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All Japanese patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with eculizumab were enrolled in post-marketing surveillance (PMS) between June 2010 and August 2019 to assess the long-term effectiveness and safety of eculizumab. The reduction in intravascular hemolysis, the change in hemoglobin (Hb) level, and the change in renal function were assessed to determine the effectiveness of eculizumab. The types and frequencies of adverse events (AEs) were assessed to determine its safety. A total of 632 patients were enrolled and the median treatment duration was 3.6 years. Treatment with eculizumab significantly reduced lactate dehydrogenase (LDH) levels and significantly increased Hb levels. These changes were maintained for up to 5 years of treatment. An estimated glomerular filtration rate ≥ 60 ml/min/1.73 m

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%.

Donor cell-derived hematological disorder (DCHD) is a rare complication of allogeneic hematopoietic stem cell transplantation (HSCT). The number of reports of DCHD has been increasing in the last decade, which likely reflects the growing number of HSCTs and the improved ability to identify the donor cell origin. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder arising in the context of clonal expansion of hematopoietic stem cells harboring a somatic mutation in phosphatidylinositol glycan anchor biosynthesis, class A. We report here a patient with adult T-cell leukemia/lymphoma, who developed PNH 7 years after umbilical cord blood transplantation. The patient has maintained complete remission with full-donor chimerism after HSCT. Thus, PNH was derived from stem cells of donor origin. The immature immune environment in the recipient after cord blood transplantation might have contributed to the rapid clonal expansion for neonatal stem cells in cord blood to develop typical symptomatic PNH in a short period. To the best of our knowledge, this is the first report in the literature of a case of PNH that developed in donor stem cells after HSCT.

Background: Fatigue is a common symptom associated with paroxysmal nocturnal hemoglobinuria (PNH). Eculizumab, a C5 inhibitor approved for treatment of PNH, has been shown to significantly alleviate fatigue, as indicated by reduced scores on the Functional Assessment of Chronic Illness Therapy-Fatigue scale (FACIT-Fatigue). FACIT-Fatigue scores range from 0-52 (higher scores indicate less fatigue); this assessment is validated for use in patients with PNH and has been used extensively both in clinical trials and in the International PNH Registry. In patients with cancer, the FACIT-Fatigue clinically important difference (CID) is estimated to be improvement of 3-5 points. This CID is commonly applied in PNH studies; however, no disease-specific CID for FACIT-Fatigue has been estimated in patients with PNH. A PNH-specific CID would be informative in evaluating changes in fatigue impact and could serve as a more robust criterion for evaluating treatment efficacy. The objective of this analysis was to determine the FACIT-Fatigue CID for patients with PNH using distribution- and anchor-based approaches and real-world data from the International PNH Registry. Methods: Adults with PNH who initiated eculizumab within 28 days of enrollment in the PNH Registry as of January 2021 with non-missing baseline FACIT-Fatigue scores were included in the analysis. FACIT-Fatigue scores were assessed at baseline and 6, 12, 24, and 36 months. Two distribution-based CID estimates were calculated using: 1) 0.5 × SD and 2) standard error of measurement (SEM). The SEM was calculated as SD−sqrt(1-α), where α represents the internal consistency measurement Cronbach's alpha. Cronbach's alpha was calculated from the 13 FACIT-Fatigue subscales. Anchor-based estimates considered 2 continuous patient-reported outcome variables: 1) European Organization for Research and Treatment of Cancer (EORTC) Global Health Status Quality of Life (QoL) summary score (quartiles; higher scores indicate better quality of life), and 2) EORTC Global Health Status Fatigue Subscale score (quartiles; lower scores indicate less fatigue). The baseline FACIT-Fatigue score was calculated for each predefined categorization of the anchors; the mean of differences in FACIT-Fatigue between adjacent categories was calculated and referenced as the anchor-based CID. Changes in anchors and high disease activity (HDA) shift from baseline to each follow-up visit were then assessed by FACIT-Fatigue score change (≤1 CID, no change, or ≥1 CID). HDA was defined as lactate dehydrogenase ratio ≥1.5 × upper limit of normal and ≥1 of the following: history of a major adverse vascular event (including a thrombotic event); anemia; or physician-reported abdominal pain, dyspnea, dysphagia, fatigue, hemoglobinuria, or erectile dysfunction. Results: 423 patients were included in the analysis (Table). The majority of patients were white or of Caucasian descent (84%); 3% were of Hispanic or Latino ethnicity. At baseline, 93% of patients had physician documentation of fatigue in their medical history (mean FACIT-Fatigue score, 29.4). The 2 distribution-based CIDs were 7 using 0.5 × SD and 5 using SEM; internal consistency was high (α=0.87). For anchor-based measurements, the CID was 8 using the EORTC QoL score and 10 using the EORTC fatigue subscale score. The percentage of patients who changed from having HDA at baseline to no HDA at eculizumab-treated follow-up visits increased over time. Using the SEM as the referent CID (owing to the high α value), the majority of these patients experienced >1 CID in FACIT-Fatigue that was sustained through 36 months (Figure). Results were similar when 0.5 × SD was used. Conclusion: Collectively, these results support the use of 5 points as the CID for FACIT-Fatigue in individual patients with PNH, which, although not necessarily the minimal value, is close to the range of CIDs reported in other diseases (3-5 points). This finding, obtained from a real-world dataset with a large number of patients, helps establish an important metric for assessment of the meaningful treatment response of patients with PNH. Of note, this CID is markedly smaller than the group average FACIT-Fatigue improvement of 10 points achieved with long-term eculizumab treatment in the pivotal blinded Phase 3 TRIUMPH study. Figure 1 Figure 1. Disclosures Cella: FACIT: Membership on an entity's Board of Directors or advisory committees. Ueda: Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Chugai Pharmaceutical: Consultancy, Honoraria, Research Funding; Alexion Pharma: Consultancy, Honoraria. Tomazos: Alexion, AstraZeneca Rare Disease: Current Employment. Gustovic: Alexion, AstraZeneca Rare Disease: Current Employment. Wang: Alexion, AstraZeneca Rare Disease: Current Employment. Patel: Alexion, AstraZeneca Rare Disease: Current Employment. Schrezenmeier: Novartis: Honoraria; Alexion, AstraZeneca Rare Disease: Honoraria, Other: Travel support, Research Funding; Roche: Honoraria; Apellis: Honoraria; Sanofi: Honoraria.

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.

Context Standard of care in PNH is based on complement C5 inhibition with anti-C5 monoclonal antibodies (MAbs), which are generally effective at treating intravascular hemolysis (IVH), but unmet needs remain. Hematological responses to anti-C5 antibody treatment can be heterogeneous, and a substantial number of patients experience residual anemia associated with breakthrough hemolysis (BTH). Iptacopan, a first-in-class, oral, small-molecule, selective, and reversible complement factor B inhibitor, inhibits both IVH and extravascular hemolysis. In an ongoing Ph2 study (NCT03439839), iptacopan has demonstrated improvements in hematological response and biomarkers of disease activity in PNH adult patients who had active hemolysis, despite eculizumab treatment. Objective APPOINT-PNH study (NCT04820530) will assess iptacopan efficacy and safety in PNH adult patients naive to complement inhibitors, including anti-C5 MAbs. Design The study comprises ≤8-wk screening; 24-wk, single-arm, open-label core treatment; and 24-wk, open-label treatment extension. On completion of week 24, patients who benefit from iptacopan treatment can enter extension. Eligible adult patients (N≈40) have confirmed PNH diagnosis (RBCs and WBCs [granulocyte/monocyte] clone size ≥10%); anemia (mean Hb 1.5× ULN). Key exclusion criteria: prior treatment with complement inhibitor; known or suspected hereditary complement deficiency; history of HSCT; laboratory evidence of bone marrow failure; active, systemic infection ≤14 days preceding study treatment; and history of recurrent, invasive infections caused by encapsulated microorganisms. Primary objective will assess hematologic response, defined as achieving sustained increase in Hb ≥2g/dL (in absence of RBC transfusions). Primary endpoint will evaluate proportion of patients achieving an increase from baseline in Hb ≥2g/dL between D126–D168 (in absence of RBC transfusions between D14–D168). Secondary endpoints include proportion of patients reaching Hb ≥12g/dL; absence of RBC transfusions; changes from baseline in Hb; changes in FACIT-Fatigue scores; changes from baseline in reticulocytes; % change from baseline in LDH; occurrence of BTH; and occurrence of major adverse vascular events (including thrombosis). Additional analyses include safety assessments; evolution of PNH clone size and C3d deposition on RBCs; and other PROs. Conclusions Ph3 studies to assess iptacopan efficacy in a wider population of PNH patients, including those naive to anti-C5 MAbs treatment, are needed. Study sponsored by Novartis Pharmaceuticals Corporation.

Introduction Crovalimab is a novel anti-complement component 5 (C5) monoclonal antibody engineered with the Sequential Monoclonal Antibody Recycling Technology (SMART-Ig; Fukuzawa et al, Sci Rep. 2017) to extend half-life and enable infrequent, subcutaneous (SC) self-administration in C5-mediated diseases. Crovalimab is being investigated as a therapy for paroxysmal nocturnal hemoglobinuria (PNH), a disease for which C5 inhibition is the standard of care. The Phase I/II COMPOSER trial (NCT03157635; Röth, et al. Blood. 2020) is a global, open-label, multicenter study of crovalimab consisting of 4 sequential parts. Parts 1, 2, and 3 assessed the pharmacokinetics (PK) and safety of crovalimab in healthy volunteers, C5 inhibitor-naive patients, and patients switched from eculizumab, respectively. Part 4 assessed an optimized crovalimab dose and regimen in naive and switched patients with PNH. Because eculizumab and crovalimab bind to different C5 epitopes, drug-target-drug complexes (DTDCs) consisting of eculizumab, C5, and crovalimab motifs can temporarily form in the circulation of patients who switch treatments. DTDCs can form in a range of sizes, from single crovalimab-C5-eculizumab motif to larger complexes with multiple motifs. Larger DTDCs are a concern because they take longer to clear and may be more likely to induce type III hypersensitivity reactions. Objectives Describe the impact of DTDC formation on the safety, PK, and pharmacodynamics of crovalimab in patients with PNH who switched from eculizumab to crovalimab and to describe the effect of crovalimab dose on DTDC size distribution and kinetics. Study Design and Methods Using data from COMPOSER Parts 1-3, a biochemical mathematical model was developed to investigate the kinetics of the formation and dissociation of DTDCs under the assumption that larger complexes are formed by the reversible binding of smaller complexes. The model was calibrated using concentration-time profiles of total C5, total crovalimab, and the concentration of eculizumab at the time of crovalimab initiation. DTDC size distributions were measured using size-exclusion chromatography coupled to enzyme-linked immunosorbent assay. Using model-based simulations, an optimized crovalimab dosing strategy was identified to reduce the formation of large DTDCs while maintaining serum concentration of crovalimab above the target level of ≈ 100 μg/mL. The optimized dose and regimen were a loading series of 1000 mg intravenously on day 1 and 340 mg SC on days 2, 8, 15, and 22, followed by maintenance dosing of 680 mg SC every 4 weeks starting on day 29. The loading dose series increased the total crovalimab dose received during the first month of treatment to reduce the formation of larger DTDCs, in line with the lattice theory of complex formation. This optimized dosing strategy was investigated in Part 4 patients who switched from eculizumab. Results In COMPOSER, 19 patients with PNH were enrolled in Part 3 and switched from eculizumab to crovalimab. DTDCs were observed in all patients from Part 3 (Figure; larger DTDCs are found in fractions 1-4 and smaller crovalimab-containing complexes, such as single motifs and single crovalimab molecules, are found in fractions 5 and 6). Two Part 3 patients experienced clinical manifestations compatible with type III hypersensitivity reactions that were ascribed to DTDCs. The DTDC size distribution in Part 4 patients, who received the optimized dosing strategy, evolved differently than in Part 3 patients, consistent with model predictions. In the switched patients from Part 4, large DTDC levels started to decrease on day 8 and continued to decrease, in contrast to Part 3, in which they started to decrease on day 15. On day 22, the mean percentage of the largest DTDCs was reduced by 56% in patients in Part 4 relative to patients in Part 3. Part 4 patients achieved and maintained serum crovalimab concentrations above ≈ 100 µg/mL throughout follow-up. Despite DTDCs being observed in all Part 4 patients who switched from eculizumab, no adverse events suggestive of a type III hypersensitivity reaction occurred. Conclusions The optimized crovalimab regimen resulted in lower concentrations of large DTDCs than in patients who received the Part 3 regimen and reduced the persistence of DTDCs in patients who switched treatment. This regimen is now being evaluated in the Phase III COMMODORE 1 (NCT04432584) and COMMODORE 2 (NCT04434092) studies. Figure Disclosures Nishimura: F. Hoffmann-La Roche Ltd: Consultancy, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; Alexion: Honoraria, Research Funding; Chugai: Consultancy. 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.. 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.. Charoin: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.. 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.. Bucher:F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Ended employment in the past 24 months, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; ANAVEON AG: Current Employment. 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.. Jordan:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ramos: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.; Genentech, Inc: Current Employment, Other: Received fellowship support from Genentech, Inc.. Arase:Osaka University: Current Employment; Chugai: Consultancy; Alexion: Research Funding; F. Hoffmann-La Roche. Ltd.: Consultancy, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Hotta: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.. Isaka:Osaka University: Current Employment; Chugai: Research Funding, Speakers Bureau; 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.. Ito: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.. Kanakura: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.; Chugai Pharmaceutical: Consultancy. Kim:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Kinoshita:Alexion: Honoraria; 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.. Morii:F. Hoffmann-La Roche Ltd: Honoraria, 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; Chugai: Honoraria, Research Funding. Panse:Apellis: Consultancy, Membership on an entity's Board of Directors or advisory committees; 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; Grunenthal: Consultancy, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Peffault De Latour:Alexion Pharmaceuticals Inc.: 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; Apellis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding. Röth:Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Biocryst: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. 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. Takamori:Alexion: Research Funding; 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.. Ueda:Chugai: Research Funding; Novartis: Honoraria; Alexion: Consultancy, Honoraria, Research Funding; 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.; Sanofi: Consultancy, Honoraria. Yoon:Kyowahako Kirin: Research Funding; Novartis: Consultancy, Honoraria; Janssen: Consultancy; 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; YuhanPharma: Research Funding; Amgen: Consultancy, Honoraria. Paz-Priel:Genentech, Inc: Current Employment; 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.. 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..

Introduction CAD is a rare, chronic type of autoimmune hemolytic anemia, where hemolysis is driven by classical complement pathway (CP) activation. Complement activation assures rapid initiation of the complement cascade as part of an early immune response and inflammation. A proinflammatory state secondary to complement activation has been demonstrated in other hemolytic disease such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome (Weitz et al. Thromb Res. 2012; Cofiell et al. Blood. 2015). Complement activation stimulates cytokine production (tumor necrosis factor, interleukin 6 [IL-6], IL-8, IL-17) via anaphylatoxins C3a and C5a and increases vascular inflammatory markers, consistent with complement-mediated inflammation (Landsem et al. Clin Exp Immunol. 2017; Cofiell et al. Blood. 2015). In patients with chronic fatigue syndrome, complement activation is associated with higher levels of fatigue (Sorensen et al. J Allergy Clin Immunol. 2003) and inflammatory cytokine activity correlates with the severity of fatigue symptoms (Montoya et al. PNAS. 2017). In addition, IL-6 plays a more dominant role in immune stimulation, particularly during chronic inflammation stress such as rheumatoid arthritis (Choy et al. Rheumatology. 2018). In CAD, CP activation and chronic inflammation may contribute to patient fatigue, apart from anemia. CP activation resulting in a proinflammatory state has not been formally studied in CAD. The interplay between complement-mediated inflammation and fatigue also has not been previously explored in patients with CAD. In the Cardinal study (NCT03347396), efficacy and safety of the humanized monoclonal anti-C1s antibody, sutimlimab (formerly BIVV009), was evaluated in patients with CAD and recent history of blood transfusion. Here we summarize the results of inflammatory cytokine expression for IL-6 and IL-10 and their relation to fatigue over the Part A 26-week treatment period of Cardinal. Methods In Cardinal Part A, patients with CAD received intravenous doses of sutimlimab on Days 0 and 7, and biweekly infusions thereafter. Patients weighing Results Mean IL-6 level (mean pg/mL [standard error of the mean (SEM)]) steadily decreased from baseline (3.21 [0.958]; normal values IL-6 Conclusions In Cardinal Part A, decrease from baseline to TAT in mean proinflammatory cytokine IL-6 and regulatory cytokine IL-10 levels were observed during sutimlimab treatment, highlighting the influence of complement inhibition on inflammation in CAD. Improved FACIT-F scores are concurrent with sutimlimab treatment and inhibition of the CP. Concurrent inverse changes were noted between select inflammatory cytokines and improvements in patients' fatigue, suggesting complement-mediated inflammation may additionally contribute to manifestation of fatigue in patients with CAD. Disclosures Weitz: Apellis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria, Speakers Bureau. Ueda:Novartis: Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria. Shafer:Sanofi: Current Employment. Su:Sanofi: Current Employment. Foster:Sanofi Genzyme: Current Employment. Storek:Sanofi: Current Employment. Patel:Sanofi: Current Employment.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients' leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1β secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.

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.

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[−]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(−) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(−) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(−) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(−) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)−CD11b+ granulocytes and CD55−CD59− glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER− granulocytes and ≥ 0.005% glycophorin A+CD55−CD59− erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (

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.

(Introduction) Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare but life-threatening stem cell disease caused by expansion of PIGA mutated clone(s). PIGA mutation abolishes the expression of GPI-anchored proteins on the cell surface including CD55 and CD59 which protect red blood cells from complement attack, resulting in complement-mediated destruction of PNH erythrocytes. Eculizumab (Ecu) effectively ameliorates the intravascular hemolysis in PNH patients by blocking complement C5 in the terminal pathway. We previously reported that 3-4 % of the Japanese patients did not respond to Ecu due to C5 polymorphism c.2654G>A (predicting p.Arg885His) and one Argentina patient with similar but different polymorphism c.2653C>T (predicting p.Arg885Cys) (N Engl J Med 370:632, 2014). Since then, we have received various consults regarding poor response cases and requests for analysis from around the world. Here, we summarize the latest series of analyses in C5 polymorphisms and propose optimal management based on these findings. (Method) Once poor response to Ecu, defined as sustained high serum LDH, was suspected with sustained high serum LDH level, peripheral blood samples with clinical data were sent to our institute after obtaining patients' informed consent. DNA was extracted from the samples, and the hot spot of C5 polymorphisms at exon 21 was sequenced by Sanger method. If no polymorphism was identified, all 41 exons of C5 were sequenced. (Results) At the initial publication (2014), 11 cases of c.2654G>A were identified out of 345 PNH patients (3.2%). As of July, 2018, a total of 22 patients were identified among roughly 600 patients treated with Ecu in Japan (3.7%). To determine the distribution of the polymorphism, a DNA panel containing 120 Han Chinese persons were previously screened, and one had the polymorphism. The same C5 polymorphism was newly identified in one among 89 patients treated with Ecu in Korea. A similar polymorphism c.2653C>T (predicting p.Arg885Cys) was also previously identified in an Argentina patient. Another polymorphism c.2653C>A (predicting p.Arg885Ser) was identified in a Dutch patient. Furthermore, a novel mutation c.2422 G>A (predicting p.Val 808 Ile) was found in a poor responder in Israel, and currently under functional analyses. (Discussion) In PNH patients treated with Ecu, serum LDH level usually drops importantly after the first loading dose, and mostly comes to upper limit of normal range after second or third loading dose. If the serum LDH level remains high, poor response should be considered. CH50 is usually not detected in Ecu responsive patients, so monitoring of CH50 level is critical to evaluate the responsiveness to Ecu. Once poor response is suspected, hot spot of exon 21 should initially be sequenced, and then the whole 41 exons of C5 may need to be sequenced. c.2654G>A was found among Han Chinese in our previous study, so it is reasonable that a Korean case had the same polymorphism considering the geography. Surprisingly, a Caucasian case with the same polymorphism has been reported (Blood Advances 1:1254, 2017) in addition to the Dutch case, underlining the importance of poor responsiveness due to polymorphisms (p.Arg885) even outside of Asia. We previously reported that Coversin, a C5 inhibitor derived from tick saliva protein, blocked hemolysis in vitro using the serum from the patients with c.2654G>A, and it was reported that post-transplant thrombotic microangiopathy was successfully treated with Coversin for the patient with the polymorphism (Blood Advances 1:1254, 2017). C5 inhibitors targeting a different epitope or having different mechanisms from Ecu as well as other upcoming complement inhibitors targeting Factor D or C3 are expected to benefit patients with C5 polymorphism and resistance to Ecu. Further analyses and clinical trials may pave the way to a second generation anti-complement drug to treat PNH patients. Figure. Figure. Disclosures Ueda: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Muus:Akari Therapeutics: Consultancy. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Nishimura:Chugai Pharmaceuticals: Consultancy, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is an uncommon type of peripheral T-cell lymphoma (PTCL) usually presenting with erythematous, subcutaneous nodules and fever [1]. It is consi...

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder caused by expansion of a clone of hematopoietic cells lacking glycosylphosphatidylinositol (GPI)-anchored membrane proteins. Multiple lines of evidence suggest immune attack on normal hematopoietic stem cells provides a selective growth advantage to PNH clones. Recently, frequent loss of HLA alleles associated with copy number-neutral loss of heterozygosity in chromosome 6p (CN-6pLOH) in aplastic anemia (AA) patients was reported, suggesting that AA hematopoiesis 'escaped' from immune attack by loss of HLA alleles. We report here the first case of CN-6pLOH in a Japanese PNH patient only in GPI-anchored protein positive (59%) granulocytes, but not in GPI-anchored protein negative (41%) granulocytes. CN-6pLOH resulted in loss of the alleles A*02:06-DRB1*15:01-DQB1*06:02, which have been reported to be dominant in Japanese PNH patients. Our patient had maintained nearly normal blood count for several years. Our case supports the hypothesis that a hostile immune environment drives selection of resistant hematopoietic cell clones and indicates that clonal evolution may occur also in normal phenotype (non-PNH) cells in some cases.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI-anchor deficiency caused by the somatic mutation of PIGA gene in a hematopoietic stem cell (PIGA-PNH). Loss of function of PIGA causes loss of GPI anchored proteins including complement regulatory proteins. Main symptoms are hemolytic anemia and venous thrombosis caused by the uncontrolled complement activation, and bone marrow failure. For X-linked PIGA, one hit of somatic mutation causes GPI-deficiency. All other genes involved in GPI-anchor biosynthetic pathway are autosomal and hits to two alleles are required to cause GPI-deficiency. In 2013, a PNH patient caused by the PIGT mutations was reported. This patient had a splice site mutation in one germ line allele and a somatic 8Mb deletion including entire PIGT. Recently, we found a Japanese patient who has a nonsense mutation in a germ line allele and a somatic 18Mb deletion in chromosome 20q including PIGT. Here we compare two patients and report that these cases of PNH are atypical in two points. One is that PIGT-PNH cases had unusual symptoms similar to auto inflammatory syndrome. Both had long lasting chronic urticaria and joint pain. Additionally, the Japanese case had recurrent aseptic meningitis and the German patient had ulcerative colitis. It should be noted that these symptoms had lasted over ten years before hemolytic attacks occurred and diagnosis as PNH was made. Urticaria, joint pain and recurrent meningitis were controllable by eculizumab, anti-C5 antibody, showing involvement of complement activation. These inflammatory symptoms are specific to PIGT-PNH and are not seen in PIGA-PNH. PIGT is involved in transfer of GPI to proteins and its defect causes accumulation of free GPI, non-protein linked GPI. In fact, FACS analysis using the antibody which recognizes free GPI revealed that GPI negative granulocytes, monocytes, and B cells from the PIGT-PNH patient, but not from PIGA-PNH patients, highly expressed free GPI. We speculate that this free GPI together with complement activation cause inflammasome activation and are establishing assay system. The other atypical nature is a mechanism of clonal expansion. In typical PNH, autoimmunity against hematopoietic stem cells causing bone marrow failure is thought to positively select GPI-deficient cells. In contrast, the somatically deleted 18Mb included a region commonly deleted in some patients with myeloproliferative noeplasms. This region contains several maternally imprinted genes, defective expression of which is likely causally related to clonal expansion in atypical PNH cases. Thus, the symptoms and mechanism of clonal expansion of PIGT-PNH are quite different from those of PIGA-PNH, so that PIGT-PNH should be categorized as an atypical PNH. Screening of PIGT-PNH among the PNH patients by detecting free GPI using FACS analysis should be feasible. Disclosures Ueda: Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma: Honoraria, Research Funding. Kanakura:Bristol Myers: Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Alexionpharma: Research Funding; Astellas: Research Funding. Kinoshita:Alexion Pharma: Honoraria.

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 Hemoglobinurea (PNH) is a rare stem cell disease caused by the expansion of PIGA mutated clone(s). PNH-type cells are deficient in the expression of GPI-anchored proteins including DAF and CD59, which protect red blood cells (RBC) from complement-mediated intravascular hemolysis. Eculizumab (Soliris®, Alexion Pharmaceuticals) is a humanized monoclonal antibody against C5 which efficiently inhibits hemolysis by blocking the terminal complement cascade. Eculizumab dramatically ameliorates several clinical symptoms, and improves the prognosis in PNH patients. However, among 345 Japanese PNH patients who were treated with eculizumab, 11 patients showed poor response. All the poor responders had a single missense C5 heterozygous mutation, c.2654G>A, which predicts the polymorphism p.Arg885His (Nishimura et al, NEJM. 2014 13;370(7):632-9). Two of those patients have already passed away due to severe complications related to PNH, and the rest of them are still suffered from various clinical symptoms including hemolytic episodes and RBC transfusion. In these circumstances, multiple new anti-complement drugs are under development in Japan. Coversin (Volution Immuno Pharmaceuticals) is a recombinant protein (16,740 Da) derived from a secreted protein in the saliva of the Ornithodoros moubata tick, and it blocks complement-mediated hemolysis at C5 level. In this study, we examined this new anti-complement agent to a PNH patient with C5 polymorphism c.2654G>A, as well as those without the polymorphism. Materials: Peripheral blood samples were collected from a poor responder to eculizumab and hemolytic PNH patients with written informed consent as approved by the Institutional Review Board of Osaka University Hospital. In vitro hemolytic assay: RBC from ABO-matched PNH patients off eculizumab treatment were washed 3 times in saline, and subsequently incubated with Mg2+ supplemented serum of the poor responder in the presence or absence of an anti-complement agent. Alternative pathway was activated by adding HCl (22:1 of 0.4M HCl) to the serum. Heat-inactivated (56°C for 30min) serum was used as a negative control. After a 24-hour incubation at 37°C, hemolysis was quantified by measuring the optical density at 405nm (OD405). The hemolytic activity was normalized against maximum hemolysis as induced by HCl (100%) and minimum hemolysis with inactivated acidified serum (0%). Results: A 41-year-old male with fatigue was diagnosed as aplastic anemia with PNH in 2008, and cyclosporine (CyA) was initiated at the dose of 150mg/day. The PNH clone expanded from 30.6% to 70.2% in granulocytes from 2008 to 2011 with elevated LDH (700 U/L) and the patient was referred to our hospital to undergo eculizumab treatment. CyA was reduced to 100mg/day and eculizumab was initiated in May 2012. Eculizumab treatment did not change the serum LDH level without any improvement of the symptoms: fatigue, abdominal pain, and periodical hemoglobinurea. A heterozygous mutation c.2654G>A was identified as the cause of the failure to eculizumab treatment, and he is still suffered from continuous intravascular hemolysis (LDH > 1400 U/L) with periodical acute hemolytic episodes, requiring frequent RBC transfusion. In the hemolytic assay, Coversin completely blocked hemolysis at the concentration of 10ug/ml, similar to the effective inhibition with hemolytic PNH patients without the polymorphism. Discussion: Eculizumab has dramatically improved the quality-of-life in the majority of the PNH patients by blocking intravascular hemolysis, but there are still some concerns; poor response due to C5 polymorphism, C3b deposition on the RBC, high cost and burden for scheduled infusion. Blocking the complement cascade at C5 level has shown to be relatively safe if meningococcal vaccination is properly performed, but still an extravascular hemolysis remains problematic at least in some cases. Inhibiting C3 amplification would resolve both intra and extravascular hemolysis, but susceptibility to infections remains a major concern. Our study showed that Coversin efficiently blocked in vitro hemolysis in the eculizumab resistant patient with C5 heterozygous mutation, c.2654G>A. Coversin might be a therapeutic option for the population of C5 polymorphism c.2654G>A in PNH patients. Our results warrant further investigation to explore new anti-complement agents for hemolytic PNH patients. Disclosures Ueda: Alexion Pharma: Research Funding. Osato:Alexion Pharma: Research Funding. Weston-Davies:Volution Immuno Pharmaceuticals (UK) Ltd: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Hayashi:Alexion Pharma: 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.

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 an acquired clonal stem cell disorder, characterized by the complement-mediated intravascular hemolysis. Eculizumab is a humanized monoclonal antibody that binds to the human C5 protein, thereby inhibiting terminal complement-mediated hemolysis. PNH patients treated with eculizumab responded with a significant reduction in hemolysis and improved in quality of life (QOL). Recent report shows that PNH-type cells were under the oxidative stress due to elevated levels of intracellular reactive oxygen species (ROS) and/or decreased antioxidant status. Fermented papaya preparation (FPP), a dietary nutriceutical derived from carica papaya, has been shown to modulate oxidative stress damage parameters in vitro and in vivo. To clarify the pathogenetic link between oxidative stress and hemolysis in PNH, the effects of each inhibitor, eculizumab and FPP, in Japanese patients with PNH were investigated independently. Methods Peripheral blood and sera were obtained from patients with PNH and healthy individuals after the obtaining informed consent. The study protocol was approved by the IRB at Osaka University Graduate School of Medicine. For ROS assay, RBCs were measured using 2’-7’-dichlorofluorescin-diacetate. The oxidative stress index (OSI) was derived from the percent ratio of the Reactive Oxygen Metabolites-derived compounds test (Diacron) and the Biological Antioxidant Potential test (Diacron) using a JCA-BM1650 analyzer (JEOL). LDH, biomarker of hemolysis, was measured by routine tests. Eculizumab was administrated according to standard schedule. FPP was taken orally 18g/day for 3 month. Health-related fatigue and QOL were also assessed using the FACIT-fatigue (Version 4A) and EORTC QLQ-C30 (Version 3) instruments. Results PNH patients without eculizumab treatment (pts w/o ecu) showed significantly higher levels in ROS compered to healthy individuals ([mean±SD] 1.13±0.18 vs. 0.84±0.10, p=0.004, n=8). Among these patients, CD59-negative RBCs showed a significant increase in ROS compared to GPI-positive RBCs in corresponding patients (1.23±0.26 vs. 1.13±0.27, p=0.03, n=6). Similarly, the OSI level was significantly higher in pts w/o ecu than healthy individuals (1.32±0.37 vs. 0.97±0.10, p=0.022, n=9) and oxidation activity in sera was significantly higher in pts w/o ecu than healthy volunteers (361.6±91.5 vs. 285.0±31.8, p=0.039, n=9), while antioxidant activity in sera was not significantly different between these 2 groups ([2440.6±191.5 vs. 2601.5±186.6, p=0.09, n=9). Interestingly, there was no significant difference in OSI level between PNH patients on eculizumab (n=8) and healthy individuals (0.84±0.10 vs. 1.07±0.45, p=0.55). Two patients who newly started eculizumab treatment showed improvement in LDH along with ROS generation ([LDH(IU/l)] Pt.I:6082-906, Pt.II:618-211; [ROS] Pt.I:1.57-1.06, Pt.II:1.50-1.40). FPP treatment (n=2) showed little effect on LDH, while it decreased ROS (Pt.I:1.72-1.47, Pt.II:1.16-1.04) and improved the scores in FACIT-fatigue and EORTC QLQ-C30. In addition, those 2 patients showed increase in ROS after discontinuation of FPP (Pt.I:1.47-2.04, Pt.II:1.04-1.19). Conclusion The RBCs and sera derived from Japanese patients with PNH were highly susceptible to oxidative stress compared to healthy individuals. Eculizumab was effective in controlling the oxidative stress, in addition to the hemolysis, suggesting that elevated oxidative stress in PNH was mainly due to the complement-mediated hemolysis. Since FPP showed little effect on hemolysis but had a potential to ameliorate the oxidative stress and improved QOL, FPP could alleviate symptoms associated with oxidative stress and may contribute to the therapeutic option for supportive therapy in PNH. Disclosures Osato: Alexion Pharma: Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hayashi:Alexion Pharma: Research Funding. Ueda:Alexion Pharma: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The pathophysiology of human aplastic anemia (AA) is immune-mediated destruction of bone marrow stem and progenitor cells. Most patients respond to immunosuppressive therapies (IST), which markedly improved survival in this disease. However, a minority of patients undergoes transformation to malignant hematologic disease, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), usually accompanied by the cytogenetic abnormality monosomy 7. Clonal evolution in AA confers a poor prognosis in the clinic but is an opportunity to assess early events in oncogenesis in the setting of inflammation and tissue regeneration. We previously reported that low mean telomere length of leukocytes at the time of diagnosis of AA (Scheinberg et al., JAMA 2010) was associated with increased risk of progression to MDS. In the current study, we directly compared acquired mutations in candidate genes and chromosomal instability, as measured by telomere length, in a cohort of AA patients that had progressed to MDS. Thirteen AA patients who developed monosomy 7 were compared with 30 AA patients who had received similar treatments but did not progress to MDS. Leukocytes telomere content was measured by qPCR in samples obtained at different time points from the diagnosis of AA and chromosome-specific telomere length was assessed by single telomere length assay (STELA) for Xp, Yp, 12q, and 17p. In the AA patients who had evolved to MDS and AML, analysis of acquired mutations in myeloid-specific genes was performed by comparison with control “germline” DNA from purified CD3 lymphocytes by exome sequencing. Cells from AA patients with clonal evolution showed marked progressive telomere attrition, 419 bp/year during the period preceding development of monosomy 7. Telomere attrition was progressive from the time of diagnosis of AA. By STELA, accumulation of very short telomere fragments was apparent at 6 months after IST. In contrast, for the AA control group, patients whose disease was stable, telomere attrition was not accelerated in serial qPCR determinations, nor was there increased accumulation of short telomere fragments by STELA. A similar pattern of increased telomere attrition was reproduced in vitro by cultivation of bone marrow cells obtained six months after IST in all AA patients who developed MDS, while none of the AA control bone marrow cultured cells developed shorter telomeres. We examined bone marrow myeloid cells at the time of monosomy 7 for acquired mutations in 125 candidate genes reported to be recurrently mutated in AML and MDS. Exome sequencing was performed using Agilent SureSelect Target Enrichment System. The raw reads were mapped to UCSC Human Genome hg19 by BWA software with default setting. With an average of 111-fold coverage on selected exon regions, somatic mutations were identified between paired samples using SAMtools and Shimmer software for SNP detection. Acquired mutations in myeloid cells were found in two cases. One patient had a heterozygous mutation in DNMT3A (K829T) present since diagnosis of AA. The other patient also had a heterozygous mutation in DNMT3A (P904S) as well as mutations in DOTL1, ASXL1, SETBP1, and STAT3. All these mutations were identifiable after IST as neutrophils recovered. Despite the presence of multiple mutations, this patient had shown a good hematologic response to IST; evolution was manifest as recurrent pancytopenia and stable marrow myeloblasts at about 5% for over 2 years after first detection of monosomy 7. The remaining 11 patients, all of whom lacked candidate gene mutations, had progressive increase in bone marrow myeloblast numbers; the only other three survivors in this cohort had received hematopoietic stem cell transplant. In conclusion, telomere shortening rather than accumulation of point mutations in hematopoietic cells preceded aneuploidy and malignant transformation at an early stage of oncogenesis in this group of patients. These results from AA may be generalizable to other cancers arising in the setting of inflammation and tissue regeneration in other organs. Identification of critically short telomeres before the development of cytogenetic abnormalities may allow for improved management of patients at risk of clonal evolution, and pharmacologic strategies to increase telomerase activity might mitigate the risk of cancer in these settings Disclosures: No relevant conflicts of interest to declare.

Donor-cell derived leukemia (DCL) is a rare but serious complication of hematopoietic stem cell transplantation (HSCT), reported in 5% of all relapses following allogeneic HSCT. Only 12 cases of DCL after umbilical cord blood (UCB) HSCT are reported in the literature. Multiple mechanisms have been proposed for DCL, including occult donor leukemia, impaired immunity, drug toxicity, or a bone marrow “leukemogenic milieu”. We propose a novel mechanism of leukemogenesis, mediated by very short telomeres of donor cells with subsequent severe telomere attrition in vivo, genomic instability, and progression to complex cytogenetics acute myeloid leukemia (AML), based on study of a patient who underwent UCB HSCT for myelodysplasia (MDS). The patient was a 44-year-old woman, subsequently shown to have a T354M mutation in GATA2, who presented at age 19 years with multiple infections and MDS. At age 41, due to progression of MDS to AML, she received induction chemotherapy and underwent a single 4/6 HLA-matched UCB HSCT. She had delayed engraftment. achieving an absolute neutrophil count of 500/ul more than 100 days post-transplant. Chimerism studies demonstrated 100% donor cell at all time-points post-transplant. Two years and eight months after HSCT she presented with pancytopenia, circulating myeloblasts, and 50% myeloblasts in bone marrow, indicating recurrence of with AML. However, cytogenetics revealed complex abnormalities, t(2p;3q) and an interstitial deletion of 5q, male cells, indicating donor origin of AML. Telomere length of the transplanted cord blood cells measured by Q-PCR showed a severe decrease in length to 7.7 kb compared with an average length of 13 kb in control UCB (n=12). Moreover the telomere length decreased precipitously to 6.6 kb 2 years after transplantation and 5.6 kb at the time of diagnosis of AML. Single telomere length assay (STELA) was used to assess chromosome-specific telomere length. Very short telomeres ( Telomere attrition occurs in the first year after HSCT, presumably due to proliferative stress on the stem cell compartment. In comparison to bone marrow, UCB have longer telomeres, but HSC number may be lower than in a BM inoculum, especially for adult recipients. In our case, initial telomere length of UCB was extremely short, for unclear reasons but possibly related to low cord blood cell viability. The long time that was needed for engraftment generated additional telomere attrition with accumulation of very short telomere and genomic instability leading to development of leukemia. Screening UCB for telomere length prior to HSCT might prove useful in identifying situations at risk for graft failure or malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1265 BACKGROUND: Acquired severe aplastic anemia (SAA) is a human disease characterized by severe pancytopenia due to marked reduction in the numbers of hematopoietic progenitors and stem cells. Immunosuppressive treatment with antithymoglobulin and cyclosporine results in improvement in blood counts in above 2/3 of patients. The most serious long-term complication in SAA is progression to myelodysplasia (MDS) and acute myeloid leukemia, usually associated with the cytogenetic abnormality monosomy 7. Clonal evolution usually requires a hematopoietic stem cell transplant and it is the major cause of morbidity and mortality. OBJECTIVE: Decreased average telomere length in leukocytes on presentation has been associated with increase risk of progression to MDS (Scheinberg et. al., 2010 JAMA, vol. 304(12):1358–64). Single telomere length assay (STELA) uses single molecule polymerase chain reaction (PCR) to amplify chromosome-specific telomeres based on the specificity of their subtelomeric region (Baird et. al., 2003 Nature Genetics, vol. 33(2):203–7). We have utilized STELA for identify SAA patients at risk of progression to MDS prior to development of the cytogenetic abnormality. METHODS: Peripheral blood samples were obtained at diagnosis and sequentially after immunosuppressive treatment from SAA patients who fulfilled entry criteria for protocols at the National Institutes of Health. Chromosome-specific PCR amplification of telomere using primers specific for Xp/Yp subtelomeric regions was performed. A non-radioactive, digoxin-labeled telomeric probe was used for Southern blotting to identify discrete bands, allowing quantification of very short telomeres. Results were confirmed using chromosome-specific primers for 2p, 11q, 12q, and 17p. RESULTS: Initially, multiple serial samples from five patients who had progressed to monosomy 7 MDS were analyzed by STELA of DNA extracted from peripheral blood leukocytes at the diagnosis of SAA and again at progression to MDS. All five patients had increase in very short telomeres ( CONCLUSIONS: This novel, non-radioactive method of quantifying chromosome-specific telomere length is capable of identifying very short telomeres in SAA patients progressing to MDS with monosomy 7. Previous studies have associated average short telomere length with chromosomal instability and progression to malignant phenotype. Identifying very short telomere in a subpopulation of circulating cells by STELA regardless of the average telomere length by qPCR is a more sensitive method. Chromosome-specific primers and analysis of end-to-end fusion of different chromosomes will allow for better characterization of the chromosomal instability leading to malignant transformation in these patients. Allogeneic stem cell transplant is the definitive treatment for MDS with monosomy 7 but requires time for identification of a suitable donor. Identification of critically short telomere before development of the cytogenetic abnormality will allow for timely management of patients at risk of clonal evolution. Therapeutic telomerase upregulation by androgens might reduce very short telomeres and potentially decrease the transformation risk. Disclosures: No relevant conflicts of interest to declare.

Background: Telomeres are repeated sequences (the hexanucleotide TTAGGG in vertebrates) located at chromosome ends of eukaryotes, protecting DNA from end joining or degradation. Telomeres become shorter with each cell cycle, but telomerase, a ribonucleoprotein complex, alleviates this attrition. The telomerase RNA component (TERC) is an essential element of telomerase, serving as a template for telomere elongation. The H/ACA domain of TERC is indispensable for telomere biogenesis. Mutations in the telomerase components allow accelerated telomere loss, resulting in various disease manifestations, including bone marrow failure. To date, this is the first detailed report of an H-box mutation in TERC that is related to human disease. Case presentation: A 26-year-old man with myelodysplastic syndrome (MDS) had very short telomeres. Sequencing identified a single heterozygous mutation in the H box of the patient's TERC gene. The same mutation was also present in his father and his son, demonstrating that it was germline in origin. The telomere length in the father's blood was shorter compared to age-matched healthy controls, while it was normal in the son and also in the sperm cells of the patient. In vitro experiments suggested that the mutation was responsible for the telomere shortening in the patient's leukocytes and contributed to the pathogenesis of bone marrow failure in our patient. Conclusion: We analyzed a mutation (A377G) in the H box of TERC in a young MDS patient who had significantly short-for-age telomeres. As telomeres protect chromosomes from instability, it is highly plausible that this genetic lesion was responsible for the patient's hematological manifestations, including marrow failure and aneuploidy in the hematopoietic stem cell compartment.