ALBERT

Description: Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder characterized by quantitative and/or qualitative von Willebrand factor (VWF) abnormalities. VWF gene is a large gene consists of 52 exons, wheras its pseudogene corresponds to exons 23-34 that makes the analysis much complicated. In this study, we performed genetic analyses of the VWF gene from 20 Japanese VWD patients, aimed to find the causative genetic mutations by using various techniques including the exome sequencing approach, and to explore the diversity of yet unknown VWD pathogenetic mechanisms. Methods: Patients' samples were collected after the written informed consent has been obtained. First, we analyzed exon 28 of VWF genes from peripheral blood genomic DNA of the patients by PCR using allele-specific primers, and analyzed DNA sequences of the patients by Sanger sequencing method. If patients show the type 3 phenotype, the possibility of deletion or insertion of the gene was taken into consideration, then MLPA method was selected. In cases the gene mutation was not detected by exon 28 sequencing or MLPA, we analyzed all exons of VWF by Sanger method. In selected patients, the exome sequencing was employed by using the next-generation sequencer Hi-Seq 2500 (Illumina) and exomes were captured using SureSelect XT Human All Exon V5+UTRs kit (Agilent Technologies). Results: Twenty VWD cases were studied. As a result of the VWF exon 28 sequencing, we identified causative gene mutation in 12 cases (10 cases were type 2, 2 cases were type 1). Gene mutations found in exon 28 were p.R1308C, p.R1315C, p.V1316M, p.R1334W, p.A1461D, p.L1503R, p.S1506L, p.R1597Q, p.G1609R. In one case and her sister of type 3, MLPA could not detect the large deletions or insertions. Sanger sequencing showed the they were compound heterozygous for an p.E2341X (c.7021G〉T) mutation in exon 41 and a p.Y2631X (c.7892-7893insA) mutation in exon 48. In another case of type 3, we used exome sequencing and found a novel homozygous gene mutation p.G2752S (c.8254G〉A) in exon 52. The mutation was confirmed by Sanger sequencing and the propositus was homozygote for the mutation, while the parents and two sons had the same mutation in the heterozygote. In the multimer analysis of plasma, multimers larger than the dimer in size were hardly visible, but there was a faint monomer band was present in the plasma. Multimeric patterns of his family were normal. Discussion: We analyzed the VWF genes of 20 VWD cases and identified causative gene mutations in 14 cases including three novel mutations (p.E2341X, p.Y2631X, p.G2752S). We previously demonstrated BOECs established from the type 3 patient (p.E2341X and p.Y2631X) reproduced the phenotype of the disease (ASH 2014). Meanwhile, G2752S mutation is located in CTCK domain, therefore possibly it affects the VWF dimer formation. However, the causative gene mutations were not detected in 6 cases (5 cases were type 1, 1 case was type 2) by exome sequencing. Although previous observations have pointed out that causative VWF gene mutations are not detectable in about 30 % of entire type 1 cases, a newer approach would be needed to elucidate molecular pathogenesis of VWD. Disclosures Suzuki: Baxalta: Honoraria; Novo Nordisk Pharma: Honoraria; Bayer Healthcare: Honoraria. Matsushita:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Asahi Kasei Pharma: Honoraria, Research Funding, Speakers Bureau; Kaketsuken: Honoraria, Research Funding, Speakers Bureau; Seamens: Speakers Bureau; Japan Blood Products Organization: Honoraria, Research Funding; Kyowa-Kirin: Honoraria, Research Funding; Novartis Pharma: Honoraria, Speakers Bureau; Sysmex: Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novo Nordisk Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai: Research Funding; Nihon Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Octapharma AG: Honoraria; CLS-Behling: Research Funding.

Description: Background FVIIIa acts as a cofactor in the intrinsic pathway in which FIXa activates FX. ACE910 is a FIXa/FX-recognizing bispecific antibody that was designed to be a replacement for FVIIIa. Because of its nature, ACE910 is not affected by FVIII inhibitor. A clinical trial is now being conducted for the potential effect in the prophylactic treatment for bleeding hemophilia A patients. Here we present the perioperative care of a patient who had incidentally suffered from appendicitis and underwent an emergency surgery during the clinical trial. Methods Plasma ACE910 concentration and FXIa-triggered thrombin generation assay (TGA) was obtained in the central measurement of the trial. An activated partial thromboplastin time (APTT), and the tissue factor (TF)-triggered TGA were conducted at our laboratory. TF-triggered TGA was performed by means of calibrated automated thrombogram (Thrombinoscope BV), in accordance with the manufacturer's instructions. We used PPP-reagent LOWTM and FluCa-KitTM in Fluoroscan Ascent FLTM (Thermo Fisher Scientific Inc.) and monitored the thrombin generation for 2 hours, set at an excitation wavelength of 390 nm and an emission wavelength of 460 nm, and ThrombinoscopeTM software (Thrombinoscope BV). ROTEM® was performed as manufactured (Tem Innovations GmbH). Case The patient is a 60-year-old man suffering from hemophilia A without inhibitors and had severe hemophilic arthropathy in the number of target joints. Even after biweekly prophylaxis had been introduced by 2000 units of rFVIII concentrates, the annualized bleeding rate remained to be 10.1 In November 2013, ACE910 was introduced by way of subcutaneous administration and the initial dose was 3 mg/kg, followed by weekly administration of 1 mg/kg. After that, he had not had any of joint or soft tissue bleeding. In the 63rd week after the initial administration, he had severe abdominal pain and diagnosed as acute appendicitis that required emergency surgery. His APTT was consistently normal since ACE910 administration, we selected to undergo the surgery without any additional FVIII replacement, although his previous product was set up to be administrated any time on demand. ACE910 had been administered as scheduled earlier on the day of the diagnosis of acute appendicitis, followed by the emergency appendectomy. Results The appendectomy was performed by pararectal incision. Although the patient's appendix was necrosed and perforated, it was easy to stop bleeding during surgery and the total amount of bleeding was only 45 mL. On postoperative day 11, a small amount of bleeding was found after the removal of drainage catheter placed subfascially, however, the bleeding stopped immediately after the bleeding site was sutured. No other issues on bleeding were found. Trough levels of plasma ACE910 concentration were maintained at 27-41 µg/mL during the period between the 12th week after the initiation of ACE910 and the time of preoperative stage. In FXIa-triggered TGA, lag time was remarkably improved after the initiation of ACE910 and remained stable throughout the course of emergency surgery (Table 1). Although peak thrombin levels were slightly decreased a week after surgery, APTT and several In-TEM values by ROTEM® remained at almost normal levels (Table 2). Discussion and Conclusion We successfully conducted the hemostatic management for appendicitis in the perioperative period without any additional administration of FVIII concentrate. The patient showed less bleeding under ACE910 prophylaxis. To date there are little information on appropriate use of FVIII concentrate in patients with acute bleeding or major surgery who are under ACE910 prophylaxis. Generally in bleeding hemophilic patients with major surgery, the loss of clotting factors due to hemodilution by fluid replacement should also be carefully monitored. In such condition, the optimum ACE910 concentration could not be well interpreted, however, the careful monitoring might be required especially in highly invasive surgeries. In our experience, TF-triggered TGA demonstrated a marginal change only between postoperative days 7 and 13, although it is not totally known whether these changes were affected by ACE910 pharmacodynamics. Further researches are needed to explore the suitable biomarkers to indicate hemostasis of hemophilic patients under the administration of ACE910. Disclosures Suzuki: Baxalta: Honoraria; Bayer Healthcare: Honoraria; Novo Nordisk Pharma: Honoraria. Kiyoi:Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Matsushita:Asahi Kasei Pharma: Honoraria, Research Funding, Speakers Bureau; Sysmex: Speakers Bureau; Octapharma AG: Honoraria; Kyowa-Kirin: Honoraria, Research Funding; CLS-Behling: Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Seamens: Speakers Bureau; Nihon Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Kaketsuken: Honoraria, Research Funding, Speakers Bureau; Eisai: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Japan Blood Products Organization: Honoraria, Research Funding; Novartis Pharma: Honoraria, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Description: Background: Type 3 von Willebrand disease (VWD) is characterized by a complete loss type of von Willebrand Factor (VWF) with the rarest disease frequency and hemorrhagic symptom is the most severe among other VWD types. The development of alloantibodies directed against VWF occurs in approximately 10% of patients with type 3 VWD. In this study, we found the VWF gene alterations and established blood outgrowth endothelial cells (BOECs) a from a Japanese type 3 VWD patient with an anti-VWF inhibitors. Case: A 5-year-old woman suffered from epistaxis, purpura and easy bruising and has been diagnosed as VWD. Her plasma level of FVIII:C was 1.8% and VWF:Ag and VWF:RCo was