Andrea Visentin

Clinical profile associated with infections in patients with chronic lymphocytic leukemia. Protective role of immunoglobulin replacement therapy

The prognosis and treatment of chronic lymphocytic leukemia (CLL) have improved significantly over the last years; however, CLL is still an incurable disease and infections are the major cause of morbidity and mortality, contributing to 25–50% of deaths.[1][1] Susceptibility to infections in CLL

Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B‐cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T‐cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro‐survival signals provided by MSCs. Competing with ATP for the nucleotide‐binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src‐kinase Lyn. Considering that most anti‐microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro‐survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival.

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines. As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.

Targeted activation of the SHP-1/PP2A signaling axis elicits apoptosis of chronic lymphocytic leukemia cells

Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn’s action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP‐9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP‐9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase‐inhibitor PP2 with a consequent decrease of MMP‐9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check‐point molecule between the BCR and CXCR4 signalling pathways.

Targeting Brutons Tyrosine Kinase in Chronic Lymphocytic Leukemia at the Crossroad between Intrinsic and Extrinsic Pro-survival Signals

Chemo immunotherapies for chronic lymphocytic leukemia (CLL) showed a positive impact on clinical outcome, but many patients relapsed or become refractory to the available treatments. The main goal of the researchers in CLL is the identification of specific targets in order to develop new therapeutic strategies to cure the disease. The Bcell receptor-signalling pathway is necessary for survival of malignant B cells and its related molecules recently become new targets for therapy. Moreover, leukemic microenvironment delivers survival signals to neoplastic cells also overcoming the apoptotic effect induced by traditional drugs. In this context, the investigation of Bruton’s tyrosine kinase (Btk) is useful in: i) dissecting CLL pathogenesis; ii) finding new therapeutic approaches striking simultaneously intrinsic as well as extrinsic pro-survival signals in CLL. This paper will review these main topics.

Alternate use of thrombopoietin receptor agonists in adult primary immune thrombocytopenia patients: A retrospective collaborative survey from Italian hematology centers

Sequential use of the TPO‐RAs romiplostim and eltrombopag in ITP patients failing either agent was retrospectively evaluated to assess efficacy and impact of clinical characteristics on outcome. Patients were grouped into 5 categories: efficacy issues: 1st TPO‐RA failure; loss of response; non‐efficacy issues: platelet fluctuations; patients preference; adverse event development. Either one TPO‐RA sequence was analyzed at 3 month and at last follow‐up. 106/546 patients on TPO‐RA underwent switch and 65% achieved, regained or maintained a short‐ term response independent of switch sequence, gender or age; lower response rates were associated with lines of previous therapy; disease duration lowers probability to respond. Clinically, patients switched for efficacy issue did not differ from those switched for non‐efficacy issues. Response was achieved/regained in 57.8% of patients switched for efficacy issues, the lowest response rates were observed in non‐responders to 1st TPO‐RA; 80% of patients switched for non‐efficacy issues maintained a response. Platelet fluctuation resolved in 44.4%. Of the 49 patients evaluable for long‐term outcome, 27 were in response on therapy; 16 discontinued the TPO‐RA for reasons other than efficacy, while only 6 were non responders. We confirm the efficacy of TPO‐RA switch; once achieved, response to the 2nd TPO‐RA seems durable.

Epidemiology and risk factors of invasive fungal infections in a large cohort of patients with chronic lymphocytic leukemia.

Invasive fungal infections (IFI) are serious, life‐threatening complications of hematological malignancies particularly in patients affected by acute myeloid leukemia and following hematopoietic stem cell transplantation. Although data on IFI in patients with acute leukemia have been updated in the past years, little information is available in patients with lymphoproliferative malignancies, particularly in chronic lymphocytic leukemia (CLL). Chronic lymphocytic leukemia is the most common leukemia in western country with an estimated incidence of 4 to 5 cases per 100 000 people per year and is characterized by a variable clinical course. Although new therapies are available to manage the leukemic process, infections are the major cause of morbidity and mortality contributing to 25% to 50% of deaths. Susceptibility to infections in CLL patients can be related to immunological defects associated with the disease (including hypogammaglobulinemia, T cell, natural killer cell, and innate immunity abnormalities) and secondary to chemoimmunotherapy. Hypogammaglobulinemia and T‐cell defects are quite common at diagnosis and become more pronounced with advanced‐stage disease. Interestingly, it has been reported that even patients with monoclonal B‐cell lymphocytosis harbor a higher risk of serious infection than the general population. Strategies to prevent bacterial infections in patients with symptomatic hypogammaglobulinemia include prophylactic antibiotics or immunoglobulin replacement therapy (IgRT). Although passive immunotherapy with immunoglobulin (IG) allows a reduction of minor and major bacterial infections, several data suggest that IgRT does not result in a decrease of mortality, and to date, no clear indications for this treatment are available. In this work, we aimed to identify the clinical and biological features of CLL patients who developed IFI. We retrospectively reviewed data from 795 subjects with CLL referred to our unit from 1983 to 2015. Invasive fungal infections were defined as infective events caused by yeast or mould that required inpatient management and/ or intravenous antifungal therapies. In this study, we included only proven and probable IFI, according to international guidelines. Time to IFI and overall survival were calculated from the date of CLL diagnosis to IFI development or death (event), respectively, or last available follow‐up (censored). Diagnostic work‐up of fungal infection included blood cultures collection, serum galactomannan (GM) antigen test for 3 consecutive

Role of miR-15a/miR-16-1 and the TP53 axis in regulating telomerase expression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), the most prevalent form of leukemia in adults in the western world, has a highly heterogeneous clinical course, and is characterized by genomic instability which gives rise to several chromosomal alterations detectable in more than 80% of CLL cases.[1][1] While

Primary neurolymphomatosis as clinical onset of chronic lymphocytic leukemia

Dear Editor, A 58-year-old woman sought neurological advice for a 3-month history of paresthesias at the lower limbs and unsteady gait. She had mild distal weakness at the legs, tactile hypoestesia up to the left knee, and absent Achilles tendon reflexes. Electrodiagnostic studies revealed axonal sensory-motor multineuropathy. Complete blood count showed a mild lymphocytosis (7000 cells/ μL). Immunophenotype detected a clonal lambda B cell population expressing CD5+, CD20dim, CD23+, and low immunoglobulin surface levels, consistent with chronic lymphocytic leukemia (CLL). Cerebrospinal fluid (CSF) was unremarkable (protein 35 mg%, 408 RBC/ μL, 1.3 WBC/μL). At CSF immunophenotype, 47 % of lymphocytes were B CD5+ cells, likely related to blood CSF contamination. Molecular analyses on circulating CLL cells showed mutated conformation of the IGVH gene of the B cell receptor (homology <98 % from the germline sequence); cytogenetic studies showed trisomy of chromosomes 12, 18, and 19 and deletion of 11q at FISH and wild-type status of TP53 and NOTCH1. The co-existence of trisomies of chromosome 12 and 19 has been recently demonstrated to be enriched in young CLL patients (median age 59 years) and to be associated with indolent clinical course [1]. Sural nerve biopsy showed endoneural perivascular infiltration of CD20 and CD5+ lymphomonocytes. Semithin sections showed reduced density of myelin fibers (Fig. 1). Total-body CT scan was negative. CLL-neurolymphomatosis was diagnosed. Before starting therapy, a further neurophysiological evaluation together with nerve ultrasound was performed. Electrodiagnostic study confirmed axonal sensory-motor multineuropathy. Nerve ultrasound revealed a preserved nerve cross-sectional area and echogenicity, but color Doppler sonography showed increased perineural and intraneural vascularity at the left tibial nerve in the popliteal fossa (Fig. 2a). The early stage and the biological low-risk profile of the disease oriented us on therapy with rituximab alone. After 8 cycles of rituximab (375 mg/m IV), paresthesias significantly decreased and gait regained functionality. At Doppler ultrasound, blood flow was absent (Fig. 2b). Neurolymphomatosis is a rare and often overlooked diagnosis [2–5]. Histological demonstration of nerve infiltration is mandatory for a certain diagnosis, but neuroimaging is increasingly gaining a diagnostic role [6–9]. Ultrasound has already been described as useful for identifying peripheral nerve involvement in B cell lymphoma [10, 11], but histological confirmation is needed, since increased nerve blood flow can be found also in active chronic inflammatory demyelinating polyradiculoneuropathy [12]. The patients described by * Chiara Briani chiara.briani@unipd.it