Kurzfassung: 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.

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: Abstract 1204 Background. Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist. Purpose. To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice. Results and Discussion. GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis. In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC. We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC. We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1. Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them. Disclosures: No relevant conflicts of interest to declare.

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: Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602

Kurzfassung: MDS are a group of myeloid neoplasms characterized by deregulated blood cell production and a high propensity to AML. Although a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS. So, in order to clarify a comprehensive registry of gene mutations in MDS, we performed whole-exome sequencing of 29 cases with MDS and related myeloid neoplasm. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 9 genes were mutated in more than 2 cases, which not only included a spectrum of known gene targets in MDS, but also affected previously unknown genes that are commonly involved in 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. In total, 219 mutations were identified in 209 out of the 582 samples of myeloid neoplasms. RNA splicing pathway mutations were highly specific to myelodysplasia, 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 and MPN. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, suggesting the importance of deregulated RNA splicing in the pathogenesis of MDS. RNA splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Splicing pathway mutations in myelodysplasia commonly affected those components of the splicing complex that are engaged in the 3′ splice site recognition, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. So, to clarify the effect of these splicing mutations on RNA splicing, we expressed the wild-type and the mutant U2AF35 or SRSF2 in HeLa cells and performed whole transcriptome analysis in these cells. The results of exon array showed that the wild-type U2AF35 promoted RNA splicing correctly, whereas the mutant U2AF35 inhibited this processes and rendered intronic sequences to remain unspliced. RNA sequencing additionally showed that the number of reads that encompassed the exon/intron junctions was significantly increased in mutant U2AF35-transduced cells. This result means that mutant U2AF35 actually induced impaired 3′-splice site recognition during pre-mRNA processing. In conclusion, our study demonstrated that abnormal RNA splicing caused by mutations of multiple genes on RNA splicing pathway is a common feature of myelodysplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5119. doi:1538-7445.AM2012-5119

Kurzfassung: Myelodysplastic syndromes (MDS) are a heterogeneous groups of myeloid neoplasms characterized by multi-lineage cytopenias of varying degrees and transition to acute myeloid leukemia (AML). At present, no curative therapeutics for MDS has been established except for allogeneic hematopoietic stem-cell transplantation, which is not applicable to the majority of the MDS patients due to their higher ages. Thus, to improve the outcome of MDS, it is essential to develop novel therapeutic agents with both high efficacy and low toxicity, and to this goal, the discovery of the key molecules for MDS pathogenesis is of particular importance. To date, a number of gene mutations have been identified and implicated in the pathogenesis of MDS, including NRAS, TP53, RUNX1, c-CBL, TET2, ASXL1, and more recently, IDH1, IDH2 and EZH2. However, in view of therapeutic targets, our current knowledge of disease causing mutations in MDS is 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. In this study, to obtain a complete registry of genetic lesions in MDS and to identify novel therapeutic targets, we performed whole exome analysis for novel mutations using high-throughput resequencing technologies, combined with large-scale screening of mutations in candidate genes using barcode-labeled DNA for a panel of ∼200 MDS samples. Whole exome analysis was performed for 20 MDS samples, where entire exon sequences were enriched by using SureSelect Human All Exon kit (Agilent Technologies) and were subjected to resequencing analysis using Genome Analizer IIx (Illumina). More than 60% of mapped reads contained exon sequences. & gt; 80% of exons were sequenced at the depth of & gt;20 and average fold-coverage was & gt;50 times. Given that the constitutive genomic DNA was 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, 8 somatic mutations and one insertions-deletion per patient were detected. Novel gene targets were also explored by resequencing barcord-labeled DNAs from 200 MDS specimens, which targeted 80 candidate genes for MDS. A number of mutations were identified, including those in IDH2, ASXL1, TET2, EZH2 and novel target genes such as PHF6, which has been reported to be mutated in T-ALL previously. 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 925. doi:10.1158/1538-7445.AM2011-925

Kurzfassung: 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 ( 〈 10 %) (p 〈 0.001) (Yoshida K, et al, unpublished data). So to obtain an insight into the genetic basis for these difference, we extensively explored spectrums of gene mutations in a set of 161 samples with MDS and related myeloid neoplasms, in which mutations of 10 genes thus far identified as major targets in MDS were examined and their frequencies were compared with regard to the species of mutated components of the splicing machinery. The mutation status of the 161 specimens was determined using the target exon enrichment followed by massively parallel sequencing. In total, 86 mutations were identified in 81(50%) in the 8 components of the splicing machinery. The mutations among 4 genes, U2AF35 (N = 20), SRSF2 (N = 31), SF3B1 (N = 15) and ZRSR2 (N = 10), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 3), PRPF40B (N = 3), U2AF65 (N = 3) and SF1 (N = 1). Conspicuously, higher frequency 4 components of the splicing machinery were mutated in 76 out of the 161 cases (47.2%) in a mutually exclusive manner. On the other hand, 172 mutations of the 10 common targets were identified among 117, including 41 TET2 (25%), 32 RUNX1 (20%), 26 ASXL1 (16%), 24 RAS (NRAS/KRAS) (15%), 22 TP53 (14%), 17 IDH1/2 (10%), 10 CBL (6%) and 10 EZH2 (6%) mutations. We examined the difference between the major spliceosome mutations in terms of the number of the accompanying mutations in the 10 common gene targets. The possible bias from the difference in disease subtypes was compensated by multiple regressions. The SRSF2 mutations are more frequently associated with accompanying gene mutations with a significantly higher number of those mutations (N=29; OR 6.2; 95%CI 1.1–35) compared with that of the U2AF35 mutations (N=14) (p=0.038). Commonly involving the E/A splicing complexes, these splicing pathway mutations lead to compromised 3' splice site recognition. However, individual mutations may still have different impacts on cell functions, which could contribute to the determination of discrete disease phenotypes. It was demonstrated that SRSF2 was involved in the regulation of DNA stability and that depletion of SRSF2 can lead to DNA hypermutability, which may explain the higher number of accompanying gene mutation in SRSF2-mutated cases than cases with other spliceosome gene mutations. In conclusion, it may help to disclosing the genetic basis of MDS and related myeloid neoplasms that highly paralleled resequencing was confirmed SRSF2 mutated case significantly overlapped common mutations. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Disseminated intravascular coagulation (DIC) is a lethal complication in patients with hematological malignancies. Although standard therapy against DIC remains to be established, soluble recombinant thrombomodulin (rTM), which serves as a receptor for thrombin, has been developed and its effectiveness for DIC was reported (Saito et al, J Thromb Haemost 2006). However, there is not enough evidence of rTM on DIC associated with hematological malignancy. Therefore we retrospectively compared outcome of hematological malignancy-related DIC treated with rTM or other conventional anticoagulant therapies. Patients and Methods One hundred and sixty-five consecutive DIC episodes in 146 patients with hematological malignancies (AML except for APL, 49; APL, 21; ALL, 19; NHL, 31; myeloma, 11; CML, 7; other; 8) hospitalized between January 2004 and May 2013 in University of Tsukuba Hospital were retrospectively analyzed. Diagnosis of DIC was based on DIC score of Japanese Ministry of Health and Labor Welfare criteria (Kobayashi et al, Bibl Haematol 1983). DIC was induced by hematological malignancy itself (h-DIC) or severe infection secondary to hematological malignancy (i-DIC) in 108 and 57 episodes, respectively. In 73 episodes, 380 units/kg/day of rTM was administered intravenously from the onset of DIC for median of 6 (range, 2-22) days. Other DIC episodes were treated with conventional anticoagulant therapy (low molecular weight heparin, 65; gabexate mesilate, 17; other anticoagulant, 10). Every anticoagulant therapy was accompanied by treatment for DIC-causing disease. We compared recovery time from DIC (the day when DIC score was decreased to 5 or less), overall survival, and severe hemorrhagic events related to the treatment, between rTM- and conventional anticoagulant-treated groups. Results Fifty-three DIC episodes were accompanied by bleeding tendency at the onset. In h-DIC, recovery from DIC was significantly more prompt in rTM-treated group, with the recovery rates of 55% (95% CI: 40 - 68) in the rTM-treated group and 33% (95% CI: 21 - 45) in the conventional therapy group at day 7 after the therapy initiation (P = 0.03, Fig.1). On day 14, the recovery from h-DIC was seen in 72% (95% CI: 56 - 83) and 60% (95% CI: 47 - 71) with the rTM and conventional therapies, respectively (P = 0.2). By contrast, recovery from i-DIC was significantly worse than that from h-DIC, and was not influenced by anticoagulant therapies. Recovery rates from i-DIC at day 14 were 27% (95% CI: 12 - 45) in the rTM-treated group and 36% (95% CI: 19 - 52) in the conventional therapy group (P = 0.6). Day 60 overall survival rates in h-DIC were 82% (95% CI: 66 - 91) and 79% (95% CI: 65 - 88) in the rTM-treated and conventional therapy groups. In i-DIC, on the other hand, 33% (95% CI: 16 - 52) and 33% (95% CI: 18 - 50) survived with the rTM and conventional therapies, respectively (Fig.2). Severe hemorrhagic events that led to discontinuation of anticoagulant therapy was significantly less in the rTM-treated group (3%; 95% CI, 0.3 - 9) compared with that in the conventional therapy group (12%; 95% CI: 6 - 20; P = 0.04). Discussion and Conclusion The recovery from h-DIC treated with rTM was more prompt compared to that with conventional anticoagulant therapy. Although the conventional anticoagulant therapy has fostered bleeding tendency, bleeding tendency was reduced after rTM administration in most of the DIC episodes. We emphasize that rTM can be an effective anti-DIC agent without causing adverse hemorrhagic event even in DIC cases with preexisting bleeding tendency. However, the outcome was still significantly worse in i-DIC secondary to hematological malignancies even after introduction of rTM. Further development of anticoagulant therapy is required for the control of i-DIC. Disclosures: Chiba: Asahi Kasei Pharma: Research Funding.