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Complement C5 inhibition is the standard of care (SoC) for patients with paroxysmal nocturnal hemoglobinuria (PNH) with significant clinical symptoms. Constant and complete suppression of the terminal complement pathway and the high serum concentration of C5 pose challenges to drug development that result in IV-only treatment options. Crovalimab, a sequential monoclonal antibody recycling technology antibody was engineered for extended self-administered subcutaneous dosing of small volumes in diseases amenable for C5 inhibition. A 3-part open-label adaptive phase 1/2 trial was conducted to assess safety, pharmacokinetics, pharmacodynamics, and exploratory efficacy in healthy volunteers (part 1), as well as in complement blockade–naive (part 2) and C5 inhibitor–treated (part 3) PNH patients. Twenty-nine patients were included in part 2 (n = 10) and part 3 (n = 19). Crovalimab concentrations exceeded the prespecified 100-µg/mL level and resulted in complete and sustained terminal complement pathway inhibition in treatment-naive and C5 inhibitor–pretreated PNH patients. Hemolytic activity and free C5 levels were suppressed below clinically relevant thresholds (liposome assay

Background: Small populations of paroxysmal nocturnal hemoglobinuria (PNH)-type cells ( Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF without malignant diseases were prospectively recruited to the study between April 1 st, 2016 and December 31 st, 2019 in Japan. Participants were excluded from the study if treated with eculizumab or ravulizumab. Peripheral blood samples were obtained with informed consent and sent to the single laboratory every 12months (mos) for 36 mos. A high-resolution flow cytometry assay known as OPTIMA method (Ann Hematol 97(12):2289-2297) was used to precisely detect a small population of GPI(-) cells, which defines ≥0.003% PNH-type granulocytes (Gran) and ≥0.005% erythrocytes as an abnormal increase. The quality of life (QOL) of the pts was assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. All other lab data and clinical information were obtained at each participating institute or hospital on sample collection, and were accumulated by the Japan PNH Study Group for analysis. Results: Total 1,985 pts were registered and 1,813 pts were eligible for analysis. Median age was 67 with 50.5% male patients. PNH-type cells were positive in 50.4% (235/466) of AA, 19.7% (70/355) of MDS, 22.3% (61/273) of suspected PNH, and 33.9% (232/685) of undiagnosed BMF. PNH-type cells were increased in nearly 30% of the pts with RCUD, RCMD, MDS-U, and 5q-, but not in RARS, RAEB-1, or RAEB-2. Time-course data of the size of PNH-type cells were available in 651 pts at 12mos and in 210 pts at 36mos. Small ( Conclusion: PNH-type cells were detected exclusively in AA and low-risk MDS, supporting the hypothesis that the increase of PNH-type cells in BMF underpin the benign immune-mediated feature of the disease. The presence of PNH-type cells predicts a better response to IST in BMF, which is consistent with previous reports. Detection of subclinical PNH-type cells was associated with an improvement of QOL scores in multiple items at 36mos. Those small populations of PNH-type cells stayed subclinical in most of the cases, but caution should be exercised in monitoring the sizes as some may evolve into clinical PNH. Figure 1 Figure 1. Disclosures Ueda: Chugai Pharmaceutical: Consultancy, Honoraria, Research Funding; Alexion Pharma: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Yonemura: Alexion Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Novartis Pharma: Honoraria. Obara: Novartis Pharma: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Alexion Pharma: Honoraria, Research Funding. Ando: Novartis: Honoraria; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Celgene: Honoraria; Astellas Pharma: Honoraria; Takeda Pharmaceutical: Research Funding. Kawaguchi: Alexion Pharma: Honoraria. Nishimura: Alexion Pharma: Consultancy; Chugai Pharmaceutical: Consultancy; Novartis Pharma: Consultancy; Roche: Consultancy; apellis pharmaceuticals: Consultancy; Biocryst: Consultancy; Sanofi: Consultancy. Nakao: Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Symbio: Consultancy; Alexion Pharma: Research Funding.

Background Inhibition of complement C5 is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH). C5 inhibition reduces transfusion requirements and improves anemia and quality of life by ameliorating intravascular hemolysis. However, current therapies require lifelong intravenous infusions at regular intervals, and breakthrough hemolysis may occur due to inadequate C5 inhibition. Crovalimab is a novel anti-human C5 antibody characterized by pH-dependent target binding, enhanced recycling by the neonatal Fc receptor, and high bioavailability, allowing for small-volume (2 × 2 mL) subcutaneous administration every 4 weeks. Part 3 of the Phase I/II COMPOSER study (Sostelly et al. Blood. 2019; Röth et al. Blood. 2020) demonstrated that in patients switched from eculizumab, enhanced crovalimab clearance occurred during the switching phase due to the formation of drug-target-drug complexes, with a risk of temporary loss of complete complement inhibition. Part 4 of COMPOSER tested a dosing regimen optimized to maintain complete complement inhibition in C5 inhibitor-naive patients and those switched from eculizumab to crovalimab. Objectives To evaluate the safety, pharmacokinetic (PK), and pharmacodynamic (PD) effects of an optimized crovalimab regimen. Study Design and Methods Crovalimab is being evaluated in the ongoing Phase I/II COMPOSER study (NCT03157635). Parts 1, 2, and 3 of COMPOSER assessed the PK and safety of crovalimab in healthy volunteers, C5 inhibitor-naive patients, and patients switched from eculizumab, respectively. In Part 4, patients received an intravenous loading series of crovalimab 1000 mg on day 1 followed by 340 mg subcutaneous on days 2, 8, 15, and 22. Maintenance dosing of 680 mg subcutaneous every 4 weeks was started on day 29 (week 5). Plasma concentrations of crovalimab, lactate dehydrogenase (LDH), and free and total C5, as well as complement activity were determined at follow-up visits. The primary endpoints were PK and PD effects of the dosing strategy. Patients were followed up for safety and efficacy, including transfusion avoidance, breakthrough hemolysis events, and hemoglobin stabilization. Results Data for 15 patients (8 naive, 7 switched) treated with the optimized crovalimab dosing strategy in Part 4 are presented here. The data cutoff was January 29, 2020. The PK profiles showed that crovalimab exposure was maintained above the target concentration (100 µg/mL) for complete complement inhibition in all patients, naive and switched. Complete complement inhibition, as measured by liposome immunoassay < 10 U/mL, was achieved immediately following the initial dose and maintained throughout the study treatment period. Consistent with these results, crovalimab-free C5 levels declined rapidly following the initial dose and remained low throughout the follow-up period. As expected based on the target-disposal properties of crovalimab, limited total C5 accumulation was observed in naive patients and a decline was seen in switched patients. Median LDH rapidly declined to ≤ 1.5-fold the upper limit of normal (ULN) in naive patients, remained ≤ 1.5-fold the ULN in switched patients, and remained below this level throughout the observation period in both patient groups (Figure). Crovalimab was well tolerated, and no serious treatment-related adverse events were observed. Conclusions These data showed that the optimized dosing strategy for crovalimab in PNH did not raise any safety concerns and suggest that the strategy results in sustained complete complement inhibition in patients naive to complement inhibitor therapy and those switched from eculizumab. These data support the continued development of crovalimab. Disclosures Peffault De Latour: Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Apellis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Sica:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding. Ramos:F. Hoffmann-La Roche Ltd: Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.; Genentech, Inc: Current Employment, Other: Fellowship support. Hernández-Sánchez:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Kim:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Kiialainen:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.. Yoon:F. Hoffmann-La Roche: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; Janssen: Consultancy; Novartis: Consultancy, Honoraria; YuhanPharma: Research Funding; Amgen: Consultancy, Honoraria; Kyowahako Kirin: Research Funding. Sreckovic:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Soubret:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ninomiya:F. Hoffmann-La Roche Ltd: Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche., Research Funding; Alexion: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Panse:F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Grunenthal: Consultancy, Membership on an entity's Board of Directors or advisory committees; Apellis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint Medicines: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai: Speakers Bureau; Pfizer: Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buatois:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Paz-Priel:Genentech, Inc: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.. Nishimura:F. Hoffmann-La Roche Ltd: Consultancy, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.; Alexion: Honoraria, Research Funding; Chugai: Consultancy. Röth:Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Biocryst: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Apellis: Consultancy, Honoraria. OffLabel Disclosure: Yes. Crovalimab is an anti-C5 monoclonal antibody being evaluated as a therapy for paroxysmal nocturnal hemoglobinuria

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder with the expansion of PIGA mutant clone(s), which is deficient in GPI-anchored proteins including CD55 and CD59. The lack of CD55 and CD59 renders PNH-type red blood cells (RBC) susceptible to complement attacks, resulting in intravascular hemolysis. Classic PNH manifests 3 major symptoms: anemia, bone marrow failure, and thrombosis. Small populations of PNH-type cells ( Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF were prospectively recruited to the study since 2011 in Japan. A high-resolution FCM assay was established to precisely detect a small population of PNH-type granulocytes (with FLAER) and RBCs (with anti-CD55 and CD59 antibodies) ≤ 0.01% of the total granulocyte or RBC population based on the Kanazawa method (Blood 2006 107:1308-1314). Six university laboratories across Japan were designated as regional analyzing centers and measured the percentages of PNH-type cells in the study population, as well as collected clinical and laboratory data. Periodic blind cross validation tests using a positive control sample containing 0.01% PNH-type cells and a negative control sample were performed to minimize inter-laboratory variations. Results: As of July 2016, 2,849 patients were enrolled in the study and 2,734 patients were analyzed. Nine hundred twelve patients (33.4%) were positive for PNH-type cells (≥ 0.005% PNH-type erythrocytes and/or ≥ 0.003% PNH-type granulocytes) and 238 (8.7%) patients had more than 1% of PNH-type cells. PNH-type cells were positive in 90/90 PNH (100%), 512/982 AA (52.1%), 132/822 MDS (16.1%), and 141/512 indistinguishable BMF (27.5%) patients. Among the MDS patients, PNH-type cells were positive in approximately 20% of patients with RCUD, RCMD, MDS-U, or 5q- syndrome, but not in any patients with RARS, RAEB-1, and RAEB-2 (Fig. 1). The serum LDH level increased in proportion to the PNH clone size of RBCs, and 63.3% of the patients possessing ≥1.0% RBCs showed LDH levels more than 1.5 times the upper limit of normal. Of 171 patients who completed submission of 3-year follow-up data, BMF patients with PNH-type cells showed a better response rate [CR+PR, 99/107 (92.5%)] to IST compared to those without PNH-type cells [44/64 (68.8%)] (P Conclusions: These interim analyses of the OPTIMA study demonstrate that our high-resolution FCM is reliable in detecting small populations of PNH-type cells and produces consistent results among different laboratories. The presence of PNH-type cells exclusively in patients with AA and low-risk MDS suggests a link between benign pathophysiology of BMF and an increase in the number of PNH-type cells. The better response of PNH-type cell-positive BMF to IST compared to BMF without PNH-type cells was consistent with previous reports. Our data, for the first time, prospectively confirms the significance of small populations of PNH cells in BMF patients in Japan and warrants further worldwide, prospective studies on non-Japanese patients with BMF. Disclosures Ueda: Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Yonemura:Alexion Pharmaceuticals: Research Funding. Obara:Alexion Pharmaceuticals: Honoraria, Research Funding. Shichishima:Alexion Pharmaceuticals, Inc. Japan: Honoraria. Ninomiya:Alexion Pharmaceuticals: Honoraria. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Kanakura:Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol - Myers: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder caused by the clonal expansion of the phosphatidylinositol glycan class A (PIGA) mutant hematopoietic stem cells, which results in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Through the high-resolution flow cytometry-based method, GPI-APs deficient blood cells (i.e., PNH-type cells) are often detectable in patients with bone marrow failure syndromes (BMF), such as aplastic anemia (AA) and low-risk types of myelodysplastic syndromes (MDS). Sugimori et al reported that when BMF patients possessed increased PNH-type cells, the patients had a good prognosis and showed a high response rate to immunosuppressive therapies, suggesting that detection of PNH-type cells is potentially useful in determining an optimal treatment for BMF patients. Thus, we conducted a nationwide, multi-center prospective observational investigation, the OPTIMA study. Methods: From July 2011, we start recruiting the patients with BMF that were diagnosed at various hematology clinics throughout Japan to the OPTIMA study. The primary endpoint of this study was to determine the prevalence of BMF patients with PNH-type cells and to clarify the clinical significance of the presence and quantitative changes of these cells with regard to the clinical features. Six different university laboratories were assigned as regional analyzing centers. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted twice a year to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Between July 2011 and May 2015, a total of 2328 patients were enrolled to this study, and we analyzed 2212 patients who were eligible for the interim analysis. Of these patients, 74 (3.3%) were diagnosed with PNH, 690 (31.2%) with AA, 592 (26.8%) with MDS, and 856 (38.7%) with undiagnosed BMF. Using high-resolution flow cytometry-based method, 755 (34.1%; 95.9% in PNH, 52.8% in AA, 18.2% in MDS, and 24.8% in undiagnosed BMT) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. Overall, 181(8.2%) patients had ≥1% of both PNH-type erythrocytes and granulocytes; the prevalence in each disease subset was 68/74 (91.9%) in PNH, 67/690 (9.7%) in AA, 22/592 (3.7%) in MDS, and 24/856 (2.8%) in undiagnosed BMF. Regarding FAB and WHO classifications of MDS subtype, no patients with RARS (0/22), RAEB-1 (0/37) or RAEB-2 (0/23) had PNH-type cells. In contrast, 20.4% (56/275) patients with RCMD, 18.3% (26/153) patients with RCUD and 50% (2/4) patients with del (5q) MDS possessed increased PNH-type cells. Blood samples from 75 (65 with and 10 without PNH-type cells) patients were analyzed three years after the first examination. Of 65 PNH (+) patients, PNH-type cells disappeared in 4 (6.2%), while the percentage remained stable in 61 (93.8%). All of the 10 PNH (-) at the enrollment were also negative for PNH-type cells in 3 years. Conclusions: A high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells below 0.01% was successfully transferred from Kanazawa University to other laboratories in Japan. Our interim analysis confirmed previous findings that PNH-type cells were detectable in patients with 52.8% of AA and 18.2% of MDS patients. Regarding FAB and WHO classifications of MDS subtype, PNH-type cells were not detected in any of MDS RARS, RAEB-1 or RAEB-2 patients. Further analysis are required to determine the clinical significance of the minimal level of PNH-type cells as well as chronological changes in the PNH-type cell percentage, especially in terms of their relation to response to immunosuppressive therapy. Disclosures Ninomiya: Alexion Pharmaceuticals: Honoraria. Ando:Eisai Co., Ltd.: Honoraria, Research Funding. Yonemura:1. Chugai Pharma, 2. Alexion Pharma, 3. Japan Blood Products Organization, 4. OHARA Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Ueda:Alexion Pharma: Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

[Background] Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells which results in the expansion of glycosylphospatidylinositol-anchored protein (GPI-AP)-deficient (PNH-type) hematopoietic cells. PNH-type blood cells are also observed in patients with bone marrow failure (BMF). PNH is conventionally diagnosed when patients have >1% of GPI-AP-deficient erythrocytes and granulocytes determined by flow cytometry. Analyses with high resolution flow cytometry by several different groups have shown that patients with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS) have small percentages of PNH-type erythrocytes, granulocytes, and/or other lineages of blood cells and that these patients respond better to immunosuppressive therapies compared with BMF patients lacking PNH-type cells. In order to determine the prevalence and clinical significance of PNH-type cells in BMF patients, we conducted a nationwide multi-center prospective observational investigation, the OPTIMA study. [Methods] From July 2011, Japanese patients with PNH, AA, MDS or BMF of uncertain origin have been prospectively enrolled into the study. Six laboratories in different cities in Japan were assigned as regional analyzing centers and measured the percentages of PNH-type cells in the study population as well as collecting clinical and laboratory data. The high-resolution flow cytometry assessments used a liquid fluorescein-labeled proaerolysin (FLAER) method and a cocktail method with anti-CD55 and anti-CD59 antibodies for the detection of PNH-type granulocytes and erythrocytes, respectively. Periodic blind cross validation tests using a standard blood sample containing 0.01% PNH-type cells and a normal control were conducted to minimize inter-laboratory variations. From analysis of 68 healthy individuals >0.003% of PNH-type granulocytes and >0.005% of PNH-type erythrocytes were considered to be abnormal (Sugimori et al, Blood, 2006). [Results] As of May 2014, flow cytometry data have been collected from 1685 patients and are included in this interim analysis. Of these patients, 65 (4%) were diagnosed with PNH, 523 (31%) with AA, 459 (27%) with MDS, and 638 (38%) with BMF of unknown etiology. Overall, 154 (9%) patients had ≥1% of both PNH-type erythrocytes and granulocytes: 63 (97%) patients with PNH; 57 (11%) with AA; 18 (4%) with MDS; and 16 (3%) with BMF of unknown etiology. In total, 545 (32%) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. These consisted of the followings; all 65 (100%) patients with PNH; 264 (51%) with AA; 76 (17%) with MDS; and 140 (22%) with BMF of unknown origin. Lactate dehydrogenase (LDH) levels ≥1.5 × upper limit of normal range were seen in 14/329 (4%) patients with 0.005-1% PNH-type erythrocytes, 23/62 (37%) patients with 1-10% PNH-type erythrocytes, and 69/71 (97%) patients with ≥10% PNH-type erythrocytes. Periodic blind validation tests revealed that inter-laboratory differences in absolute measurements of PNH-type cells were always within 0.02%. [Conclusion] A high-resolution flow cytometry-based method, based on the Kanazawa method, that enables the detection of very low percentages of PNH-type cells was successfully transferred to 6 laboratories across Japan. Our results demonstrated that the proportion of patients identified as having small percentages of PNH-type cells differed depending on diagnosis (PNH, AA, MDS, or unknown BMF) and that elevated LDH levels (>1.5 x upper limits of normal range) were more frequently associated with higher percentages of PNH-type erythrocytes. Our findings suggest that the high resolution method is helpful as a diagnostic tool in BMF syndromes, including AA, MDS, and PNH, and may prove useful in understanding the pathophysiology of these disorders. Disclosures Noji: Alexion Pharma: Honoraria. Shichishima:Alexion Pharmaceuticals, Inc; and Medical Review Company: Honoraria, Research Funding. Obara:Alexion Pharma: Research Funding. Chiba:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Hayashi:Alexion Pharma: Research Funding. Yonemura:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharma: Honoraria. Ninomiya:Alexion Pharma: Honoraria, Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by chronic complement-mediated hemolysis, leading to life-threatening events, such as thromboembolism (TE) and chronic kidney disease (CKD), and devastated quality of life (QOL). The terminal complement inhibitor eculizumab (ECU) was approved in Japan in 2010 under the indication to suppress hemolysis in PNH. We comprehensively report as post marketing surveillance (PMS) on the efficacy and safety of ECU administered from June 2010 to March 2013 in Japanese patients with PNH. In spite of complete blocking hemolysis, the degree of improvement of anemia by ECU treatment varies patient by patient. Analyzing the data of PMS, we investigated if patient characteristics, such as LDH, bone marrow function and renal function, can affect anemia in PNH patients treated with ECU. Methods: The PMS data of 242 patients were used for demographics and safety assessment. Of 242, 144 patients with ECU treatment at least for 1 year were analyzed for efficacy evaluation. We used platelet (PLT) count as a marker of bone marrow function and divided it into three groups, Results: In Japan LDH>1000 IU/L is commonly considered as ECU applicable severe PNH patients, but this was not correlated with other markers related to PNH severity. 41.2% of patients who started ECU had eGFR Conclusion: ECU treatment for 18 months reduced hemolysis and improved anemia, renal function and QOL. It is concluded that ECU is safe and well tolerated. Anemia in PNH would be caused not only by hemolysis but also modified by bone marrow as well as renal function. Disclosures Obara: Alexion Pharma: Research Funding. Ninomiya:Alexion Pharma: Honoraria, Research Funding. Chiba:Alexion Pharma: Research Funding. Usuki:Alexion Pharma: Speakers Bureau. Shichishima:Alexion Pharma: Honoraria, Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ohyashiki:Alexion Pharma: Honoraria, Research Funding. Nakao:Alexion Pharma: Honoraria, Research Funding. Ando:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharma: Honoraria, Research Funding. Nakakuma:Alexion Pharma: Honoraria. Hamada:Alexion Pharma: Employment. Shimono:Alexion Pharma: Employment. Kinoshita:Alexion Pharma: Honoraria. Ozawa:Alexion Pharma: Honoraria. Omine:Alexion Pharma: Honoraria. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a disease derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells. In some cases with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS), it is known that glycosylphosphatidylinositol-anchored protein deficient (PNH-type) cells can be often detected at low frequencies (about 0.01%) through the high-resolution flow cytometry-based methFod. Because these patient groups are reported to have a good reactivity towards immunosuppressive therapies as opposed to the other patient groups lacking PNH-type cells, detection of these cells is potentially useful in determining a treatment plan for the patients with bone marrow failure syndromes. To confirm this preliminary information, a large cohort study is needed. Method A nationwide multi-center prospective observational study (OPTIMA) was started in July 2011 to determine the prevalence of patients with bone marrow failure syndromes who carried PNH-type cells and to clarify the significance of the presence and quantitative changes of these cells with regard to the clinical features. Each of the six laboratories in different universities was assigned as a regional analyzing center. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Quality of the assay was managed in all the laboratories by periodic blind validation tests using standard blood samples containing 0.01% PNH-type cells. Until July 2013, 1214 cases were examined; 461 (38%) were positive for PNH-type cells and 141 (11.6%) had ≥1% PNH-type cells. Out of 1214, 783 patients were diagnosed to have AA (n=386), MDS (n=341), and PNH (n=56) based on the case report forms. PNH-type cells were detected in 56.2%, 19.1% and 100% of patients with AA, MDS and PNH, respectively. In a half of patients having ≥1% PNH-type cells, lactate dehydrogenase levels exceeded the ≥1.5×upper limits of normal. Conclusion Our study has successfully established the high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells (below 0.01%). Also, by implementing a uniform protocol to six individual laboratories across the country, a system has been established for the patients to undergo the detection test with equal accuracy in all of these laboratories. Further accumulation of case studies and prolonged observations are required to determine the clinical significance of the minimal PNH-type cells, especially in terms of its relation to response to immunosuppressive therapy. Disclosures: Obara: Alexion Pharmaceuticals: Honoraria. Chiba:Alexion Pharmaceuticals, In: Research Funding. Sugimori:Alexion Pharma: Honoraria. Noji:Alexion Pharmaceuticals: Honoraria. Yonemura:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals, In: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Ninomiya:Alexion Pharma: Honoraria. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals, In: Research Funding.

Background Paroxysmal nocturnal hemoglobinuria (PNH) often affects young adults, including women of childbearing age. Because PNH hemolysis increases the risk of complications such as thrombosis during pregnancy and the postpartum period, proper management should be provided for pregnant patients with PNH. Eculizumab, a C5 complement inhibitor, could be a therapeutic option for such patients. Aim/methods Japan PNH Study Group aimed to survey the current status of management of pregnant PNH patients and to propose a consensus recommendation for such a challenging situation in the treatment of PNH. Clinical data were collected from 10 pregnant PNH patients from 7 institutes and analyzed. We had consensus meetings 5 times since January 2012. Results Summary of patients' characteristics are shown in Table. Age of the patients at the time of conception ranged from 29 to 40 years old (median 30). Plasma levels of LDH at the baseline assessment were 200-2300 IU/L (median 1900). PNH clone sizes in granulocytes were 1.6-96.0% (median 81). Prophylactic anticoagulation with heparin was employed in all patients except one who showed little hemolysis due to a small size of PNH clone. Four patients received eculizumab during pregnancy with favorable efficacy. Complications including preeclampsia, hemolytic episodes and deep venous thrombosis were manageable. Newborn babies were all healthy. A negligible amount of eculizumab was detected in cord blood of three babies. Discussion Recently successful pregnancy outcomes have been reported in patients with PNH on eculizumab therapy during pregnancy, and we also experienced additional 4 cases. As eculizumab inhibits intravascular hemolysis itself, it can be expected to lower the risk of complications during pregnancy and postpartum period in women with PNH. Eculizumab is a hybrid of IgG4 and IgG2, and the latter is known to be less prone to cross the placenta. In fact eculizumab concentrations in the cord blood was much lower than therapeutic levels. Importance of anticoagulation therapy has been emphasized for the management of pregnancy with PNH. Theoretically, if intravascular hemolysis is completely inhibited by eculizumab, the risk of thromboembolic complication would become as low as that in non-PNH pregnancies. It is not clear whether the use of anticoagulant is mandatory for the pregnant patients who are receiving eculizumab, although the use of anticoagulant should not be hesitated. Conclusion Eculizumab could be safely and effectively used in pregnant patients with PNH. Taking these findings into account, we propose the following protocol for the management of a pregnancy with PNH, as shown in Figure. The patients are classified into 4 categories based on eculizumab use and the history of venous thromboembolisms or anticoagulant use. Disclosures: Usuki: Alexion Pharmaceuticals: Speakers Bureau. Urabe:Alexion Pharmaceuticals: Speakers Bureau. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Miura:Alexion Pharmaceuticals: Research Funding. Morita:Alexion Pharmaceuticals: Research Funding. Nishiwaki:Alexion Pharmaceuticals: Research Funding. Ninomiya:Alexion Pharmaceuticals: Honoraria. Gotoh:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau.

Abstract 4373 [Background] Paroxysmal nocturnal hemogloblinuria (PNH) is a hemolytic disease characterized by complement-sensitive red blood cells (RBC). Sometimes, a tiny amount of PNH-type, i.e., CD55- or CD59-negative, red blood cells as well as neutrophils can be detected in peripheral blood from patients with myelodysplastic syndrome (MDS)/ aplastic anemia (AA). It has been reported that the presence of PNH-type cells is correlated with a good response to immunosuppressive therapy foe these disease. However, the ratio of PNH-type RBCs to normal RBCs is always underestimated because PNH-type red blood cells have a shortened life span in the circulation, and there are some patients who have very low number of circulating granulocytes. [Metods] Peripheral blood was obtained from 37 patients with MDS or AA and 30 normal subjects. Mononuclear cells were then stained with antibodies against CD55, CD59, and CD71, and analyzed by a flowcytometer. Subsequently, CD55 and CD59-negative and CD71-positive reticulocytes, representing an immature fraction of reticulocytes, were identified. Sensitivity and specificity were compared for the detection of PNH-type immature reticulocytes with those evaluated by a PNH-type RBC- and granulocyte-detecting method. [Results and discussion] All 30 normal subjects were negative when the cut-off value for CD55 and CD59-negative and CD71-positive reticulocytes was set at 0.008%. With this cut-off value, PNH-type immature reticulocytes were detected in 18 out of 37 MSA/AA patients. When the cut-off values for PNH-type RBCs and granulocytes were set at 0.005% and 0.01%, respectively, in accordance with the original report by Sugimori, et al, 16 and 18 cases among 37 MDS/AA patients were positive for PNH-type RBCs and granulocytes, respectively. All the 16 cases positive for PNH-type RBCs were also positive for PNH-type granulocytes and immature reticulocytes, while 2 cases negative for PNH-type RBCs were evaluated to be positive for both PNH-type granulocytes and immature reticulocytes. All the cases positive for granulocytes were also positive for immature reticulocytes, and vice versa. These observations indicate that the method to detect minor population of PNH-type immature reticulocytes is feasible and comparable with that to detect PNH-type granulocytes, and could be superior to the PNH-type RBC-detection method in terms of sensitivity, without reducing specificity. If the evaluation of PNH-type granulocytes is difficult, PNH-type immature reticulocytes may be a good alternative to be evaluated. Disclosures: No relevant conflicts of interest to declare.

Abstract 4587 The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We previously reported that the c-Myb knock down (KD) mice exhibited anemia and thrombocytosis. To better understand the transcriptional regulatory program that accompanies the decrease of c-Myb, we performed a transcriptome analysis of megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). We found that CD9 expression was dramatically increased in the MEPs from c-Myb KD mice. CD9 belongs to a tetraspanin family and regulates cell motility and adhesion. Several reports described that CD9 is involved in platelet function by forming a complex with integrin family members including platelet fibrinogen receptor integrin aIIb-b III. To elucidate the functional contribution of CD9 to the thrombogenesis within the regulatory hierarchy mediated by c-Myb, we first examine the relation between c-Myb and CD9. We found the recruitment of c-Myb to the promoter region of CD9 by chromatin immunoprecipitation assay. A reporter assay showed that c-Myb represses CD9 promoter activity. These results indicate that CD9 is directly repressed by c-Myb. Since an agonistic antibody against CD9 stimulated megakaryocytic colony formation, we investigated the role of CD9 during megakaryopoiesis in vivo by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Consistent with this result, the recovery phase of platelet counts were delayed in the CD9-null mice after the bone marrow suppression with 5-fluorouracil, indicating the impaired platelet production. In CD9-null mice, the proliferation of megakaryocytes was promoted and circulating megakaryocytes in the peripheral blood were increased, which might compensate the PPF impairment of CD9-null megakaryocytes. Our study suggests that c-Myb suppresses CD9 expression under the steady state, while, in the stress megakaryopoiesis, CD9 is derepressed and acts to enhance platelet production. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis. Disclosures: Ninomiya: Alexion: Research Funding.

Abstract 1324 Poster Board I-346 INTRODUCTION Eltrombopag (PROMACTA®, GlaxoSmithKline) is the first non peptide, oral thrombopoietin receptor agonist which promotes the differentiation and proliferation of megakaryocytes and increases platelet counts. This study is a randomized study comprising a double-blind (DB), placebo (PBO)-controlled phase, followed by an open-label (OL) phase in previously treated Japanese patients with chronic ITP and platelet counts METHODS In the DB phase, patients were randomized into one of two treatment groups to receive either an initial dose of 12.5mg of eltrombopag or matching PBO once daily. A dose increase was allowed at Day 22 based on individual platelet count. For each patient, primary data up to Week 6 were frozen and the treatment assignment was unblinded at Week 7 before entering into the OL phase. The primary endpoint of the DB phase was to compare the proportion of patients achieving a platelet count of ≥50Gi/L and ≤400Gi/L after 6 weeks of eltrombopag or PBO. All patients completing the DB phase progressed to the OL phase. In the OL phase, patients who had received eltrombopag during the DB phase continued to receive eltrombopag for up to 26 weeks with dosage (12.5, 25 or 50mg/day) based on the individual platelet count. Patients who had received PBO during the DB phase initiated treatment with 12.5mg of eltrombopag and received eltrombopag for 26 weeks with dosage (12.5, 25 or 50mg/day) based on the individual platelet count. The primary efficacy endpoint of the long-term OL phase was to assess the ability of eltrombopag to elevate and maintain platelet counts in a target range (50-400Gi/L) during 6 months of treatment. Bleeding symptoms were also assessed subjectively and objectively at each visit. Blood samples were collected to describe the PK profile of eltrombopag. RESULTS Of 23 patients randomized, 16 had undergone splenectomy, 17 had received H. pylori eradication and 19 were receiving concomitant ITP medication at baseline. DB Phase: 23 patients were randomized to receive 6 weeks of once daily eltrombopag (n=15) or matching PBO (n=8). By Week 3, 5 of 15 (33.3%) patients receiving 12.5mg eltrombopag achieved platelet counts >50Gi/L. Three of the responders had platelet counts ≥100Gi/L at Week 3. At the end of the Week 6, 9 of the 15 patients (60.0%) receiving eltrombopag were responders (platelet count 50-400Gi/L). Three of these patients were receiving 12.5mg and the remaining 6 were receiving 25mg. All PBO patients failed to achieve a response at any point during the 6 weeks. Long-term OL Phase: During the first 3 weeks when all patients received 12.5mg of eltrombopag, 21.7% of patients achieved a platelet response of ≥50Gi/L. From Day 22 onwards a greater proportion of patients (47.8-69.6%) achieved platelet counts within the target range of 50-400Gi/L. Over the initial 3 week period a gradual rise in median platelet counts was observed and a marked increase in the median platelet count was observed from Day 22. From Day 36 until Week 26 the median platelet count was consistently within the target range of 50-400Gi/L. Eltrombopag therapy was associated with a consistent reduction in the proportion of patients with bleeding. 36.8% (7/19) had a reduction in concomitant ITP medication (corticosteroids) during the 6 months. Adverse events (AE) were reported in 22 out of 23 patients throughout the study. Nasopharyngitis was the most common AE (43%). One patient receiving eltrombopag developed a serious AE (transient ischemic attack of mild severity, considered related to study medication by the investigator) on day 10 and was withdrawn from the study. The AEs were mostly mild to moderate. There was a linear relationship between eltrombopag dose and exposure. CONCLUSION Six month treatment of low dose eltrombopag with an initial dose of 12.5mg up to a maximum dose of 50mg increased platelet counts and reduced bleeding and the use of concomitant ITP medication in Japanese patients with refractory ITP. The higher eltrombopag exposure in Japanese patients than in Caucasian patients may explain the equivalent efficacy at lower dosages of eltrombopag. Eltrombopag was well-tolerated and is an important new treatment option for patients with chronic ITP. Disclosures Miyakawa: GlaxoSmithKline: Consultancy; Nissan Chemical Industries: Research Funding; Shionogi: Honoraria; Ono Pharmaceutical: Honoraria. Ikeda:Daiichi-Sankyo: Research Funding; Tanabe-Mitsubishi: Research Funding; Chugai: Research Funding; Bayer: Research Funding; Daiichi-Sankyo: Honoraria; Bayer: Honoraria; Sanofi-Aventis: Honoraria; Takeda: Honoraria; GlaxoSmithKline: Honoraria; Kaken: Honoraria; Sumitomo: Honoraria; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Boehringer: Membership on an entity's Board of Directors or advisory committees. Koh:GlaxoSmithKline: Employment. Katsura:GlaxoSmithKline: Employment. Kanakura:GlaxoSmithKline: Consultancy; Kyowa Hakko Kirin: Research Funding; GlaxoSmithKline: Research Funding.

Abstract 1980 Poster Board I-1002 PNH is a progressively debilitating and life-threatening disease characterized by chronic complement-mediated hemolysis. The clinical complications associated with chronic hemolysis include life-threatening thrombosis, chronic kidney disease (CKD), pulmonary hypertension, organ damage, ischemic bowel disease, and hepatic failure - all of which contribute to shortened survival of patients with PNH. CKD, a consequence of hemolysis, has significant impact on survival in Japanese patients with PNH, accounting for 18% of deaths. We previously reported results from the Japanese AEGIS study, a 12-week open-label single-arm phase II study, which demonstrated that eculizumab (ecu), a humanized monoclonal antibody against the terminal complement molecule C5, significantly reduced complement-mediated intravascular hemolysis. Ecu treatment subsequently improved anemia, fatigue and renal function in 11 Japanese patients with PNH. Given the important impact on survival of renal disease, the effects of ecu on renal function were further evaluated among 27 Japanese patients with PNH enrolled in a 26-week extension of the AEGIS study. Ecu was dosed as follows: 600mg weekly for 4 weeks; 900mg one week later; and then 900mg every other week for a total of 38 weeks of therapy. Patients received meningococcal vaccine 2 weeks prior to treatment. Consistent with the previous 12 week study report, there was a sustained reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH), through the 38 weeks of ecu treatment. LDH decreased 87% from a median of 1,814 U/L at baseline to a median of 232 U/L at 38 weeks of treatment (P Disclosures: Kanakura: Alexion Pharmaceuticals: Research Funding. Ohyashiki:Alexion Pharmaceuticals: Research Funding. Shichishima:Alexion Pharmaceuticals: Research Funding. Okamoto:Alexion: Research Funding. Ando:Alexion: Research Funding. Ninomiya:Alexion: Research Funding. Kawaguchi:Alexion: Research Funding. Nakao:Alexion: Research Funding. Nakakuma:Alexion: Research Funding. Nishimura:Alexion: Honoraria, Research Funding. Kinoshita:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bedrosian:Alexion Pharmaceuticals: Employment, Equity Ownership. Valentine:Alexion: Employment, Equity Ownership. Ozawa:Alexion: Research Funding. Omine:Alexion: Consultancy, Research Funding.

Abstract 3646 Poster Board III-582 c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the AP1 super family of basic leucine zipper proteins. c-Maf has been shown to be important for the regulation of IL-4 and IL-21 gene expression in T-helper-2 cells and the formation of the lens structure. A recent report has demonstrated that the augmentation of c-Maf gene expression was observed in myeloma cell lines and bone marrow samples of multiple myeloma patients. It suggests that c-Maf plays important role in multiple myeloma pathogenesis. However, the physiological roles of c-Maf in normal hematopoiesis are largely unknown and have never been analyzed in vivo setting. In this study, we investigated that c-Maf is indispensable for hematopoesis via analyzing c-Maf deficient (c-Maf−/−) mice. In the C57BL/6J background, c-Maf−/− mice were embryonic lethal and it died between embryonic day (E) 15 and E18. c-Maf−/− embryo looked pale and had evidence of anemia. In c-Maf−/− peripheral blood at E14.5, the number of enucleated erythrocytes was decreased compared that in wild-type (WT) littermates. Flow cytometric analysis of fetal liver cells indicated that mature red blood cells were reduced in c-Maf−/− embryos. In addition, Annexin-V positive apoptotic cells were increased in c-Maf−/− fetal liver. Interestingly, there was no significant difference between WT and c-Maf−/− fetal liver cells in erythroid colony counts on the colony-forming unit assay. These results indicated that c-Maf−/− erythroid cells can develop to mature cells, and the impaired definitive erythropoiesis in the c-Maf−/− mice is due to a non-cell-autonomous effect. Immunohistochemical staining of WT fetal liver using anti-c-Maf antibody showed that c-Maf was highly expressed in macrophage but not in erythroid cells. For this reason, we hypothesized that c-Maf−/− macrophage was responsible for anemia of c-Maf−/− embryo. The erythropoietic microenvironment in fetal liver mainly consists of clusters of erythroblastic islands that made up of central macrophage and surrounding erythroblasts. The compromise of erythroblasitc islands through defect of macrophage causes anemia and embryonic lethality. To verify whether c-Maf−/− macrophage cause fail to form erythroblasitic island, we isolated the erythroblasitc island from WT and c-Maf−/− fetal liver. As a result, the number of erythroblast surrounding the macrophage in erythroblastic island was significantly reduced in c-Maf−/− mice. Moreover, in vitro reconstitution experiments showed that c-Maf−/− erythroblasts were able to form erythroblastic island with WT macrophages, while c-Maf−/− macrophages and WT erythroblasts could not form erythroblastic island. These results strongly suggest that the cause of anemia in c-Maf−/− mice is due to abnormal function of macrophages in forming erythroblastic island. Finally, to clarify the molecular mechanism in formation of erythroblastic island, we found the target gene of c-Maf in fetal liver macrophages. The DNA microarray and real time RT-PCR analyses showed that the decreased expression of vascular adhesion molecule 1 (VCAM-1), known as an adhesion molecule expressed on macrophage of erythroblastic island, was observed in c-Maf−/− fetal liver macrophage. We conclude that c-Maf is indispensable for definitive hematopoiesis and erythroblastic island formation through crucial roll in fetal liver macrophages. Disclosures: No relevant conflicts of interest to declare.

CD9 is a four transmembrane protein belonging to a tetraspanin family and regulates cell motility and adhesion. Several reports have indicated that CD9 form complexes with integrin including platelet fibrinogen receptor integrin aIIb-b III and involve in platelet function. We have previously reported that the c-myb knock down (KD) mice exhibited anemia and thrombocytopenia, and the expression level of CD9 mRNA was markdly increased in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified and agonistic antibody of CD9 stimulated megakaryocytic colony formation. These observations suggested that the CD9 expression was downregulated by c-myb. In our current study, we investigated the role of CD9 during megakaryopoiesis and platelet function by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Furthermore, after the bone marrow suppression with 5-fluorouracil (5-Fu), the recovery phase of platelet counts were delayed in the CD9-null mice. To clarify the reason of this platelet- recover delay, the number of bone marrow megakaryocyte was investigated using anti-vWF antibody staining, but the serial measurement of megakaryocyte number in CD9-null mice was not changed compared with wild-type mice. And also megakaryocyte ploidy was not changed in CD9-null mice compared with wild-type mice. Previous reports revealed that the cytoskeleton reorganization has a key role for the PPF formation, and Rac1-PAK1 signaling is important for actin restructure and aggregation prior to the cytoskeleton reorganization. The PPF formation was suppressed by Rac1 inhibitor. Although the mRNA expression levels of Rac1 in CD9-null mice were almost same as that in wild-type mice, PAK1 activation, major target of Rac1, was delayed in CD9-null mice compared with wild-type mice using thrombin-stimulating platelets. These results suggested that the delay of PAK1 activation cause the suppression of PPF formation in CD9-null mice. Our previous and current studies demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryocytosis, CD9 is upregulated and acts to induce megakaryopoiesis and platelets production through Rac1-PAK1 signaling pathway. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

In patients with paroxysmal nocturnal hemoglobinuria (PNH), lack of the GPI-anchored terminal complement inhibitor CD59 on hematopoietic stem cells and subsequently matured blood cells results in chronic intravascular hemolysis and thrombosis. The patients also show kidney disease and pulmonary hypertension in addition to disabling fatigue, abdominal pain and impaired quality of life. Eculizumab is a humanized MoAb against a terminal complement molecule C5, and has been evaluated in 2 phase III studies in North America, Western Europe and Australia. Eculizumab significantly reduced hemolysis, anemia, transfusion requirements, and thrombotic events, and improved fatigue, renal impairment and quality of life. We conducted an open-label single-arm phase II study (AEGIS) to evaluate the safety and efficacy of eculizumab in Japanese patients with PNH relative to the two phase III eculizumab studies previously reported. The AEGIS study criteria included patients with significant thrombocytopenia (platelet counts ≥ 30x109/L) and/or minimal transfusion requirements (1 or more transfusion episodes in the preceding 2 years). Eculizumab was dosed as follows: 600mg weekly for 4 weeks; 900mg one week later; and then 900mg every other week for a total of 12 weeks of therapy. Patients received meningococcal vaccine 2 weeks prior to treatment. Eculizumab was administered to 29 Japanese patients at 9 institutions. The median patient age was 47 years (range 26–70 years), median platelet count was 150x109/L (range 28–291x109/L), 45% had a history of aplastic anemia or MDS, and 48% were taking corticosteroids. Eculizumab serum levels were sufficient to completely block complement. Twenty seven out of 29 patients completed the study. Intravascular hemolysis, the primary efficacy endpoint of the trial, was rapidly and significantly reduced with eculizumab treatment. Lactate dehydrogenase (LDH) decreased 86% from a median of 1,814 U/L at baseline to a median of 244 U/L at 12 weeks of treatment ( P

The most characteristic clinical feature of PNH is intravascular hemolysis of PNH-affected RBC due to deficiency in the expression of GPI-anchored membrane proteins with complement-regulatory activity, CD55 and CD59. PNH-affected (CD55- and CD59-negative) RBC with complement sensitivity should have a shortened life span due to intravascular hemolysis, although life span of PNH-RBC separately from normal RBC could not be measured, at least clinically in PNH patients. We recently developed a sensitive flow cytometric method to analyse the PNH-phenotype (CD59-negative) in reticulocyte and whole RBC (Sato S et al: Reticulocyte-gated flow cytometric analysis of red blood cells in paroxysmal nocturnal hemogobinuria. Laboratory Hematology12: 82–85, 2006). This 2-color (RNA/CD59) flow cytometry could analyse the phenotype of reticulocyte fraction in detail, that enables to assess PNH phenotype of reticulocytes in several hematological conditions such as an emergence of RBC with PNH phenotype in aplastic anemia and a minimal residual production of PNH-RBC after hematopoietic stem cell transplantation for PNH. Analyses of PNH phenotype consecutively in total 7 patients with PNH showed differences in ratios between reticulocytes and whole RBC, where % CD59-negative population in the former was always higher than the latter due to hemolysis of CD59-negative mature RBC in the circulation. On an assumption that CD59-negative reticulocytes are not destroyed in the reticulocyte maturation time and CD59-positive RBC has a mean life span (MLS) of 120 days, we proposed a formula to estimate PNH-RBC’s MLS: W/100 = R x M / [ (100-R) x 120 + R x M ], where W, % CD59-negative whole RBC; R, % CD59-negative reticulocytes; M, MLS (day) of PNH-RBC. By this formula, PNH-RBC’s MLS was estimated at 16–45 days in the patients. It showed a poor correlation with absolute reticulocyte count or hemolysis-related laboratory data (lactate dehydrogenase (LD) etc), suggesting erythropoietic ability in response to anemia was different among PNH patients due to their various hematological backgrounds underlying PNH. Comparable degree of anemia did not induce same degree of reticulocytosis in these patients. Hence, we are proposing to use an erythropoietic ability-adjusted index, red cell turnover index (RCTI), calculated from the following formula to assess the total body hemolysis of PNH-RBC: PNH-RCTI = [(absolute reticulocyte count) x (% CD59-negative retiuclocyte) / 100] / (PNH-RBC’s MLS). PNH-RCTI is assumed to correlate with turnover rate of PNH-RBC by complement-mediated lysis in the circulation. PNH-RCTI ranged 0.88–15.3 x 103 reticulocytes/μl/day (reference range, normal RCTI 0.4–0.8). Serum LD level is a non-specifc parameter being increased by in vivo hemolysis, especially intravascular hemolysis. While PNH-RBC’s MLS did not show any significant correlation with serum LD levels (r=0.306), PNH-RCTI correlated positively with serum LD levels (r=0.704, 15 points) in 7 PNH patients. These data indicate that shortened PNH-RBC’s MLS estimated from the reticulocyte-gated flow cytometry could be a reliable parameter reflecting in vivo hemolysis in PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is clinically characterized by the complement (C’)-mediated intravascular lysis of PNH-affected erythrocytes and thrombotic diathesis. Treatment methods of intravascular hemolysis, especially on the hemolytic crisis, have not been established yet. Although several reagents have been used for the treatment of PNH hemolysis, the value of prednisone/prednisolone in treating the hemolysis of PNH is controversial; anti-C5 antibody is under clinical study. We previously demonstrated that heparin and low-molecular weight heparin (LMWH) inhibit C’-mediated hemolysis of PNH erythrocytes in vitro, by inhibiting insertion of C5b-7 into the erythrocyte membranes (Ninomiya et al: Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparon. Brit J Haematol109,875–881,2000). However, that the concentration of heparin (or even that of LMWH) required to inhibit C’-mediated lysis of PNH erythrocytes efficiently induces a profound prolongation of APTT (activated partial thromboplastin time) of plasma unables their clinical application. In the current study, we examined the inhibitory effects of heparin, LMWH (dalteparin), and N-desulfated heparin on C’-mediated lysis of PNH erythrocytes (sucrose hemolytic assay) and their anticoagulant effects on normal plasma coagulation system (APTT, TT (thrombin time), and AT-III (antithrombin)-enhancing activity). N-desulfated heparin inhibited C’-mediated lysis of PNH erythrocytes, equivalently to heparin or LMWH, on the basis of their uronic acid (UA) contents; IC50 values of heparin, LMWH, and N-desulfated heparin, was 0.8 U/ml (UA 2.9 μg/ml), 0.5 IU/ml (UA 2.3μg/ml), and 3.0μg/ml (UA 0.9μg/ml), respectively. Whereas the concentrations of heparin and LMWH, that exerted their anti-C’ activity efficiently, induced prolongations of APTT profoundly to clinically unapplicable extents, N-desulfated heparin (up to 50 μg/ml) did not show any anticoagulant activity. These data show a potential value of N-desulfated heparin for the treatment of C’-mediated intravascular lysis of PNH erythrocytes without bleeding complications due to its administration. Because anticoagulant therapy may be beneficial and indicated for the treatment of hemolysis in PNH, especially in hemolytic crisis increasing the risk of thrombotic complications, cocktail therapy of heparin (or LMWH) with N-desulfated heparin may be proposed.