ALBERT

Description: [1]  Active and passive seismic experiments in the Kii Peninsula, southwest Japan, revealed prominent structural features around the segment boundary of a megathrust earthquake associated with the subduction of the Philippine Sea Plate (PHS). A distinct reflection band in the uppermost part of the PHS shows significant lateral variation along its strike. The thicker reflection band, which corresponds to the deeper extension of the fault area of the 1944 Tonankai Earthquake, is interpreted to be a zone of high pore-fluid pressure, causing a state of conditionally stable slip on the plate boundary by the reduction in effective normal stress. A thinner reflective band corresponds to the deepest part of the rupture area of the 1946 Nankai earthquake, where plate coupling is stronger due to less effect of fluids. This along-strike structural variation controls the differences in frictional properties and the lateral limit of rupturing along the plate boundary.

Description: 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.

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

Description: Abstract 273 Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms showing a frequent transition to acute myeloid leukemia. Although they are discriminated from de novo AML by the presence of a preleukemic period and dysplastic cell morphology, the difference in molecular genetics between both neoplasms has not been fully elucidated because of the similar spectrum of gene mutations. In this regards, the recent discovery of frequent pathway mutations (45∼90%) involving the RNA splicing machinery in MDS and related myeloid neoplasm with their rare mutation rate in de novo AML provided a novel insight into the distinct molecular pathogenesis of both neoplasms. Thus far, eight components of the RNA splicing machinery have been identified as the targets of gene mutations, among which U2AF35, SF3B1, SRSF2 and ZRSR2 show the highest mutation rates in MDS and CMML. Meanwhile, the frequency of mutations shows a substantial variation among disease subtypes, although the genetic/biological basis for these differences has not been clarified; SF3B1 mutations explain 〉90% of the spliceosome gene mutations in RARS and RCMD-RS, while mutations of U2AF35 and ZRSR2 are rare in these categories (〈 5%) but common in CMML (16%) and MDS without increased ring sideroblasts (20%). On the other hand, SRSF2 mutations are most frequent in CMML (30%), compared with other subtypes (

Description: Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1β or TNF-α treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1β or TNF-α in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1β or TNF-α. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.

Description: Abstract 4011 Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect-system® (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with 〉20 depths of reading. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 295 Myelodysplastic syndromes (MDS) are a hetreogenous groups of myeloid neoplasms characterized by cytopenia of varying degrees and transition to acute myeloid leukemia (AML). MDS is one of the most frequent hematopoietic malignancies, particularly in the elderly. At present, allogeneic hematopoietic stem-cell transplantation is the only treatment that can induce long-term remission in MDS, but it is not applicable to most patients because of their advanced age and is associated with a high rate of treatment-related death and many complications such as chronic graft-versus-host disease. International Prognostic Scoring System (IPSS) is commonly used as a prognostic tool, but it is unsatisfactory from the point of view of genetic changes in MDS. Identification of the underlying genetic aberrations in MDS and the development of proper classification and targeted therapy are anticipated. To date, a number of gene mutations have been identified and implicated in the pathogenesis of MDS, including NRAS, TP53, RUNX1, cFMS, c-CBL, TET2, ASXL1, and more recently, IDH1, IDH2 and EZH2. However, only a part of MDS cases are able to be associated with these genetic changes. There are some remaining areas where copy number alterations and aUPDs are commonly observed and target genes have not been identified, and our knowledge about the genetic basis of MDS is thought to be still incomplete. Recently, next-generation resequencing technologies have been shown to be effective for the identification of disease-related gene and been successfully used to determine the genetic basis of some neoplastic disorders, such as AML and diffuse large B-cell lymphoma. More recently, the resequencing technology targeted for all protein-coding subsequences (i.e., whole exome analysis) has enabled cost-effective comprehensive mutation analysis of coding sequences, and has been successfully applied to identifying some Mendelian disorders. In this study, we performed a whole exome analysis of ten MDS patients in order to obtain a comprehensive registry of genetic lesions in MDS. Entire exon sequences were enriched by using SureSelect Human All Exon kit (Agilent Technologies) and were subjected to resequencing analysis using Illumina Genome Analizer IIx. On average, 12 gigabases (Gb) of sequence were generated per one tumor sample, in which more than 60% of mapped reads contained exon sequences. 〉 80% of exons were sequenced at the depth of 〉20 and average fold-coverage was 〉50 times. Because remission samples were difficult to obtain in MDS patients, paired CD3-positive T cells were used as a normal control. By comparing sequences in tumors and paired T cells, a number of candidate gene mutations and insertions-deletions, including those in IDH2, CKAP, TMEM146, CLEC1A, and other genes, which were validated by Sanger sequencing. Now, we are performing Sanger sequencing for some candidate genes, which were commonly mutated in more than one resequenced patients and were located within the regions of recurrent aUPDs in a cohort of 170 MDS subjects, assessing their prevalence in MDS. Our results suggested that target-capture resequencing technology is a powerful method to identify new gene mutations that are implicated in the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Background The transcription factor Hairy enhancer of split1 (Hes1) is well characterized as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play both pro- and anti-tumorigenic roles; it promotes development of T-cell acute lymphoblastic leukemia (T-ALL), while serves as a tumor suppressor for acute myeloid leukemia (AML). Hes1 has been proven as an essential mediator of Notch signaling in T-ALL development. In contrast, we reported, in the last annual meeting, that Hes1 functions as a tumor suppressor against AML development, using a mouse model of AML induced by the MLL-AF9 fusion protein. We further explored the mechanism of Hes1-mediated suppression of AML development. Methods Common myeloid progenitors (CMPs) purified from RBP-Jf/f mouse bone marrow (BM) were serially transduced with MLL-AF9 and Cre recombinase (iCre) using retroviral vectors, and transplanted into lethally irradiated syngenic mice. CMPs from Hes1-/- mouse fetal liver were also retrovirally transduced with MLL-AF9 and transplanted after multiple rounds of replating, and then, expression levels of downstream targets were evaluated by cDNA array. Next Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells and these cells were transplanted. MLL-AF9-transduced cells were treated with a hamster anti-mouse Notch agonistic antibody (Notch Ab). Results Mice transplanted with MLL-AF9/RBP-J-/- cells developed leukemia at shorter latencies than those with MLL-AF9/RBPJ+/+ cells. MLL-AF9-transduced Hes1-/- cells formed the higher number of colonies at third replating compared with MLL-AF9-transduced Hes1+/+ cells. When infused into irradiated syngenic mice, MLL-AF9/Hes1-/- cells developed leukemia at shorter latencies than MLL-AF9/ Hes1+/+ cells (MLL-AF9/Hes1-/-, 7-10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10-14 weeks, n=18; p

Description: Abstract 458 MDS and related disorders comprise a group of myeloid neoplasms characterized by deregulated blood cell production and a predisposition to AML. Although currently, a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS, because many of them are also found in other myeloid malignancies and roughly 20% of MDS cases have no known genetic changes. So, in order to clarify a complete registry of gene mutations in MDS and identify those discriminate MDS from other myeloid neoplasms, we performed whole-exome sequencing of 29 cases showing myelodysplasia. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 41 occurred in recurrent gene targets, which not only included a spectrum of known gene targets in MDS, such as TET2, EZH2, NRAS/KRAS, RUNX1, TP53 and DNMT3A, but also affected previously unknown genes that are commonly mapped to the RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery. To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms (N=582) using a high-throughput mutation screen of pooled DNA followed by confirmation/identification of candidate mutations. In total, 219 mutations were identified in 209 out of the 582 specimens of myeloid neoplasms. Mutations of the splicing machinery were highly specific to diseases showing myelodysplastic features, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML (N=151) and MPD (N=53). The mutations among 4 genes, U2AF35 (N = 37), SRSF2 (N = 56), SF3B1 (N = 79) and ZRSR2 (N = 23), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 8), PRPF40B (N = 7), U2AF65 (N = 4) and SF1 (N = 5). Interestingly, mutations in the former three genes showed clear hot spots, indicating a gain-of-function nature of these mutations. On the other hand, two thirds of the ZRSR2 mutations are nonsense or frameshift changes causing premature truncation of the protein. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, and commonly affected those components of the splicing complex that are engaged in the 3' splice site recognition during RNA splicing, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. In fact, when transduced into HeLa cells, the recurrent S34F U2AF35 mutant induced the increase in the production of unspliced RNA species and elicited the activation of the nonsense mediated decay pathway. Functionally, the U2AF35 mutants seemed to cause deregulated stem cell functions, because CD34(−) KSL cells transduced with various U2AF35 mutants invariably showed reduced chimerism in competitive reconstitution assay. In accordance with this, the S34F U2AF35 mutant lead to suppression of cell growth in a variety of cell types, including HeLa cells, in which expression of the mutant induced a G2/M cell cycle arrest and increased apoptosis. In conclusion, whole-exome sequencing unexpectedly revealed the high frequency of the splicing pathway mutations in MDS and related myeloid neoplasms, providing the first evidence indicating that compromised RNA splicing by gene mutations are responsible for human pathogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.