ALBERT

Description: Abstract 3386 It has been demonstrated that long term treatment of CML patients with Imatinib (IM) is associated with altered bone and mineral metabolism. The mechanisms that are responsible for this effect are not fully understood but an inhibition of the PDGF-Rβ (Platelet Derived Growth Factor- Receptor beta) axis has been documented. In order to evaluate if stimulation of osteoblastogenesis is a common feature of other tyrosine kinase inhibitors (TKIs) approved for the treatment of patients with CML, we evaluated the osteoblatic differentiation of Mesenchymal Stem Cells derived from adult bone marrow donors (BM-MSCs) after in vitro treatment with Dasatinib (DA), Nilotinib (NI) or Bosutinib (BO). BM-MSCs were induced to differentiate in osteoblastic cells by treatment with osteogenic medium (OM) with or without DA, NI or BO. BM-MSCs was induced to differentiate in osteoblastic cells by treatmet with osteogenic medium (0.2 mM ascorbic acid, 0.1 μ m dexamethasone and 10 mM β-glycerophosphate, OM) with or without DA 2nM, BO 5nM or NI 100nM. After 21 days of treatment, in the cultures treated with DA e NI for 21 days we have observed a significant increase of extracellular mineralization and of osteogenic markers such as bone morphogenetic protein (BMP2) (p

Description: T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients’ LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated. With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients’ plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs 〉60%: 9.1 ± 2.7 pg/ml; p

Description: Abstract 3385 Heme oxygenase 1 (HO-1) is a rate-limiting enzyme in heme degradation (from hemoproteins and hemoglobin), leading to the generation of free iron, biliverdin and carbon monoxide. Recent studies have reported that HO-1 expression may represent an important protective endogenous mechanism against physical, chemical and biological stress. The cytoprotective role of HO-1 has been demonstrated for several solid tumors and acute leukemias. In addition, HO-1 is considered to play an important role as a survival molecule in CML cells, and an overexpression of HO-1 has been found to inhibit apoptosis induced by imatinib. In our experiments, K562 cells were incubated for 24 hrs with imatinib (1 uM) alone, or with an inductor of HO-1 (Cobalt protoporphyrin, CoPP, 10uM), or with inhibitor of HO-1 activity (Tin- mesoporphyrin, TIN) and viability of cells was evaluated by the ATP-lite1step assay (PerkinElmer). As expected, addition of CoPP was able to overcome the inhibitory effect of IM on K562 cells (p

Description: Background. In the last twenty years, the outcome of multiple myeloma (MM) has markedly improved. Daratumumab is the first anti-CD38 monoclonal antibody (mAb) recently approved for the treatment of relapsed refractory multiple myeloma (RRMM). In this study we have evaluated the efficacy of daratumumab single agent and daratumumab plus pomalidomide and dexamethasone in low and high risk RRMM patients. Design and Methods. From November 2015 to March 2016, 25 and 39 RRMM patients were treated with therapy using daratumumab single agent (Group 1: median age 67, range 40-83) and daratumumab plus pomalidomide and dexamethasone respectively (Group 2: median age 71, range 45-87). In both groups, patients received daratumumab IV 16 mg/kg once a week (weeks 1-8), followed by every other week (weeks 9-24) and then once a month until disease progression or unacceptable toxicities. In Group 2 pomalidomide was administered at dosages from 1 mg to 4 mg daily according to tolerability for 21 days every 28 days, along with dexamethasone 40 mg weekly. The median time form the time of diagnosis was 7.1 years and 5.5 years in Group 1 and Group 2 respectively. In both groups, patients had received a median of 4 prior treatments. In Group 1, 80% of the patients had disease refractory to the last therapy received, 68% had disease double refractory to IMiDs and PIs, and 28% (7 patients) had GEP high risk signature. In Group 2, 78% of the patients had disease refractory to last therapy, 84% had double refractory disease and 37% (14 patients) had high GEP risk signature. Results. In the single agent group, the overall response rate (ORR) was 28%: CR 4% (1 patient), 4% VGPR (1 patient), PR 20% (5 patients). With a median follow up of 3.5 months, 48% of patients were still on treatment and 52% had discontinued treatment for disease progression. The ORR according to last GEP70 was 40% and 0% in the low risk and high risk patients respectively. All high risk patients had discontinued treatment for disease progression within the second month of treatment. In the second group, the ORR was 41%: CR 5% (2 patients), 3% VGPR (1 patient), and 33% PR (13 patients). After a median follow up of 4 months, 77% (30 patients) of patients were still on treatment, 20% (8 patients) discontinued treatment for disease progression and 3% (1 patient) of patients had discontinued treatment for adverse event grade 4. The ORR was 50% (4% CR, 4% VGPR, and 42% PR) in low risk patients and 21% (7% CR and 14% PR) in high risk patients. Infusion-related reactions were mild (54% of patients had an event of any grade, and 4% and 2% had an adverse event of grade 3 in Group 1 and Group 2 respectively). The most common non hematological adverse event of grade 3 or 4 was pneumonia (4% and 8% in Group 1 and Group 2 respectively). PFS of Group 1 and 2 are shown in Figure 1. Conclusion. Daratumumab single agent had a favorable safety profile and encouraging efficacy in patients with heavily pretreated and refractory myeloma, and the combination of daratumumab with pomalidomide and dexamethasone was well tolerated and improved the outcome in low risk patients, even in double refractory disease. Encouraging results have been observed as well in patients with high risk GEP 70 signature. Figure 1 Figure 1. Disclosures Davies: Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

Description: Background NK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56high/CD16dim/neg NK cells with low cytotoxic function and CD56dim/CD16high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56dim/CD16high/CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases. Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease. Methods In a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting. Results In relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56high/CD16neg NK cells, CD56dim/CD16neg NK cells and CD56neg/dim/CD16high. As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56high/CD16neg NK population, 4/16 (25%) with CD56dim/CD16neg NK population and 10/16 (62%) with CD56neg/dim/CD16high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62Llow/neg) and NK memory subset (CD57+/CD62Llow/neg); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56neg/dim/CD16high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6). We then evaluated the presence of cytopenia, in particular neutropenia, in our patients' cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56neg/dim/CD16high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells. Summary Although CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56neg/dim/CD16high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3778 Introduction. The EUTOS score has recently been developed by the European Leukemia-Net (ELN) to predict the achievement of an 18-month complete cytogenetic response (CCyR) and progression-free survival in imatinib-treated early chronic phase (ECP) chronic myeloid leukemia (CML) patients. The score uses the percentage of basophils and spleen size to divide patients in 2 groups of low- and high-risk. Since its publication in 2011, however, there have been conflicting reports about the efficacy of EUTOS score. Moreover, scanty data are available on the power of this scoring system to foresee optimal response to imatinib, as defined by ELN recommendations. Aims and Methods. To test the power of EUTOS score in predicting achievement of optimal response to imatinib, as defined by ELN, time to imatinib failure (TTF) and progression-free survival (PFS), we evaluated 265 ECP CML patients treated with front-line standard dose imatinib (400 mg daily) at 5 major hematology centres in the north-eastern area of Italy. Partial cytogenetic response (PCyR) and CCyR were defined as 1–35% and 0% Ph+ metaphases, respectively; major molecular response (MMR) was defined as BCR-ABL

Description: Background and objectives The standard first-line therapy for patients with DLBCL includes R-CHOP or CHOP-like regimens; although these regimens are highly effective in the majority of DLBCL patients , elderly “unfit” patients do not tolerate these schedules and usually receive palliative therapy with a consequent dismal prognosis. Several polychemotherapy regimens combining low toxicity and a substantial anti-lymphoma activity have been tested in this clinical setting. In the pre-rituximab era, VEMP (etoposide, cyclophosphamide, mitoxantrone, prednisone) polychemotherapy was investigated initially in relapsed/refractory patients and subsequently as first line therapy and displayed fairly good outcomes. In this study, we present data from an Italian single-center experience evaluating the efficacy and tolerability of the association of Rituximab with VEMP (R-VEMP) in patients not eligible for standard R-CHOP therapy or its modifications (for example R-miniCHOP) because of age and/or comorbidities. Design and Methods From October 2006 to November 2012, 34 untreated patients aged 66 years and older (median age: 79) with DLBCL (26% GC, 48% non-GC, 26% ND) were treated with a combination chemotherapy including etoposide 150 mg/m2 day 1; cyclophosphamide 650 mg/m2 day 1; mitoxantrone 12 mg/m2 day 1; prednisone 60 mg/m2 day 1-5; rituximab 375 mg/m2 day 0). Sixty-eight percent of patients had high Charlson Comorbility Index; 62% had Ann Arbor stage III/IV disease; 47% had high or intermediate-high International Prognostic Index score. Results Twenty-six patients (76%) completed the scheduled treatment (4 or 6 cycles). The Overall Response Rate (ORR) was 71%: 19 patients (56%) obtained a Complete Response (CR), 5 (15%) achieved a Partial Response (PR); 3 patients (9%) were in stable disease and 7 (20%) had a progressive disease (among these latter, 6 patients were intermediate-high or high IPI score and had extranodal disease; all of these patients had high Charlson Comorbility Index). After a median follow-up of 20 months, 15 patients (44%) maintained a CR and only one patient relapsed within 14 months after achieving a CR. In this setting of patients the median Overall Survival (OS) was 20 months (range 1-78), the Event Free Survival (EFS) was 6 months (range 1-41). The treatment was well tolerated without therapy related mortality. Among the adverse events, the most common were: grade 3-4 temporary neutropenia (71%), grade 2 transitory anemia (29%) and febrile neutropenia (20%). G-CSF was administrated to 29 patients (85%) and erythropoiesis stimulating agents to 8 patients (24%). Conclusions These data suggest that R-VEMP is a well-tolerated and highly effective regimen in elderly “unfit” patients with DLBCL and could offer a valuable alternative choice for those patients not eligible for more toxic first line protocols. Considering the high rate of serious adverse events and the difficulty to completely administer the scheduled cycles, standard R-CHOP or CHOP-like therapy is applied with much less frequency to elderly unfit patients. R-VEMP could represent a reasonable regimen that warrants to be further explored, as an additional therapeutic option in order to overcome the low survival rate observed in this subgroup of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Background and objectives. Bendamustine in combination with rituximab has demonstrated a relevant clinical activity and a good toxicity profile in patients with mantle cell lymphoma (MCL). Cytosine arabinoside (Ara-C) is a key drug in induction chemotherapy regimens of young patients with MCL. In vitro studies have shown that bendamustine increases the cytotoxic effect of Ara‐C in leukemic blasts and lymphoma cells, and the two drugs display high synergistic activity when used in consecutive combinations. Clinically, it has recently been demonstrated that rituximab + bendamustine + Ara‐C (R-BAC) combination has a remarkable activity and is well tolerated both in untreated and in relapsed/refractory patients with MCL (Visco C et al. J Clin Oncol 2013 31:1442-1449). In the present study, we report data from an Italian single-center experience evaluating the efficacy and tolerability of R-BAC association in previously untreated MCL patients both eligible and ineligible for transplantation. Design and methods. From January 2009 to November 2014, 25 newly diagnosed patients with MCL (median 67 years; range 57-83 years) were treated with immunochemotherapy according to R-BAC schedule (Rituximab 375 mg/mq day 1; Bendamustine 70 mg/mq days 2, 3; Ara-C 500 mg/mq days 2-4) x 4 or 6 28-day cycles. All patients received G-CSF prophylaxis. Ninety-six percent of patients had stage III/IV disease; MIPI score was high in 24%, intermediate in 60% and low in 12%; in one patient it was not evaluable. Results. Twenty-two patients (88%) completed the scheduled treatment (4 or 6 cycles). The ORR was 88%: CR 84% (21 patients); PR 4% (1 patient); 1 patient was in SD, but he received only 1 cycle due to toxicity and died still in SD about three years later. Seven patients (28%) underwent autologous stem cell transplantation (ASCT); seven patients (28%) received rituximab maintenance for two years. Two patients (8%) experienced disease progression during first line therapy, both had intermediate MIPI score. After a median follow up of 33 months (range 4-65 months), the OS was 80% (one patient died for unrelated causes) and the PFS was 80%. At this time point, 19 (76%) and 1 (4%) patient were in CR and in PD, respectively. The latter patient is currently undergoing salvage chemotherapy. Patients who received either maintenance immunotherapy with rituximab or consolidation with ASCT after R-BAC (overall 56%) seemed to experience longer PFS and OS. In the ASCT group the OS was 100% at a median follow up of 23 months; in the rituximab maintenance group the OS was 71% at a median follow up of 41 months. In the group of patients that received R-BAC induction therapy only, the OS was 61% at a median follow up of 26 months (p=0.14, likely due to the low number). The most common adverse events (AEs) during R-BAC were hematological: grade 3-4 neutropenia (88%), grade 3-4 thrombocytopenia (64%) and grade 3-4 anemia (36%). The numbers of common grade 3/4 non hematological AEs in the study included: febrile neutropenia (28%), acute coronary syndrome (4%), lung infection (4%) and hyperglycemia (4%). No treatment-related mortality was observed, one patient died of secondary acute myeloid leukemia four years later from induction therapy. Conclusions. The present retrospective study confirms that R-BAC as frontline regimen in MCL is well tolerated and highly effective in ASCT ineligible patients and that it can be administered also in ASCT eligible patients as induction therapy. Moreover, our data suggest that a consolidation therapy (ASCT or maintenance immunotherapy) after R-BAC induction could improve the outcome. Disclosures No relevant conflicts of interest to declare.

Description: Introduction One of the most significant novelty of the revised version of the European LeukemiaNet (ELN) recommendations for the management of chronic myeloid leukemia (CML) patients is that an earlier achievement of complete cytogenetic response (CCyR), at 6 months, and major molecular response (MMR), at 12 months, is regarded as optimal. Moreover, the prognostic value of the depth of molecular response at three months (i.e. BCR-ABL/ABL ratio ≤10%) is recognized. We have previously reported that EUTOS score is able to predict long term outcome of imatinib therapy, and that high-risk patients had a non-statistically significant lower probability to achieve all the cytogenetic and molecular endpoints defined as optimal by 2009 ELN recommendations. Aims and Methods We retrospectively evaluated our cohort of CML patients treated with front-line standard dose imatinib to test the ability of EUTOS and Sokal scores to foresee 2013 ELN-defined optimal response to therapy. A total of 314 consecutive patients treated with imatinib 400 mg daily for early chronic phase CML were analysed. Median age at diagnosis was 57 years (range: 19-85 years). According to the Sokal score there were 133 (42%) low risk, 127 (40%) intermediate risk, 52 (17%) high risk and 2 (1%) unknown risk cases, respectively. The distribution according to the EUTOS score was: 289 patients (92%) in the low-risk and 25 (8%) in the high-risk group. Partial cytogenetic response (PCyR) and CCyR were defined as 1-35% and 0% Ph+ metaphases, respectively; major molecular response (MMR) was defined as BCR-ABL