Background: EVs are secreted by cells in physiological and pathological conditions and carry selected molecules, such as proteins, DNA and RNA; specific EVs released by neoplastic cells are defined as “tumor-derived extracellular vesicles (tdEVs)”. tdEVs have been demonstrated to be involved in promoting cell growth and survival, shaping the tumor microenvironment and increasing invasiveness and metastatic activity. Thanks to their systemic availability in body fluids and their role in tumor pathogenesis, tdEVs have gained attention in the liquid biopsy area, as a possible biomarker for tumor diagnosis, prognosis and monitoring and also as possible therapeutic targets. Beyond being a target for the capture of CTCs, the Epithelial Cell Adhesion Molecule (EpCAM) was also recently employed for the detection of EVs released by tumors. Here we present a method (for research use only) based on the CellMag™ technology (Menarini Silicon Biosystems, MSB) to carry out parallel enrichment of EVs and CTCs from whole blood through an EpCAM-based magnetic enrichment. Method: Whole blood samples from stage III/IV prostate (n=5) or breast cancer patients (n=6) and healthy donors (n=4) were collected in CellRescue™ tubes (MSB). After centrifugation, EpCAM+ EVs and CTCs were enriched from the plasma and cell fraction respectively, using the CellMag™ Epithelial CTC kit (MSB); CTCs were counted at the CellTracks Analyzer II (MSB). EVs were enriched for qualitative evaluation by Transmission Electron Microscopy (TEM) and for miRNA analysis by Real-time PCR, using the TaqMan™ Advanced miRNA Human Serum/Plasma panel. Results: CTCs counts ranged from 0 to 68; TEM analysis showed a higher density of EpCAM+ extracellular microvesicles (0,1-1µm) in breast cancer patients’ samples (n=2) compared to controls (n=2). Endogenous hsa-miR-16-5p control was detected in all analyzed samples (n=9 patients’ samples, n=2 controls). One control and two patients’ samples were tested for a panel of 188 serum/plasma miRNAs. In both the breast and the prostate cancer patients a higher percentage of miRNAs was found with respect to healthy control (31%, 32% vs. 17%). Among these, several oncogenic miRNAs with diagnostic and prognostic value (such as hsa-miR-125b-5p or hsa-miR-26a-5p) were exclusively found in patients’ samples, and further confirmed to be related to ErbB2 pathway in breast cancer using the miRPathDB 2.0 database, despite the CTCs count was 0 for both these patients’ samples. Conclusions: The described multi-target liquid biopsy approach enabled the combined enrichment of EpCAM+ CTCs and plasma EpCAM+ EVs from a single blood sample, along with their molecular analysis. Citation Format: Giulia Bregola, Martina Masotti, Valentina Mattei, Martina Moras, Claudio Ratti, Francesco Picardo, Cecilia Simonelli, Nicolò Manaresi, Francesca Fontana. Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3364.

e14726 Background: CD205 is a type I transmembrane surface protein belonging to the mannose receptor family with propensity for antigen uptake/internalization. CD205 is expressed in different cancer histotypes and seems to have a role in migration and invasion. For these features CD205 is an ideal candidate to be exploited as an ADC target. MEN1309/OBT076 (herein as MEN1309) is a humanized IgG1 antibody against CD205, conjugated through a cleavable linker to DM4. It has shown impressive anti-tumor activity when tested in high/low CD205 expressing xenografts for solid tumours and NHL. MEN1309 is under assessment in Phase I trials in EU (NCT03403725) and as OBT076 in USA. Methods: A retrospective evaluation of CD205 expression in 103 triple negative breast carcinomas (TNBC), 50 pancreatic adenocarcinomas (PAC) and 100 bladder urothelial carcinomas (UC) was performed at INT in Milan, Italy. Immunohistochemistry was performed on one FFPE representative sample per case, using a commercial anti-CD205 antibody. A semiquantitative score was applied to measure CD205 expression. Absence of staining tumor cells was scored as negative. Positivity was scored based on the percentages of neoplastic cells (NCs), with intensity of cytoplasmic staining ranging from 1 (weak) to 3 (strong). Membrane staining in NCs, immunoreactivity of the intratumoral/peritumoral inflammatory component and of normal tissue were scored as absent or present. Results: TNBC: 85% were positive, 70% in > 5% of NCs; 14% showed 2+/3+ staining intensity in > 50% of NCs. 57% of cases showed a membrane positivity. PAC: 100% were positive in > 5% of NCs; 50% showed 2+/3+ staining intensity in > 50% of NCs. 98% of cases showed a membrane positivity. UC: 67% were positive, 60% in > 5% of NCs; 10% showed 2+/3+ staining intensity in > 50% of NCs. 29% of cases showed a membrane positivity. Inflammatory component and normal tissue were positive in almost all cases. Conclusions: CD205 is heterogeneously expressed in the majority of TNBC and UC and in all PAC cases. The results of this study support the clinical investigation of MEN1309 in these tumor types.

TPS1101 Background: MEN1611 is a potent, selective class I inhibitor of PI3K, a key enzyme in the transduction of various extracellular growth factor signals essential for cell survival and apoptosis. The discovery in human cancers of frequent PIK3CA mutations, that have been linked to a worse outcome in advanced HER2-positive breast cancer, makes PI3K an attractive therapeutic target. Preclinical and clinical evidences support the development of MEN1611 in combination with other agents in the context of solid tumors. Methods: B-PRECISE-01 is an open-label, multicenter, phase Ib dose escalation study in patients with PIK3CA mutant tumors, HER2-positive advanced or metastatic breast cancer which has progressed after at least 2 lines of anti-HER2 based therapy. PIK3CA mutations are assessed centrally by real-time PCR assay in DNA derived from archived tumor samples. MEN1611 will be administered orally BID for continuous 28-day cycles until disease progression, in combination with weekly IV infusions of trastuzumab. In addition, HR-positive postmenopausal patients will also be treated with fulvestrant. After the completion of the dose escalation phase (Step 1), the study will continue in an expansion cohort (Step 2) testing the Recommended Phase 2 dose (RP2D) in a total of 15 patients in each of the treatment groups. The primary study objective is to assess combination safety and select RP2D. Secondary objectives include assessment of pharmacokinetics and pharmacodynamics, preliminary clinical activity of MEN1611 in combination with trastuzumab +/- fulvestrant, and correlation with PIK3CA mutations and other relevant cancer genes mutational status. Adverse events will be graded according to NCI CTCAE v4.03. Responses will be evaluated according to RECIST v1.1. Study variables will be presented by dose-cohort and overall using appropriate descriptive statistics. The enrollment began in July 2018 at European sites and US sites will shortly participate; up to date the first dose cohort level has been achieved. Clinical trial information: NCT03767335.

Summary Background Faecal microbiota transplantation ( FMT ) is a highly effective treatment against recurrent Clostridium difficile infection. Far less evidence exists on the efficacy of FMT in treating severe Clostridium difficile infection refractory to antibiotics. Aim To compare the efficacy of two FMT ‐based protocols associated with vancomycin in curing subjects with severe Clostridium difficile infection refractory to antibiotics. Methods Subjects with severe Clostridium difficile infection refractory to antibiotics were randomly assigned to one of the two following treatment arms: (1) FMT ‐S, including a single faecal infusion via colonoscopy followed by a 14‐day vancomycin course, (2) FMT ‐M, including multiple faecal infusions plus a 14‐day vancomycin course. In the FMT ‐M group, all subjects received at least two infusions, while those with pseudomembranous colitis underwent further infusions until the disappearance of pseudomembranes. The primary outcome was the cure of refractory severe Clostridium difficile infection. Results Fifty six subjects, 28 in each treatment arm, were enrolled. Twenty one patients in the FMT ‐S group and 28 patients in the FMT ‐M group were cured (75% vs 100%, respectively, both in per protocol and intention‐to‐treat analyses; P = 0.01). No serious adverse events associated with any of the two treatment protocols were observed. Conclusions A pseudomembrane‐driven FMT protocol consisting of multiple faecal infusions and concomitant vancomycin was significantly more effective than a single faecal transplant followed by vancomycin in curing severe Clostridium difficile infection refractory to antibiotics. Clinical‐Trials.gov registration number: NCT 03427229.

Introduction: Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. The high heterogeneity of MM cells is one of the major cause of disease relapse. Detection of circulating MM cells (CMMC) from peripheral blood is a useful procedure to investigate tumor heterogeneity and provides a painless alternative to the classic bone marrow biopsy to monitor disease progression. Here we demonstrate that the synergy between CellSearch® (CS) and DEPArray™ (DA) technologies can be used to identify, isolate and characterize at the genetic level single and pure CMMCs .

Abstract Introduction Immune-checkpoint blockade has emerged as an effective therapeutic strategy in solid tumor and in hematologic malignancies, including classical Hodgkin Lymphoma (cHL). cHL represents about 11% of all malignant lymphoma and it is generally highly curable with standard frontline therapies, although about 20% of the patients will relapse or become refractory after initial treatment. The hallmark of cHL is the presence of malignant Hodgkin and Reed-Sternberg Cells (HRS) that represent only a small fraction (about 1%) of the surrounding heterogeneous immune infiltrate. Despite this extensive inflammatory microenvironment, HRS are able to escape immune surveillance using several mechanisms, including the overexpression of PD-1 ligands (PD-Ls) that bind PD-1 on reactive T-cells, inhibiting their activity and proliferation and causing ultimately T-cell exhaustion. The PD-Ls expression is upregulated in a dose-dependent manner by copy number alterations of chromosome 9p24.1, a locus encoding for PD-L1/PD-L2 as well as JAK2, which further enhances PD-Ls expression through JAK2/STAT pathway. Here we present a method for the isolation and the genetic characterization of single purified HRS, which overcomes the limitations posed by the low tumor cellularity of cHL biopsies and gives an estimation of inter-tumor and intra-tumor heterogeneity which may be useful to guide immune treatment selection. Methods FFPE tissue sections from 4 cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Since CD30 is not expressed exclusively by malignant cells, beyond the positivity to CD30 and PD-L1 HRS were selected according to morphological criteria, such as cell size and the presence of nuclei with ploidy higher than the surrounding lymphocytes. DEPArray™ NxT system (Menarini Silicon Biosystems) was used to isolate single target cells. After recovery, single cells were whole genome amplified (Ampli1™ WGA, Menarini Silicon Biosystems), and genome-wide copy-number alterations (CNAs) profiles were obtained using Ampli1™ LowPass kits (Menarini Silicon Biosystems) on Illumina® and Ion Torrent™ platforms. Results For each patient, at least 8 HRS cells and infiltrating lymphocytes were identified and isolated from lymphoid tissue using DEPArray™ NxT system. Copy-number analyses of recovered cells allowed us to precisely discriminate HRS, characterized by extensive gains and losses, from non-tumor cells, showing flat profiles as expected (Fig.1). Ploidy of HRS was automatically determined, based on best-fitting of profiles with underlying copy number levels. Hierarchical clustering showed that some alterations are highly conserved among patients, e.g. the region containing PD-L1/PD-L2/JAK2 has several copy gains in the majority of malignant cells. Interestingly, these alterations show high variable copy-number levels between different HRS even in the same patient, ranging from few copy-gains to amplifications, suggesting some level of heterogeneity. Different CNAs are also detected in regions containing genes belonging to pathways already known to be altered in cHL, like REL/NFKB and JAK/STAT pathways, which may be involved in the constitutive activation of proliferative and antiapoptotic phenotype of HRS. Conclusion Single HRS sorting combined with low-pass whole genome sequencing offer a valuable tool to uncover genetic alterations hidden by the massive cHL immune infiltrate and to estimate inter-tumor and intra-tumor heterogeneity in cHL patients. Considering that PD-Ls locus amplifications are associated with advanced stages of the disease and with a shorter progression free survival, the analysis of purified HRS could be helpful for patient stratification for the adoption of immune therapy. Disclosures Mangano: Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Garonzi:Menarini Silicon Biosystems: Employment. Lanzellotto:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Ceccolini:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fici:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Gallerani:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

e14039 Background: CD205 is a type I C-type lectin receptor normally expressed on various APC and some leukocyte sub-populations, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization. These features make CD205 ideal to be exploited as target of a novel ADC. MEN1309/OBT076 is a humanized IgG1 antibody against CD205, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. Indeed, impressive results were obtained in high and low CD205 expressing xenografts for solid tumours and NHL. In vitro experimental model describing the by-stander effect of MEN1309 was implemented. Methods:A retrospective analysis to evaluate the CD205 expression on archival NHL biopsies was performed in the CRO-National Cancer Institute, Aviano IT. An immunohistochemistry (IHC) was performed on 1 slide per case, using a commercial antibody NCL-L-DEC205 (clone 11A10, Leica Biosystems). All slides were visually evaluated and a validated staining scoring system was applied, ranging from negative score 0 to strong immunostaining score 3+. Percentages (from 0 to 100%) of negative and positive tumor cells (scored as follows: 1-25% = score 1; 26-50% = score 2; 51-75% = score 3; 76-100% = score 4) were reported for each individual slide.Evaluation of correlations between CD205 expression and clinical characteristics is ongoing. Results: 100 cases of Diffuse Large B Cells Lymphomas (DLBCL) and 33 of Follicular Lymphomas (FL) were analysed. 60% of DLBCL cases were positive for CD205 and 39% had more than 25% of positive cells (score ≥2), while the 16% showed 2+/3+ staining intensity. 79% of FL cases were CD205 positive and 66% had more than 25% of positive cells, while the 27% showed 2+/3+ staining intensity. Conclusions: CD205 is heterogeneously expressed in the majority of DLBCL and FL cases. Data on the correlation between CD205 expression and clinical features will be discussed.MEN1309 has shown anti-tumour activity in vitro and in vivo even in low antigen expressing tumours. Hence CD205 expression among NHL, even if heterogeneous, makes, both DLBCL and FL, promising settings in which to explore MEN1309 activity in an upcoming FIH trial.

TPS3100 Background: Defucosylated mAbs enhance antibody dependent cell-mediated cytotoxicity (ADCC) through an improved affinity for Fc receptors. MEN1112/OBT357 is a humanized, defucosylated mAb targeting Bst1/CD157, a GPI-anchored transmembrane protein highly expressed on blasts of AML patients either at primary diagnosis or relapse. Preclinical findings show that MEN1112/OBT357 has the potential to exert powerful ADCC against AML (Aud et al ASH 2014; Venditti et al ASH 2014). Methods: Multi-center, non-randomized, 3+3 dose escalation/expansion cohort trial of MEN1112/OBT357 intended to recruit approximately 50 adult patients (pts) with R/R AML. Refractory pts must have failed ≥ 1 cycle of cytotoxic chemotherapy or hypomethylating agents. A baseline WBC count ≤ 10 x 109/L is required (pre-treatment with hydroxyurea is permitted). Main exclusion criteria are acute promyelocytic leukemia, hematopoietic stem cell transplant within 3 months prior to screening and active central nervous system involvement. MEN1112/OBT357 is given intravenously at 5 incremental doses on Days 1, 8, and 15 in a 21-day cycle for a total of 2 cycles; monthly maintenance is allowed in pts achieving clinical benefit. Primary objective is to identify dose limiting toxicities and maximum tolerated dose of MEN1112/OBT357 in pts with R/R AML; secondary objectives include (1) clinical pharmacokinetics, (2) potential immunogenicity, (3) clinical activity (complete- and composite complete remission rate, best response rate, overall survival) in treated pts and (4) its correlation with target expression/saturation, natural killer cells status, ex vivo activity at baseline and clinical/biological AML features. AEs will be graded according to NCI CTCAE v. 4.03 guidelines. Study variables will be presented by dose-cohort and overall using appropriate descriptive statistics. Efficacy will be evaluated in each cohort in pts completing the first cycle and with ≥ 1 post-cycle assessment using modified IWG 2003 criteria. The individual study duration is 6 months. The enrolment began on December 2014. Clinical trial information: NCT02353143 Clinical trial information: NCT02353143.

Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. Bst1/CD157 is a protein belonging to the ADP-ribosyl-cyclase family expressed on monocytes and neutrophils. This antigen was shown to be also expressed in peripheral blood (PB) and bone marrow (BM) blasts of acute myeloid leukemia (AML) patients either at primary diagnosis or at relapse(1,2,3). MEN1112/OBT357 is a humanized, de-fucosylated antibody targeting Bst1/CD157 with high affinity and developed to generate antibody dependent cell-mediated cytotoxicity (ADCC) response against AML blasts. Peripheral blood (PB) and bone marrow (BM) samples of 38 AML patients (29 at diagnosis, 6 at relapse, 3 resistant), have been analyzed for the expression of Bst1/CD157 on AML blast cells by fluorescence-activated cell sorting (FACS) using a PE conjugated form of MEN1112/OBT357. Bst1/CD157 expression has been confirmed in 91% and 96% of PB and BM AML samples, respectively. Furthermore, statistical analysis demonstrated that monocyte-oriented blasts are characterized by a brighter expression of Bst1/CD157 compared to blasts of non-monocytic lineage. The efficacy of MEN1112/OBT357 in depleting AML blasts was evaluated through FACS analysis in an autologous ex vivo assay performed on whole blood. The assay was set up using blood from healthy donors exposed to 10 μg/ml Rituximab for 18 hours to induce B cell depletion. In the same conditions, the ability of 10 μg/ml MEN1112/OBT357 to induce blasts depletion was tested.In whole PB,MEN1112/OBT357 was able to deplete AML blasts in 15/32 evaluable cases (46%). In BM, MEN1112/OBT357 induced blast depletion in 9/24 evaluable cases (36%). Interestingly, higher depletion rate was observed in relapse/refractory patients. When CD16A-158Phe/Val polymorphisms were analyzed utilizing a sequence based typing (SBT) assay, it was demonstrated that AML blast depletion was independent by FcRg polymorphism. Furthermore, no significant shedding of Bst1/CD157 antigen was observed in sera from AML patients, compared to the sera from patients with other hematologic diseases or healthy donors.

BACKGROUND Serum uric acid (sUA) control is of key relevance in tumor lysis syndrome (TLS) prevention as it correlates with both TLS and renal event risk. We sought to determine whether febuxostat fixed dose achieves a better sUA control than allopurinol while preserving renal function in TLS prevention. PATIENTS AND METHODS Patients with hematologic malignancies at intermediate to high TLS risk grade were randomized to receive febuxostat or allopurinol, starting 2 days before induction chemotherapy, for 7-9 days. Study treatment was blinded, whereas daily dose (low/standard/high containing allopurinol 200/300/600 mg, respectively, or fixed febuxostat 120 mg) depended on the investigator's choice. The co-primary end points, sUA area under curve (AUC sUA1-8) and serum creatinine change, were assessed from baseline to day 8 and analyzed through analysis of covariance with two-sided overall significance level of 5%. Secondary end points included treatment responder rate, laboratory and clinical TLS incidence and safety. RESULTS A total of 346 patients (82.1% intermediate TLS risk; 82.7% assigned to standard dose) were randomized. Mean AUC sUA1-8 was 514.0 ± 225.71 versus 708.0 ± 234.42 mgxh/dl (P < 0.0001) in favor of febuxostat. Mean serum creatinine change was -0.83 ± 26.98% and -4.92 ± 16.70% for febuxostat and allopurinol, respectively (P = 0.0903). No differences among secondary efficacy end points were detected. Drug-related adverse events occurred in 6.4% of patients in both arms. CONCLUSION In the largest adult trial carried out in TLS prevention, febuxostat achieved a significant superior sUA control with one fixed dose in comparison to allopurinol with comparable renal function preservation and safety profile. CLINICAL TRIAL REGISTRATION NCT01724528.

The aim of this study was to explore the efficacy and toxicities of a combined regimen of bevacizumab plus immunotherapy and chemotherapy (BIC) and the circulating T regulatory cells (Treg) in metastatic renal cell cancer (mRCC). Nephrectomized mRCC patients were enrolled into a multicenter single-arm dose-finding study with five escalated dose levels of chemotherapy with intravenous gemcitabine and 5-fluorouracil associated with fixed intravenous doses of bevacizumab, subcutaneous low doses of interleukin-2, and interferon-α-2a. An expanded cohort (phase II study) was treated at the recommended dose for additional safety and efficacy information according to minimax Simon two-stage design. Blood samples for Treg were collected and evaluated by fluorescence-activated cell sorting (FACS) analysis on cycle 1. Fifty-one patients were entered to receive one of five dose levels. Median age was 58 years (male 67 %, pretreated 49 %): 15 patients were low risk according to Memorial Sloan-Kettering Cancer Center (MSKCC) criteria, while 27 and nine were respectively intermediate- and high-risk patients. More frequent grade 3 and 4 toxicities included nonfebrile neutropenia, thrombocytopenia, and fever. Among patients evaluable for response (49), 29.5 % had partial response and 37 % stable disease. Overall median time to progression and median overall survival were 8.8 and 22.67 months, respectively. We observed a rapid increase in the percentage of Treg after immunotherapy and a reduction after bevacizumab only in patient who obtained a partial response or stable disease. The BIC was feasible, well tolerated, and shown interesting activity. Further studies are needed to explore if Treg could have a role in clinical response in mRCC treated with bevacizumab.

Background Febuxostat, a potent and selective xanthine oxidase inhibitor, showed a significantly superior serum uric acid (sUA) control during chemotherapy (CT) in comparison to Allopurinol (Spina M. et al, ASCO 2014 and Annals of Oncology in press) when given as 120 mg oral fixed daily dosed and has been recently approved in the European Union (EU) for the prevention and treatment of hyperuricemia in adult patients undergoing CT for hematologic malignancies (HM) at intermediate- high Tumor Lysis Syndrome (TLS) risk, with recognition of the high relevance of its clinical benefit. Some pediatric HM are highly prone to develop TLS; an age-appropriate drug formulation for TLS prophylaxis is lacking in this population, resulting in an unmet medical need that Febuxostat is going to address. The similarities between adult and pediatric patients in terms of TLS pathogenesis, clinical manifestations and current management allow using pharmacokinetic (PK) information to extrapolate clinical efficacy and safety from adult to pediatric patients, as well as within the different pediatric ages (EMEA/CHMP/EWP/147013/2004).

Background: Tumor lysis syndrome (TLS) is an oncologic emergency characterized especially by elevated serum uric acid (sUA). Febuxostat (FEB) is an orally administerd selective xanthine oxidase inhibitor to reduce sUA.Methods: This was a randomized, double blind phase III trial of FEB vs Allopurinol (ALL) in terms of control of sUA level and preservation of renal function in patients undergoing chemotherapy (CT) for hematologic malignancies at intermediate to high risk of TLS. Patients were stratified according to TLS risk and sUA level to FEB or ALL starting from 2 days prior CT and continued for 7-9 days. Assigned treatment was blinded, while daily dose level (low/standard/high containing ALL 200/300/600 mg or fixed FEB 120) was upon investigator's choice. Primary endpoints were sUA area under the curve (AUC sUA1-8) and change in serum creatinine (sC) level both from baseline to Day 8, analyzed through ANCOVA including treatment and stratification factors as covariates. Secondary endpoints were response rate (sUA ≤ 7.5 mg/dL from CT start to Day 8), incidence of laboratory and clinical TLS and safety. The study was run in 79 sites in Europe and Brazil (NCT01724528).Results: 346 patients were included with similar baseline demographics in both groups. 82.1% of patients were at intermediate risk of TLS, 87.6% had a baseline sUA ≤ 7.5 mg/dl and 82.7% received standard dose level. Intention to treat (ITT) analysis: mean AUC sUA1-8 (mgxh/dl) was significantly lower in FEB arm (514.0 +/- 225.71 vs 708.0 +/- 234.42; p <0.0001). No significant difference in mean sC change(%) occurred between FEB and ALL arms (-0.83 +/- 26.98 vs -4.92 +/- 16.70 respectively, p = 0.0903). No significant difference was detected among secondary efficacy endpoints. Incidence of all adverse events (AEs) and related AEs was 67.6% vs 64.7% and 6.4% vs 6.4% in FEB and ALL arm, respectively.Conclusions: In this largest TLS prevention trial FEB proved to be significantly superior over ALL with a 28% lower exposure to sUA during CT, moreover without requirement for any dose adjustment in patients with preexisting mild or moderate renal impairment. FLORENCE study results were the basis for the FEB European approval for the TLS prevention and treatment granted in May 2015. Background: Tumor lysis syndrome (TLS) is an oncologic emergency characterized especially by elevated serum uric acid (sUA). Febuxostat (FEB) is an orally administerd selective xanthine oxidase inhibitor to reduce sUA. Methods: This was a randomized, double blind phase III trial of FEB vs Allopurinol (ALL) in terms of control of sUA level and preservation of renal function in patients undergoing chemotherapy (CT) for hematologic malignancies at intermediate to high risk of TLS. Patients were stratified according to TLS risk and sUA level to FEB or ALL starting from 2 days prior CT and continued for 7-9 days. Assigned treatment was blinded, while daily dose level (low/standard/high containing ALL 200/300/600 mg or fixed FEB 120) was upon investigator's choice. Primary endpoints were sUA area under the curve (AUC sUA1-8) and change in serum creatinine (sC) level both from baseline to Day 8, analyzed through ANCOVA including treatment and stratification factors as covariates. Secondary endpoints were response rate (sUA ≤ 7.5 mg/dL from CT start to Day 8), incidence of laboratory and clinical TLS and safety. The study was run in 79 sites in Europe and Brazil (NCT01724528). Results: 346 patients were included with similar baseline demographics in both groups. 82.1% of patients were at intermediate risk of TLS, 87.6% had a baseline sUA ≤ 7.5 mg/dl and 82.7% received standard dose level. Intention to treat (ITT) analysis: mean AUC sUA1-8 (mgxh/dl) was significantly lower in FEB arm (514.0 +/- 225.71 vs 708.0 +/- 234.42; p <0.0001). No significant difference in mean sC change(%) occurred between FEB and ALL arms (-0.83 +/- 26.98 vs -4.92 +/- 16.70 respectively, p = 0.0903). No significant difference was detected among secondary efficacy endpoints. Incidence of all adverse events (AEs) and related AEs was 67.6% vs 64.7% and 6.4% vs 6.4% in FEB and ALL arm, respectively. Conclusions: In this largest TLS prevention trial FEB proved to be significantly superior over ALL with a 28% lower exposure to sUA during CT, moreover without requirement for any dose adjustment in patients with preexisting mild or moderate renal impairment. FLORENCE study results were the basis for the FEB European approval for the TLS prevention and treatment granted in May 2015.

e20677 Background: Harmonization of clinical practice and standard of care allow multicenter pivotal trials to be successfully run both in EU and NOEU Countries, with patients’ recruitment increasing in favor of NOEU sites. Comparative data on the performance of EU vs. NOEU investigators in terms of compliance to study protocol and data collection are now offered by the FLORENCE study (a pivotal phase III study of Febuxostat vs. Allopurinol for TLS prevention in hematologic malignancies; NCT01724528) that included 75 European sites distributed in both EU (HR, CZ, DE, HU, IT, PL, RO, ES) and NOEU (RU, RS, UA) contributing with 193 and 152 randomized patients (0.35 and 0.44 patients/month/site), respectively. Methods: Compliance of sites to FLORENCE was evaluated by counting all protocol violations (APVs) and major protocol violations (MPVs) weighted for the number of randomized patients grouped by EU and NOEU sites. Distribution of APVs and MPVs was compared using a Chi Square test at 5% significant level. Additionally, distribution of the most affected MPVs subcategories, namely eligibility of randomized patients, errors in treatment intake, use of prohibited medications, compliance to stratification factors/safety data recording (SFSDR), compliance to scheduled Visits/Assessments (SVAs) and timely reporting of serious adverse events (weighted for the number of patients as per APVs and MPVs) was compared using a Fisher’s Exact test at 5% significant level. Results: APVs were 815, 32.4% in NOEU vs. 67.6% in EU sites (p = .0686). MPVs were 68, 17.7% in NOEU vs. 82.3% in EU (p < .0001), however the difference was due to only two MPVs subcategories, namely compliance to SFSDR (12.5% vs. 87.5% for NOEU and EU sites respectively; p = .0056) and to SVAs (4.2% vs. 95.8% for NOEU and EU sites respectively; p = .0001). Conclusions: Overall compliance to FLORENCE study was high with limited MPVs; NOEU sites had less MPVs affecting compliance to SFSDR and to SVAs, which may reflect difference in assigned site resources. The FLORENCE study provides the first evidence that NOEU and EU sites run a multicenter pivotal trial with high and almost comparable study compliance Clinical trial information: NCT01724528.

MEN1112 is a new humanized, defucosylated, monoclonal antibody (mAb) with high specific affinity for Bst1/CD157 antigen. Bst1/CD157 antigen expression on blood cells of acute myeloid leukemia (AML) patients and healthy donors was investigated by flow cytometry using a PE-labeled MEN1112 mAb. Twenty three patients affected with AML have been tested, 18 at diagnosis, 4 at relapse, 1 resistant. In 16 out of 23 patients both bone marrow (BM) and peripheral blood (PB) specimens were evaluated. PB and BM samples from healthy donors (N=2) were also assessed. In healthy donors and AML patients, PB and BM lymphocytes were Bst1/CD157 antigen negative whereas monocytes and neutrophils showed a distinct pattern of MEN1112 mean fluorescence intensity (MIF), with monocytes having the brightest expression. In the stem cell compartment, an intermediate level of MFI was observed (p<0.001). Prevalence of expression of the antigen on patients’ samples was over 90%. On AML blast cells from each single patient, MEN1112 expression was heterogeneous; indeed the antigen was expressed on 50%±29% and 47% ±39% of blasts in BM and PB, respectively. The anti-leukemia activity of MEN1112 on AML cell lines was tested, in vitro, by a flow cytometry-based cell depletion assay in the presence of lymphokine activated immune effector cells: a strong depletion of leukemia cells was demonstrated suggesting that MEN1112 might exert anti-leukemia activity through antibody dependent cell-mediated cytotoxicty (ADCC). The activity of MEN1112 was also tested ex vivo on whole PB showing that the antibody was able to deplete AML blasts in 9 out of 23 patients (47.4 %) with a percentage of AML blast depletion ranging between 4.3 - 66 %. In whole BM from 2 out of 11 evaluable patients MEN1112 induced 68% and 23% of AML blast depletion. Bst1/CD157 shedding assessment showed that, in the sera from AML samples, the concentration of Bst1/CD157 antigen was comparable to that measured in healthy donors. Moreover, since Fcγ receptor (CD16) genotype might be a factor contributing to the antitumor activity of the antibody, the polymorphism CD16-158Phe/Val was analyzed. Five out of 19 samples were homozygous for CD16-158 Phe; 5 were homozygous for CD16- 158 Val and 9 were heterozygous for CD16-158. MEN1112-induced blast depletion was observed for each genotype. Moreover, in an attempt to identify the determinant of MEN1112 activity, % in PB of blast (antigen positive), NK cells or residual normal cells were evaluated. Altogether, these results are promising suggesting the potential for an ADCC-mediated MEN1112 antileukemic effect and they support the clinical development of MEN1112. Disclosures Venditti: Menarini Ricerche SpA: Research Funding. Buccisano: Menarini Ricerche SpA: Research Funding. Del Principe: Menarini Ricerche SpA: Research Funding. Coppola: Menarini Ricerche SpA: Research Funding. Palomba: Menarini Ricerche SpA: Research Funding. Aureli: Menarini Ricerche SpA: Research Funding. Arriga: Menarini Ricerche SpA: Research Funding. Bellarosa: Menarini Spa: Employment. Bressan: Menarini Ricerche SpA: Employment. Manzini: Menarini Ricerche SpA: Employment. Simonelli: Menarini Ricerche SpA: Employment. Binaschi: Menarini Ricerche SpA: Employment. Amadori: Menarini Ricerche SpA: Research Funding. Sconocchia: Menarini Ricerche SpA: Research Funding.