, with an increase of intermediate and non-classical monocytes; in addition Th17/Treg ratio was found to be higher than in healthy controls, due to an increased Th17 cells percentage. Since these two features were associated with a high pro-inflammatory stimulus and correlated with the presence of concurrent autoimmune diseases, which are often reported in T-LGLL, we suggest that a strong peripheral blood inflammation is ongoing in CD8+ neutropenic patients. All these data taking together, we propose that a network takes place between LGL, monocytes and Th17 cells in CD8+ neutropenic patients. In detail, we identified that the CCL5 produced by the leukemic clone specifically stimulates the expression of IL-6 in monocytes, a cytokine that in turn is mandatory for the survival of the leukemic clone and for Th17 cells differentiation. We also reported that the above described alterations of cell subsets were partially reverted after immunosuppressive therapy, suggesting a putative role of these drugs as TME modifiers. A pro-inflammatory environment with different features was present in CD4+-LGLL patients, who were characterized by an indolent course of the disease. Similarly to CD8+ patients, they expressed a high Th17/Treg ratio, but the imbalance in this setting is sustained by a reduction of Treg cells. CD4+ LGLL patients were also found to be characterized by a peculiar over-activation of Erk in monocytes, which was reported to be indicative of Senescence Associated Secretory Phenotype (SASP), mostly composed of pro-inflammatory cytokines. Our data obtained through cytokines arrays demonstrated an overall higher level of plasma soluble factors in CD4+ patients than healthy controls, in particular soluble CD14 and Lymphotactin, which could account for TME cellular imbalances of these cases. This study emphasizes the role of inflammatory background occurring in the TME of T-LGLL patients pointing to newly reported differences between T-LGLL sub-types. In particular in symptomatic patients, we identified a putative network between TME cells and leukemic LGL, which could help defining new targets to design innovative strategies.La leucemia a grandi linfociti granulati di tipo T (T-LGLL) è un raro disordine linfoproliferativo, caratterizzato da un’espansione clonale di T-LGL. Due gruppi principali di malattia sono individuabili in base all’immunofenotipo del clone leucemico; in particolare, si diversificano per l’espressione dell’antigene CD8 (CD8+ T-LGLL) o CD4 (CD4+ T-LGLL). Mutazioni somatiche sono coinvolte nella patogenesi della malattia, in quanto tali geni mutati son stati dimostrati essere importanti per la sopravvivenza del clone leucemico e si distribuiscono in modo differente fra pazienti. Le mutazioni del gene STAT3 caratterizzano i casi CD8+ T-LGLL con un particolare immunofenotipo (i.e. CD16+/CD56-) e correlano con l’incidenza di neutropenia, la quale è la più frequente manifestazione clinica della malattia. Le mutazioni nel gene STAT5b, invece, sono state osservate nei pazienti affetti da CD4+ T-LGLL, la quale, solitamente, ha un decorso indolente. Inoltre, la sopravvivenza del clone leucemico è mediata da pathway intracellulari deregolati, i quali sono ulteriormente stimolati dal microambiente tumorale. Infatti, molte citochine pro-infiammatorie sono state descritte presenti a concentrazioni maggiori nel plasma dei pazienti, rispetto ai controlli sani, come CCL5 e IL-6. In particolare, è stato dimostrato che IL-6 sostiene la sopravvivenza del clone tramite l’attivazione della proteina STAT3. Lo stato dell’arte sul ruolo del microambiente tumorale sulla patogenesi della T-LGLL, nel sangue periferico, principalmente si basa sul ruolo di fattori solubili, ma non sono ancora disponibili dati riguardanti il ruolo di altre cellule del sistema immunitario. Per tale motivo, in questo elaborato di tesi, è stato descritto il coinvolgimento delle cellule del microambiente nella patogenesi della T-LGLL ed è stato identificato che: a) monociti sono risultati essere centrali per la sopravvivenza dei T-LGL leucemici; b) pazienti affetti da CD8+ o CD4+ T-LGLL sono caratterizzati da diversi meccanismi di sopravvivenza mediata dal microambiente. Per i pazienti CD8+, in particolare per i casi con neutropenia severa, la distribuzione delle popolazioni monocitarie è risultata essere alterata, con un incremento dei monociti intermedi e non classici; inoltre anche il rapporto Th17/Treg è sbilanciato, in quanto la percentuale dei linfociti Th17 è aumentata rispetto ai controlli sani. Entrambi i fenomeni descritti sono associati ad un alto stimolo pro-infiammatorio e correlano, inoltre, con malattie autoimmuni, le quali spesso si associano alla T-LGLL. Per tali motivi è stato ipotizzato che i pazienti affetti da CD8+ T-LGLL, manifestanti neutropenia, siano caratterizzati da una forte infiammazione nel sangue periferico. Riassumendo, è stato possibile per cui proporre una rete di comunicazione fra T-LGL leucemici, monociti e linfociti Th17. In particolare, CCL5, chemochina prodotta dal clone leucemico, stimola in modo specifico l’espressione di IL-6 nei monociti dei pazienti, la quale, a sua volta, è necessaria per la sopravvivenza del clone e per il differenziamento dei linfociti Th17. Inoltre, lo studio ha dimostrato che gli sbilanciamenti descritti erano parzialmente tornati a livelli fisiologici, dopo che i pazienti avevano terminato il ciclo di terapia con immunosoppressori, descrivendo un possibile ruolo di questi come modificatori del microambiente tumorale. Anche i pazienti CD4+ sono caratterizzati da un particolare ambiente pro-infiammatorio, nonostante manifestino solitamente un decorso indolente. In particolare, similmente ai pazienti CD8+, il loro rapporto Th17/Treg è aumentato rispetto ai controlli sani, ma lo sbilanciamento, in questo, caso è sostenuto da una riduzione dei linfociti Treg. Anche i monociti sono risultati alterati dal loro stato fisiologico; infatti riportavano una maggiore attivazione della proteina Erk, la quale è stata descritta come indicativa di senescenza cellulare, inducendo, per cui, un particolare secretoma pro-infiammatorio in queste cellule, definito con l’acronimo SASP. Dati preliminari ottenuti da analisi di arrays di citochine hanno dimostrato un’incrementata secrezione di citochine nei pazienti CD4+ rispetto ai controlli sani, in particolare la forma solubile della proteina CD14 e Lynfotactina, le quali potrebbero spiegare le alterazioni delle cellule del microambiente descritte. In conclusione, questo studio ha descritto per la prima vota il ruolo delle cellule del microambiente e la loro differenza nello stimolo pro-infiammatorio, evidenziando ulteriormente le differenze fra tipologie di T-LGLL. In particolare, per i pazienti sintomatici, è stato ipotizzato un network di comunicazione fra cellule del microambiente e clone leucemico, dal quale potrebbe essere possibile individuare nuovi target terapeutici

Dissection of the microenvironment role in T-large granular lymphocite leukemia

T Large granular lymphocytes leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of T-LGL. According to the expression on the leukemic clone of CD8- (CD8+ T-LGLL) and CD4-related determinants (CD4+ T-LGLL) two main groups of patients are identified. Somatic mutations are involved in T-LGLL pathogenesis; they have been identified in genes that are central for the survival of the leukemic clone and are differently distributed among the patients. STAT3 mutations characterize CD8+ T-LGLL cases with a discrete immunophenotype (i.e. CD16+/CD56-) and correlate with neutropenia, which represents the most relevant clinical manifestation in T-LGLL. On the other hand, STAT5b mutations can be observed in CD4+ patients, usually characterized by an indolent disease. The leukemic clone survival is mediated by several deregulated pathways that are likely under the control of external stimuli. Indeed, several pro-inflammatory cytokines are increased in patients’ plasma, including CCL5 and IL-6. This latter cytokine has been demonstrated to sustain LGL survival via STAT3 activation. The role of soluble factors have been described in the pathogenesis of T-LGLL, but no data are up to now available on the cellular compartment of the microenvironment. For this reason we studied the involvement of tumour microenvironment (TME) cells and we found that: a) monocytes are central for leukemic LGL survival; b) leukemic cells belonging to CD8+ and CD4+ T-LGLL patients take advantage from different TME-mediated survival mechanisms. In CD8+ patients, particularly in cases with severe neutropenia, monocytes were altered in their population distribution, with an increase of intermediate and non-classical monocytes; in addition Th17/Treg ratio was found to be higher than in healthy controls, due to an increased Th17 cells percentage. Since these two features were associated with a high pro-inflammatory stimulus and correlated with the presence of concurrent autoimmune diseases, which are often reported in T-LGLL, we suggest that a strong peripheral blood inflammation is ongoing in CD8+ neutropenic patients. All these data taking together, we propose that a network takes place between LGL, monocytes and Th17 cells in CD8+ neutropenic patients. In detail, we identified that the CCL5 produced by the leukemic clone specifically stimulates the expression of IL-6 in monocytes, a cytokine that in turn is mandatory for the survival of the leukemic clone and for Th17 cells differentiation. We also reported that the above described alterations of cell subsets were partially reverted after immunosuppressive therapy, suggesting a putative role of these drugs as TME modifiers. A pro-inflammatory environment with different features was present in CD4+-LGLL patients, who were characterized by an indolent course of the disease. Similarly to CD8+ patients, they expressed a high Th17/Treg ratio, but the imbalance in this setting is sustained by a reduction of Treg cells. CD4+ LGLL patients were also found to be characterized by a peculiar over-activation of Erk in monocytes, which was reported to be indicative of Senescence Associated Secretory Phenotype (SASP), mostly composed of pro-inflammatory cytokines. Our data obtained through cytokines arrays demonstrated an overall higher level of plasma soluble factors in CD4+ patients than healthy controls, in particular soluble CD14 and Lymphotactin, which could account for TME cellular imbalances of these cases. This study emphasizes the role of inflammatory background occurring in the TME of T-LGLL patients pointing to newly reported differences between T-LGLL sub-types. In particular in symptomatic patients, we identified a putative network between TME cells and leukemic LGL, which could help defining new targets to design innovative strategies

Fulminant Myocarditis: When One Size Does Not Fit All – A Critical Review of the Literature

Fulminant myocarditis, rather than being a distinct form of myocarditis, is instead a peculiar clinical presentation of the disease. The definition of fulminant myocarditis has varied greatly in the last 20 years, leading to conflicting reports on prognosis and treatment strategies, mainly because of varied inclusion criteria in different studies. The main conclusion of this review is that fulminant myocarditis may be due to different histotypes and aetiologies that can be diagnosed only by endomyocardial biopsy and managed by aetiology-directed treatment. This life-threatening presentation requires rapid, targeted management both in the short term (mechanical circulatory support, inotropic and antiarrhythmic treatment and endomyocardial biopsy) and in the long term (including prolonged follow-up). Fulminant presentation has also recently been identified as a risk factor for worsened prognosis, even long after the resolution of the acute phase of myocarditis

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

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