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The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.

The Revised International Staging System (R-ISS) stratifies patients affected by Multiple Myeloma (MM) into three distinct risk groups: R-ISS I [ISS Stage I, Standard-Risk cytogenetics and normal Lactase DeHydrogenase (LDH)], R-ISS III (ISS stage III and either high-risk cytogenetics or high LDH) and R-ISS II (any other characteristics). With the aim to verify whether the three R-ISS groups could be divided into subgroups with different prognostic factors based on the detection of Circulating Plasma Cells (CPCs) at diagnosis, in this retrospective analysis, we evaluated 161 patients with MM treated at our centre between 2005 and 2017. In all, 57 patients (33·9%) were staged as R-ISS III, 98 (58·3%) as R-ISS II and six (3·6%) as R-ISS I. CPCs were detected in 125 patients (74·4%), while in 43 patients (25·6%) no CPCs were seen. Our analysis revealed that Overall Survival (OS) and progression-free survival (PFS) rates in R-ISS II patients were higher in the subgroup without CPCs compared to the subgroup with ≥1 CPCs (OS: 44·7% vs. 16·3%, P = 0·0089; PFS: 27·8% vs. 8·1%, P = 0·0118). Our present findings suggest that the detection of CPCs at diagnosis may be used as a further prognostic biomarker to improve the risk stratification of patients with MM staged as R-ISS II.

Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.

CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer’s disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2(D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

Monoclonal antibodies (MoAbs) play a pivotal role in the treatment of chronic lymphocytic leukemia (CLL). Rituximab, a MoAb against CD20, was initially used as a single agent in the treatment of CLL before being incorporated into newer combination regimens. Integration of rituximab into chemotherapy regimens has led to an improvement in the response rate and overall survival when used in frontline therapy. Despite this, CLL remains an incurable disease, and treatment of relapsed CLL, particularly after failure of purine analog-based regimens, remains challenging. Technological advances relating to development of chimeric and humanized MoAbs are supporting the role of antibody-based regimens for many diseases, including CLL. Currently, MoAbs represent an integral component of CLL therapy. The antitumor efficacy of many therapeutic MoAbs can be potentially improved upon by their use in combination with new targeted therapy agents.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

// Elena Ciaglia 1, 2 , Giovanni Torelli 3, 4 , Simona Pisanti 1, 2 , Paola Picardi 1, 2 , Alba D’Alessandro 2 , Chiara Laezza 5, 6 , Anna Maria Malfitano 1, 2 , Donatella Fiore 2 , Antonio Christian Pagano Zottola 2 , Maria Chiara Proto 2 , Giuseppe Catapano 3 , Patrizia Gazzerro 2 , Maurizio Bifulco 1, 2 1 Department of Medicine and Surgery, University of Salerno, Baronissi Salerno, Italy 2 Department of Pharmacy, University of Salerno, Fisciano, Salerno, Italy 3 ”G.Rummo” Medical Hospital, Department of Neurosurgery, Benevento, Italy 4 Neurosurgery Unit A.O. San Giovanni di Dio e Ruggi d’ Aragona - Salerno’s School of Medicine, Largo Citta di Ippocrate, Salerno, Italy 5 Institute of Endocrinology and Experimental Oncology, IEOS CNR, Naples, Italy 6 Department of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Naples, Italy Correspondence to: Maurizio Bifulco, e-mail: maubiful@unisa.it Elena Ciaglia, e-mail: eciaglia@unisa.it Keywords: STAT3, CB1, MICA, NK cells, gliomas Received: February 18, 2015 Accepted: April 29, 2015 Published: May 11, 2015 ABSTRACT Herein we show that a majority of human brain tumor samples and cell lines over-expressed cannabinoid receptor CB1 as compared to normal human astrocytes (NHA), while uniformly expressed low levels of CB2. This finding prompted us to investigate the therapeutic exploitation of CB1 inactivation by SR141716 treatment, with regard to its direct and indirect cell-mediated effects against gliomas. Functional studies, using U251MG glioma cells and primary tumor cell lines derived from glioma patients expressing different levels of CB1, highlighted SR141716 efficacy in inducing apoptosis via G1 phase stasis and block of TGF-β1 secretion through a mechanism that involves STAT3 inhibition. According to the multivariate role of STAT3 in the immune escape too, interestingly SR141716 lead also to the functional and selective expression of MICA/B on the surface of responsive malignant glioma cells, but not on NHA. This makes SR141716 treated-glioma cells potent targets for allogeneic NK cell-mediated recognition through a NKG2D restricted mechanism, thus priming them for NK cell antitumor reactivity. These results indicate that CB1 and STAT3 participate in a new oncogenic network in the complex biology of glioma and their expression levels in patients dictate the efficacy of the CB1 antagonist SR141716 in multimodal glioma destruction. SIGNIFICANCE CB1 is implicated in the regulation of cellular processes linked to survival, proliferation, invasion and angiogenesis in several physio-pathological conditions. We shed light on previously unrecognized molecular mechanism of CB1-mediated modulation of human glioma progression and provide the first and original demonstration of CB1-STAT3 axis as a new target and predictor biomarkers of the benefit from specific therapies. Indeed CB1 antagonism capable of tumoral cell division’ control while making the glioma immunovisible and engaging the immune system to fight it may represent a hopeful alternative to other established chemotherapeutics. Because different aspects of glioma biology have been separately targeted with very limited success, we speculate that CB1 inhibitors which enclose in the same molecule cytotoxic potential and high activity to boost competent immune surveillance mechanisms, at a degree that seems to be correlated to the levels of CB1 immunoreactivity, might have profound implications for exploring new therapeutic anti-glioma actions.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput-compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.

The most frequently reported long-term toxicities of therapy in Waldenstrom macroglobulinemia (WM) are represented by second hematologic and non-hematologic cancers: therapy-related myelodysplasia/acute myeloid leukemia (t-MDS/AML), disease transformation into a diffuse large B-cell lymphoma (DLBCL), and second solid tumors (SST). The incidence of t-MDS/AML ranges from 1.2 to 8.9 % and that of transformation to DLBCL ranges from 1.4 to 10 %. The risk of SST in pretreated WM patients is 1.69 times higher than expected.

Introduction: First and second generation tyrosine kinase inhibitors, represent a new, fully biologic and targeted approach to chronic lymphocytic leukemia and allowed to obtain high responses and acceptable tolerability even in elderly and high risk patients. On the other hand, prolonged experience with these agents has raised some questions on unexpected toxicities, response quality, treatment duration and onset of resistances.Areas covered: This review analyzes the main published studies with the aim to discuss whether, in future, new agents could become a part of standard treatments or even replace the chemo-immunotherapy in chronic lymphocytic leukemia.Expert commentary: Despite different trials has been already published and many are still ongoing, follow up times are, at present, too short.A chemo-free approach surely represents a desirable goal for chronic lymphocytic leukemia, nevertheless longer observation is warranted to better define the correct use of targeted therapies.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by a dramatic appearance of premature aging. HGPS is due to a single-base substitution in exon 11 of the LMNA gene (c.1824C>T) leading to the production of a toxic form of the prelamin A protein called progerin. Because farnesylation process had been shown to control progerin toxicity, in this study we have developed a screening method permitting to identify new pharmacological inhibitors of farnesylation. For this, we have used the unique potential of pluripotent stem cells to have access to an unlimited and relevant biological resource and test 21 608 small molecules. This study identified several compounds, called monoaminopyrimidines, which target two key enzymes of the farnesylation process, farnesyl pyrophosphate synthase and farnesyl transferase, and rescue in vitro phenotypes associated with HGPS. Our results opens up new therapeutic possibilities for the treatment of HGPS by identifying a new family of protein farnesylation inhibitors, and which may also be applicable to cancers and diseases associated with mutations that involve farnesylated proteins.

The management of patients with chronic lymphocytic leukemia (CLL) has radically improved over the last few years with the addition of anti-CD20 monoclonal antibodies (MoAbs) to chemotherapy. Chlorambucil has been considered for decades as a suitable therapeutic option for frail patients. Taking into account the advantage offered by the addition of MoAbs to chemotherapy, different studies up to now have explored the feasibility of chlorambucil-based chemoimmunotherapies in treatment-naïve CLL. COMPLEMENT1 is a prospective, randomized, open-label trial evaluating the efficacy and safety of ofatumumab added to chlorambucil, compared with chlorambucil in monotherapy, in the setting of untreated patients with CLL considered unsuitable for a fludarabine-based approach. Progression-free survival was significantly longer in the chemoimmunotherapy arm when compared with the single-agent chlorambucil (22.4 months versus 13.1 months). Response rate and quality were also improved in the combination arm. Furthermore, the addition of ofatumumab did not lead to an unmanageable toxicity. While the employment of anti-CD20 antibodies represents an advantage in the treatment of the CLL symptomatic population, at present different patient selection and treatment schedules do not allow a reliable comparison between chlorambucil-based regimens. The addition of ofatumumab to chlorambucil represents a further therapeutic gain in CLL. Longer follow up and direct comparison with other MoAbs are warranted to establish the preferred first-line treatment in elderly and unfit patients.

Some compounds of a series of novel pyrrolo-1,5-benzoxa(thia)zepine, a well-known group of tubulin targeting agents, display anti-tumour effects mainly inducing cell cycle arrest and apoptosis in several human cancer models. A member of this family, pyrrolo-1,5-benzoxazepine-15 (PBOX-15), has previously shown potent pro-apoptotic activity in a variety of human tumor cell types, with minimal toxicity towards normal blood and bone marrow cells. In this study, we evaluated the PBOX-15-mediated effects in human colorectal cancer cell (CRC) lines, DLD-1 and HT-29. The compound, used at concentrations equal to or greater than 1?M, inhibited the proliferation of human CRC cells, inducing a significant cell cycle arrest in the G2/M phase. In DLD-1 cells, treatments prolonged over 48 h triggered a strong activation of the intrinsic apoptotic pathway as indicated by activation of caspase-9, caspase-3 and PARP cleavage. Moreover, nanomolar concentrations of PBOX-15, significantly improved the oxaliplatin and 5-fluouracil-induced anti-proliferative effects in DLD1 cell line. The observed synergistic interaction of both PBOX-15/Oxaliplatin and PBOX-15/5FU may involve activation of p38 MAPK and JNK pathway, which in turn significantly increased caspase-3 cleavage in DLD-1 cells, treated with PBOX-5/Oxaliplatin but not with PBOX-15/5FU. Moreover, PBOX-15/5FU-treated cells showed an increase in expression of the pro-apoptotic protein Bax. Taken together, these results show that PBOX-15 could represent a promising compound for the treatment of human CRC and a strong candidate for novel therapeutic options.

There is a significant need to develop an efficient treatment, without sacrificing efficacy, for older fit patients with chronic lymphocytic leukemia (CLL) as fludarabine, cyclophosphamide and rituximab (FCR) have very often led to treatment-related toxicities, causing delay or therapy

Bing Neel Syndrome (BNS) is defined as direct central nervous system involvement of Waldenstrom’s macroglobulinemia. BNS is usually a late event, although an incidence of 30% to 36% has been described in large series of previously untreated patients. A wide variety of clinical manifestations and radiologic findings for BNS have been reported in published data, depending on the site and type of infiltration. In addition to the radiologic findings, the diagnostic approach includes lymphoplasmacytic cell quantitation and flow cytometric analysis of the cerebrospinal fluid (CSF). Recently, the evaluation of MYD88 L265P mutation in the CSF has been proposed as a possible diagnostic biomarker for BNS. We describe the case of a 58-year-old patient with BNS. The detection of MYD88 L265P mutation in the CSF contributed to the diagnosis and to the sequential monitoring of minimal residual disease. In the future, the use of CSF sequential molecular monitoring could play an important role in treatment decisions.

Vascular events represent the most frequent complications of thrombocytemias. We aimed to evaluate their risk in the WHO histologic categories of Essential Thrombocytemia (ET) and early Primary Myelofibrosis (PMF). From our clinical database of 283 thrombocytemic patients, we selected those with available bone marrow histology performed before any treatment, at or within 1 year from diagnosis, and reclassified the 131 cases as true ET or early PMF, with or without fibrosis, according to the WHO histological criteria. Vaso-occlusive events at diagnosis and in the follow-up were compared in the WHO-groups. Histologic review reclassified 61 cases as ET and 72 cases as early PMF (26 prefibrotic and 42 with grade 1 or 2 fibrosis). Compared to ET, early PMF showed a significant higher rate of thrombosis both in the past history (22% vs 8%) and at diagnosis (15.2% vs 1.6%), and an increased leukocyte count (8389 vs 7500/mmc). Venous thromboses (mainly atypical) were relatively more common in PMF than in ET. Patients with prefibrotic PMF, although younger, showed a significant higher 15-year risk of developing thrombosis (48% vs 16% in fibrotic PMF and 17% in ET). At multivariate analysis, age and WHO histology were both independent risk-factors for thrombosis during follow-up; patients >60 yr-old or with prefibrotic PMF showed a significantly higher risk at 20 years than patients

Introduction Fludarabine, cyclophosphamide and rituximab (FCR) and bendmaustine rituximab (BR) combinations have both been evaluated as salvage regimens in Waldenstrom's Macroglobulinemia. FCR exerts good quality of responses but there are some concerns regarding its use mainly for tardive myelosuppression and long term toxicity. BR showed to be effective with an excellent short term toxic profile but in literature there are no data on long term follow-up and toxicity. The aim of this study was to evaluate long term outcome and long term toxicity of patients treated with FCR and BR. Patients and Methods We analyzed 87 relapsed and refractory Waldenstrom's Macroglobuklinemia patients enrolled in two retrospective Italian multicenter studies in which FCR or BR were administered as salvage regimens. Patients treated with both bendamustine and fludarabine were excluded from the study. For each treatment group we compared: clinical and disease characteristics, Progression free survival (PFS) defined as progression or death for any cause from the beginning of salvage treatment; Event free survival (EFS) defined as progression or development of major infection, solid tumor, secondary MDS/AML, DLBCL, or death due to any cause. Results Of the 87 patients, 37 had received FCR and 50 BR. The two groups of patients did not differ in sex, median age, median number of prior treatments, previous therapy with alkylating agents, disease status, median IgM level, presence of adenopathies and/or splenomegaly at CT performed before salvage treatment. The significant differences between the two groups were: higher number of patients >70 years in the BR group (P=0.01) and lower number of patients previously treated with monoclonal antibody in the FCR population (P Conclusions FCR and BR as salvage regimens led to similar outcome in terms of PFS and EFS. Considering that FCR in respect to BR exerts long lasting responses the main issue remains its long term toxicity profile. The future challenge will be to assess the long term effect of the new targeted agents. Disclosures Ferrero: Mundipharma: Other: Speakers Honoraria; Celgene: Other: Speakers Honoraria.

Introduction: Transformation of Marginal Zone Lymphoma (MZL) into an aggressive histology is uncommon phenomenon that can occur at any time after diagnosis and is expected to have a detrimental impact on prognosis. Biological and clinical knowledge on transformed Marginal Zone Lymphoma (tMZL) is poor and no standard treatment is established in the Rituximab era for these patients (pts). We retrospectively analyzed all consecutive biopsy proven tMZL in two Italian Hematological Divisions from 2002 to 2014 and we focused on post-transformation treatment and outcome. Methods: The dataset included 378 MZL pts diagnosed between 2002 and 2014 at Division of Hematology in Pavia and in Milan (Niguarda Hospital): 204 pts (53.9%) by extranodal MALT lymphomas, 113 pts (29.8%) by splenic MZL, and 61 pts (16.3%) by nodal MZL. Histological transformation (HT) was defined as transformation into an aggressive lymphoma at any time from previous MZL diagnosis; only cases with biopsy confirmed HT were comprised in the present analysis, while cases with only clinical suspicion of transformation were not counted as tMZL. Results: HT was documented in 18 of the 378 pts (4.8%), 6.5% in nodal MZL, 7.0% in splenic MZL and 2.9% in MALT. Histology at transformation was Diffuse Large B-Cell lymphoma in all but one case; the remaining pt was diagnosed as high-grade B-cell lymphoma, unclassifiable. CD20 was negative only in one Rituximab-naïve pt. Median time from first diagnosis to HT was 31 months (range: 10-124) and median number of previous therapies was 1 (range 0-1); pts received first line therapy listed in table 1. Median age at transformation was 68 years (range: 46-85), M/F ratio was 0.8. In the tMZL population, first diagnosis was nodal MZL in 4 pts (22%), splenic MZL in 8 pts (45%) and MALT in 6 pts (33%). At first diagnosis of MZL, 72% of t-MZL pts had stage IV disease, 17% had B symptoms, 11% had elevated LDH and ECOG performance status was lower than 2 in all the cases. HCV serology was positive in 5/17 cases; HCV status was not available for one pt. At HT disease stage was III or IV in 14 pts (78%), B symptoms were present in 7 pts (39%), LDH and beta2microglobulin were both elevated in 7 pts (39%) and ECOG performance status was lower than 2 in all the cases. Pts received post-HT treatment listed in table 2. At time of analysis 6 pts died (33%), and the main cause of death was progressive disease. With a median post-transformation follow-up of 16.6 months (range: 2-98), the 2-years Progression-Free Survival (PFS) was 45,4 % and the 2-years Overall Survival (OS) was 56.75%. No correlation was found between the following characteristics and survival: MZL type at first diagnosis, stage, symptoms, LDH and ECOG at HT, number and types of pre-HT therapies. Conclusions: This large cohort confirms that HT is a relatively rare and early event in MZL. At present time, we did not identify any feature predictive of outcome for transformed MZL. Chemotherapy in combination with Rituximab showed to be an effective treatment for tMZL. Table 1 First line treatment N. of patients % Therapy strategy CVP 7 39 Anthracycline-containing regimen 2 11 Chlorambucil monotherapy 5 28 Splenectomy 1 5.5 H.pylori eradication 1 5.5 Watch and wait strategy 2 11 Rituximab incorporation Included in first line therapy 6 33 Maintenance 0 0 Response ORR (%) 67 CRR (%) 33 Table 2. Post-HT treatment N. of patients % Therapy strategy CHOP regimen 16 89 Platinum-containing regimen 1 5.5 Missing 1 5.5 Rituximab incorporation Included in first line therapy 17 94 Maintenance 0 0 ASCT consolidation 3 17 Response ORR (%) 61 CRR (%) 59 Disclosures Rusconi: Roche: Honoraria.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

INTRODUCTION. Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy, mainly characterized by peripheral cytopenias and splenomegaly. The current standard of treatment for HCL are Nucleoside Analogs (NA) cladribine and pentostatin, which produce remarkably high remission rates and durable responses. Aim of this study was to evaluate efficacy, short- and long-term toxicity of NA in HCL pts treated outside clinical trials. PATIENTS AND METHODS. We retrospectively analyzed 86 HCL patients (pts) treated with NA between 1996 and 2015 in two Hematologic Centers in Italy. The study was conducted in accordance to the Helsinki Declaration of 1964, as revised in 2000. Cladribine and pentostatin were administered according to standard schedules. Response criteria published by Jones et al. (Br J Haematol 2012) were retrospectively applied. Molecular assessment of BRAF-V600E mutation before and after NA therapy using quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay (sensitivity 0.1%) was performed in 10 pts. RESULTS. The median follow-up of pts (71 males and 15 females, median age 53 years) was 5.8 years (range 0.5-28). During the disease course, 86 pts were treated with NA (cladribine n=76, 88%; pentostatin n=10, 12%); 59 pts received NA front-line (cladribine in 56/59 pts, 95%). Among the other 27 pts, receiving NA as second or subsequent line of therapy, 25 had been previously treated with interferon. Median time from diagnosis to the first NA was 3.3 months (range 0-315). Hematological toxicity was observed in 53 of 77 evaluable pts (69%), and was not significantly different with cladribine (72%) or with pentostatin (37%) (p=0.1). Grade 3-4 neutropenia, anemia and thrombocytopenia were observed in 50 (65%), 7 (9%) and 7 (9%) pts respectively. Extra-hematological toxicity was reported in 48 of 79 evaluable pts (61%). The incidence of extra-hematological toxicity with cladribine (63%) and pentostatin (37%) was not statistically different (p=0.2). Grade 3-4 febrile neutropenia was observed in 24 pts (30%); 9 pts (11%) had grade 3-4 infections; 4 pts (5%) had grade 3-4 skin toxicity, 1 pt (1%) had grade 3 hepatic toxicity. Four of 86 pts (5%) developed a second malignancy (prostatic adenocarcinoma n=2, colon adenocarcinoma n=1, diffuse large B cell lymphoma n=1). The median time from NA to second malignancy was 58 months (range 49-111). Four of 86 pts (5%) developed a skin cancer (basal-cell carcinoma n=3, squamous cell carcinoma n=1), after a median time of 59 months (range 16-126). Response was assessed at a median time of 3 months after the end of therapy. Overall Response Rate (ORR) and Complete Remission (CR) Rate were respectively 93% and 63% in the entire cohort, and 98% and 72% in pts treated with NA front-line. The allelic burden of BRAF before and after therapy with NA was available in 10 cases; none of the pts in clinical CR after NA achieved a complete molecular response (Table 1). The median Progression-Free Survival (PFS) for pts treated with NA frontline was 6.5 years. There was a trend toward a longer PFS in pts receiving NA as first-line therapy as compared to those treated in second or subsequent lines (p=0.05). PFS curves according to type of NA and line of therapy are shown in Figure 1. The 5- and 10-year Overall Survival (OS) rates were 96% (95% CI: 85% - 99%) and 90% (95% CI 76%-96%) respectively. OS was similar in pts treated with cladribine orpentostatin (p=0.49). CONCLUSIONS. This study shows that: i) NA are associated with an acceptable toxicity, neutropenia and febrile neutropenia being the main complications of therapy; ii) timing of response assessment may account for a CR rate that is slightly lower than reported in the literature, confirming the importance of waiting at least 4 months for response evaluation as recommended by current guidelines; iii) the persistence of molecular disease also in pts achieving a clinical CR supports the implementation of consolidation strategies aimed at eradicating minimal residual disease to prolong disease-free survival. Table 1. BRAF allelic burden before and after therapy Patient Age (years) Sex Line of Therapy Allelic Burden (%) Clinical Response Pre- Post- 1 33 F 1 21,8 0,1 CR 2 36 M 2 5,4 0,4 CR 3 39 M 1 24,2 0,7 CR 4 54 M 1 10,8 0,3 PR 5 42 F 1 44 1,2 CR 6 53 F 1 7,9 0,2 NV 7 56 M 1 6,5 0,3 CR 8 54 M 1 12,2 0,2 CR 9 32 M 1 35,8 9,5 PR 10 38 M 2 54,4 12,2 PR Figure 1. Progression-free survival according to type of NA and line of therapy Figure 1. Progression-free survival according to type of NA and line of therapy Disclosures Arcaini: Gilead: Consultancy, Other: Advisory Board; Celgene: Consultancy, Other: Advisory Board; Roche: Consultancy, Other: Advisory Board.

Background: Indolent non follicular B-Cell Lymphomas (INFL) are a heterogeneous group of lymphomas and include small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphomas (LPL) and marginal zone lymphomas of splenic (SMZL), nodal (NMZL) and extranodal (ENMZL) subtypes. In 2010 the NF10 study was started by the Fondazione Italiana Linfomi as a prospective registry specifically devised for investigating the prognosis of this group of lymphomas. We provide a preliminary report describing registered cases with an emphasis on initial therapy. Methods: The registration of consecutive adult patients with newly diagnosed INFL and no exclusion criteria is ongoing at a dedicated website via secure HTTP protocols. For the purposes of the study in addition to the conventional INFL subtype, the category of disseminated MZL and CD5- low grade lymphoma were also considered. So far the study has been activated in 65 centers in Europe and South America. Results: Between July 2010 and July 2015, 665 cases have been registered. The current report is based on 395 cases that have been validated. Forty-seven (12%) cases were registered as SLL, 76 (19%) as LPL, 59 (15%) as CD5-low grade and 213 (54%) as MZL, including 73 (18%) SMZL, 18 (5%) NMZL, 81 (21%) ENMZL or 41 (10%) disseminated subtypes. Median age was 67 years (range 29-94), 53% of patients were males; Ann Arbor stage was III-IV in 79%; 14% had B symptoms, 7% had ECOG performance status > 1, lactate dehydrogenase and b2-microglobulin were elevated in 70% and 54% of cases, respectively. Six percent of cases were HCV positive (HCV+ rate was 7.5% among MZL cases). Regarding HBV infection, 21% of patients were HBcAb-positive and 3% of patients were HBsAg-positive. Immediate systemic therapy was planned in 50% of patients. SMZL, SLL and CD5-low grade were the subtypes with the lower rates of immediate therapy (44%, 46% and 24% respectively) whereas ENMZL were addressed to systemic therapy in 67% of cases. When systemic therapy was prescribed rituximab (R) was used in 88%. In 81% of patients R was combined to cytotoxic therapy including alkylating agents in 40%, CHOP-like in 18%, bendamustine in 17% and fludarabine in 6%. ENMZL and CD5-low grade had the highest rates of R-alkylating use (61% and 64%); SMZL and MZL were frequently treated with R-CHOP like regimens (35% and 40%). Young age at diagnosis (less than 60 years) and increased b2-microglobulin were more frequently associated with patients requiring immediate systemic therapy. With 22 months of median follow up, 2-year progression free survival and overall survival (OS) were 88% (95CI: 83-92) and 95% (95CI: 91-97) respectively; the initial choice of deferring immediate therapy did not impact on OS. Conclusions: We provide a complete report on the initial approach to patients with INFL showing that immediate therapy is required in half of the cases with a heterogeneous approach among INFL subtypes. The majority of patients requiring therapy was treated with the combination of R and alkylating agents. The NF10 study confirms that a web-based world-wide cooperation allows the collection of a relevant and complete data set, providing a platform for future prognostic and pathobiological studies in order to identify novel and more efficient therapeutic targets. Disclosures Luminari: Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ferrero:Mundipharma: Other: Speakers Honoraria; Celgene: Other: Speakers Honoraria. Gaidano:Celgene: Research Funding; Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards.

Anna Maria Frustaci, Alessandra Tedeschi, Paola Picardi, Roberto Cairoli, Marco MontilloDepartment of Hematology, Niguarda Cancer Center, Niguarda Ca' Granda Hospital, Milan, Italy Abstract: Treatment aim for chronic lymphocytic leukemia has been radically changed over the past years from providing only a palliative approach to reaching disease eradication and improving survival. Ofatumumab is a monoclonal humanized antibody with peculiar in vitro and in vivo properties, at present approved for double fludarabine and alemtuzumab refractory chronic lymphocytic leukemia. Its efficacy in this subset of patients, who typically have an unfavorable prognosis, facilitated its use in different Phase II and III trials. Ofatumumab as single agent or combined with chemotherapeutic or biologic agents, led to sundry results in the setting of both previously treated or untreated patients. Its role in maintenance therapy is also under investigation. Further advances concerning ofatumumab administration as first line therapy in combination with chlorambucil, came recently from the COMPLEMENT 1 study. Results from this trial will open the door to new perspectives of its use in treatment-naïve patients. Ofatumumab was well tolerated in almost all the studies, with the main adverse events relating mostly to infusion reaction. Hematologic toxicity, especially neutropenia, was also common. A significant improvement in patients' quality of life was reported following ofatumumab treatment and this was mainly due to its effect on constitutional symptoms. Nevertheless, some concerns remain regarding the long-term efficacy of the drug in terms of response duration and survival. The real strength of this drug needs to be confirmed by further studies and direct comparative trials. Keywords: ofatumumab, chronic lymphocytic leukemia, refractory, alemtuzumab, fludarabine, high risk