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Background: The third-generation tyrosine kinase inhibitor ponatinib has demonstrated high clinical efficacy in the setting of patients with resistant chronic phase chronic myeloid leukemia (CML), also inducing deep molecular responses. However, ponatinib-related cardiovascular toxicities make management challenging, especially of those patients with CML with previous cardiovascular comorbidities. Case Report: We report on the efficacy of ponatinib treatment used as fourth-line therapy in a 55-year-old woman affected by significant comorbidities (mainly cardiovascular) present before the diagnosis of CML. Ponatinib therapy induced a rapid and excellent clinical response, with the achievement of a durable deep molecular response that allowed us to propose a strategy of treatment discontinuation in order to reduce drug-related toxicities. Conclusion: A strategy of a treatment-free interval might represent a useful clinical tool in those patients with CML who achieve a durable deep molecular response but are also affected by significant comorbidities in order to minimize the risk of ponatinib-related toxicities.

To understand neutrophil impairment in the progression from MGUS through active MM, we investigated the function of mature, high-density neutrophils (HDNs), isolated from peripheral blood. In 7 MM, 3 MGUS and 3 healthy subjects by gene expression profile, we identified a total of 551 upregulated and 343 downregulated genes in MM-HDN, involved in chemokine signaling pathway and FC-gamma receptor mediated phagocytosis conveying in the activation of STAT proteins. In a series of 60 newly diagnosed MM and 30 MGUS patients, by flow-cytometry we found that HDN from MM, and to a lesser extend MGUS, had an up-regulation of the inducible FcγRI (also known as CD64) and a down-regulation of the constitutive FcγRIIIa (also known as CD16) together with a reduced phagocytic activity and oxidative burst, associated to increased immune-suppression that could be reverted by arginase inhibitors in co-culture with lymphocytes. In 43 consecutive newly-diagnosed MM patients, who received first-line treatment based on bortezomib, thalidomide and dexamethasone, high CD64 could identify at diagnosis patients with inferior median overall survival (39.5 versus 86.7 months, p = 0.04). Thus, HDNs are significantly different among healthy, MGUS and MM subjects. In both MGUS and MM neutrophils may play a role in supporting both the increased susceptibility to infection and the immunological dysfunction that leads to tumor progression.

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM-MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM-MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

Recent reports identify the ratio between absolute neutrophil count (ANC) and absolute lymphocyte count (ALC), called neutrophil to lymphocyte ratio (NLR), as a predictor of progression-free survival (PFS) and overall survival (OS) in various malignancies. We retrospectively examined the NLR in a cohort of 309 newly diagnosed multiple myeloma (MM) patients treated upfront with novel agents. NLR was calculated using data obtained from the complete blood count (CBC) at diagnosis and subsequently correlated with PFS and OS. The median NLR was 1.9 (range 0.4–15.9). Higher NLR was independent of international staging system (ISS) stage, plasma cell infiltration or cytogenetics. The 5-year PFS and OS estimates were, respectively, 18.2 and 36.4 % for patients with NLR ≥ 2 versus 25.5 and 66.6 % in patients with NLR < 2. Among younger patients (age

In newly diagnosed myeloma patients, upfront autologous transplant (ASCT) prolongs progression-free survival 1 (PFS1) compared with chemotherapy plus lenalidomide (CC+R). Salvage ASCT at first relapse may still effectively rescue patients who did not receive upfront ASCT. To evaluate the long-term benefit of upfront ASCT vs CC+R and the impact of salvage ASCT in patients who received upfront CC+R, we conducted a pooled analysis of 2 phase III trials (RV-MM-209 and EMN-441). Primary endpoints were PFS1, progression-free survival 2 (PFS2), overall survival (OS). A total of 268 patients were randomized to 2 courses of melphalan 200 mg/m2 and ASCT (MEL200-ASCT) and 261 to CC+R. Median follow-up was 46 months. MEL200-ASCT significantly improved PFS1 (median: 42 vs 24 months, HR 0.53; P

BRIT1 (BRCT-repeat inhibitor of hTERT expression), also known as microcephalin (MCPH1), is a crucial gene in the complex cellular machine that is devoted to DNA repair and acts as a regulator of both the intra-S and G2/M checkpoints. The most important role of BRIT1/MCPH1 in the regulation of cell cycle progression appears to be the G2/M checkpoint. The K562 and peripheral blood cells of chronic myeloid leukemia (CML) patients at diagnosis were found to downregulate BRIT1/MCPH1. However, we could not find any correlation between bcr/abl activity and the BRIT1/MCPH1 level. In order to study the genomic instability of CML cells, we evaluated the ability of these cells to arrest mitotic division after exposure to hydroxyurea, a known genotoxic agent. We showed that CML cells continue to proliferate without the activation of the G2/M cell cycle checkpoint arrest or of the apoptotic mechanism. This behavior may predispose the cells to accumulate genomic defects. In conclusion, we found that CML cells have a low BRIT1/MCPH1 level and show a defective G2/M arrest, confirming that these cells have a constitutive genomic instability.

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.

Objectives: We recently identified the ratio between absolute neutrophils count and absolute lymphocyte count, NLR ≥ 2, combined to ISS as a predictor of progression free survival (PFS) and overall survival (OS) in patients younger than 65 years with symptomatic Multiple Myeloma (MM). We retrospectively examined the NLR-ISS in 165 consecutive smoldering Myeloma (sMM) accessed our Center between January 2004 and June 2014. Methods: NLR was calculated using data obtained from the complete blood count (CBC) at diagnosis and subsequently correlated with time to treatment (TTT) for symptomatic MM. All patients underwent to bone marrow evaluation to estimate plasma cells infiltration (BMPC) and cytogenetic alterations detectable by FISH, Magnetic Resonance Imaging (MRI) to detect bone lesions, serum free-lite chain evaluation (sFLC). Patients with bone marrow plasma cells >60% or lytic lesions at MRI were excluded from further analysis. Results: We identified 127 patients with sMM defined accordingly to the updated IMWG 2015 guidelines. The median NLR was 1.7 (range 0.6-10.5), lower than the value previously found for MM 1.9 (range 0.4-15.9, p=0.005. Higher NLR was independent from ISS stage, BMPC amount, high-risk FISH and sFLC. Using NLR ≥ 2 we could not predict TTT. Indeed, in univariate analysis only BMPC ≥ 30% (p=0.003), albumin 3.5 g/L (p=0.0001), ratio of uninvolved/involved sFLC (p=0.0002), immunoparesis (p=0.016) and LDH (p

Background: VSIG4 acts as a co-inhibitory ligand to negatively regulate T cell proliferation and cytokine production by arresting cell cycle at G0/G1 phase and upregulating tolerance-inducing p27KIP-1. VSIG4 expression is restricted to macrophages. VSIG4 fusion protein also inhibits T cell production of Th1, Th2, and Th17 cytokines. Thus, we hypothesized that endogenous VSIG4 impairs helper T cell functions and then inhibits the subsequent antibody response. Materials and Methods: Mice were immunized subcutaneously with 100 g ovalumin (OVA) in a 1:1 emulsion with phosphate-buffered saline (PBS) and CFA in 100 l PBS. OVAspecific IgM or IgG subclasses were determined using culture supernatants or sera from immunized mice with OVA-coated plates and horseradish peroxidase-conjugated anti-mouse IgG1, -IgG2a, -IgG2b, and -IgG3. To study the VSIG4 role in antibody class switching via regulation of helper T cells, BALB/c mice were immunized intraperitoneally with 500 g of 2,4,6-Trinitrophenyl hapten (TNP)Keyhole Limpet Hemocyanin (KLH) for 14 days. Isolated B cells from TNP-KLH-immunized mice and purified CD4+ T cells from BALB/c wild-type or VSIG4 KO mice immunized s.c. with OVA/CFA were cocultured in the presence of TNPOVA (50 ng/ml) for 5 days. Culture supernatants were collected, and ELISA was performed on a TNP-CGG (100 g/ml)-coated plate to measure anti-TNP IgM and IgG titers. Results: Isotype switching of OVA-specific antibody subclasses to IgG1, IgG2a, IgG2b, and IgG3 was enhanced in OVA-immunized VSIG4 KO mice. 2,4,6-TNPe KLH-primed B cells cocultured with OVAprimed CD4+ T cells from OVA-immunized VSIG4 KO mice in the presence of TNP-OVA showed enhanced isotype switching to IgG subclasses compared to those cocultured with cells isolated from OVA-immunized wild-type (WT) mice. Furthermore, the levels of CD40L expression, the frequency of memory CD4+ T cells, and the production of isotype switching-inducing cytokines increased significantly in OVA-primed CD4+ T cells from VSIG4 knockout (KO) mice. T cells from OVA-specific T cell receptor transgenic mice produced more IFNwhen cocultured with macrophages from VSIG4 KO mice compared to WT mice. Conclusion: Our results demonstrate that macrophage-associated VSIG4 plays a negative role in helper T cell-dependent isotype switching by inhibiting helper T cell activation and differentiation, and suppressing the isotype switching-inducing cytokine production in antigen-primed CD4+ helper T cells.

We recently proposed that Th1 and Th2 cytokines exert opposite effects on the pathogenesis and clinical outcome of organ-specific autoimmunity by altering the expression of genes involved in target cell survival. Because a Th2 response against tumors is associated with poor prognosis, we investigated the ability of IL-4 to protect tumor cells from death receptor- and chemotherapy-induced apoptosis. We found that IL-4 treatment significantly reduced CD95 (Fas/APO-1)- and chemotherapeutic drug-induced apoptosis in prostate, breast, and bladder tumor cell lines. Analysis of antiapoptotic protein expression revealed that IL-4 stimulation resulted in up-regulation of cellular (c) FLIP/FLAME-1 and Bcl-xL. Exogenous expression of cFLIP/FLAME-1 inhibited apoptosis induced by CD95 and to a lesser extent by chemotherapy, while tumor cells transduced with Bcl-xL were substantially protected both from CD95 and chemotherapeutic drug stimulation. Moreover, consistent IL-4 production and high expression of both cFLIP/FLAME-1 and Bcl-xL were observed in primary prostate, breast, and bladder cancer in vivo. Finally, primary breast cancer cells acquired sensitivity to apoptosis in vitro only in the absence of IL-4. Thus, IL-4 protects tumor cells from CD95- and chemotherapy-induced apoptosis through the up-regulation of antiapoptotic proteins such as cFLIP/FLAME-1 and Bcl-xL. These findings may provide useful information for the development of therapeutic strategies aimed at restoring the functionality of apoptotic pathways in tumor cells.

Background A combination of bortezomib (1.3 mg/m2), melphalan (5 mg/m2), and dexamethasone (40 mg) (BMD), with all three drugs given as a contemporary intravenous administration, was retrospectively evaluated. Patients and methods Fifty previously treated (median 2 previous lines) patients with myeloma (33 relapsed and 17 refractory) were assessed. The first 19 patients were treated with a twice-a-week (days 1, 4, 8, 11, ‘base’ schedule) administration while, in the remaining 31 patients, the three drugs were administered once a week (days 1, 8, 15, 22, ‘weekly’ schedule). Results Side-effects were predictable and manageable, with prominent haematological toxicity, and a better toxic profile in ‘weekly’ schedule (36% versus 66% in ‘base’ schedule). The overall response rate was 62%. After median follow-up of 24.5 months (range 2.7–50 months), the median progression-free survival (PFS) was 21.6 with no difference between the two schedules and the median overall survival (OS) was 33.8 months. Independently from the adopted schedule, we found that also in a cohort of relapsed/refractory patients achieving at least partial remission improved PFS (35.2 versus 9 months) and OS (unreached median versus 18 months). Conclusion Taken together, our observations suggest that BMD is an effective regimen in advanced myeloma patients with acceptable toxicity.

Several reports have clearly demonstrated that the activation of Nuclear factor-kappa B (NF-kB) is essential for the pathogenesis of MM because it regulates the expression of many proteins involved in cell survival, proliferation, and resistance to chemotherapeutic drugs. We therefore analyzed by immunohistochemistry, immunofluorescence and western blot analysis the nuclear localization of NF-κB in MM cells derived from 60 different patients with MM at presentation and in relapse, as well as in three myeloma cell lines (XG1, RPMI 8226, KMS-18). Surprisingly, nuclear localization (the active form) of NF-κB was detected in only one MM sample from a refractory MM patient and in two samples from relapsed patients, while all the other samples, including the MM cell lines, exclusively express the cytoplasmic (inactive) form of NF-κB. We next analyzed the NF-κB status in mesenchymal cells from MM patients and we found that NF-κB was clearly present in the nucleus. We have also analyzed the sensitivity of MM primary cells to different doses of the proteasome inhibitor Bortezomib, which is described to antagonize NF-κB activity. We found a consistent dose- and time-dependent antitumor activity against both chemoresistant and chemosensitive myeloma cells in all the samples analyzed, independently of NF-κB localization. In conclusion, contrary to the current dogma that indicates a constitutive activation of NF-kB in MM cells, we demonstrated that in a steady state, especially in patients at diagnosis, NF-kB resides in the cytoplasm (the inactive form) but the cells maintain the sensitivity to Bortezomib, thus indicating that block of IkB degradation is not the only mechanism responsible for proteasome inhibitor induced-apoptosis.

CD95 (Fas/Apo-1) is a broadly expressed death receptor involved in a variety of physiological and pathological apoptotic processes. Since its discovery, defects in CD95/CD95L system have been proposed as major pathogenic factors responsible for impaired immunological tolerance to self antigens and autoimmunity. Later, analysis of altered sensitivity to CD95-induced apoptosis in cells targeted by the immune response has revealed an unexpected role for CD95 and CD95L in organ-specific autoimmunity. CD95 has been shown to be expressed and functional in virtually all cell types that are target of the organ-specific autoimmune response. Here we review some of the major findings concerning the role of CD95 in autoimmunity, in dysfunctions due to increased or decreased CD95-induced apoptosis.