Kurzfassung: [Background] Nestin-expressing cells (NeC) have been characterized as one of many types of bone marrow (BM) microenvironmental cells, including endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, etc. Recent studies have gradually provided information about anatomy and functions of each of these cells. Nevertheless, subcellular signaling and transcriptional regulations in individual miciroenvironmental cells have poorly been demonstrated. On a different line of studies, it has been suggested that NOTCH signaling in BM microenvironmental cells affects hematopoiesis; despite this, information is limited whether NOTCH signaling plays a role in NeC. It is of note that nestin was originally identified in neural stem cells (NSC), that NOTCH signaling is known to play a pivotal role in the NSC, and that the BM NeC could be derived from neuroectoderm. This potential linkage urged us to investigate whether and how downregulation of NOTCH signaling in BM NeC affects hematopoiesis. [Method] Mice with an rbpj-flox allele were crossed with those with a CreERT2/GFP (Green Fluorescent Protein) transgene under the nestin promoter. At 8-12 weeks, tamoxifen was intraperitoneally injected for 4-12 weeks to delete the rbpj gene only in NeC (rbpj cKO mice). In this experimental system, GFP is expected to be expressed as a surrogate marker for the rbpj gene deletion, by the deletion of stop codon inserted at 5' to the cDNA of GFP. Then, transplantation assays were performed using rbpj-null BM cells as a donor to reconstitute hematopoiesis in the wild-type mice, or using rbpj-null mice as recipients to see reconstitution of hematopoiesis from wild-type BM cells. The effect of splenectomy was investigated in the untransplanted and transplanted conditions. The littermate rbpjnull/wt orrbpjwt/wtmice were used as controls. [Results] GFP was detected in 0.1-0.5% of flow cytometry (FCM)-sorted CD45(-) cells only after tamoxifen injection. Deletion of rbpj was specifically confirmed in GFP-positive BM and spleen cells. Tamoxifen induced mild splenomegaly in rbpj cKO mice compared with littermate control mice. Enlarged spleen showed preserved follicular architecture but increased CD71(+)Ter119(+) mature erythroid cells in the red pulp. In contrast, BM of rbpj cKO mice bearing mild splenomegaly demonstrated marked decrease in the CD71(+)Ter119(+) mature erythroid cells without obvious anemia. There was a substantial animal-to-animal variation in the phenotypes; however, the strength of phenotypes was correlated with the frequency of GFP-positive cells in the BM, suggesting that the phenotypic variation was a result of the efficiency of rbpj gene deletion. We hypothesized that the rbpj cKO mice would develop anemia if spenectomized, because extramedullary erythropoiesis in spleen might compensate the defective BM erythropoiesis. However, tamoxifen did not cause significant anemia in splenectomized rbpj cKO mice. In these mice, reduction of the CD71(+)Ter119(+) mature erythroid cells in BM was significantly milder than nonsplenectomized mice. In transplantation analysis, the recipient rbpj cKO mice transplanted with BM cells from wild-type mice showed a reduction in CD71(+)Ter119(+) mature erythroid cells and mild splenomegaly, as were seen in rbpj cKO mice without transplantation. The phenotypes were again erased by the splenectomy. The recipient wild-type mice transplanted with BM cells from rbpjcKO mice did not show any phenotypes. [Discussion] Rbpj cKO in NeC induced impaired erythroid differentiation in BM together with mild splenomegaly. We confirmed that these phenotypes were caused by rbpj cKO in BM NeC by transplantation experiments. Surprisingly, such BM erythropoiesis impairment was reversed by splenectomy. This unexpected finding uncovered the presence of a previously unidentified balance controller between BM and spleen erythropoiesis. Hematopoietic stem cell-autonomous NOTCH signaling has been shown to be dispensable for adult murine hematopoiesis; however, NOTCH signaling in BM-NeC is responsible for control of balance of erythropoiesis at the BM and the spleen. Disclosures Obara: Alexion Pharmaceuticals: Honoraria, Research Funding.

Kurzfassung: Background: Studies on germline variants responsible for cancer predisposition provide an important clue to the understanding of genetic basis of cancer and also help better prediction and management of relevant cancers. As for myeloid neoplasms, only a handful of genes, including RUNX1, CEBPA, GATA2, ETV6, and ANKRD26, have been implicated in early onset familial acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), although they are rarely seen in sporadic cases. Recently, using whole exome sequencing of familial AML/MDS, we have reported novel AML/MDS predisposing gene, DDX41, an encoding dead-box helicase gene. Conspicuously, the onset of AML/MDS was over 60 in most of the affected cases, raising a possibility that the genetic predisposition might be obscured and many cases could be diagnosed with sporadic AML/MDS. In this study, we investigated germline DDX41 mutations in sporadic cases with AML/MDS and the incidence and mutation types were compared between Asian and Western patients. Patients and Methods: We performed targeted sequencing of DDX41 in patients from Asian (N = 239) cohort of AML/MDS, where the origin of the detected variations was determined by using matched germline DNA. The result was compared to those obtained from the Western cohort (N = 1,034) in terms of frequency and type of mutation. The effect size of the mutations was estimated by calculating odds ratios of each variant for AML/MDS development using the data for DDX41 variants in Asian and Western population from the ExAC (Exome Aggregation Consortium) database (http://exac.broadinstitute.org) as controls. Results: Germline and somatic DDX41 mutations were found in 12 (5.0%) and 10 (4.7%) of sporadic cases with AML/MDS from the Asian cohort, as compared to 8 (0.8%) and 10 (1.0%) from the Western cohort. All the patients with germline variants were aged over 40 year-old with a median of 68.5, confirming the late onset of the disease also in the sporadic cases with germline variants. Eight of the 12 germline variants (67%) in the Asian cohort were accompanied by an additional somatic mutation, as compared to 2 of the 8 (25%) in the Western cohort. Biallelic involvement was demonstrated in selected cases (N = 2). In total, 8 and 3 germline variants were observed in the Asian and the Western cohorts, respectively, without no common variants between both cohorts, of which the predominant variants included p.A500fs (n=5; 42%) and p.E7X (n=2; 17%) in the Asian cohort and p.F140fs (n=6; 75%) in Western cohort. In contrast, a prominent hotspot mutation involving a highly conserved amino-acid within the helicase domain (p.R525H) was commonly observed in both cohorts, accounting for 55% of all the somatic mutations. These germline variants as a whole showed significant enrichment in AML/MDS cases compared to the respective control population (OR 〉 171, 95% confidence interval (CI): 51-730 for the Asian variants and more than 21.7, 95%CI: 8.4-50 for the Western variants), although the enrichment of individual variants showed substantial variations, suggesting different effect size among these variants: the odds ratio was 19.5 (p 〈 0.001) for p.F140fs, and 92.4 (p 〈 0.001) for p.A500fs. p.E7X was detected in 2 out of 239 cases with MDS/AML, whereas not in the control Asian population. Conclusion: We demonstrated frequent germline variants of DDX41 among sporadic cases with AML/MDS from different ethnic populations. Having common ancestral origins in different ethnic populations, these alleles are found in the general population at very low frequencies ( 〈 1 in 4000), accounting for the largest congenital risk for the development of sporadic AML/MDS therein (3-5% of all sporadic AML/MDS). The onset was typically over 40 years of age and frequently accompanied by an additional somatic mutation most likely in the unaffected allele, showing a prominent hotspot at p.R525. The germline variants seem to be dominant and caused premature truncation of the protein, leading to loss-of-function in most cases, whereas somatic mutations were typically missense variants not totally abrogating protein function, suggesting the importance of less than haploinsufficiency but more than null function for leukemogenesis. At the meeting, an extended result from more than 1000 Asian cases will be presented. Disclosures Kiyoi: Kyowa-Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Pfizer Inc.: Research Funding; Novartis Pharma K.k.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Company, Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., LTD.: Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Yakult Honsha Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Teijin Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; MSD K.K.: Research Funding. Miyazaki:Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Chugai: Honoraria, Research Funding; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Naoe:Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Usuki:Boehringer Ingelheim: Other: personal fees, Research Funding; Shionogi: Other: personal fees; Fujimoto Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; SymBio Pharmaceutical: Other: personal fees, Research Funding; Eisai: Research Funding; Otsuka Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Other: personal fees, Research Funding; Shire: Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; Novartis: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; MSD: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Taiho Pharmaceutical: Other: personal fees, Research Funding; Fuji Film RI Pharma: Other: personal fees; Chugai Pharmaceutical: Other: personal fees; GlaxoSmithKline: Other: personal fees, Research Funding; Bristol-Myers Squibb: Other; Astellas: Research Funding. Miyawaki:Astellas Pharma Inc.: Consultancy, Other: personal fees; Ohtsuka Pharma Co, LTD.: Other: Safety Data Committee.

Kurzfassung: Background: Nestin-expressing cells (NeC) have been characterized to consist of hematopoietic stem cell (HSC) niche in the mouse bone marrow (BM). Decreases of BM NeC have been reported in myeloproliferative neoplasms (MPN) in humans and in the mouse model of MPN. These lines of information further emphasize the importance of the NeC for the maintenance of normal hematopiesis. Nevertheless, NeC appear to be heterogenous; nestin is reported to be expressed in multiple types of BM stromal cells distinct from each other, with regard to the anatomical localization and the cell-surface antigen expression pattern. One type is reported to be localized adjacent to sinusoids and another type surrounding arterioles. A subset of endothelial cells also appears to be a candidate of NeC in the BM. It is thus critical to define the identities of distinct subsets of BM NeC. Furthermore, each subset of NeC needs to be studied in the human BM from normal subjects and those with BM diseases to understand pathophysiologic significance of NeC in patients. Myelodysplastic syndromes (MDS) are a clonal disease characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. In this disease, anormalities of BM microenvironment have been repeatedly reported; however, consensus in detail has not been reached. Purpose: To define the identities of distinct subsets of NeC in the BM from normal human subjects and to explore their abnormalities in MDS. Methods:Formalin-fixed paraffin-embedded BM biopsy samples from lymphoma patients without BM involvement (designated normal) and from MDS patients were immunostained with antibodies against six markers: nestin, CD34, laminin, α-smooth muscle actin (αSMA), glial fibrillary acidic protein (GFAP), and neurofilament heavy chain (NFH). Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed. The microscopic analysis of IHC-stained samples involved 10 randomly selected fields of view at 400× magnification, where the numbers of NeC and CD34-positive spindle-shaped cells were counted for quantitative analysis, as well as the association of these two types of cells was evaluated. IF samples were analyzed by a confocal laser scanning microscope using 10 randomly selected fields of view at 63× magnification. Then, nestin-, GFAP-, and NFH-stained areas were measured using the confocal LAS AF software for quantitative analysis. Results:NeC were found at multiple locations in distinct contexts in the normal human BM. A majority of NeC were present in association with the arterior/arteriolar structures. These artery/arteriole-associated NeC were distributed at each of the three layers; the intimal layer inside the laminine-stained basement membrane, the tunica media epressing αSMA, and the adventitial layer outside the αSMA-stained structure. The NeC located at the intimal layer expressed the highest level of nestin. The NeC were present in a close proximity with the CD34-expressing endothelial cells, although whether the endothelial cells co-expressed nestin and CD34 was unclear. The NeC at the other layers showed relatively lower levels of nestin expression. We identified NeC which did not associate with the vascular structures, albeit at a low frequency and with weak nestin staining in the normal human BM. In MDS BM, there was a significant increase in the NeC that were unassociated with the vascular structures. A portion of these increased NeC co-expressed GFAP. These cells potentially represented Schwann cells, because some of them surrounded the NFH-stained structure. Consistent with this, GFAP- and NFH-stained areas were increases in the MDS BM, together with the nestin-stained areas when measured by the confocal LAS AF software. Discussion: Multiple subsets of NeC were identified in the normal human BM as well as in the MDS BM. It is yet elusive whether each subset of NeC has a HSC niche function. In MDS BM, there was an increase in a distinct subset of NeC. The origin of these cells was elusive, but the Shwann cells normally present along with the arterial/arteriolar structures could be a candidate, because in the normal BM, a portion of GFAP-expressing cells along with the vascular structures expressed nestin. It should be elucidated whether the increased sympathetic nervous structure is involved in the pathophysiology of MDS. Disclosures Obara: Alexion Pharmaceuticals: Honoraria, Research Funding.

Kurzfassung: Background: Bone marrow (BM) fibrosis is common in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), its pathogenesis remains unclear. It is also unknown whether and how the pathophysiology of BM fibrosis in MDS and MPN are similar or different, while it is anticipated that altered BM microenvironment plays a key role. Methods: To visualize various BM microenvironmental cells in MDS or MPN patients as compared to those in control subjects, we utilized multicolor immunostaining using a tyramide signal amplification system and a fluorescence spectrum analyzer. Paraffin-embedded BM biopsy samples from 47 patients with MDS (26 without and 21 with fibrosis), 10 with primary myelofibrosis or myelofibrosis secondary to essential thrombocythemia (MPN-F), and 17 with non-Hodgkin lymphoma without BM involvement (as control) were immunostained with antibodies for mesenchymal/neuronal cell markers. Gene mutations were analyzed by targeted sequencing. Results: In control samples, C-X-C motif ligand 12 (CXCL12) was stained mainly in cells at perivascular areas and the periphery of a minor proportion of adipocytes. These patterns did not differ in MDS without fibrosis from control. However, CXCL12-stained non-adipose cells were significantly increased in MDS with fibrosis (MDS-F) [median (range), 5190 (2117-14299) μm2/mm2 as compared to 628 (464-1331) μm2/mm2 in control; p=0.00453]. These cells tended to be increased in also MPN-F though statistical significance was not reached [6096 (1098-24458) μm2/mm2; p=0.06304] . The localization of these cells was not confined to the perivascular areas in both diseases. There were no apparent differences in the degree or the pattern of staining for CXCL12 between MDS-F and MPN-F. We also focused on Schwann cells and Nestin-expressing stromal cells because these cells have been independently reported to function as an important hematopoietic stem cell niche. Neurofilament heavy chain (NFH) or neuron-specific class III beta-tubulin-positive nerve fibers (Tuj1) surrounded by glial fibrillary acidic protein (GFAP)-positive Schwann cells were rarely observed in control samples. This observation was true of MDS without fibrosis. However, GFAP-positive Schwann cells were significantly increased in MDS-F and MPN-F [median (range), 39457 (1325-60654) um2/mm2 and 75951 (19879-326216) um2/mm2, as compared to 6258 (1325-14842) um2/mm2 in control; p=0.0017 and 0.0034, respectively]. When the section was stained for Nestin, Schwann cells in MPN-F did not show positive signals except for one case. In contrast, Nestin was frequently stained in the GFAP-positive Schwann cells of MDS-F (9 of 21 MDS-F, 42.9%); particularly 5 of 7 cases (71.4%) in which fibrosis was judged as severe (MF-3), showed positive signals. One exceptional case of MPN-F that showed Nestin-positivity in increased Schwann cells was a triple-negative case (no mutations in JAK2, CALR, or MPL); instead, mutations were found in NRAS, TET2, BCOR, and SMC1A, indicating that this case may have a feature of MDS. Conclusion: Abnormal increases in CXCL12-expressing mesenchymal cells and nerve fiber-surrounding Schwann cells were visualized by multicolor fluorescence in MDS-F and MPN-F. Schwann cells in MDS-F and MPN-F showed a remarkable difference in the expression of Nestin. It is to be elucidated whether the increase in the Schwann cells has a role in the pathogenesis/pathophysiology, and if so, whether those Schwann cells with distinct phenotypes have differential roles in these two disease conditions. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership.

Kurzfassung: 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.

Kurzfassung: 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 ( 〈 1.0%) have been found in approximately 60% of aplastic anemia (AA) and in 20% of low-risk myelodysplastic syndrome (MDS) patients with no clinical or biochemical evidence of hemolysis. The presence of increased PNH-type cells in bone marrow failure (BMF) patients was shown to predict better response to immunosuppressive therapy by retrospective (Blood 2006 107:1308-1314) and prospective (Br J Haematol 2014 164:546-54.) studies. To ascertain the clinical significance of PNH-type cells, we conducted a nationwide, multicenter, prospective, observational clinical study named OPTIMA in Japan. 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 〈 0.001, Chi-square test) (Fig. 2). 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.