ALBERT

Description: Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009). To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease. Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl’s Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted. Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms. Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61. Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443. Disclosures: No relevant conflicts of interest to declare.

Description: Background AFM13 is a bispecific TandAb antibody for the treatment of Hodgkin lymphoma (HL) and other CD30+ malignancies. AFM13 specifically targets both CD30, which is the antigen identifying HL tumor cells, and CD16A, for the recruitment of NK cells. Our preclinical data demonstrate a specific and potent anti-tumor activity mediated by the engagement of NK immune effector cells. Methods AFM13 was investigated in an open-label single-arm phase I dose escalation trial in patients with relapsed/refractory HL. The objective of this study was to evaluate the safety, tolerability, pharmacokinetics, immunogenicity, anti-tumor activity, and maximum tolerated dose (MTD). Each patient received 4 weekly doses of AFM13. Seven dose levels, from 0.01 to 7.0 mg/kg, were escalated in cohorts of 3 patients. In addition, one cohort of 4 patients received AFM13 twice a week at 4.5 mg/kg for 4 weeks. Results Twenty-eight (28) HL patients were enrolled, and these heavily pre-treated patients had a median number of six (6) prior therapies. 14/28 patients were refractory to their most recent therapy. 9/28 patients had a prior therapy with brentuximab vedotin. AFM13 was safe and well-tolerated, and did not reach the MTD. One dose-limiting toxicity (DLT) was observed at 0.5 mg/kg, and this dose cohort was expanded to 6 patients. No further DLTs were observed. The most frequent adverse events were infusion-related reactions (headache, fever, fatigue and myalgia) that often occurred after the first administration. Nearly all adverse events were short-lived. According to the Cheson criteria, 2 patients achieved partial responses and 13 patients achieved stable disease. In addition, AFM13 was active in brentuximab vedotin relapsing/refractory patients, with 7 out of 9 patients achieving stable disease after AFM13 treatment. The responses of the 13 patients who received AFM13 at and above the 1.5 mg/kg dose level were further analyzed for pharmacodynamic/anti-tumor activity correlations. In this group the drug exhibited substantial anti-tumor activity, with 8 out of the 13 patients experiencing a reduction in both tumor volume (as assessed by CT scan) and tumor activity (as assessed by FDG-PET scan imaging). Furthermore, 3 out of the 13 patients (23%) exhibited a reduction in tumor volume of more than 50%. The on-mechanism activity of the drug was further substantiated by the NK cell activation and soluble CD30 clearance pharmacodynamic parameters. Statistically significant correlations between increasing anti-tumor activity parameters (i.e. tumor volume reduction and FDG-PET SUV reduction) and the pharmacodynamic biomarkers were observed as: i) a dose-dependent increase in the activation of NK cells, and ii) a dose-dependent reduction in soluble CD30 levels. Variations in SUV were correlated with the number of activated NK cells (i.e. CD69+ cells) at baseline and 24 hours after the start of therapy with AFM13. The number of activated NK cells peaked 12 hrs after dosing, and dropped after 48 hrs. A decrease in SUV was observed at the highest level of activated NK cells, and was accompanied by a concomitant reduction in soluble CD30 levels. Furthermore, AFM13 exhibited a half-life of 1 day, representing a substantial increase relative to that of the alternative bispecific tandem scFv that is currently in clinical evaluation for hematological malignancies. Conclusions AFM13 has demonstrated good safety and encouraging anti-tumor activity. Furthermore, there is a solid scientific rationale suggesting that during phase I the anti-tumor potential of AFM13 was not fully realized. A compelling hypothesis for further exploration is whether we may detect much stronger activity, in refractory/relapsing HL patients, in a phase II study employing a pK-optimized regimen with an extended duration of therapy. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. von Tresckow:Novartis: honoraria for acting as a consultant: Consultancy; Takeda Pharma GmbH: reimbursement of congress, travel, and accommodation costs and honoraria for preparation of scientific educational events: Honoraria. Topp:Affimed Therapeutics AG: Consultancy. Ravic:Affimed Therapeutics AG: Consultancy. Hucke:Affimed Therapeutics AG: Consultancy. Engert:Affimed Therapeutics AG: Consultancy.

Description: Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.

Description: Background: MCL is a distinct lymphoma entity with improved outcome achieved by the introduction of rituximab, high dose Ara-C and autologous stem cell transplantation (ASCT) into the first line therapy. The outcome of the relapsed patients (pts) remain however poor and there is little data on the outcome after subsequent relapses and there is no information on secondary MIPI prognostic value. Aim: To analyze the outcome of the MCL patients after first line treatment failure and to evaluate the prognostic role of the sec MIPI which is MIPI calculated at the time of relapse/progression. Methods: This analysis is a part of the Lymphoma project in which consecutive lymphoma patients are registered since the year 1999. Altogether 519 newly diagnosed MCL patients were registered in 5 university centers and 9 regional departments between 1999 and 2011. Patients who were treated with rituximab as part of the first line treatment (n=388) were included into the analysis. The diagnoses were confirmed according to WHO classification in the reference pathology centers. The median follow up is 4.5 years. Results: The whole cohort consists of 261 males and 127 females (2.1:1) with median age 65 y (28-87), the majority of pts had advanced disease (CS IV in 81.6% pts), PS ECOG ≥ 2 in 23.6% pts, elevated LDH in 52.5% of pts. The MIPI risk profile was as follows: low risk 21.7%, intermediate risk 27.2% and high risk in 51.1%. All pts received rituximab as part of the induction, 48.7% pts received CHOP, 5.7% alternation of CHOP and HD Ara-C, 26.2% intensive induction with HD Ara-C, 10.3% CVP, 6.4% FC. High dose therapy with ASCT was performed in 23.9% of pts. The ORR was 89.0% with 63.8 CR/CRu, 6.3% had stable disease and 4.9% were primary progressive. The PFS and OS were 2.9 y and 5.5 y with significant impact of MIPI risk (p

Description: Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.

Description: Abstract 2182 Introduction: NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined. Methods: The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes. Results: The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested. Conclusion: Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells with immunoligands ex vivo will be discussed. Disclosures: Engert: Affimed Therapeutics AG: Honoraria, Research Funding.

Description: Abstract 4669 Two similarly designed prospective, uncontrolled, open-label clinical trials were planned to assess the safety and efficacy of a new intravenous immunoglobulin (IVIG), IGIV3I 10% Grifols, in subjects with chronic immune thrombocytopenia (ITP) in the USA, Canada and Europe (Spain, Russia and the United Kingdom). Subjects were candidates to be enrolled if they had a diagnosis of chronic ITP and a baseline platelet count ≤ 20 × 109/l. Eligible subjects received treatment with IGIV3I 10% Grifols 1g/kg for 2 consecutive days or 0.4 g/kg for 5 consecutive days. The primary efficacy endpoint was the response rate, defined as the proportion of subjects reaching or exceeding the threshold value of 50 × 109/l on or before day 8 (American study) or on day 30 (European study) where day 1 is the day of the first infusion. Secondary efficacy endpoints were the time to response, defined as the number of days elapsed from day 1 to achievement of the threshold value; duration of response, defined as the number of consecutive days with a platelet count known to be ≥50 × 109/l; the maximum (peak) platelet count and regression of haemorrhagic diathesis for those subjects presenting with bleeding signs at baseline. Safety endpoints included adverse events (AEs), physical examinations, vital signs and clinical laboratory parameters monitoring. A total of 27 subjects (18 adults and 9 pediatrics) have been enrolled, i.e. administered with at least one infusion of the product at any dose, in the study. Twenty-four (24) subjects (89%) were considered responders. This proportion was higher in pediatric subjects (9/9, 100%) than in adult subjects (15/18, 83%). The mean time to response was 1.6 days (Standard Deviation (SD) 0.9); the mean duration of response was 14.0 days (SD 12.1) and the mean maximum platelet count was 263 × 109/l (SD 195.6). Twenty-three (23) subjects (all the pediatric subjects 9/9 and 14/18 adults) presented with some sign of bleeding at baseline. All subjects (23/23, 100%) experienced an improvement in the haemorrhagic diathesis, regardless of some of them being considered non-responders according to the platelet count criterion. A total of 92 AEs potentially related to study drug were reported. The most common of these were headache (25 events), nausea (8 events), pyrexia (7 events) and chills (6 events). Only 1 AE was reported as definitely related to study drug, an event of palmar erythema. Two serious AEs (SAE) potentially related to the study drug were reported in 2 subjects. One of them was an unexpected laboratory alteration which consisted in leukopenia and decreased hemoglobin and advised to maintain the subject hospitalized for further observation during the weekend. The reaction was transient, without complications or other clinical symptoms and total recovery of the normal laboratory values. The second SAE was an event of thrombosis in the right humeral vein, in a patient with a number of predisposing medical conditions. At the time of discharge the patient's overall condition was satisfactory. Analysis of AEs, clinical laboratory values, physical assessments and vital signs did not indicate any safety concerns for subjects receiving the study drug and are in line with those expected for the ITP population treated with IVIG. Both primary and secondary efficacy endpoints show a good response to the product in terms of rapid elevation of platelet counts to an hemostatic level in line with what is expected for an IVIG product. Moreover, the improvement of the baseline bleeding diathesis even in patients considered as non-responders according to the platelet count criterion is highly suggestive of the clinical efficacy of the product. Overall, these results indicate that treatment with the new IGIV3I 10% Grifols is safe and efficacious for rapidly increasing platelet counts in chronic ITP subjects and improving their bleeding diathesis. Disclosures: Khojasteh: Grifols S.A.: Research Funding. Kato:Grifols S.A.: Research Funding. Parikh:Grifols S.A.: Research Funding. Singleton:Grifols S.A.: Research Funding. Tebbi:Grifols S.A.: Research Funding. Cromwell:Grifols S.A.: Research Funding. Fu:Grifols S.A.: Research Funding. Kessler:Grifols S.A.: Research Funding. Letzer:Grifols S.A.: Research Funding. Ritchie:Grifols S.A.: Research Funding. Sexauer:Grifols S.A.: Research Funding. Saxena:Grifols S.A.: Research Funding. Sirpal:Grifols S.A.: Research Funding. Torres:Grifols S.A.: Research Funding. Loriya:Grifols S.A.: Research Funding. Kovaleva:Grifols S.A.: Research Funding. Sandoval:Grifols S.A.: Research Funding. Sanz:Grifols S.A.: Research Funding. Julià:Grifols S.A.: Research Funding. Montañés:Grifols S.A.: Employment. Navarro:Grifols S.A.: Employment.

Description: A minimal hemoglobin (Hb) of 12.5 g/dL is required to protect blood donors from iron-deficiency and anemia and ensure collection of an adequate red cell product. The effects of whole blood or red blood cell (RBC) donation on donor Hb concentration and iron stores have been extensively studied. These changes have not been well characterized in platelet donors. Because platelet donation can occur as frequently as every 72 hours up to 24 times per year, tubes taken for donor testing (approximately 50 mL) at each donation may result in the loss of blood volume equivalent to 2-3 units of whole blood (500 mL each) in frequent donors. We hypothesized that iron deficiency and its associated thrombocytosis is underappreciated in platelet donors. To test this hypothesis, we proposed to 1) analyze the degree of iron deficiency / depletion in platelet donors, 2) assess the correlation between pre-donation platelet count with iron stores, and 3) evaluate the effect of platelet donation frequency on erythropoiesis- and iron-related parameters in white males age 40-65 years, typically representative of the platelet donor pool. Eligible donors were selected from a donor pool who had not donated whole blood / RBCs in the previous 12 months prior to study enrollment. Prospective participants with a history of iron-related pathology (e.g. iron deficiency, hereditary hemochromatosis, anemia, bleeding, or abnormal colonoscopy findings) were excluded from the study. Eligible donors who had not donated any blood products in the prior 12 months were enrolled as controls. Analysis of circulating RBC parameters, serum iron, serum ferritin, serum transferrin concentration and saturation, serum hepcidin, and soluble serum TfR1 were performed and correlations analyzed. The “TfR1 Ferritin Index” (i.e. log(sTfR1/ferritin)) was also evaluated, representing iron restricted erythropoiesis in the absence of frank iron deficiency. Statistical significance was measured using a student t-test; data is presented as mean ± s.e.m. and p

Description: Abstract 4656 A 91-year-old woman with past medical history of hypertension presented with hematuria. There were no ecchymosis, Petechiae or other obvious active bleeding. Her hemoglobin was 11.4 g/dl on presentation hematuria got worse and her hemoglobin drops to 7.6 g/dl over next 48 hours and she was hemodynamically unstable. She was transferred to the Medical Intensive Care Unit for resuscitation with IV fluids and PRBCs. Coagulation tests revealed a prolongation of activated partial thromboplastin time of more than 100 seconds (control 33 seconds) which could not be corrected with mixing normal plasma. Diagnosis of acquired factor VIII inhibitor was considered and recombinant activated factor VII (rFVIIa) was initiated. The factor VIII activity level was reduced to less than 1%. Bethesda assay demonstrated the presence of a factor VIII inhibitor at 103.8 Bethesda units per ml (BU/ml), other coagulation studies were with in normal range. CT scan of her abdomen showed retroperitoneal hematoma. rFVIIa was started at 50 units/kg body weight every 3 hours and subsequently increased to 200 units/kg. She was simultaneously started on steroids. Her hematuria did not improve in spite of high dose rFVIIa. On day 4 rFVIIa was tapered and switched to 50 units/kg FEIBA (Factor eight inhibitor bypass agent). She also received Rituximab 375 mg/m2. We continued FEIBA until day 7 but her hematuria did not improve, she required more than 10 units of Packed Red Blood Cells PRBCs during this period. On day 7 we decided to start plasmapheresis as there were some case reports of using plasmapheresis with or without immunoadsorption columns (which are currently not available in US). We started plasmapheresis and gave her 2 doses of IVIG (Immunoglobulin). Her pre and post plasmapheresis inhibitor levels were 104 BU/ml and 54 BU/ml respectively. Her urine turned pink and her Prbc demand decreased. A second plasmapheresis was done 2 days later showed significant decrease of inhibitor level from 80 BU/ml to 14.5 BU/ml. Her hematuria resolved by next day. We continued her on FEIBA for three more days she did not have hematuria and she did not require any PRBCs. CT scan of abdomen showed decrease in size of retroperitoneal hematoma. Cyclophosphamide 1000 mg was given for induction of immune tolerance followed by high dose factor VIII (100 IU/KG) as per Bonn protocol. Her factor VIII levels and factor VIII inhibitor levels were checked every day before and after Factor VIII infusion. Her inhibitor level is ranging between 14–16 BU/ml she is not bleeding any more and her abdominal hematoma is resolved. Her pre and post transfusion factor VIII levels ranges between 30–40% and 120–140%. respectively. Patient is still getting factor VIII everyday. Role of plasmapheresis is not very well defined in acquired Factor VIII inhibitor patients. Acquired hemophilia is a rare autoimmune disorder in which the patient develop an autoantibody directed against coagulation factor VIII leading to a clinically bleeding diathesis. There are few case reports in literature showing efficacy of Plasmapheresis in this disorder. This is a rare condition and it is very difficult to find large randomized trial to establish a standard of care. Patient mentioned above did not respond to rFVIIa or FEIBA. In our observation plasmapheresis with IVIG proved to be an effective method of rapidly reducing the inhibitor level. In case of life threatening bleeding we need to reduce the inhibitor level quickly. We also observed that once inhibitor level was low bleeding stopped. Immune induction therapy with cyclophosphamide followed by high dose factor VIII was successful in maintaining low inhibitor level. Disclosures: Kessler: Grifols S.A.: Research Funding.

Description: Introduction Transfusions of packed red blood cell (pRBC) units to infants in Neonatal Intensive Care Units (NICUs) may predispose neonates to healthcare-associated infections (HAIs). We compared neonatal pRBC transfusion practices at four NICUs (Morgan Stanley Children’s Hospital of NewYork-Presbyterian, Columbia University Medical Center, Komansky Children’s Hospital of Weill Cornell Medical Center, Christiana Care Health System, and Children’s Hospital of Philadelphia). In addition, because prolonged refrigerator storage of pRBC may further predispose to HAIs, we tested the hypothesis that an increased mean RBC storage age was associated with a greater risk of infections. Design The interdisciplinary NICU Antimicrobial Prescribing (iNAP) study was conducted in four Level III NICUs from May 2009 to April 2012 to assess HAIs and improve antimicrobial prescribing in NICUs. Eligible infants were admitted