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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.

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.

// 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.

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.

Waldenstrom’s macroglobulinemia is characterized by lymphoplasmocytic cells accumulation predominantly in bone marrow, secreting immunoglobulin M monoclonal protein. There is not a standard of care as disease is rare and there are no large randomized trials to address treatment. Asymptomatic patients should be observed. In symptomatic patients treatment should be individualized considering patient fitness and disease characteristics. In elderly unfit patients single agent treatment may be still considered an option. In younger and fit patients immunochemotherapy should be considered the standard of care as recent data showed an improvement in quality of responses, progression-free and overall survival. In this overview are reported the most significant clinical trials that may help in treatment decision.

The survival of patients with chronic lymphocytic leukemia (CLL) has significantly improved in the last 30 years. The introduction of purine analogs in the treatment armamentarium followed by the combination of these compounds with alkylating agents first improved response rates and progression-free survival (PFS) when compared with alkylating agent-based therapy only. However, the great advance arrived with the development of a chemoimmunotherapeutic approach comparing this with chemotherapy alone demonstrating an improvement not only in terms of response rate and PFS but also, for the first time, in the rate of overall survival (OS). The last decade brought significant achievements in the understanding of CLL pathogenesis leading to the development of new agents targeting cell surface, intracellular pathways, and tumor microenvironment. As traditional chemotherapy is associated with acute and long-term toxicity, interest in these non-chemotherapeutic treatments has been constantly growing. The challenge will be to develop a rationale for non-chemotherapeutic approaches using these new agents as monotherapy, or in combination, with the aim of obtaining an individualized strategy based on disease characteristics and even patient basis. Hopefully, an increasing participation of ultra high-risk CLL patients in clinical trials evaluating the efficacy of these new treatments may lead to reasonable success, if not cure, in this unfavorable setting.

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

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.

Background: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. Methods: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. Results: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less a ggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. Conclusion: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (

iPA is a naturally occurring nucleoside with an isopentenyl moiety derived from the mevalonate pathway and a well-established anti-tumor activity. In analogy to the unique specificity for phosphoantigens, such as IPP, shown by human Vγ9Vδ2 T cells, here, we report for the first time the ability of iPA to selectively expand and directly target human NK cells. Interestingly, submicromolar doses of iPA stimulate resting human NK cells and synergize with IL-2 to induce a robust activation ex vivo with significant secretion of CCL5 and CCL3 and a large increase in TNF-α and IFN-γ production when compared with IL-2 single cytokine treatment. Moreover, iPA promotes NK cell proliferation and up-regulates the expression of specific NK cell-activating receptors, as well as CD69 and CD107a expression. Accordingly, this phenotype correlates with significantly greater cytotoxicity against tumor targets. At the molecular level, iPA leads to a selective, potent activation of MAPK signaling intermediaries downstream of the IL-2R. The effect results, at least in part, from the fine modulation of the FDPS activity, the same enzyme implicated in the stimulation of the human γδ T cells. The iPA-driven modulation of FDPS can cause an enhancement of post-translational prenylation essential for the biological activity of key proteins in NK signaling and effector functions, such as Ras. These unanticipated properties of iPA provide an additional piece of evidence of the immunoregulatory role of the intermediates of the mevalonate pathway and open novel therapeutic perspectives for this molecule as an immune-modulatory drug.

The role of high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) in the treatment armamentarium of aggressive B- and T-cell non-Hodgkin lymphoma (NHL) is still a matter of debate. In the pre-Rituximab era, the PARMA study demonstrated the superiority of HDT/ASCT over conventional salvage chemotherapy in chemosensitive, relapsed patients. Subsequently, HDT/ASCT has become a standard approach for relapsed NHL. With the advent of Rituximab in the landscape of NHL, transplantation as part of first-line therapy has been challenged. However, no benefit in terms of disease-free or overall survival of HDT/ASCT over standard therapy was shown when Rituximab was added to both arms. Moreover, the superiority of HDT/ASCT over conventional salvage therapy in patients relapsing from first-line therapy including Rituximab was not confirmed. From these disappointing results, novel strategies, which can enhance the anti-lymphoma effect, at the same time reducing toxicity have been developed, with the aim of improving the outcome of HDT/ASCT in aggressive NHL.In T-cell lymphoma, few publications demonstrated that consolidation of complete remission with HDT/ASCT is safe and feasible. However, up to one-third of patients may never receive transplant, mostly due to progressive disease, and relapse still remains a major concern even after transplant.

Recruitment of neoplastic T cells to skin is a critical step in the pathogenesis of mycosis fungoides (MF) lesions. Cutaneous T-cell attracting chemokine (CTACK)/CCL27 attracts memory T cells to skin, resulting in increased cutaneous expression. The interactions between neoplastic cells and skin immune system require further elucidation. CTACK/CCL27 expression and density of dendritic cells (DC), CD8+ and CD4+ lymphocytes were investigated in skin lesions of 52 early-stage MF patients treated by interferon (IFN)-alpha in combination with photochemotherapy (psoralen plus ultraviolet A, PUVA). Skin lesion biopsies obtained at diagnosis and after treatment were studied by immunohistochemistry. Initial CTACK/CCL27 expression was abnormal/suprabasal in 36 patients. Normal/basal CTACK/CCL27 expression tended to correlate with a high DC density and low CD4+ cell density in the neoplastic infiltrate. Treatment induced a significant increase in CTACK/CCL27 expression (chi(2) test: P=0.004). Overall, 33 patients relapsed [median event-free survival (EFS), 46 months] during follow-up (median, 92.5 months, range, 43-165). Normal/basal CTACK/CCL27 expression at the end of treatment correlated with lower rates of recurrence and a longer median EFS (111 months vs. 39 months with suprabasal expression; log rank test: P=0.031). CTACK/CCL27 overexpression in early-stage MF might thus be related to a balance between neoplastic cells and immunomodulant DC. Normal CTACK/CCL27 expression after treatment designates a subset of patients with favorable disease behavior. The mechanisms underpinning CTACK/CCL27 overexpression after therapy in the remaining patients, who are at greater risk of recurrence, warrant further investigation.

Objectives: The therapy of advanced, relapsed or refractory primary cutaneous lymphomas is often unsatisfactory. Recent data indicate a favourable pharmacokynetic, pharmacodynamic and toxicity profile of pegylated liposomal doxorubicin (Peg-Doxo) in primary cutaneous T-cell lymphomas, while in primary cutaneous B-cell lymphomas (PCBCLs), the drug efficacy has never been assessed so far. Methods: We performed a prospective phase II pilot clinical trial of Peg-Doxo monotherapy (20 mg/m2) in PCBCLs. One patient had a marginal zone B-cell lymphoma and four were affected by diffuse large B-cell lymphoma-leg type, all with widespread nodular lesions. Results: All the patients achieved a complete response (CR = 100%) in a short period of time (median 3 months), even when pretreated with radio-chemotherapy. Two experienced a relapse. At follow-up, one patient died for progressive disease; four are in CR after 5, 52, 63 and 69 months. As concerning the toxicity profile, the treatment was well-tolerated, no one decreased or delayed the dose. The haematological toxicity was mild with only one case of grade III neutropenia; a patient showed a grade I neurotoxicity. Dermatological toxicity, in particular the palmar–plantar erythrodysesthesia, did not occurred, probably because of both the low dosages of Peg-Doxo monotherapy and the oral prophylaxis with pyridoxine. Conclusions: In spite of the small number of patients, it emerges that monochemotherapy with Peg-Doxo has a significantly high clinical activity and a good safety profile in PCBCLs, even in aggressive forms, compared with other therapeutic regimens, which are completely reviewed. It suggests the need of further investigations in this field.

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

Background Despite the prediction of a fairly benign clinical course, a subset of patients with low-risk myelodysplastic syndromes (MDS) have a more aggressive disease and shorter overall survival. The DNA repair and folate pathway genes play an important role in prognosis and progression in both solid and hematological cancers, and their expression has been known to be associated with polymorphism of genes. However, the impact of polymorphisms of these genes on MDS patients outcome has not yet been demonstrated. The aim of this study was to investigate the association between the polymorphisms of genes encoding main proteins of BER system (XRCC1, XRCC3 and APE1) and folate-metabolizing enzymes (TS, MTHFR) and survival in IPSS low-intermediate1 MDS patients. Methods the study was designed according to the Schoenfeld design for biomarkers, assuming the presence of an unfavorable pharmacogenetic profile (one or more adverse genotypes) in at least one-third of the study population. Accordingly, 10 events in 54 patients would allow the detection of an Hazard Ratio (HR) >6 associated with the group having unfavorable genotypes (80% power and 5% type I error for a two-tailed test). We thus prospectively genotyped 54 MDS patients (median age 75 years) with IPSS low (n=23) or intermediate-1 (n=31) treated with best supportive care only. Genomic DNA was isolated from 1ml of peripheral blood by means of commercially available kits. Polymorphisms were determined by PCR-HRM (High Resolution Melting) assay and restriction digests of PCR products. All samples were analyzed for the following polymorphisms: XRCC1 194 (rs1799782 C/T, Arg/Trp) and 399 (rs25487 G/A, Arg/Gln), XRCC3 241 (rs861539 C/T, Thr/Met), TS5'-UTR (2R/3R and rs183205964 G/C) and 3'-UTR Ins/Del (rs11280056 6bp+/6bp-), MTHFR 677 (rs1801133 C/T, Ala/Val) and 1298 (rs801131 A/C, Gln/Ala), APE1 148 (rs1130409 T/G, Asp/Glu). The characteristics and laboratory features of MDS patients with each polymorphisms were compared using Х2-test and Mann-Whitney test. The associations between polymorphisms status and survival were assessed using Kaplan-Meier method and Log-rank test. For the multivariate survival analysis, Cox proportional hazard models, was used to identify the genotypes fitted as indicator variables. Before performing clinical correlations, genotype frequencies were checked for agreement with those expected under the Hardy-Weinberg equilibrium. Results The frequencies of genotypes of studied gene polymorphisms in patients with low/Int-1 risk are listed in Table 1. At univariate analysis, a significantly shorter survival was associated with XRCC1 399 GG, TS3’-UTR -6/-6, TS5’-UTR 2R/3G, 3C/3G, 3G/3G and MTHFR 677 TT variant alleles. In multivariate analysis, using a stepwise logistic regression model, patients with TS3’-UTR -6/-6, XRCC1-399 GG, TS5’-UTR 2R/3G, 3C/3G, 3G/3G and MTHFR- 677 TT unfavorable genotypes presented with an Hazard Ratios of 4.65, 7.07, 11.44 and 67.12, respectively, if compared to the reference group of variant alleles (P=.058, P=.024, P=.026 and P=.000). Accordingly, we performed an exploratory analysis to investigate the effect on survival arising from the combination of the unfavorable genotypes to each other. As a fact, 3-years OS was 33% for those patients with ≥2 variant alleles, as compared to 62.5%, and 100%, respectively, for those with 2 or 0/1 variant alleles, suggesting that patients with a higher number of variant alleles had a shorter survival. Conclusions The mutational status of BER, TS and MTHFR genes predicts the overall survival of patients with low-Int-1 IPSS MDS treated with best supportive care only. If confirmed on larger series, these polymorphisms could help to identify a subset of low-risk MDS patients with shorter survival, who might benefit from an early therapy with hypometilating agents. Acknowledgments The study was supported in part by AIL Pesaro Onlus. Disclosures: No relevant conflicts of interest to declare.

Background We previously demonstrated (Visani et al, Blood 2011) the safety of a new conditioning regimen with bendamustine, etoposide, cytarabine, and melphalan (BeEAM) prior to autologous stem cell transplant (ASCT) in resistant/relapsed lymphoma patients (EUDRACTnumber2008-002736-15). Furthermore, this regimen showed significant anti-lymphoma activity (80% CR). At the time of publication (2011), disease type (NHL versus HL) and disease status at transplant (chemosensitive versus chemoresistant) were the only statistically significant variables influencing PFS (p=0.01; p=0.007). However, median follow-up for surviving patients was short (18 months), therefore, it was not possible to draw final conclusions on the efficacy. Aims We evaluated the efficacy of the BeEAM regimen in terms of disease-free (DFS) and overall survival (OS) after a median follow-up of 41 months. Methods Forty-three patients (median age 47 years, range 18-70) with resistant/relapsed NHL (28) or Hodgkin lymphoma (HL, 15) were consecutively enrolled in the study. Twenty-one patients had primary refractory disease, whereas 22 had relapsed disease, 5 of whom where in second or subsequent relapse, at the time of enrolment. The study was designed according to Fleming’s method. The primary objective of the study was to determine the 36-months event free survival rate. We fixed the lowest acceptable rate as 40% and the successful rate as 60%, with a significance level a=0.05 and a power 1-b =0.80. At the time of publication, the median follow-up was 18 months, and therefore it was not possible to establish if we had met the primary end-point of the study. Results we updated the follow-up at 41 months after transplant. Thirty-one out of 43 patients are still in CR (72%), as documented by both PET and CT scan. Two patients with HL were refractory and rapidly died, whereas 10/43 patients (23%) relapsed after a median time of 7.5 months (range:3-23) from transplant. Five patients died (3 NHL, 2 HL), whereas 5 patients are still alive after relapse. Median PFS and OS were still not reached. Conversely, 3-year PFS was 75%, allowing our study to met its primary end-point. Interestingly, disease type (HL versus NHL) at transplant is no longer influencing PFS (p=0.7), and still does not influence OS (p=0.1). On the other hand, disease status at transplant (chemosensitive vs chemoresistant) is still a strong predictor of both PFS and OS (p=0.03 and p=0.009, respectively). At present, one patient developed myelodisplasia after transplant. No other late effects were observed up to now. Conclusions The new BeEAM regimen met the primary end-point of the study and confirms its safety, after 41 months of follow-up. Interestingly, NHL and HL were not statistically different in terms of both PFS and OS at 41 months of observation. These data confirm the high efficacy of this regimen in heavily pretreated non-Hodgkin, as well as Hodgkin lymphoma. Acknowledgments supported in part by AIL Pesaro Onlus. Disclosures: Ocio: Onyx: Consultancy, Research Funding; Novartis: Consultancy; Array Biopharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding.

Abstract 3953 Oral bexarotene, a novel retinoid that selectively activates retinoid × receptors, is approved for the treatment of cutaneous manifestations of refractory or relapsed cutaneous T cell lymphomas (CTCL) as monotherapy. Phase II/III trials have demonstrated a greater than 50% response rate in patients with all stages of CTCL who were refractory or intolerant to the previous therapy but the recommended bexarotene dose of 300 mg/m2/day is associated with significant side-effects. Novel studies are looking for synergistic therapies combining lower bexarotene doses in order to reach the same or even better outcomes while avoiding a negative impact on the quality of life. Early studies with bexarotene and psoralen plus ultraviolet A photochemotherapy (PUVA) show promise. Synergy between bexarotene and PUVA may derive from retinoid-induced reductions in epidermal thickness, which consequently improves UVA penetrations, and/or from retinoid-specific enhancement of immune functions. The aim of this study was to assess the efficacy and safety of a 2 stage-protocol (induction and maintenance) with low dose bexarotene (Targretin) and PUVA in refractory and/or resistant patients with Mycosis Fungoides (MF) and Sézary Syndrome (SS). A prospective, phase 2 clinical study was conducted between February 2007 and May 2010 by two Italian academic hospital centers. We enrolled 15 patients with stages I through IV MF/SS; all patients had failed PUVA or several systemic regimens. Response to treatment was assessed with clinical endpoints such as remission of symptoms (pruritus, pain, sleep disturbance) and signs (erythema, scaling, thickness of plaques, presence of nodules, tumours, peripheral lymphadenopathy). Complete remission (CR) was defined as the disappearance of all clinical evidence of disease for at least 4–6 weeks, partial remission (PR) as 50% or more decrease in extent or severity of disease. Overall response (OR) rate was defined as the combination of CR/PR. Patients with MF/SS were started with bexarotene (150 mg/day) and PUVA (three times weekly). Increments of bexarotene to a maximum of 300 mg/day were scheduled. Patients who achieved CRs and PRs during the induction remained on the same dose in the maintenance period, whereas the frequency of PUVA was gradually reduced. A preventive therapy to lower triglycerides and elevated thyroid hormone levels in the blood, was administered in all cases; therapeutic dietary and lifestyle changes were often recommended. Primary endpoint were OR rate at the end of induction and maintenance; secondary endpoints were safety and event-free survival (EFS). Kaplan-Meyer method and long-rank test was used for statistical analysis. All 15 patients (8 M and 7F) were evaluable for response; ten patients had early-stage (IB) and 5 had advanced stage (2 IIB, 1 IIIA, 1 IIIB, 1 IV). The median age was 68 years (34-81). The OR rate was 93% with 33% CR after induction period and 93% with 47% CR at the end of the protocol. A response was observed even in 1 case with SS and in 2 cases with MF in large cell transformation. Significantly, patients treated previously with PUVA monotherapy achieved better and faster CR compared with those treated with more than one systemic therapy (83% and 22% respectively; χ2 test, P=0.041; median time to maximal response 4 weeks and 8 weeks respectively, P=0.007). Median EFS at 37 months, for the whole group was 27 months. All patients have been closely monitored for adverse events during treatment; 2 of 15 patients (13%) had mild grade I-II asthenia, hypercholesterolemia and central hypothyroidism, 2 patients developed elevations of CPK (grade 3). With regard to hematologic toxicity, leukopenia was mild (grade I) and occurred in 3 patients (20%). In conclusion, our study have demonstrated a greater than 90% response rate in patients with all stages of CTCL refractory or intolerant to the previous therapies; the efficacy is more pronounced in patients previously treated with PUVA monotherapy. Starting treatment at lower dose of bexarotene followed by dose escalation may improve the efficacy and safety of both bexarotene and PUVA. Moreover the additional maintenance phase seems to improve the OR and length of remissions. Disclosures: Leoni: Celgene: Honoraria.

Abstract 3072 Thromboembolic disease (TE) is a major cause of morbidity and mortality in Essential Thrombocythemia (ET). Recently JAK2V617 mutational status and allele burden seem to add prognostic significance to standard risk factors; furthermore it was reported an interesting but not confirmed association between baseline leukocytosis and thrombosis. The aim of the study was to evaluate such factors in a series of patients with ET. Study subjects were recruited from the Ancona Hematology Clinic database of ET patients; clinical and laboratory data were collected at time of diagnosis and correlated with the occurrence of TE. In addition, qualitative and quantitative PCR for JAK2V617F was performed on stored samples of peripheral blood or bone marrow in a subset of study patients. Qualitative JAK2V617F analysis was done according to standard PCR-based methods and quantitative JAK2V617F was determined with a commercially available Taqman assay. Standard statistical methods were applied to test significance of associations between thrombosis and other variables. A total of 218 ET patients were included in the study (median age 59, range 18–90; 71 males and 147 females). At diagnosis the median of platelets, hemoglobin and leukocytes were 776×109/L, 13.9g/dL, and 8.4 ×109/L respectively. At diagnosis a history of remote thrombosis was recorded in 27 patients (12,4%). Of the 122 patients analysed for JAK2 mutational status, 70 (57.4%) were JAK2 wild-type and 52 (42.6%) were JAK2V617F positive. Quantitative analysis was carried out in 48 cases: 6 patients (11.5%) were homozygotic and 42 (80.8%) were heterozygotic. At diagnosis, a total of 17 patients (7.8%) had TE: 8 (3.7%) arterial thrombosis (AT) and 9 (4.1%) venous thrombosis (VT). the AT included acute coronary syndromes (ACS; n=4), transient ischemic attack/cerebrovascular accidents (TIA/CVA; n=3), and peripheral arterial thrombosis (PAT; n=1). The VT included deep vein thrombosis/pulmonary embolism (DVT/PE; n= 4), abdominal vein thrombosis (AVT; n=4) and dural sinus thrombosis (n=1). During follow-up 25 patients experienced TE: 13 VT (6%) and 12 AT (5.5%). Post diagnosis VT included.: DVT/PE (n= 4), AVT (n=4), superficial vein thrombosis (TVS; n=3), retinal vein thrombosis (n=1) and dural sinus thrombosis (n=1). Post diagnosis AT included TIA/CVA (n=6), ACS (n=3) and PAT (n=3). Gender, age, hemoglobin, platelets, leucocyte count, splenomegaly, increased cellularity of bone marrow, bone marrow fibrosis and common vascular risk factors did not affect the probability of thrombotic events at diagnosis and during follow-up. On univariate analysis a significant increase of thrombosis as presenting manifestations, was registered in patients with a history of remote thrombosis (OR= 10.0, 95% CI 4,1-24,5; p Subjects with both JAK2 mutation and a history of remote thrombosis have a OR of 24.44 (95% CI 3.07–194.66; p=0.01) in comparison with wild-type patients without a history of thrombosis. During follow-up, the probability of vascular events was predicted only by a history of remote thrombosis (OR=2.3, 95% CI 1.01–5.35; p=0.05), and neither by JAK2V617F mutational status (OR=1.24, 95% CI 0.43–3.62; p=0.76). The present analysis suggests that the thrombotic risk is higher in JAK2V617F positive patients and is further increased by a past history of thrombosis. The opportunity of combine such criteria must be defined in prospective trials. Disclosures: Leoni: Celgene: Honoraria.

Combination therapy is commonly used in the treatment of advanced and aggressive cutaneous T-cell lymphomas (CTCL), like Mycosis Fungoides (MF) and Sézary Syndrome (SS). Alemtuzumab, the humanized anti-CD52 antibody primarily developed for the treatment of B-CLL, has been also administered effectively in patients with CTCL as monotherapy, but it might be an ideal candidate for combination therapy either with a biological immune response modifier like interferon (IFN)-α, or with a drug known to be particularly active in CTCL like gemcitabine. No data on the activity of combination schemes including alemtuzumab in CTCL seem to be available. The aim of this study was to explore the clinical activity of alemtuzumab in two combination modalities, including either IFN-α or gemcitabine. We enrolled 5 patients, 4 males and 1 female, affected by SS/erythrodermic MF, with a median age of 57 yrs (43–74) and a median disease history of 21 mo.s (9–95). Two patients were treated with both combination schemes. Before starting therapy, all patients except one had a rapidly progressive disease, relapsed or refractory to previous treatments, and had previously received three or more systemic chemotherapy regimens. Most patients had reduced performance status and all had severe itching. Four patients received alemtuzumab in combination with IFN-α: subcutaneous alemtuzumab was administered at 3 mg on day 1, 10 mg on day 3, followed by 15 mg on alternating days for 7 total doses; after a further 3 months of treatment with IFN-α (3 MU three times weekly) the patients received reinduction with alemtuzumab (10 mg weekly for another month). Three patients received alemtuzumab in combination with gemcitabine: subcutaneous alemtuzumab was given at 10 mg on days 1, 8 and 15 of a 28-day schedule in combination with gemcitabine at a dose of 1200 mg/m2 intravenously (median number of cycles=4). Trimethoprim/sulphamethoxazole (twice daily 2 times per week) and valacyclovir (500 mg twice daily) were administered from day 2 until at least 2 months after alemtuzumab discontinuation. The response (complete/CR, partial/PR, stable disease/SD) was assessed in skin, lymph nodes and peripheral blood, by using the SWAT criteria and by comparing the absolute number of circulating Sezary cells (SC) detected by flow-cytometry before and after treatment. The overall response (OR) was determined as the worse result obtained in the different sites. The OR rate was 57.1% (4 PR vs 3 SD), higher in the group treated with alemtuzumab and gemcitabine (66.7%: 2 PR vs 1 SD) than in the group treated with alemtuzumab and IFN-α(50%, 2 PR and 2 SD). The better response rate obtained in the group treated with gemcitabine was due to the higher activity in the skin (2 CR, 1 SD vs 2 PR, 2 SD) and in the lymph nodes (3 PR vs 3 PR, 1 SD). The circulating SC count was low at the end of therapy (median, 164, range, 24–530), with a lower median value in the group treated with gemcitabine (131), than in the group treated with IFN-α (303). Itching, self-assessed on a 0 to 10 visual analog scale, was significantly reduced from a median score of 9 before treatment, to 1 at the end of therapy. Both combination treatments were well tolerated. Infusion-related adverse events were not reported. Grade 3–4 hematologic toxicity was observed only in one case treated by alemtuzumab and gemcitabine (grade 3 leukopenia). One patient from the group treated with alemtuzumab and IFN-α had infectious complications (CMV pneumonitis and pulmonary aspergillosis). Two patients treated with gemcitabine showed CMV detectable viral load in peripheral blood without any occurrence of complications. All patients treated with alemtuzumab and IFN-α experienced a progressive disease, respectively at 5, 9, 11, 12 months, whereas the 3 patients treated with alemtuzumab and gemcitabine are still in PR at 4, 10, 12 months (median overall PFS: 9 mo.s). At last follow-up, 3 patients are still alive with disease, while two had died, one for unrelated causes and the other for progressive disease. Although the present series is small, our observations point towards a potential role of alemtuzumab in combination schemes in heavily pretreated CTCL patients, as the response rate (57%) appears better than that reported in the literature for alemtuzumab alone (OR 33%), and the toxicity profile of both combination seems to be fairly acceptable. Moreover, gemcitabine might be a better candidate than IFN-α for combination therapy with alemtuzumab, since the progression-free survival seems to be prolonged.

The oral anticoagulant therapy (OAT) in oncologic patient is often problematic because both the haemorragic risk and the thrombotic risk, i.e. the recurrence of venous thromboembolism, are increased. Many studies have been conducted in patient with solid neoplasms; it remains still unclear whether these results could be entirely extrapolated to patients with haematological malignancies. Our aim was to analyse the clinical outcomes and the control of anticoagulation, measured as % time in target INR range, in the patients with cancer compared to patients without cancer; moreover we focused on patients with haematological neoplasms. A total of 500 patients were recruited in 6 years, regardless of the indication for OAT; 146 (29%) of them were affected by cancer (47 haematological and 99 solid neoplasms); those being treated with warfarin for less than 1 month were excluded from this analysis. It resulted that patients affected with cancer had a worse anticoagulation control, compared to those without cancer (55% vs 60%; p

Pegylated liposomal doxorubicin (PLD) is approved in breast cancer, ovarian cancer, Kaposi’s sarcoma. Its skin-tropism suggests a role in the therapy of primary cutaneous lymphomas (PCLs). Among the cutaneous toxicities of PLD, the palmar-plantar erythrodysesthesia (PPE) is characterized by tingling, dysesthesia, erythema, swelling and, in severe cases desquamation, crusting, ulceration and necrosis. PPE is dose- and schedule-dependent and is usually reversible. Different pathophysiologic mechanisms are suggested: PLD, excreted from sweat glands, penetrates into the corneum stratum with local oxidative and inflammatory reactions; direct cytotoxic effect; extravasation of PLD from the deep microvessels through the local pressure. At our knowledge no specific reports on PPE in patients (pts) with PCLs treated with PLD were presented. We report 31 PCL pts treated with PLD: 26 affected by primary cutaneous T-cell lymphomas (PCTCL) and 5 by primary cutaneous B-cell lymphomas (PCBLC). Among PCTLC, 21 pts (16 M, 5 W, median age 67 yrs) received PLD in monotherapy at the dosage of 20 mg/m2 every 3–4 weeks. They were affected by relapsed or refractory PCTCLs: Mycosis Fungoides (MF) (52%), transformed MF in large cell lymphoma (20%), Sèzary Syndrome (SS) (14%), peripheral T-cell lymphoma unspecified (PTCL–U) (14%). After 6 courses, 47% achieved a CR, with ORR of 81%. The median time to the max response was 3 mo.s. After a median follow-up of 36 months 5 pts had progression of disease (PD) and 5 relapsed. To date 9 pts are alive and 12 dead, 4 for PD and 8 for non-related causes. Median OS, EFS and PFS are 36, 17 and 19 mo.s respectively; OS, EFS and PFS rates at 66 mo.s are 33%, 16% and 37%, respectively. Among the remaining 5 PCTLC pts one with transformed MF in large cell lymphoma was treated with PLD in monotherapy and then intensified with bortezomib and dexamethasone obtaining a VGPR. Another pt with PTCL-U received PLD plus rituximab with a CR and the last 3 pts (MF, MF transformed and SS) underwent CBVD scheme obtaining a PR. Among the 5 PCBCLs a woman was affected by marginal zone lymphoma (PCMZL) and 4 men by large B-cell lymphoma (PCLBCL) (all in stage: T3,N0,M0). All pts showed a CR with PLD monotherapy, in a short period (median 2,5 mo.s), even when heavily pretreated. One of them experienced a relapse. One died for PD; to date the others are still in CR after 5, 52, 63 and 69 mo.s. PLD was well tolerated. In PCTLC adverse effects were observed in 6 pts (23%) and grade III–IV toxicity occurred in 2 (8%): capillary leakage syndrome and neutropenia. Nobody required dose modifications or delays. Grade II gastrointestinal toxicity was present in 2 pts. Only two showed a grade I PPE. In these cases we delayed the dose up to 1 week, obtaining a complete resolution, thus returning to the original interval. Even in the five pts affected by PCBCL, PLD was well tolerated and no one decreased or delayed the dose. A pt showed a reversibile grade I neurotoxicity; the hematological toxicity was mild with one case of grade III neutropenia treated with G-CSF. Nobody experienced PPE. PPE is frequently reported in pts with cancer treated with PLD: about 50% off all pts who received the currently approved dose of 50 mg/m2 every 4 weeks experienced PPE and about 20% grade III PPE. When PPE develops, PLD dose reduction, schedule modification, ultimately drug withdrawal are considered. In our pts affected by PCLs, PPE occurred rarely, with mild grade (2 case of grade I). It is probably due to low dosages of Peg-Doxo, compared to the commonly used in other solid neoplasms. Besides oral prophylaxis with pyridoxine 300 mg daily was administered from the beginning of the treatment until 1 month after its discontinuation in all pts, as suggested by some authors. This is the first report of PLD in PCBCL; as regarding the other few studies (Wollina, Cantonetti, Di Lorenzo, Lybaek, Quereux) concerning PLD in PCTLC the incidence of PPE resulted always low. In conclusion, it emerges that PLD shows a significantly high clinical activity and a good safety profile in PCLs. Our data seem to confirm the dose dependent toxicity effect of PLD, since three-four weekly schedule of PLD at the dose of 20 mg/m2 has less PPE compared to the classical schedule of 50 mg/m2 every 4 weeks. Some studies in ovarian and breast cancer demonstrated that PLD dose intensity reduction can prevent PPE. The administration of prophylactic pyridoxine seems to be useful in the reduction of the incidence and severity of PPE.

Background: Recruitment of neoplastic T-cells in skin is a critical step in the pathogenesis of Mycosis Fungoides (MF) lesions. The chemokine CCL27 attracts memory CCR10-positive T cells to the skin. Increased CCL27 serum levels and skin epidermal expression have been observed in MF patients compared to normal controls, but the interactions between neoplastic cells and the skin immune system needs to be further elucidated. Aims: We investigated whether CCL27 expression in MF is related to the density of dendritic cells (DC), CD8+ lymphocytes and CD4+ neoplastic lymphocytes; moreover, whether it influences the clinical response to therapy, it is modifiable by therapy itself and if the changes eventually observed can predict the frequency of recurrences and event-free survival. Material and methods: CCL27 epidermal immunohistochemical staining was performed in 52 early stage-MF patients, 34 M/18 F, treated by PUVA plus interferon a for 14 months, with a complete follow-up data. Specimens were obtained at diagnosis and at the end of treatment. Formalin-fixed/paraffin-embedded tissue sections were immunostained with anti-CCL27, CD1a, CD8, CD4 antibodies. CCL27 immunoreactivity was graded as + (faint/moderate staining, limited to epidermal basal layer, as in normal skin); ++ (faint/moderate staining in the suprabasal layers; strong staining up to the lower two-thirds of epidermis); +++ (strong staining of full-thickness epidermis). Density of CD1a-positive DC and CD8+ lymphocytes was graded as + (few/isolated cells), ++ (small, ≤5 cell clusters), +++ (larger, >5 cell clusters). Density of CD4+ lymphocytes was graded as + (perivascular infiltrate), ++ (discontinuous subepidermal band), +++ (continuous band). Statistical analysis was also performed (c2 test for tables of contingency; event-free survival curves (EFS) by Kaplan-Meier method). Results: At diagnosis, CCL27 expression was similar to normal skin in 16 cases and abnormal/suprabasal in 36, with 12 +++ cases. A normal/basal CCL27 expression tended to be correlated with a high DC density and with a low neoplastic infiltrate density (although P=NS). CCL27 at diagnosis was not correlated with clinical response (50 complete remission/CR, 2 partial remission). Treatment induced a significant CCL27 increase (suprabasal in 42 cases with 24 +++ cases; c2 test: P=0.004). Skin CCL27 expression at the end of treatment, but not at diagnosis, was related to recurrence and influenced EFS. During follow-up (median, 92.5 mo.s; range, 43–165), 33 patients relapsed (median EFS, 46 mo.s). A normal/basal CCL27 expression at the end of treatment, was correlated with a lower incidence of disease recurrence (3/9 compared to 30/41 with suprabasal expression; c2 test: P=0.022) and a longer median EFS (111 mo.s vs 39 mo.s with suprabasal expression; log rank test: P=0.031). Conclusions: Increased CCL27 expression in early-stage MF lesions might be related to a balance between neoplastic cells and immunomodulant dendritic cells. CR induction in almost all patients by treatment is not correlated with a CCL27 reduction, but a normal CCL27 expression after treatment designates a subset of patients (about 20%) with a favourable behaviour. The mechanisms involved in the increased CCL27 expression after therapy in the remaining 80% patients, which have a higher probability to recur, need to be further investigated.

Patients with primary cutaneous lymphomas (PCLs) are treated with multiple therapeutic regimens, which may increase the risk of subsequent solid and haematological neoplasms. The aim of our study was to assess the incidence of other malignancies in our series of PCLs. From March 1994 to January 2007, 272 patients with PCLs (179 M, 93 F, median age 65 yr, range 14–88) were referred to our center for staging, treatment and follow-up. The clinical charts were reviewed to detect the incidence of malignancies occurred before or after the diagnosis of PCL or concomitantly. The series was composed by 228 patients (150 M, 78 F, median age 66 yrs) with T-cell lymphomas (202 Mycosis Fungoides/MF, 10 Sézary Syndrome/SS, 9 CD30+ PCL, 7 non MF/non CD30+ T cell PCL); 43 patients (28 M, 15 F, median age 60 yrs) with B-cell lymphomas (25 Follicular/FL, 14 marginal/MZL, 3 Leg-type, 1 Lymphoblastic) and one patient with CD4+/CD56+ hematodermic neoplasm. Chemotherapy was administered to 48 patients. During follow-up 12 patients died for the disease and 24 for other causes. A second tumor was observed in 41 patients (15%): 6 of them experienced more than one neoplasms: overall we observed 48 malignancies, 38 solid and 10 haematological. The other neoplasms appeared similarly before (20) and after (21) the diagnosis of PCL; in 7 cases they were diagnosed simultaneously. Solid tumours (17 preceding, 4 concurrent, 17 subsequent) were diagnosed in: skin (11), colon (5), lung (4), breast (3), CNS (3), bladder (2), liver (2), kidney (2), uterus (2), testis (1), prostate (1), stomach (1), thyroid (1). The haematological malignancies (3 preceding, 3 concurrent, 4 subsequent) were: B-cell lymphomas (4), acute myeloid leukemias (3), plasmocytoma (1), T-cell lymphoma (1), Hodgkin’s lymphoma (1). Among the six patients with more than one adjunctive neoplasms one patient had lung and kidney carcinoma preceding PCL; two patients a preceding carcinoma (skin and bladder, respectively) and subsequently a lung carcinoma; other two patients showed both a preceding and a concurrent neoplasm (skin and colon carcinoma, B-cell lymphoma and skin carcinoma, respectively). Finally a patient had a preceding skin carcinoma, a concurrent nodal Hodgkin’s lymphoma and a subsequent nodal B-cell lymphoma. So we have reported 48 other neoplasms in 41 patients within 272 PCLs (15%). The occurrence of the other malignancy was not related to the B/T phenotype of PCLs, as it was observed in 35/228 (15.4%) T-cell lymphomas (32 MF, 2 SS, 1 non MF/non CD30+ T cell lymphoma) and in 6/43 (14%) B-cell lymphomas (3 FL, 3 MZL; χ2 test: P=0.88). The interval of occurrence was longer for tumors preceding (median 60 mo.s, range 8–180) than for tumors following PCL (median 45, range 6–122). The administration of chemotherapy for PCL was not associated with an increased incidence of second neoplasm(χ2 test, P=0.77). Multicentric studies might help in elucidating the role of genetic and immunitary factors in the pathogenesis of multiple neoplasms in patients with PCLs.