Etsuko Sekimoto

A Single-Chain Fv Diabody against Human Leukocyte Antigen-A Molecules Specifically Induces Myeloma Cell Death in the Bone Marrow Environment

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma.

Bleeding tendency in chronic neutrophilic leukemia.

To define the clinical and pathologic features and natural history of chronic neutrophilic leukemia (CNL), as defined by the World Health Organization (WHO), Elliott critically reviewed the literature and identified a total of 34 patients meeting strictly defined WHO criteria for CNL [1]. Of the 34 patients, 9 died of intracranial hemorrhage and 3 demonstrated easy bruising. Elliot indicated that a disproportionate number of reports of intracranial hemorrhage must be considered in the context of an association with chemotherapy-induced thrombocytopenia during treatment of progressive disease. Furthermore, he described that excess hemorrhagic events were not a significant finding in chronic stable-phase disease in the absence of chemotherapy-induced thrombocytopenia. Here, we describe platelet dysfunction in a patient with CNL complicated by bleeding tendency. At the time this patient showed bleeding symptoms, she had not received chemotherapy and her platelet count was normal. On 20 August 2003, an 83-year-old Japanese woman was referred to our department for examination of bleeding tendency. She had a 3-year history of recurrent purpura and occasional nasal bleeding. Neither the patient nor any members of her family had a history of bleeding tendencies. On physical examination, purpura was detected on the face, back and extremities. A CT scan of the abdomen demonstrated splenomegaly. Peripheral blood showed: hemoglobin, 9.7 g/dl; platelets, 284 9 10/l; leukocytes, 29.0 9 10/l with 0.2% myelocytes, 0.6% metamyelocytes, 2.0% stabs, 90.8% segments, 1.0% eosinophils, 2.2% basophils, 1.2% monocytes and 2.0% lymphocytes. Neutrophil alkaline phosphatase (NAP) score was 352 (normal, 190–370). The serum concentration of granulocyte colonystimulating factor (G-CSF) was under 10.0 pg/ml. Immunoelectrophoresis did not show any M protein in serum or urine. The bone marrow was grossly hypercellular with marked myeloid hyperplasia and 1.0% blasts. There were no dysplastic features, minimal fibrosis, normal megakaryocytes and erythropoiesis. Chromosomal analysis demonstrated a normal karyotype. Reverse transcriptasepolymerase chain reaction (RT-PCR) studies failed to demonstrate the presence of BCR-ABL fusion. The patient was diagnosed as having CNL. Routine hemostatic tests demonstrated: bleeding time, more than 8 min (normal, 1–5 min); prothrombin time, 14.9 s (normal, 10.0–13.5 s); activated partial thromboplastin time, 39.8 s (normal, 25.0–40.0 s); plasma fibrinogen level, 295 mg/dl; plasma FDP concentration, 4.1 lg/ml (normal, below 10 lg/ml). Factor VIII activity was 180% (normal, 62–145%), von Willebrand factor antigen was 136% (normal, 50–150%), and ristocetin cofactor activity was 133% (normal, 50–150%). Activities of factor XIII and a2-plasmin inhibitor were 76% (normal, over 70%) and 86% (normal, 85–118%), respectively. Platelet aggregation induced by ADP, collagen, epinephrine or ristocetin was decreased when compared to that in the control. In contrast, arachidonic acid induced platelet aggregation was normal (Fig. 1). Similar patterns of abnormal platelet aggregation were repeatedly demonstrated on three different occasions. Flow cytometric analyses of her platelets for CD41 and CD42b expression showed normal patterns. These findings suggested that this patient had storage pool disease. In this disease, platelet aggregation induced by ADP, collagen, or epinephrine is impaired, but that induced by arachidonic T. Shigekiyo (&) J. Miyagi M. Chohraku K. Kawauchi E. Sekimoto A. Shirakami H. Shibata Department of Internal Medicine, Tokushima Prefectural Central Hospital, 1-10-3 Kuramoto-cho, Tokushima 770-0042, Japan e-mail: shigekiyo@tph.gr.jp

Treatment of acquired amegakaryocytic thrombocytopenic purpura with romiplostim

A standard treatment for acquired amegakaryocytic thrombocytopenic purpura (AATP) has not yet been established. Once AATP is diagnosed, a therapeutic trial of cyclosporine, with or without antithymocyte globulin (ATG), is indicated [1]. Allogeneic bone marrow transplantation (BMT) is considered to be an appropriate treatment for patients with refractory or progressive AATP, who are relatively young and have matched siblings [2]. Recently, successful treatment of AATP with a thrombopoietin (TPO) receptor agonist, eltrombopag, has been reported [3]. We herein describe a patient with AATP refractory to other therapies, including eltrombopag, who was successfully managed with another TPO receptor agonist, romiplostim. This publication was approved by the ethics committee of Tokushima Prefectural Central Hospital. A 55-year-old male was referred to us for thrombocytopenia in December 2004. He had a past history of myocardial infarction at 51 years of age, and had therefore been taking numerous drugs, such as aspirin, nicorandil, quinapril hydrochloride, carvedilol, candesartan cilexetil, azosemide, spironolactone, and allopurinol. He was not a heavy drinker. A physical examination revealed many petechiae and ecchymoses on the extremities and the abdominal wall. The peripheral blood results were as follows: Hb, 13.2 g/dl; RBC, 4.19 10/ml; WBC, 7.6 10/ml (seg., 63.5%; eos., 7.0%; baso., 1.5%; mono., 7.5%; lym., 23.0%) and PLT, 4 10/ml. His blood chemistry values, including total bilirubin, AST, ALT, ALP, LDH, BUN and creatinine, were all within the normal limits. The prothrombin time, activated partial thromboplastin time, plasma fibrinogen level, and plasma FDP concentration were all normal. The platelet-associated IgG level was 57.6 ng/10 cells (reference range, 5–25 ng/10 cells). Tests for rheumatoid factor and antinuclear antibody were negative. Screening assays for the human immunodeficiency virus, human T-cell leukemia virus type-1, hepatitis B virus, hepatitis C virus and human parvovirus B19 (IgM) were negative. Bone marrow aspiration showed normal cellularity and the absence of megakaryocytes, with a normal appearance of erythroid and myeloid elements (Figure 1A and B). A bone marrow cytogenetic study showed normal karyotypes. The patient was therefore diagnosed with AATP. Unfortunately, a bone marrow biopsy was not carried out. Of the drugs that the patient had received, both aspirin and allopurinol had previously been given to patients with AATP [1, 3, 4]. However, since it was impossible to show that none of the other drugs were associated with AATP, all medications were discontinued [5]. Pulse therapy with methylprednisolone (1 g/day 3), followed by prednisolone (1 mg/kg/day), was conducted at the same time, but his platelet count remained low for one month. Cyclosporine (5 mg/kg/day) was initiated in January 2005, but his thrombocytopenia had not improved after two months. Horse ATG (1 g/day 5) was administered in March 2005. His platelet count oscillated between 15 10/ml and 470 10/ml in 35–39-day intervals, but became consistently low in December 2005. Bone marrow aspiration in March 2006 gave the same results as in December in 2004 (data not shown). Rabbit ATG (0.5 g/day 5) was administered in March 2006. The change in his platelet count was similar to that after the administration of horse ATG. Danazol (300 mg/day) treatment was initiated in October 2006, but his thrombocytopenia had not improved after three months. Although he was referred to a hospital for allogeneic BMT in May 2007, he was refused a transplant because of his age, complications and the absence of related donors. Cyclophosphamide hydrate (1 and 2 g) was intravenously administered in June and August 2007, respectively, with no improvement of his thrombocytopenia. Since successful treatment of AATP with rituximab (375 mg/m intravenously, weekly, for two consecutive weeks) in a patient with systemic lupus erythematosus was reported, he was given rituximab (375 mg/m) twice in November 2008 [6]. However, his platelet count did not increase at all. When a test dose of horse ATG was given in March 2009, he developed hypotension and a skin rash. We therefore abandoned a second course of both horse and rabbit ATG. Intravenous immunoglobulin was not tested due to its potential risk of thrombosis [7]. Eltrombopag was initiated at 12.5 mg/day in September 2011, and was increased by 12.5 mg/day every two weeks. After one month of treatment Keywords

Type 2B-like acquired von Willebrand syndrome

More than 300 cases of acquired von Willebrand syndrome (AVWS) have been reported, but it is thought that this number reflects only the tip of the iceberg [1]. Data from an international registry has shown that AVWS may be associated with lymphoproliferative (48%) or myeloproliferative (15%) disorders, neoplasia (5%), immunological (2%), cardiovascular (21%) and miscellaneous conditions (9%) [2]. AVWS usually mimics von Willebrand disease (VWD) type 1 or 2A [3, 4]. To our knowledge, only two cases of type 2B-like AVWS have been reported previously [4, 5]. They were both associated with plasma cell dyscrasia. Here we show a new case of type 2B-like AVWS. On 2 November 2000, a 74-year-old man was referred to our hospital for the follow-up of monoclonal gammopathy of undetermined significance (MGUS). Serum immunoelectrophoresis showed two kinds of M protein, IgG (j) and IgG (k). The serum immunoglobulin levels were IgG 2289 mg/dl, IgA 175 mg/dl, and IgM 63 mg/dl. He remained relatively well until 24 July 2008, when he developed a high fever. A diagnosis of hydronephrosis and urinary tract infection due to prostatic hypertrophy was made and after transurethral resection of the prostate, which was performed on 22 August 2008, macrohematuria persisted for 4 weeks despite administration of tranexamic acid. There was no other mucocutaneous bleeding. His serum IgG level had hardly changed up to that point. Routine coagulation tests demonstrated: prothrombin time, 11.4 s (normal 10.0–13.5 s); activated partial thromboplastin time, 48.5 s (normal 25.0–40.0 s); plasma fibrinogen level, 298 mg/dl. Factor VIII activity (FVIII:C) was 17% (normal 50–150%), von Willebrand factor antigen (VWF:Ag) was 11% (normal 50–150%) and von Willebrand factor ristocetin cofactor activity (VWF:RCo) was below 10% (normal 50–150%). Activities of factor IX, factor XI and factor XII were all normal. Bleeding time was 2.0 min (normal 1.0–5.0 min). The platelet count was consistently normal. Platelet aggregation induced by ADP, collagen or epinephrine was increased when compared to that in the control. Ristocetin-induced platelet aggregation (RIPA) was also enhanced, showing 70% aggregation with 0.3 mg/ml ristocetin compared with absent aggregation in a normal control (Fig. 1). His platelet-rich plasma (PRP) aggregated spontaneously without the addition of any agonists. All aggregation studies were performed with an aggregometer (MC Medical, Tokyo, Japan) in siliconized glass cuvettes at 37.0 ± 0.5 C with constant stirring at 1000 ± 100 rpm. Plasma VWF multimeric analysis showed that large multimers were absent and medium multimers were markedly decreased. Since these results T. Shigekiyo (&) E. Sekimoto A. Shirakami H. Yamaguchi H. Shibata S. Ozaki Department of Internal Medicine, Tokushima Prefectural Central Hospital, 1-10-3 Kuramoto-cho, Tokushima 770-0042, Japan e-mail: shigekiyo@tph.gr.jp

Identification of a homozygous missense mutation (p.Cys379Gly) in the D1 domain of von Willebrand factor propeptide in a family with type 2A (IIC) von Willebrand disease

1. McCarthy M, Gane E, Pereira S, et al. Liver transplantation for hae‐ mophiliacs with hepatitis C cirrhosis. Gut. 1996;39:870‐875. 2. White GC 2nd, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost. 2001;85:560. 3. Shima M, Fujimura Y, Nishiyama T, et al. ABO blood group genotype and plasma von Willebrand factor in normal individuals. Vox Sang. 1995;68:236‐240. 4. Vlot AJ, Koppelman SJ, Bouma BN, Sixma JJ. Factor VIII and von Willebrand factor. Thromb Haemost. 1998;79:456‐465. 5. Shahani T, Covens K, Lavend’homme R, et al. Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII. J Thromb Haemost. 2014;12:36‐42. 6. Fahs SA, Hille MT, Shi Q, Weiler H, Montgomery RR. A condi‐ tional knockout mouse model reveals endothelial cells as the prin‐ cipal and possibly exclusive source of plasma factor VIII. Blood. 2014;123:3706‐3713. 7. Chiu KW, Nakano T, Chen KD, et al. Pyrosequencing to identify ho‐ mogeneous phenomenon when using recipients/donors with dif‐ ferent CYP3A5*3 genotypes in living donor liver transplantation. PLoS One. 2013;8:e71314. 8. Hove WR, van Hoek B, Bajema IM, Ringers J, van Krieken JH, Lagaaij EL. Extensive chimerism in liver transplants: vascular en‐ dothelium, bile duct epithelium, and hepatocytes. Liver Transpl. 2003;9:552‐556. 9. Kleeberger W, Rothamel T, Glockner S, Flemming P, Lehmann U, Kreipe H. High frequency of epithelial chimerism in liver transplants demonstrated by microdissection and STR‐analysis. Hepatology. 2002;35:110‐116. 10. Katagiri H, Kushida Y, Nojima M, et al. A distinct subpopulation of bone marrow mesenchymal stem cells, muse cells, directly commit to the replacement of liver components. Am J Transplant. 2016;16: 468‐483.

Inheritance of von Willebrand disease Vicenza in a Japanese family

1. Bowyer A, Key N, Dalton D, Kitchen S, Makris M. The coagulation laboratory monitoring of Afstyla singlechain FVIII concentrate. Haemophilia. 2017;23:e469-e470. 2. Mikaelsson M, Oswaldsson U. Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products. Semin Thromb Hemost. 2002;28: 257-264. 3. Sommer JM, Moore N, McGuffie-Valentine B, Bardan S, Buyue Y, Kamphaus GD, et al. Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories. Haemophilia. 2014;20:294-300. 4. Turecek PL, Romeder-Finger S, Apostol C, Bauer A, Crocker-Buque A, Burger DA, et al. A worldwide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE. Haemophilia. 2016;22:957-965. 5. Viuff D, Barrowcliffe T, Saugstrup T, Ezban M, Lillicrap D. International comparative field study of N8 evaluating factor VIII assay performance. Haemophilia. 2011;17:695-702. 6. Ingerslev J, Jankowski MA, Weston SB, Charles LA, ReFacto Field Study P. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using onestage clotting assays. J Thromb Haemost 2004;2:623-628. 7. CSL Behring. AFSTYLA Prescribing Information. 2017. 8. St Ledger K, Feussner A, Kalina U, Metzner H, Horn C, Stowers A, et al. Characteristics of rVIIISingleChain in the onestage and the chromogenic substrate assay: results of an international field study. Haemophilia. 2016;22(Suppl. 4):59. 9. St Ledger K, Feussner A, Kalina U, Horn C, Metzner HJ, BensenKennedy D, et al. International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories. J Thromb Haemost. 2018;16:555-564. 10. Peyvandi F, Oldenburg J, Friedman KD. A critical appraisal of onestage and chromogenic assays of factor VIII activity. J Thromb Haemost. 2016;14:248-261.

Fibrinogen Tokushima II: a new case of congenital dysfibrinogenemia with a γ methionine-310 to threonine substitution

The first case of congenital dysfibrinogenemia with a c methionine (Met)-310 to threonine (Thr) substitution and consequent N-glycosylation at c asparagine (Asn)-308, fibrinogen Asahi, was reported by Yamazumi et al. [1] in 1989. To our knowledge, only two cases of congenital dysfibrinogenemia with this mutation have subsequently been reported [2, 3]. Here, we report a new case of congenital dysfibrinogenemia with this mutation. This case is characteristic in two points. The first is that the proband received fibrinogen replacement therapy. The second is that her affected son showed no bleeding tendency. A 86-year-old woman (Fig. 1a; II-2) was referred to us in late May 2011, as her plasma fibrinogen level was very low on a preoperative examination. She had a past history of persistent bleeding after tooth extraction, but had shown neither hypermenorrhea nor excessive bleeding post-partum. She also had suffered no miscarriages. Her family history was negative for bleeding or thrombotic tendencies. There was no consanguinity. Routine hemostatic tests revealed a platelet count of 180 9 10/ll, bleeding time 1.5 min (reference range 1–5 min), prothrombin time 19.5 s (reference range 10.0–13.5 s), activated partial thromboplastin time 31.7 s (reference range 25.0–40.0 s), functional fibrinogen concentration below 50 mg/dl (reference range 160–360 mg/dl), and plasma FDP concentration 1.6 lg/ml (reference range below 10 lg/ml). Her thrombin time and reptilase time were over 600.0 s (reference range 12.0–14.6 s) and over 600.0 s (reference range 22.9–33.9 s), respectively. Her immunological fibrinogen concentration was 422 mg/dl (reference range 228–388 mg/dl). Functional and immunological fibrinogen concentrations were determined by the thrombin time method and single radial immunodiffusion, respectively. Reptilase time was measured using Pefakit Reptilase Time (Pentapharm, Basel, Switzerland). Her 66-year-old son (Fig. 1a; III-1) had a functional fibrinogen concentration of below 50 mg/dl and immunological fibrinogen concentration of 434 mg/dl. Her 58-year-old daughter (Fig. 1a; III-2) had a functional fibrinogen concentration of 293 mg/dl and immunological fibrinogen concentration of 315 mg/dl. These results suggested that congenital dysfibrinogenemia had been transmitted in this family. To elucidate the genetic basis of congenital dysfibrinogenemia in this family, we first examined the proband’s fibrinogen gene. This study was approved by the ethics committee of Tokushima Prefectural Central Hospital. Informed consent was provided according to the Declaration of Helsinki. Genomic DNA was isolated from whole blood, and 23 exons were amplified under standard PCR conditions. The primer sequences used for PCR were the same as those used by Terasawa et al. [4]. The PCR products were directly sequenced on an ABI 3730 9 1 DNA Analyzer. The sequence determined for exon 8 of the c-chain gene is shown in Fig. 1b. The nucleotide at position 7581, numbered according to the GenBank database, of the c-chain gene was heterozygous for T and C. A single T to C substitution results in replacing wild-type Met at residue 310 by Thr. No other mutation was found in either T. Shigekiyo (&) E. Sekimoto A. Shirakami H. Yamaguchi H. Shibata S. Ozaki Department of Internal Medicine, Tokushima Prefectural Central Hospital, 1-10-3 Kuramoto-cho, Tokushima 770-0042, Japan e-mail: shigekiyo@tph.gr.jp