ALBERT

Description: Background: ImMucin is a 21-mer long peptide therapeutic vaccine encoding the entire signal peptide (SP) domain of the MUC1 tumor-associated antigen, which is over expressed by most hematological tumors including multiple myeloma (MM). Results from first in man phase I/II study (VAXIL-001) demonstrated that 100 micrograms (ug) ImMucin combined with 250ug hGM-CSF is highly tolerable and can induce a robust, diversified MUC1 SP-specific T cell and B cell immunity in most patients, across major histocompatibility complex barrier. ImMucin vaccination also led to a significant decline in soluble MUC1 (sMUC1) in 9/10 patients with abnormal prevaccination levels, accompanied with disease stabilization. Methods: This current follow–up phase I/II study investigated the long term safety, quality of Life (QOL) (primary endpoint) and efficacy (both immunity and clinical response a secondary endpoint), of ImMucin, obtained in VAXIL-001 study, in participant MM subjects that didn’t required further treatment post vaccination. Patients were being evaluated once every 3 months in the first year and twice a year at the following years till progression or Q4-2015. QOL easement was assessed every visit using the SF-36 questionnaire. Immunomonitoring analysis included; CD62L+effecter memory marker, ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells and anti-MUC1 SP antibody production. Clinical response was assessed according to the international myeloma working group response criteria sMUC1 levels were also evaluated every visit. Results: Long- term safety was encouraging, with no evidence for any adverse events. Additionally, the average QOL score was maintained throughout the study. Immunity, determined by INF-g production by both CD4+ and CD8+ T-cells and anti-MUC1 SP antibodies, was maintained for 6-10 and 10-12 months post vaccination respectively. The predominant T-cell phenotype during the post vaccination period was characterized with CD62L+ CD4 and CD8+ T-cells. At 13-41.3 months after the completion of the VAXIL-001 study (measured for first and last patients, respectively), 12/15 patients are alive. Median time from first vaccination was 24 months (range 2.5-41.3), at which time 10/15 patients had a PD. Disease progressed during the vaccination period (up to week 26) in 4/15 patients and during the follow up period in 6/15 patients. Notably, 5/15 patients maintained their CR (n=3) or SD (n=2). Notably, clinical response was associated with low-intermediate PDL-1 bone marrow (BM) levels prior and post vaccination, while high PDL-1 BM levels were associated with temporary response and progression. Moreover, sMUC1 levels have moderately increased during the follow up period (x〉600pg/ml), though didn’t reach initial prevaccination values. Conclusions ImMucin demonstrated an encouraging short and long-term safety profile. Vaccination induced a remarkable anti-MM immune response. However, immunity was transient suggesting a need for boosting. Interestingly, durable disease stabilization was achieved in third of the patients, continuing despite loss of immune response in peripheral blood. Moreover, the encouraging responses to subsequent therapies employed at clinical progression, suggest this novel approach to be potentially valuable in the setting of maintenance and/or early biochemical progression. Disclosures Carmon: Vaxil BioTherapeutics Ltd. : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kovjazin:Vaxil BioTherapeutics Ltd. : Employment. Shapira:Vaxil BioTherapeutics Ltd. : Consultancy.

Description: Introduction: Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy; more than 90% are B cell lymphomas and express CD-20 antigen. Rituximab® (RTX) is an IgG1κ chimeric anti-CD20 mAb that binds specifically to the CD20-antigen on B lymphocytes. Our group previously reported that 99mTc-RTX represents a promising molecular imaging agent for NHL [1]. When used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor/blood and tumor/normal tissue ratios with renal clearance. The development of radiolabeled Fab´s directed against specific targets may become a new strategy for NHL staging and surveillance. Objective: To radiolabel Fab´s (RTX) with 99mTc and to perform its chemical and biological evaluation. Methodology: We performed antibody fragmentation with papain and, once purified, fragments were identified by MaldiTOF/TOF and derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl2.2H2O was added to Fab´s (RTX)-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity was determined by HPLC. The in-vitro radiochemical stability of the radiolabeled Fab´s were analyzed in saline and serum up to 4 h. In-vitrobinding and competition assays were performed using Ramos and Raji cell lines up to 90 min. Biodistribution studies were evaluated in normal Balb/c mice and in Raji tumor-bearing Nude mice at 0.5 and 1 h. Results: Radiochemical purity of radiolabeled Fab´s were ≥90%. The in-vitro radiochemical stability studies showed that the radioconjugate was stable and no significant transchelation was detected. In-vitro binding and competition assays confirm that after its derivatization and radiolabeling, Fab´s (RTX) retained its specificity of binding to CD-20 antigen. This results confirm that Fab´s (RTX) affinity for CD20+ NHL cells remained unaffected after its derivatization. In-vivobiodistribution studies show that radiolabeled Fab´s has renal uptake with neglectable uptake in other organs, indicating that the primary route of clearance is renal. Lymph-node/muscle ratios of 4.00 and 2.55 at 0.5 and 1 h post injection, respectively. Conclusions: Fab´s (RTX) were easily and rapidly labeled demonstrating good stability and radiochemical purity. Based on lymph-node uptake and lymph-node/muscle ratios, 99mTc-HYNIC-Fab´s (RTX) may be useful for tumor molecular imaging agent for NHL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy and the fifth leading cause of cancer death in the world; over 90% are B cell lymphomas and express CD-20 surface antigen [1]. CD-20 antigen is a transmembrane protein of 33-37 kDa protein located on the surface of pre-B and mature lymphocytes B [2, 3]. It is expressed in 90% of B-cell NHL [4]. Anti-CD20 antibody (Rituximab®), a specific chimeric monoclonal antibody directed against CD20 on B lymphocytes and the first monoclonal antibody approved for the treatment of CD20+ NHL. The development of new agents directed against specific targets may improve the sensitivity and specificity of imaging for its staging. Objective To radiolabel Rituximab with 99mTc and evaluate its properties as a potential imaging agent for NHL. Methodology Rituximab was derivatized with succinimidyl-hydrazinonicotinamide (Suc-HYNIC) and MALDI TOF/TOF was used to confirm the level of HYNIC conjugation to Rituximab. This antibody was radiolabeled with 99mTc using a mixture of Tricine/SnCl2.2H2O. Radiochemical purity was determined by ITLC-SG, size exclusion chromatography and HPLC. In-vitro stability was studied in solution; serum and L-Cysteine up to 24 h. In-vitro binding and competition assays were performed with Ramos and Raji NHL cell lines up to 120 min and were analyzed by laser confocal microscopy. Ramos and Raji cells were incubated with buffer to evaluate any degree of autofluorescence. Biodistribution studies were performed in normal Balb/C mice at 1, 4, 24 and 48 h (n = 5). Results HYNIC- Rituximab was efficiently labeled with 99mTc. The in-vitro radiochemical stability studies of the radiolabeled antibody showed that the complexes formed were stable and no significant transchelation was detected. In-vitro binding and displacement assays confirm that after derivatization and labeling, Rituximab retained its specificity of binding to CD-20 antigen. Immunoreactivity of HYNIC-Rituximab to Ramos and Raji cell lines was determined by direct immunofluorescence. We observed a remarkable cell membrane staining of the NHL cells. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). After its incubation with buffer, auto fluorescence was discarded. These results show that Rituximab´s affinity for NHL cells remained unaffected after its derivatization. Biodistribution studies were performed to quantify localization and clearance of the radiolabeled antibody complex in normal tissues. Significant accumulation of radioactivity was found in liver, indicating that the primary route of clearance was hepatic. Other major uptake was not found after evaluating the rest of the organs at the observed time points. Conclusions 99mTc-HYNIC-Tocilizumab was easily and rapidly labeled, with radiochemical purities greater than 90%, retaining its specificity of binding. Our results indicate that 99mTc-HYNIC-Rituximab represents a promising molecular imaging agent for NHL. Acknowledgments ANII, Roche Laboratories, Pro.In.Bio. PEDECIBA Química References [1] Swerdlow AJ. Epidemiology of Hodgkin’s disease and nonHodgkin’s lymphoma. Eur J Nucl Med Mol Imaging 2003; 30 Suppl 1:S2–12. [2] Einfield DA, Beown JP, Valentine MA, et al. Molecular cloning of the human cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 1988; 7, 711-717. [3] Valentine MA, Meier KE, Rossie S, et al. Phosphotylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase. C J Biol Chem 1989; 264, 11282-11287. [4] Tedder TF, Boyd AW, Freedman As, et al. The B cell surface molecule B1 is functionally linked withBcell activation and differentiation. J Immunol 1985; 135, 973-979. Disclosures: No relevant conflicts of interest to declare.

Description: Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4+ or CD8+ subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Description: Background: Treatment of splanchnic vein thrombosis (SVT) is challenging, due to the increased risk of bleeding and potentially life-threatening complications. Current recommendations are based on evidence from the treatment of venous thrombosis in usual sites, but small observational studies in SVT population suggest that the bleeding risk may offset the benefit of anticoagulant treatment in this setting. The aim of this study was to evaluate the safety of vitamin K antagonists (VKAs) in SVT patients. Methods: We retrospectively included SVT patients treated with VKAs and followed by 37 Italian Anticoagulation Clinics until June 2013. All documented bleeding and thrombotic events were reviewed by a Central Independent Adjudication Committee. The primary outcome was the incidence of major bleeding (MB), according to the ISTH definition, during VKA treatment. Vascular events, including both arterial and venous thrombosis, and mortality were also documented. Results: 375 patients with SVT on VKA treatment were included (median age 53 years; 54.7% males). The most common risk factors were: hematological diseases (21.6%), hepatic cirrhosis (15.2%), solid cancer (10.7%), recent abdominal surgery (8.0%) and intra-abdominal inflammation or infection (6.7%); in 37.1% SVT was unprovoked. The therapeutic INR target range was 2.0-3.0 in 353 patients (94.1%). During a median VKA treatment duration of 1.98 years, 15 MB events occurred, corresponding to an incidence rate of 1.24 (95% CI, 0.75-2.06) per 100 patient-years. All bleeding patients were receiving warfarin with INR target range 2.0-3.0 and almost two thirds of bleeding complications occurred with therapeutic INR. One MB was fatal, corresponding to a case-fatality rate of 6.7%. Gastrointestinal bleeding represented 40% of all MB events. At multivariate analysis, adjusted for age and sex and stratified by Center, the presence of esophageal varices emerged as independent predictor of MB (HR 4.9; 95% CI, 1.4-17.1), while inflammatory bowel diseases were borderline statistically significant (HR 15.2; 95% CI, 0.99-233.1). The incidence rate of vascular events on treatment was 1.37 (95% CI, 0.84-2.23) per 100 patient-years, including 9 venous thrombosis and 7 arterial thrombosis. The mortality rate was 0.83 (95% CI, 0.45-1.54) per 100 patient-years. Conclusions: In designated SVT patients, oral anticoagulant treatment was safe, with a reported incidence of major bleeding less than 2% per year and a reasonable case-fatality rate. It is therefore of utmost importance to accurately select which SVT patients are suitable to be prescribed with VKAs. Disclosures Ageno: Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; STAGO: Honoraria. Rodeghiero:GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Suppremol: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Abstract 4459 Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disorder characterized by the translocation t(9,22)(q34;q11), resulting in the Philadelphia chromosome (Ph) and the bcr-abl fusion gene. A small proportion of Ph+ CML patients (5–10%) show variant Ph translocations involving one or more chromosomes in addition to chromosomes 9 and 22. Its generation mechanism and clinical significance remains unclear. We report a novel four-way translocation detected in a newly diagnosed chronic phase CML patient. Case presentation A 51 year-old white women with no significant past medical history presented with a two-week upper left abdominal pain. She denied weight loss, fever, night sweats, fatigue or thrombosis. She has 11 siblings. On physical examination an enlarged spleen was detected, 7 cm below left costal margin. No hepatomegaly was found. Peripheral blood counts on admission showed WBC 286 × 109/l, with immature myeloid forms (21%bands, 31%neutrophils, 3%lymphocytes, 5%basophiles, 1%eosinophils, 16%metamyelocites, 14%myelocytes, 6% promyelocytes, 1% blast cells), hemoglobin 11,8 g/dl and platelet count of 986 ×109/l. LDH= 1094 IU/L. Uric acid, renal and hepatic functions were normal. Abdominal ultrasonography showed homogeneous splenomegaly (185 mm) with no focal lesions. Bone marrow aspirate demonstrated increased cellularity with myeloid hyperplasia without remarkable basophilia. Trephine demonstrated hypercellularity without fibrosis and 1% blast cells. Conventional cytogenetic analysis by GTG-banding revealed a variant Ph chromosome positive in 100 % of metaphases analysed involving chromosomes 1 and 17 inaddition to chromosomes 9 and 22: 46 XX, t (1;17; 9; 22) (p?35;q24;q34;q11)[20]. No normal cells nor additional numerical and structural chromosomal changes were detected at diagnosis. A fragment of 1605 bp was amplified with primers BCR-P1F and ABL-P1R. The sequence analysis of the amplified PCR product showed a complex BCR-ABL rearrangement formed by exon 8 of the BCR (NM_004327) and exon 2 of the ABL genes (NM_005157), with a 148 bp insertion corresponding to the whole exon 2 of the MAST2 (NM_015112, Homo sapiens microtubule associated serine/threonine kinase 2) gene. Bone marrow cytogenetical evaluation at four months do not show Philadelphia chromosome on 20 cells analysed. Interphase FISH with DUAL Colour Dual Fusion probe has a normal pattern on 300 nuclei: 2R2G. It corresponds to a complete cytogenetic response. Chronic phase CML was diagnosed and cytoreductive treatment with hydroxyurea 1,5 g/day, fluids, thromboprophilaxis and allopurinol was initiated. A good control of blood counts was obtained, as well as a complete reduction of the enlarged spleen and regression of left abdominal pain. Once cytogenetic/molecular confirmed diagnosis, treatment with Imatinib 400 mg/day was started, and hidroxiurea was discontinued. She achieved complete hematologic response at 1 month. Splenomegaly was no longer detected. At 2 months she developed neutropenia (1100/mm3) and thrombocytopenia (55000/mm3) so Imatinib was held. Two weeks later, after hematologic recovery, Imatinib was re initiated but in the following control neutropenia and thrombocytopenia were again detected. Discussion About 90–95% of CML have the characteristic t(9;22)(q34;q11.2) reciprocal translocation that results in the Ph chromosome [der(22q)], the cytogenetical hallmark of this entity. The remaining cases can have variant translocations that involve 3, 4 or more chromosomes and its occurrence is not associated with disease evolution. This patient shows a four way translocation involving chromosomes 1 and 17 in addition to chromosome 9 an d 22. It is controversial whether variant translocations result in a different disease phenotype. There is no impact on the cytogenetic or molecular response or outcome, so a special approach is not recommended. Conclusion This is a novel four way translocation described in a chronic phase CML patient, with a good response to standard therapy. Complete haematological and cytogenetic response at 4 months of treatment with Imatinib was achieved. Up to now, a differential approach does not appear to be mandatory in CML with variant translocations. However, in this patient alternative tyrosine kinase inhibitor may be of benefit due to hematologic toxicity attributable to Imatinib. Disclosures: No relevant conflicts of interest to declare.

Description: Background Anti-multiple myeloma (MM) vaccinations have recently emerged as a strategy to eradicate MM cells in patients with residual tumor mass. However, most vaccination studies reported transient immune responses, involving CD8+ T-cells or antibodies responses in patients with predefined MHC repertoire. Signal peptide (SP) domains, a 13-50 amino acid long peptides, have been demonstrated to present an exceptionally high number of antigen specific MHC class I, class II and B-cell epitopes per sequence length. Therefore, they are suitable to induce a robust immune response combining CD8+, CD4+ T-cells and antibodies across HLA barrier. Following this rationale we developed ImMucin, A 21-mer therapeutic vaccine encoding the entire SP domain of the MUC1 tumor-associated antigen (TAA), which is over expressed by most carcinomas and hematological tumors including MM. Methods A phase I/II study to assess the safety (primary endpoint) and efficacy (secondary endpoint) of ImMucin was conducted in MM subjects with stable or progressive residual asymptomatic MUC1+ disease who have already had upfront auto-SCT. Patients recruited into the study initially received 6 bi-weekly vaccination of intra-dermal 100ug ImMucin plus 250ug hGM-CSF. Based on immune and clinical response eligible patient could receive additional 6 bi-weekly vaccination of 100ug or 250ug ImMucin plus 250ug hGM-CSF. Immunomonitoring analysis included; ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells, HLA-A2.1 tetramer binding in CD8+ T-cells, proliferation and antibodies production. Positive response in these assays vs. pre vaccination levels were; x2 increase in IFN-g and 〉0.5%+ T-cells, any increase in tetramer binding levels, x2 increase and stimulation index (SI)〉2 and any increase in anti-ImMucin titer respectively. Results 15 patients, median age 58 years participated in the study. The median number of prior therapies was 2. Median time from diagnosis and from auto-SCT to vaccination were 25 months (12-143) and 15 months (3-134) respectively. 9 patients had post auto-SCT residual MM and 6 had an evidence for biochemical progression. 11 patients completed the vaccination program while 4 discontinued the program due to PD. ImMucin was shown to be safe with no vaccine related grade ≥3 adverse events (AEs). Patients had temporary, grade 1,2 AEs, including injection related rash (n=8), fatigue (n=6), weakness, self-resolving fever and muscle pain (n=5). IFN-g production response to ImMucin was highly positive in all patients with 4-80 folds increase for CD4+ and 18-80 folds increase for CD8+. Mean baseline and peak post vaccination levels for ImMucin-specific CD4+ T-cells were 0.21% and 4.07%, respectively (P

Description: Posttransplantation human herpesvirus-8 (HHV8)/Kaposi sarcoma herpesvirus (KSHV) primary infection and/or reactivations are associated with uncommon and sometimes fatal, neoplastic, and non-neoplastic diseases. HHV8-related clinical manifestations notably range from Kaposi sarcoma (KS) to either primary effusion lymphoma or multicentric Castleman disease B-cell malignancies, and from polyclonal HHV8-positive plasmacytic lymphoproliferative disorders to bone marrow failure and peripheral cytopenias, associated or not with hemophagocytic syndromes, and to acute hepatitis syndromes. We reviewed the patient series reported in the literature and summarized clinical management aspects, in terms of diagnosis, follow-up, and treatment. We described typical clinical presentations and histopathologic diagnostic features of these diseases, and we discussed the role of HHV8-specific serologic, molecular, and immunologic assays, particularly focusing on recent data from HHV8-specific T-cell monitoring in posttransplantation KS patients. We finally discussed actual therapeutic options, namely, the reduction or discontinuation of immunosuppressive therapy or the switch from calcineurin inhibitors to mTOR inhibitors, as alternatives to antineoplastic chemotherapy, along with the use of antiherpesvirus agents as prophylactic or therapeutic measures, and treatment with rituximab in posttrans-plantation multicentric Castleman disease patients and non-neoplastic HHV8-associated syndromes.