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Ilaria Laurenzana,1,* Stefania Trino,1,* Daniela Lamorte,1 Marco Girasole,2 Simone Dinarelli,2 Angelo De Stradis,3 Vitina Grieco,4 Maddalena Maietti,5 Antonio Traficante,5 Teodora Statuto,4 Oreste Villani,6 Pellegrino Musto,6 Alessandro Sgambato,7 Luciana De Luca,4,* Antonella Caivano4,* 1Laboratory of Preclinical and Translational Research, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 2Institute for the Study of the Structure of Matter, National Research Council (CNR), Rome, Italy; 3Institute for Sustainable Plant Protection, National Research Council (CNR), Bari, Italy; 4Laboratory of Clinical Research and Advanced Diagnostics, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 5Unit of Clinical Pathology, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 6Hematology and Stem Cell Transplantation Unit, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 7Scientific Direction, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy*These authors contributed equally to this workCorrespondence: Antonella CaivanoLaboratory of Clinical Research and Advanced Diagnostics, IRCCS CROB, Rionero in Vulture, 85028, Potenza, ItalyTel +39 0972 726395Fax +39 0972 726482Email antonella.caivano@crob.itIntroduction: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established.Methods: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR.Results: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters.Conclusion: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.Keywords: extracellular vesicles, biomarkers, hematological malignancies, nanoparticle tracking analysis, flow cytometry, digital PCR

In the era of precision medicine, liquid biopsy is becoming increasingly important in oncology. It consists in the isolation and analysis of tumor-derived biomarkers, including extracellular vesicles (EVs), in body fluids. EVs are lipid bilayer-enclosed particles, heterogeneous in size and molecular composition, released from both normal and neoplastic cells. In tumor context, EVs are valuable carriers of cancer information; in fact, their amount, phenotype and molecular cargo, including proteins, lipids, metabolites and nucleic acids, mirror nature and origin of parental cells rendering EVs appealing candidates as novel biomarkers. Translation of these new potential diagnostic tools into clinical practice could deeply revolutionize the cancer field mainly for solid tumors but for hematological neoplasms, too.

Multiple myeloma (MM) is characterized by the abnormal proliferation of clonal plasma cells (PCs) in bone marrow (BM). MM-PCs progressively occupy and likely alter BM niches where reside hematopoietic stem and progenitor cells (HSPCs) whose viability, self-renewal, proliferation, commitment, and differentiation are essential for normal hematopoiesis. Extracellular vesicles (EVs) are particles released by normal and neoplastic cells, such as MM cells. They are important cell-to-cell communicators able to modify the phenotype, genotype, and the fate of the recipient cells. Investigation of mechanisms and mediators underlying HSPC-MM-PC crosstalk is warranted to better understand the MM hematopoietic impairment and for the identification of novel therapeutic strategies against this incurable malignancy. This study is aimed to evaluate whether EVs released by MM-PCs interact with HSPCs, what effects they exert, and the underlying mechanisms involved. Therefore, we investigated the viability, cell cycle, phenotype, clonogenicity, and microRNA profile of HSPCs exposed to MM cell line-released EVs (MM-EVs). Our data showed that: (i) MM cells released a heterogeneous population of EVs; (ii) MM-EVs caused a dose-dependent reduction of HSPCs viability; (iii) MM-EVs caused a redistribution of the HSPC pool characterized by a significant increase in the frequency of stem and early precursors accompanied by a reduction of late precursor cells, such as common myeloid progenitors (CMPs), megakaryocyte erythroid progenitors (MEPs), B and NK progenitors, and a slight increase of granulocyte macrophage progenitors (GMPs); (iv) MM-EVs caused an increase of stem and early precursors in S phase with a decreased number of cells in G0/G1 phase in a dose-dependent manner; (v) MM-EVs reduced the HSPC colony formation; and (vi) MM-EVs caused an increased expression level of C-X-C motif chemokine receptor type 4 (CXCR4) and activation of miRNAs. In conclusion, MM cells through the release of EVs, by acting directly on normal HSPCs, negatively dysregulate normal hematopoiesis, and this could have important therapeutic implications.

Background Acute promyelocytic leukemia (APL) is characterized by fusion of PML/RARα genes as a result of t(15;17)(q24;q21). APL is now one of the curable hematological malignancies thanks to molecularly targeted therapies based on all-trans retinoic acid (ATRA) and arsenic trioxide (ATX). Extramedullary (EM) relapse is a rare event in APL, ear involvement being even more infrequent, with only six cases so far described. About 30–35% of patients with newly diagnosed APL have additional cytogenetics abnormalities, whose prognostic significance is still controversial. The most common additional aberration is trisomy 8 or partial gain 8q. Case presentation We describe here a novel unbalanced translocation der(3)t(3;8)(q29;q23.3-q24.3) associated with 8q partial gain in a 41 year-old man affected by APL in molecular remission after first line treatment, who had a responsive EM relapse in the auditory canal. Conclusions EM relapse is a rare event in APL and ear involvement is even more infrequent. To our knowledge, this is the first reported case of APL with a new der(3)t(3;8)(q29;q23.3-q24.3) and 8q partial gain associated with t(15;17)(q24;q21). Despite the recurrence of the disease at EM level, the clinical outcome of this patients was favorable.

Azorella glabra Wedd. (AG) is traditionally used to treat gonorrhea or kidney&rsquo, s problems. The antioxidant, antidiabetic, anticholinesterase and in vitro antitumor activities of AG extracts were recently reported. The aim of this work was to investigate anti-leukemic properties of AG chloroform fraction (AG CHCl3) and of its ten sub-fractions (I-X) and to identify their possible bioactive compounds. We determined their in vitro antioxidant activity using 2,2&rsquo, azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), nitric oxide (NO) and superoxide anion (SO) assays, and their phytochemical profile by spectrophotometric and LC-MS/MS techniques. I-X action on two acute myeloid leukemia (AML) cell lines viability, apoptosis and cell cycle were evaluated by MTS, western blotting and cytofluorimetric assays. Different polyphenol, flavonoid and terpenoid amount, and antioxidant activity were found among all samples. Most of I-X induced a dose/time dependent reduction of cell viability higher than parent extract. IV and VI sub-fractions showed highest cytotoxic activity and, of note, a negligible reduction of healthy cell viability. They activated intrinsic apoptotic pathway, induced a G0/G1 block in leukemic cells and, interestingly, led to apoptosis in patient AML cells. These activities could be due to mulinic acid or azorellane terpenoids and their derivatives, tentatively identified in both IV and VI. In conclusion, our data suggest AG plant as a source of potential anti-AML agents.

// Antonella Caivano 1 , Francesco La Rocca 1 , Ilaria Laurenzana 1 , Tiziana Annese 2 , Roberto Tamma 2 , Ubaldo Famigliari 3 , Vittorio Simeon 1 , Stefania Trino 1 , Luciana De Luca 1 , Oreste Villani 4 , Simona Berardi 5 , Antonio Basile 5 , Angelo Vacca 5 , Giuseppe Saglio 6 , Luigi Del Vecchio 7, 8 , Pellegrino Musto 9 , Daniela Cilloni 6 1 Laboratory of Pre-clinical and Translational Research, Scientific Institute of Research and Cure (IRCCS), Referral Cancer Center of Basilicata (CROB), Rionero in Vulture, Italy 2 Department of Human Anatomy, Histology and Embryology, University of Bari Medical School, Bari, Italy 3 Division of Pathology, Department of Oncology, St Luigi Hospital, Turin, Italy 4 Department of Onco-Hematology, IRCCS-CROB, Rionero in Vulture, Italy 5 Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy 6 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy 7 CEINGE-Biotecnologie Avanzate s.c.a r.l and Medical Biotechnologies, Federico II University, Naples, Italy 8 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 9 Scientific Direction, IRCCS-CROB, Rionero in Vulture, Italy Correspondence to: Antonella Caivano, email: caivanoa@libero.it Keywords: angiogenesis, bone marrow endothelial cells, receptor tyrosine kinase, EphA3, multiple myeloma Received: September 02, 2016 Accepted: March 01, 2017 Published: March 10, 2017 ABSTRACT This study investigates the role of ephrin receptor A3 (EphA3) in the angiogenesis of Multiple Myeloma (MM) and the effects of a selective target of EphA3 by a specific monoclonal antibody on primary bone marrow endothelial cells (ECs) of MM patients. EphA3 mRNA and protein were evaluated in ECs of MM patients (MMECs), in ECs of patients with monoclonal gammopathies of undetermined significance (MGECs) and in ECs of healthy subjects (control ECs). The effects of EphA3 targeting by mRNA silencing (siRNA) or by the anti EphA3 antibody on the angiogenesis were evaluated. We found that EphA3 is highly expressed in MMECs compared to the other EC types. Loss of function of EphA3 by siRNA significantly inhibited the ability of MMECs to adhere to fibronectin, to migrate and to form tube like structures in vitro , without affecting cell proliferation or viability. In addition, gene expression profiling showed that knockdown of EphA3 down modulated some molecules that regulate adhesion, migration and invasion processes. Interestingly, EphA3 targeting by an anti EphA3 antibody reduced all the MMEC angiogenesis-related functions in vitro . In conclusion, our findings suggest that EphA3 plays an important role in MM angiogenesis.

Multiple myeloma (MM) is characterized by a clonal proliferation of neoplastic plasma cells (PCs) in bone marrow (BM) and the interplay between MM PCs and the BM microenvironment, which plays a relevant role in its pathogenesis. In this important cross-talk, extracellular vesicles (EVs) are active. EVs, including small and medium/large EVs, are lipid bi-layer particles released in circulation by normal and neoplastic cells. A selected cargo of lipids, proteins, and nucleic acids is loaded into EVs, and delivered locally and to distant sites, thus influencing the physiology of recipient cells. In the 'liquid biopsy' context, EVs can be isolated from human biofluids proving to be powerful markers in cancer. Areas covered: Here, we summarize the recent advances on EVs in MM field. Expert commentary: EVs from MM PCs: i) enhance malignant cell proliferation and aggressiveness through an autocrine loop; ii) are able to transfer drug resistance in sensitive-drug cells; iii) stimulate angiogenesis; iv) increase the activity of osteoclasts; v) have immunosuppressive effects. In addition, EVs from MM stromal cells also promote MM cell proliferation and drug resistance. Finally, we underline the importance of EVs as MM potential biomarkers in 'cancer liquid biopsy' and as a potential new therapeutic target.

Background Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative therapeutic approach for different hematological malignancies (HMs), and epigenetic modifications, including DNA methylation, play a role in the reconstitution of the hematopoietic system after AHSCT. This study aimed to explore global DNA methylation dynamic of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) from donors and their respective recipients affected by acute myeloid leukemia (AML), acute lymphoid leukemia (ALL) and Hodgkin lymphoma (HL) during the first year after transplant. Methods We measured DNA methylation profile by Illumina HumanMethylationEPIC in BM HSPC of 10 donors (t0) and their matched recipients at different time points after AHSCT, at day + 30 (t1), + 60 (t2), + 120 (t3), + 180 (t4), and + 365 (t5). Differential methylation analysis was performed by using R software and CRAN/Bioconductor packages. Gene set enrichment analysis was carried out on promoter area of significantly differentially methylated genes by clusterProfiler package and the mSigDB genes sets. Results Results show significant differences in the global methylation profile between HL and acute leukemias, and between patients with mixed and complete chimerism, with a strong methylation change, with prevailing hyper-methylation, occurring 30 days after AHSCT. Functional analysis of promoter methylation changes identified genes involved in hematopoietic cell activation, differentiation, shaping, and movement. This could be a consequence of donor cell “adaptation” in recipient BM niche. Interestingly, this epigenetic remodeling was reversible, since methylation returns similar to that of donor HSPCs after 1 year. Only for a pool of genes, mainly involved in dynamic shaping and trafficking, the DNA methylation changes acquired after 30 days were maintained for up to 1 year post-transplant. Finally, preliminary data suggest that the methylation profile could be used as predictor of relapse in ALL. Conclusions Overall, these data provide insights into the DNA methylation changes of HSPCs after transplantation and a new framework to investigate epigenetics of AHSCT and its outcomes. Electronic supplementary material The online version of this article (10.1186/s13287-019-1245-6) contains supplementary material, which is available to authorized users.

Background: Thecross talk between neoplastic cells and microenvironment is mediated by direct cell-to-cell contacts, secretion of soluble factors and release of extracellular vesicles (EVs). EVs deriving from tumor cells in chronic lymphocytic leukemia (CLL) may affect the surrounding microenvironment, playing a key role on survival of neoplastic clone. Of note, EVs are increased in CLL patients compared to normal subjects. In this study, we performed a comprehensive characterization of serum EVs from previously untreated CLL patients, investigating, in particular, phenotype and absolute number, in order to test their possible prognostic significance. Patients and methods: Serum samples of 131 newly diagnosed CLL and from 28 healthy subjects were analyzed. One milliliter of serum was processed with serial ultracentrifugations. Each sample of EV-enriched pellet, from patients and controls, was freshly analyzed by FACS Calibur (Becton Dickinson BD) cytometer using Cell Quest software (BD). The system was calibrated using standard microbeads with a diameter of 0.3-0.9-3 μm to define the size limit for microvesicles (MV), a subtype of EVs. To determine the number of MV/μL serum, TruCOUNT beads (BD) were added immediately prior to analysis by flow cytometry. MV morphology was characterized by transmission electron microscope (TEM). MV were then labeled with fluorochrome-conjugated monoclonal antibodies (anti-CD19, CD3, CD94, CD20, CD2, CD56, CD52, CD37) and their specific isotypic controls. Finally, MV were correlated with the main clinical and biological disease's characteristics, including clinical outcome. Results: Flow cytometric analysis of MVs showed a size within 1mm, based on forward and side scatter evaluation and the use of standard beads. The analysis was carried out on MV population isolated by the gating strategy. MV were also visualized by TEM, showing a spheroid morphology. We found a significantly higher mean number of MV in CLL patients with respect to healthy subjects (p Conclusions: Our study indicates that: (i) MV number is higher in CLL patients as compared to normal controls; (ii) CD19 and CD37 are the most represented B-cell antigens on CLL derived MV; (iii) total MV levels are associated with high tumor burden; (iv) total MV levels predict for TTT in Rai 0 patients, as well as for TTT and OS in all stage patients. These observations suggest that MV may represent a new biomarker for CLL. Disclosures D'Arena: Janssen-Cilag: Honoraria. Musto:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Smenamides are an intriguing class of peptide/polyketide molecules of marine origin showing antiproliferative activity against lung cancer Calu-1 cells at nanomolar concentrations through a clear pro-apoptotic mechanism. To probe the role of the activity-determining structural features, the 16-epi-analogue of smenamide A and eight simplified analogues in the 16-epi series were prepared using a flexible synthetic route. The synthetic analogues were tested on multiple myeloma (MM) cell lines showing that the configuration at C-16 slightly affects the activity, since the 16-epi-derivative is still active at nanomolar concentrations. Interestingly, it was found that the truncated compound 8, mainly composed of the pyrrolinone terminus, was not active while compound 17, essentially lacking the pyrrolinone moiety, was 1000-fold less active than the intact substance and was the most active among all the synthesized compounds.

Primary plasma cell leukemia (pPCL) is a rare and very aggressive variant of multiple myeloma (MM). Specific clinical, biological and molecular patterns distinguish pPCL from MM. Here, we performed a genome-wide methylation analysis by high-density array in 14 newly diagnosed pPCL patients along with 60 MMs, and 5 patients affected by monoclonal gammopathy of uncertain significance (MGUS). Our analysis revealed a global hypomethylation profile associated with pPCL. Additionally, differential methylation patterns were found related to distinct chromosomal aberrations and DIS3 mutations, affecting genes with roles in bone metabolism, cell migration, transcription regulation or DNA damage response. When compared with MM patients, pPCL showed a distinct methylation profile mostly characterized by hypomethylated probes specific for genes involved in several processes like cell adhesion and migration. Furthermore, decreasing methylation levels were evidenced for genes significantly modulated in the progressive phases of plasma cell dyscrasias, from MGUS to MM and pPCL. Overall, our data provide new insights into the molecular characterization of pPCL, thus being potentially useful in the prognostic stratification or identification of novel molecular targets.

The bone marrow (BM) microenvironment in hematological malignancies (HMs) comprises heterogeneous populations of neoplastic and nonneoplastic cells. Cancer stem cells (CSCs), neoplastic cells, hematopoietic stem cells (HSCs), and mesenchymal stromal/stem cells (MSCs) are all components of this microenvironment. CSCs are the HM initiators and are associated with neoplastic growth and drug resistance, while HSCs are able to reconstitute the entire hematopoietic system; finally, MSCs actively support hematopoiesis. In some HMs, CSCs and neoplastic cells compromise the normal development of HSCs and perturb BM-MSCs. In response, “reprogrammed” MSCs generate a favorable environment to support neoplastic cells. Extracellular vesicles (EVs) are an important cell-to-cell communication type in physiological and pathological conditions. In particular, in HMs, EV secretion participates to unidirectional and bidirectional interactions between neoplastic cells and BM cells. The transfer of EV molecular cargo triggers different responses in target cells; in particular, malignant EVs modify the BM environment in favor of neoplastic cells at the expense of normal HSCs, by interfering with antineoplastic immunity and participating in resistance to treatment. Here, we review the role of EVs in BM cell communication in physiological conditions and in HMs, focusing on the effects of BM niche EVs on HSCs and MSCs.

Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.

Primary plasma cell leukemia (pPCL) is a rare and aggressive variant of multiple myeloma (MM) which may represent a valid model for high-risk MM. This disease is associated with a very poor prognosis, and unfortunately, it has not significantly improved during the last three decades. New high-throughput technologies have allowed a better understanding of the molecular basis of this disease and moved toward risk stratification, providing insights for targeted therapy studies. This knowledge, added to the pharmacogenetic profile of new and old agents in the analysis of efficacy and safety, could contribute to help clinical decisions move toward a precision medicine and a better clinical outcome for these patients. In this review, we describe the available literature concerning the genomic characterization and pharmacogenetics of plasma cell leukemia (PCL).

Mesenchymal stem cells (MSCs) are a heterogeneous cellular population containing different progenitors able to repair tissues, support hematopoiesis, and modulate immune and inflammatory responses. Several clinical trials have used MSCs in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent hematopoietic stem cell (HSC) engraftment failure, reduce aplasia post chemotherapy, and to control graft versus host disease (GvHD). The efficacy of MSCs is linked to their immune suppressive and anti-inflammatory properties primarily due to the release of soluble factors. Recent studies indicate that most of these effects are mediated by extracellular vesicles (EVs). MSC-EVs have therefore therapeutic effects in regenerative medicine, tumor inhibition, and immune-regulation. MSC-EVs may offer specific advantages for patient safety, such as lower propensity to trigger innate and adaptive immune responses. It has been also shown that MSC-EVs can prevent or treat acute-GvHD by modulating the immune-response and, combined with HSCs, may contribute to the hematopoietic microenvironment reconstitution. Finally, MSC-EVs may provide a new potential therapeutic option (e.g., transplantation, gene therapy) for different diseases, particularly hematological malignancies. In this review, we will describe MSC and MSC-EVs role in improving allo-HSCT procedures and in treating GvHD.

Purpose: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. Methods: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. Results: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. Conclusions: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

Lin28A is a highly conserved RNA-binding protein that concurs to control the balance between stemness and differentiation in several tissue lineages. Here, we report the role of miR-128a/Lin28A axis in blocking cell differentiation in acute myeloid leukemia (AML), a genetically heterogeneous disease characterized by abnormally controlled proliferation of myeloid progenitor cells accompanied by partial or total inability to undergo terminal differentiation. First, we found Lin28A underexpressed in blast cells from AML patients and AML cell lines as compared with CD34+ normal precursors. In vitro transfection of Lin28A in NPM1-mutated OCI-AML3 cell line significantly triggered cell-cycle arrest and myeloid differentiation, with increased expression of macrophage associate genes (EGR2, ZFP36 and ANXA1). Furthermore, miR-128a, a negative regulator of Lin28A, was found overexpressed in AML cells compared with normal precursors, especially in acute promyelocytic leukemia (APL) and in ‘AML with maturation’ (according to 2016 WHO classification of myeloid neoplasms and acute leukemia). Its forced overexpression by lentiviral infection in OCI-AML3 downregulated Lin28A with ensuing repression of macrophage-oriented differentiation. Finally, knockdown of miR-128a in OCI-AML3 and in APL/AML leukemic cells (by transfection and lentiviral infection, respectively) induced myeloid cell differentiation and increased expression of Lin28A, EGR2, ZFP36 and ANXA1, reverting myeloid differentiation blockage. In conclusion, our findings revealed a new mechanism for AML differentiation blockage, suggesting new strategies for AML therapy based upon miR-128a inhibition.

Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. The hypothesis that TRAP1 plays a regulatory function on the BRAF pathway, arising from the observation that BRAF levels are decreased upon TRAP1 interference, was tested in human breast and colorectal carcinoma in vitro and in vivo. This study shows that TRAP1 is involved in the regulation of BRAF synthesis/ubiquitination, without affecting its stability. Indeed, BRAF synthesis is facilitated in a TRAP1-rich background, whereas increased ubiquitination occurs upon disruption of the TRAP1 network that correlates with decreased protein levels. Remarkably, BRAF downstream pathway is modulated by TRAP1 regulatory activity: indeed, TRAP1 silencing induces (i) ERK phosphorylation attenuation, (ii) cell-cycle inhibition with cell accumulation in G0–G1 and G2–M transitions, and (iii) extensive reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-knockdown cells identified cell growth and cell-cycle regulation as the most significant biofunctions controlled by the TRAP1 network. It is worth noting that TRAP1 regulation on BRAF is conserved in human colorectal carcinomas, with the two proteins being frequently coexpressed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed activity against the TRAP1 network and high cytostatic potential in BRAF-mutated colorectal carcinoma cells. Therefore, this novel TRAP1 function represents an attractive therapeutic window to target dependency of BRAF-driven tumors on TRAP1 translational/quality control machinery. Cancer Res; 74(22); 6693–704. ©2014 AACR.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). Leukemogenesis is characterized by rapid growth and impaired differentiation of leukemic cells, which progressively occupy and likely alter bone marrow (BM) niche where normal HSPCs reside [Kumar B., et al. Leukemia. 2018]. The homing and adhesion of AML cells to the BM niche is critical for its pathogenesis and involves many adhesion molecules, chemokine receptors, their ligands and signaling cascades. In the healthy context, the CXC chemokine receptor type 4 (CXCR4) expressed on HSPCs and its ligand, stromal derived factor-1 alpha (SDF-1α) produced by BM niche, are key mediators of HSPC-BM niche interaction inducing the homing, quiescence and survival of HSPCs, and the regulation of the HSC pool amount [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. In AML context, where CXCR4/SDF-1α axis is deregulated, growth, BM niche-homing and adhesion of leukemic cells are altered also inducing AML relapse/resistance [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. Despite recent improvements in therapeutic approaches, only a fraction of AML patients is cured. Therefore, it is crucial to identify new molecular mechanisms driving the AML pathogenesis. Extracellular vesicles (EVs) are lipidic bi-layer membrane particles released by normal and neoplastic cells. They shuttle a complex molecular cargo which reflects that of the origin cell [Caivano A., et al. Tumor Biol 2015; Caivano A., et al. Cell Oncol 2017] and which can be transferred to target cells altering their biological functions [De Luca L., et al. Oncotarget 2016; De Luca L., et al. Int J Mol Sci. 2017]. In particular, neoplasm derived EVs promote a tumor-supporting environment in non-malignant cells favoring cancer proliferation [Laurenzana I., et al. Stem Cells Int. 2018; De Luca L., et al. Expert Rev Mol Diagn. 2019]. We studied the effect of leukemia derived-EVs (LEVs) on healthy umbilical cord blood HSPCs to explore their potential role in dysregulating normal hematopoiesis. Specifically, the proliferation and differentiation, the quantitative and qualitative colony-forming features and the SDF-1α /CXCR4 mediated-migration of HSPCs induced by LEVs were investigated. EVs were isolated from AML cell lines by centrifugation steps and analyzed for AML surface markers by flow cytometry. Next, they were co-incubated with healthy CD34+ sorted cells for 24 hours. After treatment, cell count, apoptosis, HSPC-specific cluster of differentiation marker (CD10, CD34, CD38, CD45RA, CD90, CD123) expression [Karamitros D., et al. Nat Immunol. 2018], in vitro colony-forming unit cell assay, CXCR4 levels and SDF-1α mediated-migration assay were assessed. Our data showed: i) LEVs displaying specific AML surface markers; ii) a similar number of CD34+ cells in LEV treated-HSPCs, including HSCs, compared with controls; iii) no differences in apoptosis between the two groups; iv) an increase of multipotent progenitor (MPP, p=0.01) associated with a reduction of lymphoid-primed multipotent progenitors (LMPP, p=0.01) in treated cells; v) an increase of common myeloid progenitors (CMP, p=0.01), together with reduction of granulocyte macrophage progenitors (GMP, p=0.01) and megakaryocyte erythroid progenitors (MEP) in treated group; vi) a reduction of colony-forming ability, including erythroid, granulocytic and megakaryocytic colonies (p=0.03), in treated cells; vii) decreased levels of CXCR4 in LEV treated-HSPCs, that were accompanied by reduced HSPC migration mediated by SDF-1α. Altogether, this study suggests that AML derived-EVs can dysregulate the normal hematopoiesis promoting an increase of less differentiated precursors (MPP and CMP) which is associated with a reduction of more committed myeloid progenitors (GMP and MEP) (Figure 1). Furthermore, AML derived-EVs seem to induce less attraction of HSPCs in BM niche by interfering with SDF-1α/CXCR4 axis. Strategies to block EV production and secretion, as well as EV induced-reprogramming, could be novel exciting therapeutic approaches in AML. Disclosures Musto: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The clinical course of chronic lymphocytic leukemia (CLL) may be complicated at any time by autoimmune phenomena.The most common ones are hematologic disorders, such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). Pure red cell aplasia (PRCA) and autoimmune agranulocytosis (AG) are, indeed, more rarely seen. However, they are probably underestimated due to the possible misleading presence of cytopenias secondary to leukemic bone marrow involvement or to chemotherapy cytotoxicity. The source of autoantibodies is still uncertain, despite the most convincing data are in favor of the involvement of resting normal B-cells. In general, excluding the specific treatment of underlying CLL, the managementof these complications is not different from that of idiopathic autoimmune cytopenias or of those associated to other causes. Among different therapeutic approaches, monoclonal antibody rituximab, given alone or in combination, has shown to be very effective.

The tumor suppressor p53 is a canonical regulator of different biological functions, like apoptosis, cell cycle arrest, DNA repair, and genomic stability. This gene is frequently altered in human tumors generally by point mutations or deletions. Conversely, in acute lymphoblastic leukemia (ALL) genomic alterations of TP53 are rather uncommon, and prevalently occur in patients at relapse or with poor prognosis. On the other hand, p53 pathway is often compromised by the inactivation of its regulatory proteins, as MDM2 and ARF. MDM2 inhibitor molecules are able to antagonize p53-MDM2 interaction allowing p53 to exert tumor suppressor transcriptional regulation and to induce apoptotic pathways. Recent preclinical and clinical studies propose that MDM2 targeted therapy represents a promising anticancer strategy restoring p53 dependent mechanisms in ALL disease. Here, we discussed the use of new small molecule targeting p53 pathways as a promising drug target therapy in ALL.

TLRs are main actors of the innate immune response against HPV. There are very few studies on the role of TLRs mediated HPV clearance in Head and Neck oncology. Our aim was to evaluate whether TLR4 expression identifies HPV infection and/or HR-HPV integration status in oral and oropharyngeal cancers. By immunohistochemistry we assessed TLR4 levels in OSCC/OPSCC. To detect viral integration or episomic status In situ hybridization for HPV-DNA and Pyro-sequencing techniques have been performed. The relationship between TLR4 expression with HPV infection status has been investigated. ISH HPV positive samples have reported lower levels of TLR4 intensity than negative samples (p = .002). There was no statistical correlation between TLR4 intensity and PCR HPV results (p more than 0.0.5). Point-biserial correlation coefficient revealed significant association between TLR4 expression and HR-HPV integration status (p = .0001) and between TLR4 expression index and HR-HPV infection (p = .001). These data have shown that TLR4 down-regulation is strongly associated to both HPV-16 infection and its integration into the host DNA.

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

Molecular targeted therapies are based upon drugs acting on tumors by interfering with specific targets involved in growth and spread of cancer. Many targeted therapies were approved by Food and Drug Administration as standard treatment, others were introduced into preclinical or clinical studies on hematological malignancies (HMs). The development of drug-resistance in some HMs and the lack of effective treatments in other ones emphasized the need for searching new molecular targets and therapeutic agents. The aim of this study was to evaluate the effects of 4c pyrazolo[3,4-d]pyrimidine compound, a Src inhibitor, on lymphoid and myeloid neoplasms. Here, we demonstrated its ability to reduce cell viability, induce apoptosis and cell cycle arrest in lymphoid cell lines such as Jurkat, SKMM1, Derl-2/7, and myeloid cell lines, such as Jurl-MK1. Moreover, we reported a high expression of a Src kinase, Fyn, in these cell lines compared to healthy subjects. This study was a starting point to investigate 4c pyrazolo[3,4-d]pyrimidine compound as a drug for HMs and Src kinases as its potential molecular targets.

The use of extracellular vesicles (EVs) from body fluids as “liquid biopsies” is emerging as a promising approach for the diagnosis, prognosis and therapeutic monitoring of cancer patients. MicroRNA-155 (miR155), a non-coding transcript of the B-cell integration cluster (BIC) gene, has been reported to play a critical role in the pathogenesis of several types of hematologic malignancies (HMs) in which high miR155 levels have been found. At yet, however, the EV miR155 level and its putative clinical relevance in sera of HM patients have not been reported. EVs from sera of representative patients with eight different HMs and healthy subjects (controls) were isolated using differential centrifugation. The identity and quality of the EVs were verified by atomic force and transmission electron microscopy. The EV miR155 levels were measured by quantitative RT-PCR. The sensitivity, specificity and area under the curve (AUC) of differences in EV miR155 levels were determined using ROC curve analyses. We found that the EV miR155 levels were significantly higher in chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and Waldenstrom’s macroglobulinemia (WM) cases compared to controls. Conversely, we found that the EV miR155 levels were significantly lower in myelodysplastic syndrome (MDS) and multiple myeloma (MM) cases. No differences were found in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) or Hodgkin’s Lymphoma (HL) cases compared to controls. EV miR155 ROC curve analyses revealed significantly different patterns in CLL and AML cases compared to controls, and in AML cases compared to MDS cases (p = 0.004, p = 0.01 and p = 0.04, respectively). In addition, we found that high EV miR155 levels correlated with high white blood cell counts in AML patients. Our data indicate that EV miR155 may serve as an attractive new, non-invasive diagnostic biomarker in human hematologic malignancies.

Hematopoietic stem cells (HSC), including umbilical cord blood CD34+ stem cells (UCB-CD34+), are used for the treatment of several diseases. Although different studies suggest that bone marrow mesenchymal stem cells (BM-MSC) support hematopoiesis, the exact mechanism remains unclear. Recently, extracellular vesicles (EVs) have been described as a novel avenue of cell communication, which may mediate BM-MSC effect on HSC. In this work, we studied the interaction between UCB-CD34+ cells and BM-MSC derived EVs. First, by sequencing EV derived miRNAs and piRNAs we found that EVs contain RNAs able to influence UCB-CD34+ cell fate. Accordingly, a gene expression profile of UCB-CD34+ cells treated with EVs, identified about 100 down-regulated genes among those targeted by EV-derived miRNAs and piRNAs (e.g. miR-27b/MPL, miR-21/ANXA1, miR-181/EGR2), indicating that EV content was able to modify gene expression profile of receiving cells. Moreover, we demonstrated that UCB-CD34+ cells, exposed to EVs, significantly changed different biological functions, becoming more viable and less differentiated. UCB-CD34+ gene expression profile also identified 103 up-regulated genes, most of them codifying for chemokines, cytokines and their receptors, involved in chemotaxis of different BM cells, an essential function of hematopoietic reconstitution. Finally, the exposure of UCB-CD34+ cells to EVs caused an increased expression CXCR4, paralleled by an in vivo augmented migration from peripheral blood to BM niche in NSG mice. This study demonstrates the existence of a powerful cross talk between BM-MSC and UCB-CD34+ cells, mediated by EVs, providing new insight in the biology of cord blood transplantation.

Gene expression profiling (GEP) of normal thyroid tissue from 43 patients with thyroid carcinoma, 6 with thyroid adenoma, 42 with multinodular goiter, and 6 with Graves-Basedow disease was carried out with the aim of achieving a better understanding of the genetic mechanisms underlying the role of normal cells surrounding the tumor in the thyroid cancer progression. Unsupervised and supervised analyses were performed to compare samples from neoplastic and non-neoplastic diseases. GEP and subsequent RT-PCR analysis identified 28 differentially expressed genes. Functional assessment revealed that they are involved in tumorigenesis and cancer progression. The distinct GEP is likely to reflect the onset and/or progression of thyroid cancer, its molecular classification, and the identification of new potential prognostic factors, thus allowing to pinpoint selective gene targets with the aim of realizing more precise preoperative diagnostic procedures and novel therapeutic approaches. STATEMENT OF SIGNIFICANCE This study is focused on the gene expression profiling analysis followed by RT-PCR of normal thyroid tissues from patients with neoplastic and non-neoplastic thyroid diseases. Twenty-eight genes were found to be differentially expressed in normal cells surrounding the tumor in the thyroid cancer. The genes dysregulated in normal tissue samples from patients with thyroid tumors may represent new molecular markers, useful for their diagnostic, prognostic and possibly therapeutic implications.

Multiple myeloma (MM) is the second most common hematologic malignancy and, although the development of novel agents has improved survival of patients, to date, it remains incurable. Thus, newer and more effective therapeutic strategies against this malignancy are necessary. Plant extracts play an important role in anti-tumor drug discovery. For this reason, in the investigation of novel natural anti-MM agents, we evaluated the phytochemical profiles, in vitro antioxidant activity, and effects on MM cells of Azorella glabra (AG) Wedd. Total polyphenols (TPC), flavonoids (TFC), and terpenoids (TTeC) contents were different among samples and the richest fractions in polyphenols demonstrated a higher antioxidant activity in in vitro assays. Some fractions showed a dose and time dependent anti-proliferative activity on MM cells. The chloroform fraction (CHCl3) showed major effects in terms of reduction of cell viability, induction of apoptosis, and cell cycle arrest on MM cells. The apoptosis induction was also confirmed by the activation of caspase-3. Importantly, the CHCl3 fraction exhibited a negligible effect on the viability of healthy cells. These results encourage further investigations on AG extracts to identify specific bioactive compounds and to define their potential applications in MM.

Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 mu m) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Extracellular vesicles (EVs) are a heterogeneous group of particles, between 15 nanometers and 10 microns in diameter, released by almost all cell types in physiological and pathological conditions, including tumors. EVs have recently emerged as particularly interesting informative vehicles, so that they could be considered a true “cell biopsy”. Indeed, EV cargo, including proteins, lipids, and nucleic acids, generally reflects the nature and status of the origin cells. In some cases, EVs are enriched of peculiar molecular cargo, thus suggesting at least a degree of specific cellular packaging. EVs are identified as important and critical players in intercellular communications in short and long distance interplays. Here, we examine the physiological role of EVs and their activity in cross-talk between bone marrow microenvironment and neoplastic cells in hematological malignancies (HMs). In these diseases, HM EVs can modify tumor and bone marrow microenvironment, making the latter “stronger” in supporting malignancy, inducing drug resistance, and suppressing the immune system. Moreover, EVs are abundant in biologic fluids and protect their molecular cargo against degradation. For these and other “natural” characteristics, EVs could be potential biomarkers in a context of HM liquid biopsy and therapeutic tools. These aspects will be also analyzed in this review.

Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Introduction According to WHO classification, large granular lymphocyte leukemia (LGL leukemia) includes three entities: T-cell LGL leukemia, chronic NK lymphoproliferative disorders (NK-CLPD) and aggressive NK cell leukemia. Since no standard therapies for immature NK cell neoplasms have been established so far and the overall outcomes are dismal, new therapeutic options are needed. A pyrazolo[3,4-d]pyrimidine library of compounds have already been studied in a previous work by Tintori et al. It has been demonstrated that one of these compounds proved to be efficient against some member of Src-family of nonreceptor protein-tyrosine kinases, in particular against Fyn. Its abnormal activity has been shown to be related to various hematological malignancies. In this context we verified the effect of the pyrazolo[3,4-d]pyrimidine 4c compound on LGL leukemia cells and we investigated its mechanism of action. Methods The pyrazolo[3,4-d]pyrimidine 4c compound was tested in a time course MTS test (24, 48, 72 h) at different concentrations (0.1, 0.5, 1, 2, 4, 8, 10 µM) on NK leukemia cells (KHYG1 and NK92), hepatosplenic gamma-delta T cell lymphoma cells (Derl-2 and Derl-7), acute T cell leukemia cells (Jurkat), chronic myeloid leukemia cells (Jurl-MK1), acute myeloid leukemia cells (OCI), multiple myeloma cells (SKM-M1), cervical cancer cells (HeLa) and healthy stem cells (CD34+). Fyn mRNA and protein expression was evaluated in all cell lines by quantitative RT-PCR and western blotting (WB). Fyn mRNA levels were evaluated in cells from 10 healthy controls, 10 T-LGL leukemia, 5 GD-LGL leukemia and 8 NK-CLPD patients by qRT-PCR. Apoptosis and cell cycle studies were performed on KHYG1 after treatment with this compound at 4 µM for 24 h using cytometric analysis by labeling cells with annexin V and propidium iodide. Apoptosis pathway was investigated using proteome profiler human apoptosis array kit; pro-caspase 3, activated caspase 3, Akt and p70S6 kinase were validated by WB. Fyn immunoprecipitation was conducted on treated KHYG1 at 4 µM for 24 h. Results The pyrazolo[3,4-d]pyrimidine 4c compound reduced cell viability in KHYG1 cells (50% of reduction after 24 h of treatment at 4 µM), in Derl-2 cells (about 50% after 72 h at 4 µM), in Derl-7 (about 40% after 72 h at 8 µM), in Jurkat cells (50% after 72 h at 2 µM), in NK92 cells (40% after 72 h at 4 µM). No differences in cell viability were observed in Jurl-MK1, OCI, SKM-M1, HeLa and CD34+. To evaluate Fyn expression level, we performed qRT-PCR and WB showing that both transcript and protein levels were highest in NK and T cells (KHYG1>NK92>Jurkat>Derl-7>Derl-2) compared to other cells. Furthermore, we investigated the viability reduction in treated KHYG1 cells demonstrating that pyrazolo[3,4-d]pyrimidine 4c compound induced 45% of apoptosis caspase-3 mediated and cell cycle arrest in G2/M phase. In these cells we detected a reduction of Fyn phosphorylation after treatment and we also observed a decrease of total levels of Akt and p70S6 kinase, other two protein involved in apoptotic process and cell proliferation. Interestingly, qRT-PCR data showed a significant over-expression of Fyn mRNA level also in T-LGL, GD-LGL and NK-CLPD patients compared to healthy controls (p Conclusion In this study we demonstrated a higher Fyn expression in LGL leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target. Disclosures Musto: Novartis: Honoraria; Celgene: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Sandoz: Consultancy; Janssen: Honoraria; Mundipharma: Honoraria; Roche: Honoraria.

Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases. Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1–positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. ©2011 AACR.

Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

INTRODUCTION: Extracellular vesicles (EVs), including exosomes, shedding vesicles (microvesicles: MVs) and apoptotic bodies, are membrane vesicles of 40-3,000 nm that are released from many cell types, such as red blood cells, platelets, lymphocytes, dendritic cells, endothelial cells and tumour cells. They have a key role not only in the regulation of normal physiological processes, but also in the pathology underlying several diseases. Recently, it was discovered that EVs circulate in bodily fluids of cancer patients and could serve as potential diagnostic biomarkers. However, the presence and role of peripheral circulating EVs in hematological malignancies (HMs) remain unclear. The aim of this study was to investigate EVs in peripheral blood of HM patients, defining the EVs count, size and phenotype. METHODS: Peripheral blood samples were obtained prior to treatment from 87 subjects: 6 Chronic Lymphocytic Leukemia (CLL), 10 Non-Hodgkin's Lymphoma (NHL; 5 follicular, 5 diffuse large B-cell), 6 Waldenstrom's Macroglobulinemia (WM), 6 Hodgkin's Lymphoma (HL), 6 Multiple Myeloma (MM), 5 Acute Myeloid Lukemia (AML), 19 Myeloproliferative Neoplasms [(MPNs; 5 Essential Thrombocythemia (ET), 5 Polycythemia Vera (PV), 5 Primary Myelofibrosis (PMF), 4 Chronic Myelogenous Leukemia (CML)], 5 Myelodysplastic Syndrome (MDS) and 28 healthy controls. EVs were isolated from serum of peripheral blood by ultracentrifugation steps. For calculation of counts, TruCOUNT beads were added immediately prior to analysis by flow cytometry (FACS). For size assessment, Megamix beads with specific size (0.3,0.9, 3.0 micrometer) were used. For phenotype characterization, a panel of specific antibodies (anti CD59, CD61, CD62E, CD19, CD20, CD38, CD138, CD13, CD30) were employed in a FACS analysis. RESULTS: MVs count was significantly higher in patients with CLL, WM, HL, MM, AML and CML (median values 890, 3227, 1218, 914, 656 and 1275 MVs/microliter of serum, respectively) compared to healthy controls (median value 270 MVs/ microliter of serum, ***P In CLL and MDS, through the number of cases was small, MVs counts directly correlated with Rai stage and R-IPSS risk, respectively. As regards vesicles volume, all HMs generated a distinct population of MVs with smaller size when compared with controls. As expected, MVs were consistently Annexin V positive and expressed a common membrane protein, CD59, an ubiquitous complement regulator. Part of MVs originated from platelets (median value 400 CD61 positive/ microliter of serum) and endothelial cells (median value 60 CD62E positive/ microliter of serum). Interestingly, MV phenotype was disease specific: a relevant amount of MVs expressed surface specific proteins related to malignancy. In particular, we found MVs positive for CD19 and/or CD20 in CLL, WM and NHL, CD38 and/or CD138 in MM, CD13 in AML, MPNs and MDS, CD30 in HL. These markers were not significantly expressed on the surface of healthy subject MVs. CONCLUSIONS: In this study, for the first time, serum MVs in a panel of HMs were analysed. We found that patients with various types of HMs release elevated peripheral blood MV levels selectively displaying markers of the underlying malignancy. MVs could be therefore useful as potential novel biomarkers in HMs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Multiple myeloma (MM) remains an incurable malignancy despite important recent advances in treatments. Neo-vascularization entails a crucial aspect of interactions between neoplastic plasma cells (PCs) and their microenvironment. Without it, MM would be unable to grow and progress, and would probably regress to a low-mass steady-state comparable to monoclonal gammopathy of undetermined significance (MGUS). To overcome drug resistance and improve clinical response to novel therapeutic approaches halting both PC growth and the increased bone marrow (BM) microvascular density are needed. In this setting, monoclonal antibodies against MM-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate membrane target structures expressed across all MM cells. The Eph receptors, a large family of receptor tyrosine kinases, have been implicated in many processes involved in malignancy, including alteration of the tumour microenvironment, and in angiogenesis, in both of which EphA3 likely plays an active role. Interestingly, the over-expression of EphA3 is sufficient to confer tumorigenic potential, although probably further mechanisms can occur to abnormally activate the receptor. A first-in-class engineered IgG1 antibody targeting the EphA3 was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EphA3 over-expressing hematological myeloid malignancies refractory to conventional treatment. Methods We investigated the EphA3 role in MM patients in order to define whether it may represent a potential new molecular target for a novel therapeutic approach with a specific anti EphA3 monoclonal antibody. The EphA3 expression was studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with MGUS (MGECs), of control subjects (normal ECs). Moreover, the effects of anti EphA3 antibody in MM were studied in vitro and in vivo in a MM xenograft mouse model. After written informed consent, BM aspirates were collected from 26 MM and 6MGUS patients. Normal ECs were derived from 5 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed both mRNA and protein levels of EphA3 in normal ECs, MGECs and MMECs and in MM cell lines by absolute RT-PCR and by WB coupled to immunofluorescence and FACS analysis respectively. Immunoistochemistry was also performed on MM BM biopsies. The biological effects of EphA3 targeting were studied in vitro silencing (siRNA) the EphA3 mRNA in MMECs and using the anti EphA3 antibody testing them in series of in vitro functional assays including viability, apoptosis, adhesion, migration, wound healing and angiogenesis tests. We further examined the inhibitory capacity of anti-EphA3 Ab on tumor growth in SCID mice bearing MM tumor cell xenografts. Finally, we assessed morphology, vessel density, and apoptosis of excised xenotransplanted tumors. Results Briefly, our data showed that EphA3 mRNA and protein levels are progressively increased from ECs to MGECs, reaching the highest values in MMECs. EphA3 stained intensely and diffusely MM microvessels and PC in MM BM biopsies. The EphA3siRNA MMECs revealed a protein level reduction of approximately 80% when compared to the control. We not detected viability or apoptotic defects, whereas in vitro adhesion, migration and angiogenesis inhibition was evident when compared to the not silenced counterpart. The anti EphA3 antibody inhibited MMECs migration and reduced in vitro MM angiogenesis. In particular, tumour masses developed in xenograft mice treated with anti-EphA3 Abs were smaller in size and showed foci of ischemic-hemorrhagic necrosis, in association with a significant (P < 0.05) reduction in the number of intact tumor microvessels. The proliferative activity was not significantly different from that observed in tumors from untreated or control isotype treated mice, while the apoptotic index was significantly (P < 0.05) increased in comparison with tumors from both groups of mice. Conclusions In this study we have characterized the role of the EphA3in MM patients, providing in vitro and in vivo experimental evidences that support the possibility of using EphA3 as a new molecular target for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3580 Introduction: Although treatment with tyrosine kinase inhibitors (TKIs) has revolutionized the management of adult patients with BCR-ABL1 -positive acute lymphoblastic leukemia (ALL) and significantly improved response rates, relapse is still an expected and early event in the majority of them. It is usually attributed to the emergence of resistant clones with mutations in BCR-ABL1 kinase domain or to BCR-ABL1 -independent pathways but many questions remain unresolved about the genetic abnormalities responsible for relapse after TKI and chemotherapy-based regimens. Patients and methods: In an attempt to better understand the genetic mechanisms responsible for this phenomenon, we have analyzed matched diagnosis-relapse samples from 30 adult BCR-ABL1 -positive ALL patients using high resolution Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI, n=15 pairs and Genome-Wide Human SNP 6.0, n= 15 pairs). Genetic differences were analyzed in terms of copy number changes and loss of heterozygosity (LOH) events. 20 patients were enrolled in clinical trials of GIMEMA AL Working Party and treated with imatinib alone or in combination with conventional chemotherapy (40%) or dasatinib as frontline therapy (60%). The median age at diagnosis was 54 years (range 23–74) and the median blast cell count was 97% (range 60–99). The median time to relapse was 27 months (range, 9–104). 10 patients were treated according to the GMALL trials, a high-dose chemotherapy based protocol in combination with imatinib. The median age at diagnosis was 65 years (range 19–79) and the median leucocyte count was 37300/μl (range 5000 – 220000/μl). The median time to relapse was 9.8 months (range, 3 – 25). Results: First, we compared diagnosis and relapse samples for the presence of macroscopic (> 1.5 MB) copy number alterations (CNA). Novel acquired macroscopic CNAs were detected in 7/20 (35%) TKI relapse cases and included losses of 3p12-p14, 5q34, 9q34, 10q24 and 12p13-p12 and gains of 1q, 9q34-q33 and 22q and in 4/10 (40%) chemotherapy-relapse cases and included losses of 9p21 and 12q21–22 and gains of all chromosome 8 or part of it in 2 patients. Since no common patterns of acquired alterations were observed, it is likely that relapse may be due to a more generalized genetic instability rather than to specific mechanisms. Moreover, chemotherapy did not select resistant clones with higher number of alterations. 8/20 (40%) TKI resistant cases and 4/10 chemotherapy resistant patients harbored the same CNAs present in the matched diagnosis sample (losses of 9p21 in 7 cases, 7p and 22q11 in single cases and gains of chromosomes 1q, 4, 8q, 17q and 21), indicating a common clonal origin. In contrast, in 5/20 (25%) TKI resistant cases and 4/10 (40%) chemotherapy resistant patients macroscopic CNAs present at diagnosis were lost at relapse (losses of chromosomes 7, 11q, 14q, 15q, 16q and 19p and gains of 5q, 8q, 9q34 and 22q11). Thereafter, we compared diagnosis and relapse samples for microscopic CNAs (< 1.5 MB). The alteration most frequently acquired at relapse was loss of the tumor suppressor CDKN2A (53% vs 33 % of diagnosis). Other common acquired CNAs at relapse included gains of ABC transporter genes, such as ABCC1, ABCC6 (1q41) and BCL8 (15q11); losses affected EBF1 (5q33) and IGLL3 (22q11) genes involved in B-cell development, BTG1 (12q21) involved in cell cycle regulation and CHEK2 (22q12) involved in DNA repair. Interestingly, for all relapse cases analysis of IKZF1 deletions, identified in 80% of patients, demonstrated a clonal relationship between diagnostic and relapse samples, suggesting that this alteration is not acquired with relapse but it is maintained with fidelity from diagnosis working as a marker of disease. The majority (92%) of relapse samples harbored at least some of the CNAs present in the matched diagnosis sample, indicating a common clonal origin. Conclusions: Genomic copy number changes evolving from diagnosis to relapse have been identified demonstrating that a diversity of alterations contributes to relapse and with the most common alterations targeting key regulators of tumor suppression, cell cycle control, and lymphoid/B cell development. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, Ateneo RFO grants, PIO program, Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Ottmann:Novartis Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.