Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy

Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

Dexamethasone scheduling in MNC culture. The supplement was added in standard medium until the detection of MSC colonies (n = 16 CB units) or alternatively added for the first week of MNC culture only (n = 34). (DOCX 120 kb

Multiple large osteolytic lesions in a patient with systemic mastocytosis: a challenging diagnosis

Patients with advanced variants of Systemic Mastocytosis may develop destructive bone lesions when massive mast cell (MC) infiltrates are present. Finding of large osteolyses in indolent systemic mastocytosis, typically characterized by low MC burden, should prompt investigations for an alternative explanation

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

P09.01 Adoptive cell therapy of hematological malignancies using cytokine-induced killer cells retargeted with monoclonal antibodies

Background Cytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI). Materials and Methods CIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent. Results The combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone. Conclusions Here we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies. References Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311. The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato Antonio Disclosure Information A. Dalla Pieta: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None

P09.13 Optimization of a GMP-grade large-scale expansion protocol for cytokine-induced killer cells using gas-permeable static culture flasks

Background Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Materials and Methods CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. Results CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. Conclusions We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications. Disclosure Information A. Ventura: None. P. Palmerini: None. A. Dalla Pieta: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML

Role of blood cells dynamism on hemostatic complications in low-risk patients with essential thrombocythemia

Patients with essential thrombocythemia (ET) aged less than 60 years, who have not suffered a previous vascular event (low-risk patients), may develop thrombotic or hemorrhagic events. So far, it has not been possible to identify useful markers capable of predicting which of these patients are more likely to develop an event and therefore who needs to be treated. In the present study, we analysed the relationship between vascular complications and longitudinal blood counts of 136 low-risk ET patients taken over a sustained period of time (blood cells dynamism). After a median follow-up of 60 months, 45 out of 136 patients (33%) suffered 40 major thrombotic and 5 severe hemorrhagic complications. A total number of 5,781 blood counts were collected longitudinally. Thrombotic and hemorrhagic events were studied together (primary endpoint) but also separately (thrombotic alone = secondary endpoint; hemorrhagic alone = tertiary endpoint). The primary endpoint showed no significant association between platelet and WBC count at diagnosis and risk of any event (platelet, p = 0.797; WBC, p = 0.178), while Hb at baseline did show an association (p = 0.024). In the dynamic analysis with Cox regression model, where the blood count values were studied by time of follow-up, we observed that the risk for Hb was 1.49 (95% CI 1.13-1.97) for every increase of 1 g/dL, and that this risk then marginally decreased during follow-up. WBC was associated with an increased risk at baseline for every increase of 1 7 10(9)/L (hazard ratio (HR) 1.07, 95% CI 1.01-1.13, p = 0.034), the risk was stable during follow-up (HR 0.95, p = 0.187 at 60 months). Also, for each increment at baseline of 100 7 10(9) platelets/L, HR was increased by 1.08 (95% CI 0.97-1.22, p = 0.159) and decreases during follow-up. In conclusion, this study is the first to evaluate in ET low-risk patients, the risk of developing a thrombotic/hemorrhagic event considering blood counts over time. Overall our study shows that the risk changes over time. For example, the risk associated with WCC is not linear as previously reported. An interesting new finding is that PLT and even Hb contribute to the risk of developing vascular events. Future treatments should take into consideration these findings and aim to control all parameters over time. We believe this early study may help develop a dynamic analysis model to predict thrombosis in the single patient. Further studies are now warranted to further validate our findings

Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy

Interleukin 7 requirement for survival of T-cell acute lymphoblastic leukemia and human thymocytes on bone marrow stroma.

We explored the role of interleukin-7 (IL-7) in the bone marrow (BM) stroma-mediated survival of primary T-cell acute lymphoblastic leukemia (T-ALL) cells and normal thymocytes. We presented evidence that IL-7 has a major role in the enhanced survival mediated by BM stroma both in T-ALL cells and thymocytes