Daria Sollazzo

Decreased expression of indoleamine 2,3-dioxygenase 1 in dendritic cells contributes to impaired regulatory T cell development in immune thrombocytopenia

Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.

Circulating CD4+CD161+CD196+ Th17 cells are not increased in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder in which, for still unknown reasons, platelet surface proteins become antigenic and stimulate the immune system to produce autoantibodies and self-reactive cytotoxic T lymphocytes. These findings result in immune-induced platelet destruction

Have splenectomy rate and main outcomes of ITP changed after the introduction of new treatments? A monocentric study in the outpatient setting during 35 years

In the last years, rituximab (RTX) and agonists of the thrombopoietin receptor (TPO‐R) eltrombopag and romiplostim have provided new treatment options in persistent and chronic immune thrombocytopenia (ITP). Here, we analyzed the changes in therapeutic choices over time and their impact on clinical outcomes in a cohort of 557 ITP outpatients followed at the “L. and A. Seràgnoli” Institute of Hematology, Bologna, Italy, from 1980 to 2015. Overall 397 patients (71%) required front‐line corticosteroids, mainly prednisone. Over the decades, splenectomy was delayed from second to third‐line, but was steadily used in around 15–25% of patients refractory or relapsing after first‐line treatment. Consensually, RTX and TPO‐R agonists emerged as second and third‐line therapy of choice, respectively. Splenectomy was associated with the best response rates and the lower incidences of relapse, while the relapse rate after RTX was comparable to that observed with corticosteroids and other immunosuppressive agents. The introduction of TPO‐R agonists gave an alternative to the administration of immunosuppressive drugs and probably contributed to moderate the incidence of infectious complications that remained stable over the decades, despite an increasing use of RTX from the 2000s onwards. Overall responses were similar over time, with over 97% achieving a response in all time‐periods. However, the cumulative risk of bleeding significantly decreased [14.3% (1980–89) vs. 7% (1990–99) vs. 5.6% (2000–09) vs. 0.2% (2010–15)] (P < 0.001), mainly thanks to the optimization of front‐line corticosteroids therapy and to the wider availability of second and third‐line therapies. Am. J. Hematol. 91:E267–E272, 2016.

Risk Factors for Infections In Myelofibrosis: Role of Disease Status and Treatment. A multicenter study of 507 patients

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3–4, 45%) for an incidence rate of 3.9% per patient‐year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate‐2 IPSS category (HR 1.8, 95%CI:1.2–2.7; P = 0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1–2.5; P = 0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX‐treated patients (44% vs. 20%, P < 0.001). In conclusion, IPSS‐category and splenomegaly, emerged as the main risk factors for infections in MF. RUX‐treated patients experienced significantly more infection episodes; however, future prospective studies are needed to isolate the confounding contribution of other risk factors such as disease stage. Am. J. Hematol. 92:37–41, 2017.

Molecular profile of CD34 + stem/progenitor cells according to JAK2V617F mutation status in essential thrombocythemia

Molecular profile of CD34+ stem/progenitor cells according to JAK2V617F mutation status in essential thrombocythemia

Bleeding in essential thrombocythaemia: a retrospective analysis on 565 patients

large cell content and proliferative rate of the tumour (Table I). MIR21 was also over-expressed in 7 of the 15 SMZL samples (median = 2Æ8), including 3 cases with an aggressive clinical presentation (B symptoms) and 6 cases with histological aggressiveness. In contrast, the eight cases with neither morphological nor clinical aggressiveness did not exhibit overexpression of MIR21 (median = 0Æ7) (Mann Whitney test: P = 0Æ02). Conversely, MIR21 over-expression was associated with a good prognosis in DLBCL (Lawrie et al, 2008) and Roehle et al (2008) reported that down regulation of MIR21 in DLBCL was associated with an inferior survival. In contrast, the present study suggests an association of MIR21 overexpression with aggressiveness of SMZL. In conclusion, only a small number of miRNAs seem to have a distinct expression pattern in SMZL when compared to nontumoural spleens. MIR21 overexpression appears to correlate with disease aggressiveness, indicating its potential role in marginal zone transformation.

The CD47 pathway is deregulated in human immune thrombocytopenia

OBJECTIVE A novel mechanism of platelet destruction involving the CD47/ signal regulatory protein-α (SIRPα) system has recently been suggested in a mouse model of immune thrombocytopenia (ITP). The CD47 molecule serves as ligand for SIRPα receptor and as receptor for thrombospondin acting as antagonistic to phagocyte activity and a regulator of apoptosis, respectively. In this study, we evaluated if the CD47/SIRPα axis may be involved in the apoptosis and clearance of platelets in human ITP. MATERIALS AND METHODS Using flow cytometry, we characterized whether expression of CD47 on fresh and in vitro-aged platelets- and of SIRPα receptor on CD14-derived dendritic cells (DCs), macrophages, circulating DCs, and monocytes is reduced is ITP; whether the in vitro platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRPα in ITP; and whether platelets are more susceptible to the CD47-induced death signal in ITP. RESULTS We demonstrated that low platelet count in ITP is not due to increased phagocytosis associated with decreased expression of CD47 on the platelet surface and, despite reduced SIRPα expression, blockage of SIRPα on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs. In contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages, and platelets are not healthy because they show increased apoptosis and are resistant to CD47-induced death signal. CONCLUSIONS Our results demonstrate that the CD47 pathway in ITP patients abnormally modulates platelet homeostasis.

The choice of second-line therapy in steroid-resistant immune thrombocytopenia: role of platelet kinetics in a single-centre long-term study.

Splenectomy is a time‐honoured well established approach for patients with steroid‐resistant immune thrombocytopenia (ITP). However, due to the more recent availability of therapeutic options alternative to splenectomy, such as rituximab and agonists of the thrombopoietin‐receptor, the choice of second‐line therapy is challenging. Platelet kinetics has been widely used to predict response to splenectomy. We describe the outcome of 70 chronic ITP patients who performed a platelet kinetic study after failure of front‐line corticosteroids and subsequently underwent open splenectomy. After a median follow‐up from surgery of 20 years, 62 (88.5%) patients responded to splenectomy and 9 patients (13%) relapsed. Achieving a complete response (CR) significantly predicted a higher probability long‐term stable response. The pattern of platelet sequestration was predominantly splenic in 52 patients (74%), predominantly hepatic in 12 patients (17%), and diffuse in 6 (9%). Patients with nonsplenic (diffuse and hepatic) sequestration showed significantly lower overall responses compared to patients with splenic captation (P = 0.002). A nonsplenic sequestration significantly correlated with lower CR rate and, among CR patients, predicted an increased risk of relapse. Also, the probability of stable responses in nonsplenic uptake patients was substantially lower than in patients with splenic uptake (85% vs. 50%, P = 0.0083). Platelet life span and platelet turnover did not correlate with response and relapse rate. Overall, splenic sequestration was able to predict not only a better quality, but also a higher durability of the responses. However, it should be enphasized that the response rate and duration of response even in patients with nonsplenic uptake were similar or even superior to those reported in patients treated with rituximab as first option. Am. J. Hematol. 89:1047–1050, 2014.

Crucial factors of the inflammatory microenvironment (IL-1β/ TNF-α/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study

Along with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of Myelofibrosis (MF). Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived circulating CD34+ cells. We found that, regardless mutation status, IL-1β or TNF-α increases the survival of MF-derived CD34+ cells. In addition, along with stimulation of cell cycle progression to the S-phase, IL-1β or TNF-α ± TIMP-1 significantly stimulate(s) the in vitro clonogenic ability of CD34+ cells from JAK2V617 mutated patients. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α + TIMP-1 decreased the erythroid compartment of the CALR mutated patients. Megakaryocyte progenitors were stimulated by IL-1β (JAK2V617F mutated patients only) and inhibited by TNF-α. IL-1β + TNF-α + C-X-C motif chemokine 12 (CXCL12) ± TIMP-1 highly stimulates the in vitro migration of MF-derived CD34+ cells. Interestingly, after migration toward IL-1β + TNF-α + CXCL12 ± TIMP-1, CD34+ cells from JAK2V617F mutated patients show increased clonogenic ability. Here we demonstrate that the interplay of these inflammatory factors promotes and selects the circulating MF-derived CD34+ cells with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach.

Circulating Calreticulin Is Increased in Myelofibrosis: Correlation with Interleukin-6 Plasma Levels, Bone Marrow Fibrosis, and Splenomegaly.

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease.