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Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

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.

Data characterizing the safety and effectiveness of eculizumab in patients with paroxysmal nocturnal hemoglobinuria (PNH) are limited. We describe the safety and effectiveness of eculizumab in PNH patients enrolled in a post-marketing surveillance study. Types and frequencies of observed adverse events were similar to those reported in previous clinical trials and no meningococcal infection was reported. Effectiveness outcomes included the reduction of intravascular hemolysis, the change in hemoglobin (Hb) level, the withdrawal of transfusion and corticosteroids, the change of renal function, and overall survival. The effect of eculizumab on intravascular hemolysis was demonstrated by a reduction in lactate dehydrogenase levels at all measurements after baseline. Significant increases in Hb levels from baseline were also observed after 1 month’s treatment with eculizumab (p < 0.01). Of those who were transfusion-dependent at baseline, the median number of transfusions decreased significantly from 18 to 0 unit/year after 1 year of treatment with eculizumab (p < 0.001). An increase in Hb and a high rate of transfusion independence were observed, especially in patients with platelet count ≥150 × 109/L. Approximately 97 % of patients showed maintenance or improvement of renal function. Overall survival rate was about 90 % (median follow-up 1.9 years). These results suggest an acceptable safety profile and favorable prognosis after eculizumab intervention.

In the original publication of this article, Tables 2, 3 and 4 were published incorrectly. The corrected tables 2, 3 and 4 are given in the following pages.

Chronic hepatitis C virus (HCV) infection conditions are proposed to be an HCV syndrome of hepatic, extrahepatic or systemic disorders with malignancies such as a malignant lymphoma (ML).[1] HCV is...

In paroxysmal nocturnal hemoglobinuria (PNH), various symptoms due to intravascular hemolysis exert a negative impact on patients’ quality of life (QOL). To determine clinical factors related with improvements in QOL in PNH patients treated, we analyzed changes in QOL scales in PNH patients treated with eculizumab based on data collected from post-marketing surveillance in Japan. Summary statistics were obtained using figures from QOL scoring systems and laboratory values, and evaluated by t test. One-year administration of eculizumab improved the most QOL items in comparison with the baseline. In particular, significant improvement of EORTC QLQ-C30 was observed in fatigue, dyspnea, physical function, and global health status. Canonical correlation analysis revealed a high correlation between QOL and laboratory values. Changes in serum lactate dehydrogenase (LDH) and hemoglobin showed strong correlations with QOL improvement. Quality of life improvement was independent of patients’ baseline characteristics of co-occurrence of bone marrow failure (BMF), or the degree of LDH. In this analysis, we found that the degree of QOL improvement was independent of the baseline LDH before eculizumab treatment and of co-occurrence of BMF. Paroxysmal nocturnal hemoglobinuria patients who have not received eculizumab treatment due to mild hemolysis may benefit from eculizumab treatment.

PNH is an acquired stem cell disorder of a clonal nature characterized by complement-mediated intravascular hemolysis negative for the direct antiglobulin test (DAT, Coombs test), a thrombotic tendency, bone marrow dysfunction, and infrequent development of leukemia. Hemolysis and thrombosis are currently accepted as ascribable to deficiency of a series of glycosylphosphatidylinositol (GPI)-linked membrane proteins including decay-accelerating factor (DAF, CD55) and CD59 with complement-regulatory activity on blood cells. The deficiency is due to a synthetic defect of GPI caused by mutations of genes (virtually PIGA) involved in the steps of GPI biosynthesis. It has been suggested that PNH bone marrow failure is closely associated with acquired aplastic anemia, and it is considered to be immune mediated. For the diagnosis of PNH, identification of the three major clinical manifestations of hemolysis, thrombosis, and bone marrow dysfunction, as well as the detection of a simultaneous lack of multiple GPI-linked membrane proteins in individual blood cell lineages, reflecting the defect of GPI and mutations of GPI biosynthesis genes like PIGA in hematopoietic stem cells, is needed. Flow cytometry detects all blood cells lacking any GPI proteins, while conventional hemolytic tests such as the Ham’s acidified serum test (the Ham test) and the sugar-water test detect only PNH erythrocytes susceptible to autologous complement. The evaluation of hemolysis, thrombosis, and underlying bone marrow disorder virtually defines PNH classification and disease severity.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis via complement (C)-mediated hemolysis, thrombosis, and bone marrow failure. Treatment protocol of PNH is proposed from the views of degree of clinical severity; of predominant pathophysiology, described above; and of response to medication therapy. PNH patients with lower degree of clinical severity or complete remission with below 1 % of PNH-type granulocytes and erythrocytes should be observed without medication. With the progression of clinical severity and main pathophysiology and increase of above 10 % of PNH-type erythrocytes, medication therapy should be initiated because of appearance of various subjective symptoms. Although it is certain that eculizumab treatment is predominant for intravascular hemolysis, the therapy develops breakthrough hemolysis, extravascular hemolysis of C3-bound erythrocytes, and poor response to eculizumab treatment. It is controversial whether or not hemolysis in classic PNH patients should be treated with prednisolone because of its adverse effects. Then, administration of a human plasma-derived haptoglobin product may be suitable for uncontrolled hemolysis triggered by eculizumab treatment and hemolytic attack. Immunosuppressive therapy with cyclosporine A (CSA) and/or antithymocyte globulin and androgens in PNH with hypocellular bone marrow are useful as well as those for aplastic anemia and a combination of filgrastim and CSA or recombinant human erythropoietin administration results in a trilineage response of hematopoiesis or a response of anemia, respectively. As thrombotic events rarely occur even in PNH patients without receiving eculizumab and with less than 50 % of PNH granulocytes or in PNH patients receiving eculizumab, indication of warfarin should be carefully investigated. Supportive therapies, including blood transfusions, and therapies for complications, such as iron deficiency anemia and renal dysfunction, are also needed. Hematopoietic stem cell transplantation is curative therapy for PNH, but indication of it is now limited predominantly to PNH with severe bone marrow failure.

Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear.We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients.Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab.The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

High-dose chemotherapy followed by autologous stem cell transplantation (auto-SCT) is widely used as a salvage therapy in the treatment of refractory malignant lymphoma. To investigate the safety and feasibility of a high-dose MCNU, carboplatin, etoposide and cyclophosphamide (MCVC) regimen, we conducted a prospective multicenter trial. Thirty patients with relapsed/refractory/poor-risk non-Hodgkin lymphoma (NHL n = 27) or Hodgkin lymphoma (HD n = 3) were uniformly treated with an MCVC regimen and underwent auto-SCT. The median follow-up duration of the surviving patients was 67 months (56-133 months). The major toxicities were anorexia (94 %), diarrhea (80 %), nausea (79 %), febrile neutropenia (70 %), alopecia (67 %) and mucositis (60 %). Three patients developed severe left ventricular dysfunction, and two patients developed severe sinusoidal obstructive syndrome (SOS). Of these patients, two died without disease progression. Treatment-related mortality was 6.6 %. Late-onset adverse events including two cases of cytomegalovirus pneumonia and one of interstitial pneumonia were observed. In DLBCL (n = 13) and transformed FL (n = 2) patients, OS and EFS at 3 years were 72 and 46 %, respectively. These results suggest that the MCVC regimen followed by auto-SCT is a feasible and tolerable therapy for relapsed/refractory malignant lymphoma. However, cardiac toxicity due to high-dose cyclophosphamide and development of SOS can occur and should be carefully monitored. Further follow-up is needed to evaluate the long-term efficacy and safety of this regimen.

Pregnancy with paroxysmal nocturnal hemoglobinuria (PNH) is associated with significant risk of complications, such as life-threatening thrombosis. Recently, eculizumab has come into clinical use and revolutionized the treatment of PNH. However, clinical information regarding eculizumab use for PNH during pregnancy is limited. The present report describes pregnancies with PNH treated with eculizumab that were registered with the Japan PNH study group and reviews the literature. In case 1, the patient received eculizumab throughout pregnancy and delivered a healthy neonate at term, although breakthrough hemolysis occurred at 20 weeks of gestation. In case 2, the patient discontinued eculizumab before pregnancy and developed preeclampsia at 27 weeks of gestation. She received eculizumab and delivered a preterm, but healthy, neonate by cesarean section. In case 3, the patient received eculizumab from 18 weeks of gestation and delivered a healthy neonate at term without any complications. Reports of 11 pregnant women treated with eculizumab were identified in the literature. Of 14 pregnancies, including our own cases, breakthrough hemolysis and preeclampsia occurred in five and two cases, respectively. There were no thrombotic complications, maternal or neonatal deaths, or fetal structural abnormalities. Thus, eculizumab appears to be safe and effective for managing PNH during pregnancy.

Two patients with paroxysmal nocturnal hemoglobinuria (PNH) and parvovirus B19 (PVB19) infection are reported. One was infected by PVB19-contaminated blood transfusion, whereas the other had become infected naturally. Both patients completely recovered after transient pancytopenia, but showed severe and intermediate neutropenia (0.216 and 0.768 × 109/l, respectively) at the nadirs of aplastic crises. In the literature, PNH cases also showed neutropenia (0 and 0.54 × 109/l) at aplastic crises. When patients with PNH are infected by PVB19, they may show severe neutropenia and mild thrombocytopenia in addition to severe reticulocytopenia. Therefore, these patients might have to be treated with granulocyte colony-stimulating factor to be able to recover from severe neutropenia.

Transfusion-related acute lung injury (TRALI) is a clinical syndrome characterized by bilateral pulmonary edema in association with transfusions. We encountered a 23-year-old woman with acute lymphoblastic leukemia, in whom TRALI without anti-human leukocyte antigen class I and anti-granulocyte antibodies developed following allogeneic bone marrow transplantation. TRALI improved mainly in association with treatment of saline and ventilation support after several days, but graft-versus-host disease and thrombotic microangiopathy developed, resulting in death due to multiple organ failure. This case indicates that TRALI can also occur following allogeneic bone marrow transplantation.

We describe a rare case of a 68-year-old man with B-cell chronic lymphocytic leukemia (B-CLL) who developed autoimmune hemolytic anemia (AIHA) and anti-phospholipid syndrome (APS). On admission, he was diagnosed as having B-CLL and AIHA on the basis of CD5-positive B-lymphocytes infiltrated into his bone marrow and a positive Coombs test, respectively. Although the symptoms of B-CLL and AIHA were improved by chemotherapy, he developed deep-vein thrombosis, which was probably caused by a lupus anticoagulant. It was interesting that the thrombosis due to APS occurred following a decrease of hemolysis from AIHA after intensive chemotherapy.

Thrombocytopenia due to immune mechanisms is rare and difficult to manage in elderly patients. We describe a case of an 89-year-old female with severe immune thrombocytopenia (ITP) who rapidly improved by pulse therapy with cyclophosphamide. She was admitted to our hospital because she had arthralgia in both sides of her femoral region since January 1999, aphthous stomatitis and ecchymosis of the leg since April 1999, and bloody phlegm in July 1999. On admission, her peripheral blood count revealed severe thrombocytopenia (0.1 x 10(4)/microl). Her megakaryocyte count from bone marrow was increased to 512/microl without abnormal cells. Systemic lupus erythematosus was suspected because of strong positive protein in the urine in addition to the clinical and hematological findings described above, but she was negative for all the autoantibodies examined. Finally, she was diagnosed as having ITP on the basis of high platelet associated immunoglobulin G in addition to hematological and physical findings and she was treated with prednisolone. It was difficult to maintain her platelet count with only prednisolone, but 600 mg of cyclophosphamide rapidly increased her platelet count in spite of tapering the prednisolone. In September 2000, her platelet count was kept within normal limits by administration of 15 mg/day of prednisolon. It is suggested that immunosuppressive therapy for ITP using high-dose cyclophosphamide is useful in elderly patients as well as in juvenile adult patients.

Seventy-eight patients multitransfused using leukocyte-reduction filters at bedside were prospectively studied to determine the rate of post-transfusion alloimmunization and its risk to platelet transfusion. HLA- and HPA-antibodies were detected by standard lymphocytotoxicity test (LCT), antihuman globulin-lymphocytotoxicity test (AHG-LCT) and mixed passive haemagglutination method (MPHA).Of these 78 patients, all of whom had a negative antibody screening on admission, 11 (14%) developed HLA antibodies but only one (1%) developed an HPA antibody. HLA antibodies were detected in 9 (24%) of 38 patients with previous transfusion, pregnancy or both but in only 2 (5%) of 40 patients with no presensitization (p

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, progressive hematopoietic stem cell disorder characterized by chronic complement-mediated hemolysis leading to life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody, inhibits terminal complement activation, reduces hemolysis, decreases the risk of thrombosis, and improves renal function and quality of life in PNH patients. The long-term efficacy and safety of eculizumab in Japanese patients were assessed in a 2-year extension to a 12-week, open-label study (AEGIS). Eculizumab treatment led to an immediate and sustained reduction in intravascular hemolysis (P < 0.001) and red blood cell transfusions (P = 0.0016) compared with baseline levels. There were no reports of thromboembolism during eculizumab treatment. The majority of patients had stable (56 %) or improved (41 %) renal function and an improved quality of life (P = 0.015), with sustained reductions in fatigue and dyspnea. Eculizumab was well tolerated; no deaths or serious hemolytic events were reported, and the rate of infections declined over time. There were no significant differences in the response to eculizumab in patients with or without bone marrow dysfunction. These results demonstrate that eculizumab is an effective, well-tolerated long-term treatment for Japanese PNH patients and leads to continued amelioration of some hemolytic complications.

A patient who had myelodysplastic syndrome (MDS) and dilated cardiomyopathy (DCM) had a transient improvement of cardiac function after peripheral blood stem cell transplantation (PBSCT). When he was admitted to hospital for PBSCT, a chest X ray showed cardiomegaly, and Tc-99m quantitative gated single photon emission computed tomography (QGS) showed increases in left ventricular (LV) volumes and a decrease in LV ejection fraction (LVEF). A coronary angiogram showed no evidence of coronary artery disease. Left ventriculography showed similar findings as QGS, and the findings from a myocardial biopsy were compatible with DCM. Three months after a successful allo-PBSCT with his brother as the donor, the cardiomegaly had been attenuated, the LV volumes decreased and LVEF increased on the QGS images. However, 10 months later, his cardiac function had deteriorated. The changes in cardiac function did not correlate with the hematological changes, such as the hemoglobin level. (Circ J 2004; 68: 958 - 960)

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.

T-cell prolymphocytic leukemia (T-PLL) is characterized by a post-thymic immunophenotype, salient chromosome abnormalities, and an aggressive clinical course. However, cases in which these features are absent have been occasionally reported in Japan. Here, clinical and biological features of 13 T-PLL cases, diagnosed between 1992 and 2009 in the Tohoku region of Japan, were compared with three Western series. Median age was 64 (range 40–78) years old, and the male to female ratio (12:1) was higher than that of the Western series (P

To clarify the clinical outcome of peripheral T-cell lymphomas (PTCLs), we conducted a retrospective review comparing the outcomes of patients with PTCL (nodal peripheral T-cell lymphoma, unspecified, n=34 ; angioimmunoblastic T-cell lymphoma, n=12) to those with diffuse large B-cell lymphoma (DLBCL, n=48). All patients received CHOP-based chemotherapy without rituximab. PTCL patients presented at a more advanced clinical stage (91% vs. 65%, P

A 52-year-old Japanese woman was referred to our hospital because of fever and coxalgia. She had a white blood cell count of 241 × 102/ μL with 59.6% blasts, which had a high nuclear/cytoplasmic ratio and variably condensed nuclear chromatin. Flow cytometry and chromosomal analysis of bone marrow cells indicated positive findings of CD10, CD19, CD34, HLA-DR antigens and t(9; 22)(q34; q11.2), respectively. No rearrangements of bcr/abl in peripheral blood neutrophils were found by fluorescence in situ hybridization, suggesting that she had B-acute lymphoblastic leukaemia with Ph chromosome. Blood glucose and HbA1c (glycated haemoglobin) levels on admission were 23.4 mmol/L and 21.0%, respectively. The results of 1.5 anhydro-d-glucitol and glycoalbumin tests revealed that she certainly had diabetes mellitus (DM). Insulin therapy was initiated. Her high level of HbA1c also suggested the possibility that the patient suffered from haemoglobinopathies in addition to DM. Sequencing analyses of α1-, α2- and β-globin genes were all normal. The patient achieved complete remission (CR) by one month after her first course of chemotherapy, and the HbA1c level decreased to 10.4% following insulin therapy and chemotherapy, which were initiated when she attained CR. Her extremely high HbA1c level was due mainly to DM. Also, suppression of erythropoiesis by proliferation of leukaemic cells and latent iron deficiency might have partially contributed to the increased HbA1c. This could result in a transient but extremely high HbA1c level. To our knowledge, this is the first report of an acute leukaemia patient who expressed an extremely high level of HbA1c.

A case of a child with paroxysmal nocturnal hemoglobinuria (PNH) is characterized by an increased sensitivity of the erythrocyte to hemolytic action of complement. The widely used Ham test may not always be reliable. Recently, a panel of monoclonal antibodies has become available to detect various glycosylphosphatidylinositol (GPI)-linked proteins by flow cytometry (FCM)1 and the deficiency of GPI-anchored proteins on the various kinds of cell membranes is implicated as the pathogenesis of PNH. We diagnosed a case of a child with PNH by FCM and complement lysis sensitivity (CLS) test, which showed the increased sensitivity of PNH erythrocytes to complement. His diagnosis was delayed because of Ham test negativity and rarity of PNH cases in children.

A 62-year-old woman with multiple myeloma, who has been in complete remission (CR) for 10 years, is reported. The patient was treated with conventional chemotherapy, including nitrosourea derivatives. Five patients with myeloma, including the present case, who have survived for 10 years or more in CR and on whom detailed clinical descriptions were published, are reviewed. Their disease condition represented ‘a cure’ or ‘a state extremely close to cure’. The review indicates the following favorable prognostic factors common to these patients: age ≤65 years and a rapid response to treatment. Progressive bone destruction and/or lytic changes at disease onset is perhaps not a bad prognostic factor in myeloma patients with excellent outcome.

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody that inhibits terminal complement activation, has been shown to reduce hemolysis in PNH patients. The pivotal open-label, 12-week phase II registration study (AEGIS) was designed to evaluate the efficacy and safety of eculizumab in Japanese patients with PNH. This trial achieved its primary endpoint of reducing intravascular hemolysis with high statistical significance. Twenty-seven of the 29 patients responded to eculizumab treatment, resulting in an 87% reduction in hemolysis (P 0.0001) and subsequent improvement in anemia (P = 0.0003) despite reduction in transfusion requirements (P = 0.006). Fatigue and dyspnea significantly improved within 1-2 weeks of eculizumab treatment and the improvement was independent of changes in hemoglobin. Chronic kidney disease (CKD) was common (66%) and eculizumab treatment improved CKD in 41% of patients at 12 weeks (P 0.001). Elevated thrombotic risk was evident in Japanese PNH patients and eculizumab treatment normalized D: -dimer levels in 45% of patients with elevated D: -dimers at baseline (P 0.001). The AEGIS results demonstrate that eculizumab is effective, safe and well tolerated in Japanese patients with PNH.

T-prolymphocytic leukaemia (T-PLL) is a rare dis-order with a poor prognosis. A 69-year-old man was diagnosed as having a small-cell variant of T-PLL according to the French–American–British classification by haematological, immunological and ultrastructural studies, although the cells had a CD7− phenotype and no chromosomal abnormality. He had no symptoms or organomegaly. The number of his lymphocytes, 53.7 × 109/l at the time of diagnosis, gradually decreased without therapy, and he was in complete remission 39 months later. A rearranged band in the T-cell antigen receptor-β gene, which was detected at the time of diagnosis, decreased or disappeared. This is the first report of a T-PLL case with spontaneous complete remission.

To clarify whether measurement of serum haptoglobin (Hp) has impact on understanding pathophysiology in bone marrow failure (BMF) syndromes, we investigated concentrations of serum Hp by nephelometric procedure in 156 Japanese patients with BMF, including 54 aplastic anemia (AA), 50 paroxysmal nocturnal hemoglobinuria (PNH), and 52 myelodysplastic syndromes (MDS) patients. The frequencies with low concentrations of serum Hp (

Summary A 47-year-old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement-sensitivity lysis tests, 80% of her erythrocytes were markedly complement-sensitive (PNH-III). Laser cytofluorimetry with a monoclonal antibody against decay-accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF-negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patient's erythrocytes. When the patient's serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

We describe the first patient with hereditary spherocytosis (HS) known to have developed splenic infarction following infectious mononucleosis (IM). An 18-year-old Japanese man was referred to our hospital in November 2004 because of continuous fever and icterus. He had undergone cholecystectomy at the age of 14 years. On patient admission in November 2004, a physical examination showed marked hepatosplenomegaly, icterus, and jaundice. He had a white blood cell count of 14.9 x 10(9)/L with 9.5% atypical lymphocytes, a red blood cell count of 2.93 x 10(12)/L, and a hemoglobin concentration of 7.8 g/dL. Microspherocytes were observed in the patient's peripheral blood smear, and immunoglobulin M antibody to Epstein-Barr virus (EBV) viral capsid antigen was detected. The patient's diagnosis was HS with IM. On day 4 of admission, the patient complained of severe abdominal pain. Abdominal computed tomography scanning revealed findings of splenic infarction. Two months after the occurrence of splenic infarction, a splenectomy was performed. A pathohistologic examination of the resected spleen revealed no evidence of thrombosis or arterial occlusion. We assume that the cause of splenic infarction was insufficient blood flow to oxygenate the entire spleen during the acute enlargement of the spleen.

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.

Host-derived Langerhans cells (LCs) are crucial antigen-presenting cells that cause graft-vs.-host disease after allogeneic haematopoietic stem cell transplantation (HSCT). However, chimaerism of LCs after allogeneic HSCT is largely unknown in humans. We here report a case that developed dermatopathic lymphadenitis accompanied by an accumulation of donor-derived LCs in the second month after allogeneic HSCT with reduced-intensity conditioning. This is the first case to show that donor LCs have the ability to migrate into draining lymph nodes and replace host LCs early after HSCT in humans.

To determine and directly compare the clinical course of white and Asian patients with paroxysmal nocturnal hemoglobinuria (PNH), data were collected for epidemiologic analysis on 176 patients from Duke University and 209 patients from Japan. White patients were younger with significantly more classical symptoms of PNH including thrombosis, hemoglobinuria, and infection, while Asian patients were older with more marrow aplasia. The mean fraction of CD59-negative polymorphonuclear cells (PMN) at initial analysis was higher among Duke patients than Japanese patients. In both cohorts, however, a larger PNH clone was associated with classical PNH symptoms, while a smaller PNH clone was associated with marrow aplasia. Thrombosis was significantly more prevalent in white patients than Asian patients, and was associated with a significantly higher proportion of CD59-negative PMN. For individual patients, CD59-negative populations varied considerably over time, but a decreasing PNH clone portended hematopoietic failure. Survival analysis revealed a similar death rate in each group, although causes of death were different and significantly more Duke patients died from thrombosis. Japanese patients had a longer mean survival time (32.1 yr vs. 19.4 yr), although Kaplan-Meier survival curves were not significantly different. Poor survival in both groups was associated with age over 50 years, severe leukopenia/neutropenia at diagnosis, and severe infection as a complication; additionally, thrombosis at diagnosis or follow-up for Duke patients and renal failure for Japanese patients were poor prognostic factors. These data identify important differences between white and Asian patients with PNH. Identification of prognostic factors will help the design of prospective clinical trials for PNH.

Efficient collection of progenitor cells is essential for PBPC transplantation. Two apheresis machines (Amicus, Baxter Healthcare; and Spectra, Gambro BCT, software version 4.7) were compared prospectively by a crossover trial.Apheresis collections were performed for two consecutive days on patients for autologous and donors for allogeneic PBPC transplantation. The patients and donors, receiving a G-CSF, were randomized into two groups. In Group I, PBPCs were collected by the Amicus on the 1st day and the Spectra on the 2nd day, and the reverse order was used with Group II. A total of 60 apheresis procedures of 30 (16 in Group I and 14 in Group II) among 40 patients and donors enrolled were performed and evaluated.The nucleated cell counts, MNC counts, CD34+ PBPC counts, and amounts of CFU-GM collected per procedure were similar with the Amicus and the Spectra. On the other hand, the decrease of peripheral blood platelet counts of patients and donors was more prominent from using Spectra than Amicus (p0.0001). Components collected by the Amicus had fewer platelets than those collected by the Spectra (p0.0001). The efficiencies of collecting nucleated cells, MNCs, and CD34+ PBPCs were not different between the machines (p0.05). However, the efficiency of collecting platelets was significantly higher with Spectra than with Amicus (p0.0001). The Amicus took longer than the Spectra to process the same volume (p0.05).Amicus is superior to Spectra in avoiding apheresis-induced thrombocytopenia caused by platelets contaminating the collected samples. Therefore, the Amicus is useful for patients with thrombocytopenia or with a less-than-normal platelet count.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by PIG-A mutations. To assess clonal expansion in PNH over time, patients with serial testing by flow cytometry (interval of at least one year) were analyzed. This study involved 164 patients with PNH from Duke (DP) and 151 patients from Japan (JP). The mean fraction of PMNs deficient in CD59 (mean±SE) was 75.2±4.2% in DP and 40.0 + 8.3% (n=21) in JP at the initial analysis, and were 74.1 ±4.7% and 50.7 + 8.6% at the latest analysis (P=NS). However, in individual cases, the proportion of affected PMNs varied considerably from -84% to +98%. Some DP and JP pts, however, had a diminution in their CD59-deficient PMNs over time, which was associated with the development of overall hematopoietic failure (P=.04 for DP, P=.05 for JP). These data illustrate the complexity of clonal expansion in PNH, and the marked variability over time. GPI-deficient populations vary considerably over time for individual patients, but a decreasing PNH clone indicates impending hematopoietic failure. Correlation between clonal expansion or diminution and the development or recovery from aplasia will help our understanding of the natural history of PNH.

PNH, which is characterized by intravascular hemolysis and venous thrombosis, is an uncommon disorder of hematopoietic stem cells with an acquired somatic mutation of PIG-A. An understanding of the natural history of PNH is essential to improve therapy. To determine and to directly compare the clinical courses of PNH patients from the USA and Japan, data were collected for 385 patients with PNH. There were 176 patients from Duke (DP) and 209 patients from Japan (JP). The mean age at diagnosis (mean ± SE) was 32.8 ±1.2 years (4-80) in DP, and 45.1 ±1.3 years (10-86) in JP. The incidence of various chnical events was analyzed. These analyses provide important diagnostic and long-term data on two large cohorts of PNH patients, and identify important differences between Caucasian and Asian patients with PNH. Caucasians tend to be younger and have classical symptoms of PNH including thrombosis, while Asians tend to be older and have symptoms of aplasia. Multivariate analysis of factors influencing survival was also undertaken. By this study, estimates of PNH prognostic factors may provide a better understanding of the complications of the disease so that therapeutic interventions may be sought. Further, by noting differences in the two populations, possible genetic modifiers of the course of the disease may be identified for further investigation.

[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.

Cobra venom factor (CVF) transiently activates polymorphonuclear leukocytes (PMN) by complement activation, followed by rapid complement depletion and gradual reversal of PMN activation. Utilizing these sequential changes caused by CVF, the individual and combined effects of complement and PMNs on myocardial infarct size (IS) were investigated. Rats were treated with CVF, and/or anti-PMNs. Complement was depleted, but circulating PMNs were being activated at 4 h after CVF administration, and at 36 h after, complement was depleted, but PMNs were in a near basal condition. Under anesthesia, the rats had a 30-min coronary occlusion followed by 6 h of reperfusion. The IS was assessed by tetrazolium staining. CVF, as well as anti-PMNs, reduced myeloperoxidase (MPO) activity in the risk area and the reduced MPO resulted in a reduced IS, which was also the effect of anti-PMNs, but complement depletion by CVF, during which circulating PMNs were activated, failed to reduce the IS despite low MPO activity. These results suggest that complement and the condition of PMNs each play a role in determining the IS, and ischemic reperfusion injury might be produced even by relatively low myocardial MPO activity. (Jpn Circ J 2001; 65: 659 - 666)

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 4588 Background: High-dose chemotherapy (HDCT) followed by autologous hematopoietic stem cell transplantation (auto-HSCT) has been shown to be appropriate approach compared with standard salvage therapy in patients with chemo-sensitive relapsed/refractory malignant lymphoma. Although a regimen consisting of high dose ranimustine (MCNU), carboplatin, etoposide and cyclophosphamide (MCVC) is widely used in clinical practice in Japan as HDCT followed by auto-HSCT for refractory malignant lymphoma, there have been few studies that tried to clarify the details of this therapy. We have conducted a multicenter prospective study involving 7 centers in Japan in order to investigate safety, feasibility and efficacy of MCVC regimen followed by auto-HSCT. Method: This study was carried out according to the principles set out in the Declaration of Helsinki, 1975, and the protocol and informed-consent forms were approved by the institutional review board before the initiation of the study. All patients provided written informed consent. Between July 2007 and June 2009, 30 patients with relapsed/refractory/poor-risk non-Hodgkin lymphoma (NHL n=27) or Hodgkin lymphoma (HD n=3) were uniformly treated with MCVC regimens and underwent auto-HSCT as follows: ranimustine (MCNU) 200mg/m2 i.v. on day-8 and -3 (total dose 400 mg/m2), carboplatin 300mg/m2 i.v. on day-7 to -4 (total dose 1200mg/m2), etoposide (VP-16) 500mg/m2 i.v. on day -6 to -4 (total dose 1500 mg/m2), and cyclophosphamide 50mg/kg i.v. on day-3 and -2 (total dose 100mg/kg). Hematopoietic stem cells were reinfused on day 0 and G-CSF administrated until hematological recovery. Result and discussion: Median age at transplantation was 51 (range, 18–62). Four (13%) of these patients were older than 60 years. Diffuse large B-cell lymphoma was the most main histologic subtype in these patients (43%). At transplantation, 16 (53%) were in CR and the others showed PR (n=12, 40%) or PD (n=2, 7%). Median number of CD34+ cells reinfused was 4.8 × 106/kg (range, 2.0–11.2 × 106/kg). All patients achieved prompt engraftment after auto-SCT; that is, the median numbers of days to achieve engraftment of neutrophils (>500/μ l) and platelets (>20000/μ l) were 10 and 13, respectively. The median amounts of red blood cell and platelet transfusions after auto-HSCT were 4 units and 50 units, respectively. The major toxicities (>50% in patients) were appetite loss (all grade 97%, grade 3/4 70%), febrile neutropenia (grade 3/4 70%), nausea/vomiting (all grade 80%, grade 3/4 56%), diarrhea (all grade 77%, grade 3/4 17%), alopecia (all grade 63%) and mucositis (50%). Treatment-related mortality (TRM) was 3.3% with one death caused by sinusoidal obstructive syndrome. With median post-transplantation follow up times of 15 months, median over all survival was not reached and median event-free survival was 18.9 months (95% CI, 14.3–23.7 months). These data suggested that the time of bone marrow recovery from transplantation and the incidence of febrile neutropenia of the MCVC were similar to that of the BEAM and BEAC. The incidence of nausea/vomiting, diarrhea and mucositis seemed to be more frequent with the MCVC than the BEAC and BEAM, and the incidence of TRM of MCVC seems to be lower than that of the BEAC and BEAM regimens. Conclusion: These outcomes suggest that the MCVC regimen followed by auto-SCT is a feasible, tolerable and effective therapy for relapsed/refractory malignant lymphoma. Disclosures: No relevant conflicts of interest to declare.

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 1987 Poster Board I-1009 Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposed a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also must proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase a chance that additional genetic mutations are acquired, in turn leads to step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3' untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation of PIG-A mutant cells. We have analyzed the expression of HMGA2 by quantitative RT-PCR in peripheral blood and bone marrow from healthy volunteers and PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers in the peripheral blood (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p Disclosures: Shichishima: Alexion Pharmaceuticals: Research Funding. Kinoshita:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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

Acquired bone marrow failure (BMF) syndromes include aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS). In this study, to clarify what the frequencies of borderline PNH patients with lower proportions of glycosylphosphatidylinositol (GPI)-protein-deficient erythrocytes and no apparent clinical symptoms of PNH, such as visible hemoglobinuria and thrombotic events, are and what the significance of serum haptoglobin (Hpl) in patients with BMF is, we examined flow cytometry of erythrocytes with expression of CD59 and quantified the concentrations of serum Hpl in 142 Japanese patients with BMF, including 54 AA, 36 PNH, and 52 MDS patients. The diagnosis and grading of the severity of AA were based on the criteria of the International Agranulocytosis and Aplastic Anemia Study Group (Blood, 1987) and that of Frickhoten et al (N Engl J Med, 1991), respectively. A patient with over 1% of CD59− erythrocytes, determined by flow cytometry, was judged to have PNH erythrocytes. The diagnosis and phenotypes of MDS were determined according to the French-American-British criteria (Br J Haematol, 1982). The concentrations of serum Hpl were measured by the nephelometric procedure, developed by Van Lente et al (Clin Chem, 1979), with some modifications. The IgG bound to erythrocytes on the cell surfaces was measured by immunoradiometoric assay, developed by Jeje et al (Transfusion, 1984), with some modifications. The proportions of CD59− erythrocytes from 32 healthy individuals, AA patients, MDS patients, and PNH patients were 0.047 ± 0.052% (mean ± standard deviation), 0.143 ± 0.183%, 0.103 ± 0.126%, and 37.529 ± 33.442%, respectively. Thirteen (36.1%) of 36 PNH patients had lower proportions (range, 1–10%) of CD59− erythrocytes and no apparent clinical symptoms. Subsequently, the concentrations of serum Hpl in 436 Japanese healthy individuals, AA patients, MDS patients, and PNH patients were 152.4 ± 72.7 mg/dl (range, 42.6–309 mg/dl), 127.6 ± 130.4 mg/dl (range, 7–551 mg/dl), 73.2 ± 74.3 mg/dl (range, 3–430 mg/dl), and 13.2 ± 24.6 mg/dl (range, 2–130 mg/dl), respectively. There were significant differences in the values between AA and MDS (p

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder, in which almost all hematopoietic cells lack glycosylphosphatidylinositol (GPI)-anchored proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities at the level of hematopoietic stem cells. It has been reported that several minor PNH clones in patients with aplastic anemia and myelodysplastic syndromes occur at random during the clinical course (Okamoto M et al, Leukemia, 2006), while one or more major PNH clones in patients with typical PNH continue to exist during their clinical courses (Nishimura J et al, Blood, 2002). To clarify how PNH clones in patients with PNH expand during their clinical courses, we examined sequence analyses of the PIG-A gene using peripheral blood granulocytes from PNH patients with high (>10%; n=5) and low (