Integration of B-cell receptor-induced ERK1/2 phosphorylation and mutations of SF3B1 gene refines prognosis in treatment-na\uefve chronic lymphocytic leukemia

ADP-ribosyl Cyclase 1; Disease Progression; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Variable[no abstract available

Caratterizzazione di una sottopopolazione mieloide umana analoga ai promielociti e dotata di attività immunosoppressoria

The ability of the immune system to avoid self-reactivity and autoimmune diseases is achieved by central and peripheral tolerance. Immune tolerance is susteined by different population, one of which is represented by myeloid-derived suppressor cells (MDSC). MDSC are a heterogeneous population of cells consisting of myeloid progenitor cells and immature myeloid cells that are able to suppress both adaptative and innate immunity. In pathological conditions, such as cancer, various infectious diseases and autoimmune diseases, the release of soluble factors induce the increase of myelopoiesis and a partial block of the maturation of myeloid cells. These soluble factors are also believed to be involved in the mobilization and activation of MDSC. MDSC was found in tumour-bearing mice but also in cancer patients. While the phenotype of murine MDSC is well defined by specific markers, like Gr-1, CD11b e IL4Rα, the immunophenotype of human MDSC is still not well defined. In recent years, both in tumour-bearing mice and in cancer patients, MDSC was identified with granulocytes or monocytes features. In this work we demonstrated that human MDSC can be induced in vitro by cytokine treatment of myeloid progenitors. Our data show that the combination of G-CSF and GM-CSF induce the accumulation of immature myeloid cells (BM-MDSC) with features resembling those we identified in the blood of cancer patients. We also demonstrated that BM-MDSC are able to suppress T cells proliferation and that this suppression is accompanied by the downregulation of CD3ζ and CD3ε chains and by the reduction of IFN-γ secreted by activated lymphocyte. Since BM-MDSCs consist of a heterogeneous population, we focused our research to identify the subpopulation of BM-MDSC characterized by the suppressive activity. We demonstrated that human MDSC are immature cells blocked in a particular stage of differentiation. This cells, identifided by the phenotype CD16-/CD11blow/-, showed morphological features of ex-vivo-bone marrow isolated promyelocytes but, unlike these, they are smaller, less grainy, and are able to suppress T lymphocyte proliferation also by CD3ε downregulation. We also observed that the suppressive subpopulation is composed by other two fractions that distinguish each other by a different emission in the red wavelenght, a different grain and a different ability to suppress T lymphocyte proliferation. In the second section of this work we focused in the evaluation of chemotherapy on the accumulation and the functionality of MDSC, one of the main population involved in tumor immunosuppression and in the failure of immunotherapy. Our data showed that, in analogy to the results obtained in mice, 5-fluorouracil is able to remove the suppressive effect of BM-MDSCs on T lymphocyte proliferation. These observation allow us to suggest the use of chemotherapy like adiuvant in immunotherapy approaches.Il sistema immunitario è in grado di bloccare il riconoscimento degli antigeni self e quindi lo sviluppo di risposte autoimmunitarie mediante il mantenimento della tolleranza immunitaria. Tale fenomeno viene sostenuto dall’azione di diverse popolazioni cellulari tolerogeniche, una delle quali è rappresentata cellule soppressorie di derivazione mieloide (MDSC). Le MDSC sono una popolazione molto eterogenea composta da cellule mieloidi immature caratterizzate dalla capacità di sopprimere sia la risposta immunitaria innata che quella adattativa. È stato ipotizzato che in condizioni patologiche, quali infezioni, malattie autoimmunitarie e neoplasie, il rilascio di diversi fattori di crescita induca l’aumento della mielopoiesi ed il blocco maturativo delle cellule mieloidi che si accumulano in uno stato di immaturità. Tali fattori solubili sono inoltre ritenuti coinvolti nella mobilizzazione e nell’attivazione delle MDSC. Le MDSC sono state identificate sia nel modello murino che in pazienti affetti da tumore. Mentre il fenotipo delle MDSC murine è facilmente identificabile mediante l’uso di marcatori specifici (Gr-1, CD11b e IL4Rα), le caratteristiche immunofenotipiche delle MDSC umane non sono ancora state definite. Nonostante questo, sia nel modello murino che nei pazienti affetti da tumore, le MDSC si sono dimostrate avere caratteristiche talvolta granulocitarie e talvolta monocitarie. In questo lavoro abbiamo dimostrato che è possibile indurre, in vitro, la generazione di MDSC umane a partire da precursori midollari coltivati in presenza di diversi fattori solubili, tra i quali il G-CSF ed il GM-CSF. Il nostro studio ha dimostrato che l’uso combinato di G-CSF e di GM-CSF permette di indurre l’accumulo di cellule mieloidi immature (BM-MDSC) con caratteristiche simili a quelle da noi identificate nel sangue di pazienti affetti da tumore. Le BM-MDSC sono in grado di inibire la proliferazione di linfociti T attivati sia con mitogeni che con allo-antigeni. Inoltre abbiamo dimostrato che la soppressione mediata dalle BM-MDSC è associata sia alla diminuzione dell’espressione delle catene ζ ed ε del CD3 che alla riduzione della produzione di IFN-γ secreto dagli stessi linfociti T. Considerando l’elevata eterogeneità delle BM-MDSC, in questo lavoro abbiamo cercato di identificare quali sottopopolazioni di BM-MDSC fossero responsabili dall’attività soppressoria. Mediante esperimenti di sorting cellulare abbiamo dimostrato, che le BM-MDSC sono cellule immature ascrivibili ad un preciso stadio di differenziazione. Queste cellule, definite dal fenotipo CD16-/CD11blow/-, presentano infatti caratteristiche morfologiche simili ai promielociti isolati dal midollo ex-vivo ma, a differenza di queste, sono caratterizzate da una minore granulosità, da maggiori dimensioni e dalla capacità si sopprimere la proliferazione linfocitaria accompagnata anche dalla diminuzione dell’espressione della catena ε del CD3 espressa sulla superficie dei linfociti T. Abbiamo inoltre evidenziato l’eterogeneità di questa frazione dimostrando che, al suo interno, si possono identificare altre due sottopopolazioni caratterizzate da una diversa fluorescenza nella lunghezza d’onda del rosso, da una diversa granulosità e da una diversa attività soppressoria. Nella seconda parte del lavoro ci siamo concentrati sulla valutazione degli effetti della chemioterapia sull’accumulo e sulla funzionalità delle MDSC, una delle principali popolazioni cellulari coinvolte nell’immunosoppressione associata ai tumori e responsabili dell’inefficacia delle terapie immunoterapiche. I dati da noi ottenuti dimostrano che, come osservato nel modello murino, l’aggiunta del 5-fluorouracile a basse dosi è in grado di indurre l’eliminazione dell’attività soppressoria delle BM-MDSC nei confronti dei linfociti T. Queste osservazioni permettono di suggerire il possibile utilizzo di alcuni chemioterapici come adiuvanti nei trattamenti immunoterapici, in quanto in grado di eliminare una delle popolazioni coinvolte nell’immunosoppressione associata ai tumori

Autoimmune haemolytic anaemia in mantle cell lymphoma : an insidious complication associated with leukemic disease

AIHA; MCL; autoimmune haemolytic anaemia; indolent MCL; mantle cell lymphoma; Aged

Evans syndrome secondary to chronic lymphocytic leukaemia: presentation, treatment, and outcome

Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome.Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome

Integrated single cell network profiling data of ERK signaling and mutations of SF3B1 gene refine prognosis in chronic lymphocytic leukemia

Introduction: Extracellular signal-regulated kinase (ERK) is a major pathway downstream of the B-cell receptor (BCR) and its activation is a significant, independent predictor of shorter clinical progression in chronic lymphocytic leukemia (CLL). Recently identified driver genetic lesions, most likely favored by BCR signals, have reshaped the genetic landscape and added a further level of complexity in CLL. Despite the established driving role of BCR signaling and gene mutations in pathobiology and clinical behavior of CLL, little is known about the clinical influence of integrated BCR responses and genetic alterations. In this study, we investigated the clinical impact of integrated BCR response of ERK and gene mutations in CLL. Methods: Peripheral blood cell samples at diagnosis from 152 CLL patients were analyzed in this study. ERK phosphorylation was analyzed using Single Cell Network Profiling (SCNP), a flow cytometry-based assay that allows signaling analysis at the single cell level. NOTCH1, SF3B1, TP53, MYD88, and BIRC3 gene mutations were analyzed by direct DNA sequencing. Univariate and multivariate models for time to first treatment (TTFT) were generated using Cox proportional hazards regression. TTFT curves estimated using the Kaplan-Meier method for the respective groups of patients were compared using the log-rank test. Results: BCR stimulation with anti-IgM induced a signaling response of ERK (anti-IgM\u2192p-ERK) that was significantly higher in UM subset (P=0.0020), in CD38-positive CLL (P=0.0059), in treated patients (P=0.0003), and in mutated- SF3B1 cells (P=0.0098). Univariate analysis identified increased anti-IgM\u2192p-ERK (P=0.001), UM-IGHV (P<0.0001), positive CD38 (P=0.001), mutated SF3B1 (P<0.0001), as significant, independent predictors of shorter TTFT. In multivariate analysis, only increased anti-IgM\u2192p-ERK (P=0.03) and SF3B1 mutation (P=0.0001) were independent significant parameters of prognosis. Integrating anti-IgM\u2192p-ERK data and SF3B1 mutations stratified patients in three independent prognostic categories (P<0.0001) and identified an intermediate-risk group that included patients with low p-ERK and mutated SF3B1 or high p-ERK and wild-type SF3B1. Consistent with the results in the whole patient set, integrating anti-IgM\u2192p-ERK and SF3B1 mutation in an unselected group of Binet stage A patients (n=110) identified an intermediate-risk group including patients with low p-ERK and mutated SF3B1 or high p-ERK and wild-type SF3B1 (P=0.0002). Conclusions: These data reveal that integrated dynamic ERK signaling and SF3B1 mutations independently predict disease progression risk in CLL and identify a novel risk-group of patients, thus providing complementary prognostic information and suggesting a functional synergy between BCR-induced ERK signaling and altered SF3B1

Epstein-Barr virus DNA load in chronic lymphocytic leukemia is an independent predictor of clinical course and survival

The relation between Epstein-Barr virus (EBV) DNA load and clinical course of patients with chronic lymphocytic leukemia (CLL) is unknown. We assessed EBV DNA load by quantitative PCR at CLL presentation in mononuclear cells (MNC) of 220 prospective patients that were enrolled and followed-up in two major Institutions. In 20 patients EBV DNA load was also assessed on plasma samples. Forty-one age-matched healthy subjects were tested for EBV DNA load on MNC. Findings were validated in an independent retrospective cohort of 112 patients with CLL. EBV DNA load was detectable in 59%, and high ( 652000 copies/\ub5g DNA) in 19% of patients, but it was negative in plasma samples. EBV DNA load was significantly higher in CLL patients than in healthy subjects (P < .0001). No relation was found between high EBV load and clinical stage or biological variables, except for 11q deletion (P = .004), CD38 expression (P = .003), and NOTCH1 mutations (P = .05). High EBV load led to a 3.14-fold increase in the hazard ratio of death and to a shorter overall survival (OS; P = .001). Poor OS was attributable, at least in part, to shorter time-to-first-treatment (P = .0008), with no higher risk of Richter's transformation or second cancer. Multivariate analysis selected high levels of EBV load as independent predictor of OS after controlling for confounding clinical and biological variables. EBV DNA load at presentation is an independent predictor of OS in patients with CLL

Epstein-Barr virus DNA load in chronic lymphocytic leukemia is an independent predictor of clinical course and survival

The relation between Epstein-Barr virus (EBV) DNA load and clinical course of patients with chronic lymphocytic leukemia (CLL) is unknown. We assessed EBV DNA load by quantitative PCR at CLL presentation in mononuclear cells (MNC) of 220 prospective patients that were enrolled and followed-up in two major Institutions. In 20 patients EBV DNA load was also assessed on plasma samples. Forty-one age-matched healthy subjects were tested for EBV DNA load on MNC. Findings were validated in an independent retrospective cohort of 112 patients with CLL. EBV DNA load was detectable in 59%, and high ( 652000 copies/\ub5g DNA) in 19% of patients, but it was negative in plasma samples. EBV DNA load was significantly higher in CLL patients than in healthy subjects (P < .0001). No relation was found between high EBV load and clinical stage or biological variables, except for 11q deletion (P = .004), CD38 expression (P = .003), and NOTCH1 mutations (P = .05). High EBV load led to a 3.14-fold increase in the hazard ratio of death and to a shorter overall survival (OS; P = .001). Poor OS was attributable, at least in part, to shorter time-to-first-treatment (P = .0008), with no higher risk of Richter's transformation or second cancer. Multivariate analysis selected high levels of EBV load as independent predictor of OS after controlling for confounding clinical and biological variables. EBV DNA load at presentation is an independent predictor of OS in patients with CLL