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Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although several reports described gene mutations in AEL, genotype phenotype correlations have not fully been elucidated with little knowledge about feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 121 adult AEL cases with the median age of 60 (23-87), using whole genome/exome sequencing of 35 cases, followed by targeted-capture sequencing of 387 genes together with 1,279 SNP loci for copy number measurements in all cases. Among these, 21 were also analyzed by RNA sequencing. Genetic profiles of these AEL cases were compared to those of 409 cases with non-erythroid AML (non-AEL) including 195 cases from The Cancer Genome Atlas. Six patient-derived xenografts (PDX) were established from AEL with JAK2 and/or EPOR focal gain/amplification/mutation. PDX cells were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 genomic groups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype (48/121; 40%) and showed very poor prognosis. Remarkably, almost all the PEL cases (12/13; 92%) were categorized into Group A. Conspicuously, 75% of PEL cases with TP53 mutation had focal gain/amplifications/mutations of JAK2 (5/12; 42%), EPOR (7/12; 58%), and ERG/ETS2 (1/12; 8%) loci on chromosomes 9p, 19q, and 21q, respectively, while 34% of MEL cases with TP53 mutation had focal gain/amplifications/mutations of JAK2 (2/29; 7%), EPOR (7/29;24%), and ERG/ETS2 (7/29;24%) loci, frequently in combination. Group B was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in non-AEL, whereas NPM1-mutated patents in this group lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in non-AEL (15/101; 15%). All cases in Group C (n=22, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. Prominently, 68% (17/25) of STAG2-mutated AEL cases had KMT2A-PTD, which was rarely found in non-AEL cases. The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, RUNX1 and TET2. We also identified recurrent loss-of-function USP9X mutations in this group, which were previously reported in ALL with an upregulated JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only in those cases with JAK2 or EPOR focal gain/amplification/mutation, but also in AEL without these amplifications, suggesting that aberrantly upregulated STAT5 activation might represent a common molecular signature of AEL. Survival analysis revealed that TP53 mutation is a poor prognostic factor in AEL and non-AEL and no statistically significant difference between AEL and non-AEL with TP53 mutation. Intriguingly, 19p gains/amplifications were associated with a significantly poor prognostic prognosis in TP53-mutated AEL cases. Based on this finding, we evaluated the effect of a JAK inhibitor, ruxolitinib, on 6 PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest , ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with 4 PDX models with STAT5 downregulation, although the other 2 models were resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2 and/or EPOR. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a therapeutic role of JAK inhibition was suggested. Disclosures Nakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Yoda: Chordia Therapeutics Inc.: Research Funding. Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Miyazaki: Sumitomo-Dainippon: Honoraria, Research Funding; Astellas: Honoraria; Chugai: Honoraria; Abbvie: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Daiichi-Sankyo: Honoraria; Kyowa-Kirin: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria. Usuki: Alexion: Speakers Bureau; Eisai: Speakers Bureau; MSD: Speakers Bureau; PharmaEssentia: Speakers Bureau; Yakult: Speakers Bureau; Mundipharma: Research Funding; Astellas-Amgen-Biopharma: Research Funding; Nippon Boehringer Ingelheim: Research Funding; Takeda: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding; Ono: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Sumitomo Dainippon: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Symbio: Research Funding, Speakers Bureau; Gilead: Research Funding; Abbvie: Research Funding; Nippon shinyaku: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Research Funding, Speakers Bureau; Brisol-Myers Squibb: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy. Ohyashiki: Novartis Pharma: Other: chief clinical trial; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ganser: Celgene: Honoraria; Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Heuser: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Karyopharm: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Thol: Astellas: Honoraria; Abbvie: Honoraria; Novartis: Honoraria; Jazz: Honoraria; BMS/Celgene: Honoraria, Research Funding; Pfizer: Honoraria. Shih: PharmaEssentia Co: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Ltd: Research Funding; Ltd: Research Funding; Novartis: Research Funding. Takaori-Kondo: Celgene: Research Funding; Bristol-Myers K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Research Funding. Ogawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although gene mutations in AEL have been described in several reports, genotype phenotype correlations are not fully understood with little knowledge about the feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 105 AEL cases with the median age of 60 (23-86), using targeted-capture sequencing of commonly mutated genes in myeloid neoplasms, together with 1,279 SNPs for copy number measurements. Among these 105 cases, 13 were also analyzed by RNA sequencing. Genetic profiles of these 105 AEL cases were compared to those of 775 cases with non-erythroid AML (NEL) including 561 cases from The Cancer Genome Atlas and Beat AML study. An immature erythroid cell line (TF1) and three patient-derived xenografts (PDX) established from AEL with JAK2 and/or EPOR amplification. Cell line and samples from patients were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 subgroups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype and showed very poor prognosis. Remarkably, all PEL cases were categorized into Group A. Conspicuously, 80% of PEL cases had amplifications of JAK2 (6/10; 60%), EPOR (7/10;70%), and ERG (6/10;60%) loci on chromosomes 9p, 19q, and 21q, respectively, frequently in combination, although they were rarely seen in NEL cases. All cases in Group B (n=19, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. To further characterize this subgroup, we compared genetic profiles of STAG2-mutated AEL and NEL. Prominently, 70% (14/20) of STAG2-mutated cases in AEL had KMT2A-PTD, whereas it was found only in 8.8% (3/34) of NEL. CEBPA mutations were also more common in AEL (6/21; 29%) than NEL (4/34; 12%). While Group C was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in NEL, NPM1-mutated patents in this subgroup lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in NEL (25/209; 12%). The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, U2AF1 and KMT2C. Recurrent loss-of-function mutations in USP9X were unique to this subtype, although USP9X mutations have been reported in ALL with upregulation of JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only those cases with JAK2 or EPOR amplification, but also those without, suggesting that aberrantly upregulated STAT5 activation might represent a common defect in AEL. Based on this finding, we evaluated the effect of a JAK inhibitior, ruxolitinib, on an AEL-derived cell line and three PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest, ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with TF1 and 2 PDX models with STAT5 downregulation, although the other model was resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2, EPOR and/or ERG. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a role of JAK inhibition was suggested. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Miyazaki:Astellas Pharma Inc.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Takaori-Kondo:Celgene: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; MSD: Honoraria. Kataoka:Asahi Genomics: Current equity holder in private company; Otsuka Pharmaceutical: Research Funding; Takeda Pharmaceutical Company: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding. Usuki:Alexion: Research Funding, Speakers Bureau; Apellis: Research Funding; Novartis: Research Funding, Speakers Bureau; Chugai: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Ganser:Novartis: Consultancy; Celgene: Consultancy. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Ruxolitinib is used for drug efficacy test using patient-derived xenografts established from acute erythroid leukemia.

Background Copy-number alterations (CNAs) and gene mutations are hallmarks of cancer genomes, and they are implicated in the development of myeloid neoplasm. However, their relationships have not been fully examined. To address this issue, we have recently developed a novel, next-generation sequencing-based platform for copy-number analysis, which enabled us to detect mutations and CNAs simultaneously. We applied this platform to around 2,000 cases with myeloid neoplasms. Aims We aimed at delineating the landscape of CNAs and their relationships with gene mutations in myeloid neoplasms. Methods We examined 2,101 cases with myeloid neoplasms by whole-exome sequencing (WES) or targeted deep sequencing. Excluding 116 samples showing low qualities of copy-number signals, we performed subsequent analysis on the remaining 1,985 cases with myelodysplastic syndromes (MDS, n = 1,102), myelodysplastic/myeloproliferative neoplasms (MDS/MPN, n = 140), de novo acute myeloid leukemia (de novo AML, n = 448), and secondary AML (sAML, n = 295). In copy-number analysis, total copy numbers and allele-specific copy numbers (ASCNs) were quantified based on sequencing depths and allelic ratios on genome-wide probes. Copy-number signals were corrected for multiple biases (e.g. GC content, ASCN, and fragment length). We also validated the performance of this platform through comparison with SNP-array karyotyping data in 115 de novo AML cases. CNAs longer than 5 Mb were regarded as arm-level CNAs, and those shorter than 5 Mb were regarded as focal CNAs. Results In total, we identified 4,141 CNAs (52.9 % of cases with at least one CNA), and 3,863 mutations (73.9 % of cases with at least one mutation). Most frequent alterations included -7/del(7q) (13.2 %), del(5q) (11.4 %), trisomy 8 (7.2 %), and del(20q) (5.2 %), and mutations of TET2 (12 .3 %), TP53 (11.3 %), ASXL1 (10.1%), and DNMT3A (9.9 %). To evaluate the difference of copy-number landscapes between de novo AML and myelodysplasia (MDS, MDS/MPN, and sAML), we compared the frequencies of CNAs between them. Uni-parental disomy (UPD) of 13q (FLT3) and 11p (WT1), and amplifications of 11q, 13q, and 21q (ERG) were more enriched in de novo AML, while der(1;7), UPD of 11q (CBL), and del(20q) were enriched in myelodysplasia, suggesting differential involvements of CNAs. We next analyzed the correlations between CNA profiles and prognosis in cases with myelodysplasia. Since TP53 status implies a large impact on both patients' prognosis and CNA profiles, we separately analyzed TP53-positive (n = 53) and negative (n = 686) cases with available survival data. In TP53-negative cases, -7/7qLOH (Hazard ratio(HR): 2.28, q < 0.001), and UPD of 11q (CBL) (HR: 2.60, q = 0.0034) significantly correlated with shorter overall survivals (OS), while, in TP53-positive cases, amp(11q), +19, and amp(21q) were marginally associated with shorter OS. To delineate the relationships between CNAs and mutations, we interrogated correlations between both lesions among MDS cases without TP53 alterations (n = 937). A number of significant correlations were detected, such as those between trisomy 8 and del(20q) with U2AF1 mutations (q < 0.05, for each), and monosomy 7 and amp(21q) with mutations of RUNX1 and NRAS (q < 0.01, for each). These correlations were also revealed in clustering analysis based on CNA and mutation profiles, which identified 5 unique clusters: Cluster 1 (n = 171) with trisomy 8, del(20q), and mutations of U2AF1 and ETV6, Cluster 2 (n = 43) with monosomy 7, amp(21q), and mutations of NRAS, SETBP1, and RUNX1, Cluster 3 (n = 19) with amp(1q) and amp(3q), Cluster 4 (n = 127) with those of SF3B1, TET2, and DNMT3A, and Cluster 5 (n = 50) with those of SRSF2, STAG2, ASXL1, and RUNX1. The remaining 527 cases were not assigned into any cluster due to lack of significantly correlated alterations. Finally, the temporal relationships of coexisting alterations were estimated based on their cell fractions; monosomy 7 had significantly greater cell fractions (P = 0.031) and is predicted to precede NRAS mutations, while the cell fractions of U2AF1 mutations tended to be greater than those of trisomy 8 (P = 0.063), suggesting their implications in different stages of disease progression. Conclusion An integrated analysis of CNAs and mutations in >2,000 cases revealed the impacts of CNAs on disease characteristics and provided novel insight into the interplay between CNAs and mutations in the pathogenesis of MDS. Figure Disclosures Atsuta: CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Kanda:Celgene: Consultancy, Research Funding; Novartis: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Taiho: Research Funding; Asahi-Kasei: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Ono: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; CSL Behring: Research Funding; Taisho-Toyama: Research Funding; Tanabe Mitsubishi: Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Takara-bio: Consultancy, Honoraria; Novartis: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Pfizer: Research Funding; Asahi-Kasei: Research Funding; Alexion: Consultancy, Honoraria; CSL Behring: Research Funding; Takara-bio: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Taiho: Research Funding; Celgene: Consultancy, Research Funding; Tanabe Mitsubishi: Research Funding; Taisho-Toyama: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Otsuka: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties; Novo Nordisk: Consultancy. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Usuki:Boehringer-Ingelheim Japan: Other: Received Research ; Daiichi Sankyo: Other: Received Research ; SymBio Pharmaceuticals Limited.,: Other: Received Research ; Novartis: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Takeda Pharmaceutica: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau; Celgene Corporation: Other: Received Research , Speakers Bureau; Sumitomo Dainippon Pharma: Other: Received Research , Speakers Bureau; Pfizer Japan: Other: Received Research ; Stellas Pharma: Other: Received Research ; Otsuka: Other: Received Research ; Kyowa Kirin: Other: Received Research ; GlaxoSmithKline K.K.: Other: Received Research ; Sanofi K.K.: Other: Received Research ; Shire Japan: Other: Received Research ; Janssen Pharmaceutical K.K: Other: Received Research . Imada:Bristol-Meyer Squibb K.K.: Honoraria; Celgene K.K.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria; Nippon Shinyaku Co.,Ltd.: Honoraria. Takaori-Kondo:Kyowa Kirin: Research Funding; Pfizer: Honoraria; Janssen: Honoraria; Chugai: Research Funding; Takeda: Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria, Research Funding. Kiguchi:Celltrion, Inc.: Research Funding; Astellas Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; MSD CO., Ltd.: Research Funding; Novartis Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myeres Squibb Co., Ltd.: Research Funding; Janssen Pharmaceutical Co., Ltd.: Research Funding; Celgene Co., Ltd.: Research Funding; SymBio Pharmaceutical Co., Ltd.: Research Funding; Taiho Pharmaceutical Co., Ltd.: Research Funding; Tejin Co., Ltd.: Research Funding; Sanofi K.K., Ltd.: Research Funding. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Ogawa:Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy.

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia (AML) characterized by erythroid predominance and dysplasia, which is morphologically classified into two subtypes: pure erythroid and myeloid/erythroid leukemias (PEL and MEL). Clinically, AEL exhibits aggressive and rapid clinical course, which is considered to be linked to frequent TP53 mutations and complex karyotypes found in this subtype. Although multiple sequencing studies were conducted to explain such phenotypic variety and to search promising therapeutic targets, neither the diagnostic discrimination of PEL and MEL subtypes nor the establishment of standard therapeutic strategy has fully been successful. To clarify genetic characteristics and to identify novel therapeutic targets, we comprehensively characterized the mutation and copy number alteration (CNA) profiles and assessed differential impacts of the frequent driver genetic abnormalities on disease phenotypes and clinical outcomes. We initially analyzed 105 cases with AEL (PEL; n=10, MEL; n=69, and unspecified; n=26). Among these, 9 cases were serially analyzed at 2-6 time points. All samples were obtained according to protocols approved by the ethics board of each participating institution. As control, 907 cases with non-erythroid AML (NEL) distinguished by the low fraction ( Most frequently observed were mutations in TP53 (39%), STAG2 (20%), and NPM1 (15%), and KMT2A-partial tandem duplication (PTD) (18%) in AEL. On the basis of mutational profiles, consensus clustering divided AEL into 4 subgroups. Group A (n=41, 39%) was defined by TP53 mutations into which all PELs were categorized. In CNA analysis, amplification in 9p, 19p, and 21q were significantly more enriched in PEL than NEL. Intriguingly, amplification of 9p, 19p, and 21q lesions commonly included JAK2, EPOR, and ERG loci, respectively. These genes were affected by amplification or mutations more frequently in AEL than NEL (Figure 1). Group B (n=19, 18%) was enriched for mutations in STAG2, which was significantly more frequent in MEL than in PEL (P In summary, our findings suggest that AEL is classified into 4 major subgroups having unique genetic and clinical features. PEL and MEL are genetically distinct subtypes, which was also highlighted by comparison to NEL. Frequent involvement of EPOR/JAK/STAT pathway suggests therapeutic indication of JAK inhibition for AEL especially in the most aggressive type of AEL cases with TP53 mutations. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria; Dainippon-Sumitomo: Honoraria; Nippon-Shinyaku: Honoraria; Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria. Takaori-Kondo:Pfizer: Honoraria; Chugai: Research Funding; Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Heuser:Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Ogawa:Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

We conducted a prospective randomized study to assess the optimal postremission therapy for adult acute myeloid leukemia in patients younger than 65 years in the first complete remission. A total of 781 patients in complete remission were randomly assigned to receive consolidation chemotherapy of either 3 courses of high-dose cytarabine (HiDAC, 2 g/m2 twice daily for 5 days) alone or 4 courses of conventional standard-dose multiagent chemotherapy (CT) established in the previous JALSG AML97 study. Five-year disease-free survival was 43% for the HiDAC group and 39% for the multiagent CT group (P = .724), and 5-year overall survival was 58% and 56%, respectively (P = .954). Among the favorable cytogenetic risk group (n = 218), 5-year disease-free survival was 57% for HiDAC and 39% for multiagent CT (P = .050), and 5-year overall survival was 75% and 66%, respectively (P = .174). In the HiDAC group, the nadir of leukocyte counts was lower, and the duration of leukocyte less than 1.0 × 109/L longer, and the frequency of documented infections higher. The present study demonstrated that the multiagent CT regimen is as effective as our HiDAC regimen for consolidation. Our HiDAC regimen resulted in a beneficial effect on disease-free survival only in the favorable cytogenetic leukemia group. This trial was registered at www.umin.ac.jp/ctr/ as #C000000157.

Various prospective trials have been performed to assess the roles of allogeneic hematopoietic cell transplantation (allo-HCT) and chemotherapy in patients with acute myeloid leukemia (AML) in first complete remission (CR1). However, the results have not always been consistent, and there has been a limited evaluation of quality of life (QOL) in these postremission strategies. We performed a Markov decision analysis that enabled us to compare survival outcomes with a QOL evaluation using a database of 2029 adult AML patients who achieved CR1. The Markov decision model compared 2 strategies: allo-HCT or chemotherapy in CR1. Patients who had intermediate- or unfavorable-risk AML had a longer life expectancy when they received allo-HCT in CR1 than patients treated with chemotherapy alone. Likewise, patients who had a suitable related donor who received allo-HCT in CR1 had a longer life expectancy. The life expectancy was shortened to a greater degree by adjustment for QOL in the allo-HCT group. Nevertheless, QOL-adjusted life expectancies in most of the subgroups remained longer in the allo-HCT group than in the chemotherapy group. Our results showed that older patients with a related donor and younger patients with unfavorable cytogenetics benefited the most from allo-HCT in CR1.

Introduction: In recent decades, a considerable number of tumor antigens have been characterized, including the leukemia-associated antigen, Wilm's tumor 1 (WT1). WT1 is found to be expressed at low levels in various normal tissues such as gonads, kidney and the hematopoietic system. However, it is highly overexpressed in various hematological malignancies and solid tumors, including in the majority of acute myeloid leukemia (AML) cases. OCV-501 is a human leukocyte antigen (HLA) class II-restricted helper peptide derived from the WT1 protein. This Phase II trial was conducted to evaluate the efficacy and safety of OCV-501 in elderly patients with AML (ClinicalTrials.gov identifier: NCT01961882). Methods: This multicenter, randomized, double-blind, placebo-controlled trial was conducted in Japan, the Republic of Korea and Taiwan between October, 2013 to November, 2017. The key eligibility criteria for this trial were patients with AML, aged 60 years or older, who achieved first complete remission within one or two courses of standard induction therapy, completed more than one course of standard consolidation therapy, and not scheduled for hematopoietic stem cell transplantation. Patients were assigned to receive either OCV-501 monotherapy or placebo. Subcutaneous administration of OCV-501 3mg or placebo was given once weekly up to the eighth administration, and once biweekly from the ninth administration onwards up to a duration of 2 years, followed by post-treatment observation period. The primary endpoint was disease-free survival (DFS) that was defined as the time from randomization to AML relapse or death from any cause, whichever came first, within the observation period. Secondary endpoints were overall survival (OS), quality of life (QOL; The European Organization for Research and Treatment of cancer [EORTC] QLQ-C30), and performance status (Eastern Cooperative Oncology Group performance status [ECOG PS]). Safety, including any occurrence of adverse events (AEs) was evaluated. Results: A total of 133 patients who had achieved first remission after prior induction and consolidation therapies were randomized to receive either OCV-501 (OCV-501 arm, N=68) or placebo (placebo arm, N=65). The median age of the patients was 67 years (range, 60 - 85) and most of them had good performance status (ECOG PS score 0 - 1). The WT1 mRNA level at Day 1 was Conclusion: There was no significant difference in DFS between OCV-501- and placebo-treated patients. OCV-501 was found to be generally safe and well-tolerated in elderly AML patients. Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical KK: Honoraria; Celgene KK: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Kiguchi:Teijin Co., Ltd.: Other: Grant; Sanofi K.K., Ltd.: Other: Grant; Celltrion, Inc.: Other: Grant; Taiho Pharmaceutical Co., Ltd.: Other: Grant; SymBio Pharmaceuticals, Ltd.: Other: Grant; Takeda Pharmaceutical Co., Ltd.: Other: Personal fees; Ono Pharmaceutical Co., Ltd.: Other: Personal fees; Astellas Pharmaceutical Co., Ltd.: Other: Grant; Celgene Co., Ltd.: Other: Grant and personal fees; Janssen Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Novartis Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Nippon Shinyaku Co., Ltd.: Other: Grant and personal fees; Mochida Pharmaceutical Co., Ltd.: Other: Personal fees; Eisai Co., Ltd.: Other: Personal fees; MSD Co., Ltd.: Other: Grant and personal fees; Daiichi Sankyo Pharmaceutical Co., Ltd.: Other: Grant; Chugai Pharmaceutical Co., Ltd: Other: Grant; Bristol Myers Squibb Co., Ltd.: Other: Grant and personal fees; Pfizer Co., Ltd.: Other: Personal fees; Otsuka Pharmaceutical Co., Ltd.: Other: grant and personal fees; Kyowa Hakko Kirin Co., Ltd.: Other: Grant and personal fees. Miyawaki:Novartis Pharma KK: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Consultancy; Astellas Pharma Inc.: Consultancy. Heike:Sumitomo Dainippon Pharma Co., Ltd.: Other: Advisor; Otsuka Pharmaceutical Co., Ltd.: Other: Advisor. Mitsuki:Otsuka Pharmaceutical Co., Ltd.: Employment. Yoshida:Otsuka Pharmaceutical Co., Ltd.: Employment. Liew:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Astellas Pharma Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

Germline mutations of DEAD-box helicase 41 (DDX41) have recently been implicated in adult-onset myeloid neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, with their prevalence being largely based on the studies from Caucasian populations, the prevalence of DDX41 pathogenic variants in other ethnicities and their risks for myeloid neoplasms have not been fully investigated. Moreover, the clinicopathological features of DDX41-mutated myeloid neoplasms and their genetic profiles have not been elucidated either. To address these issues, we investigated germline DDX41 variants among a large cohort of Japanese patients (n=1,609) with sporadic myeloid neoplasms including MDS (n=1,102), myelodysplastic/myeloproliferative neoplasms (n=14), myeloproliferative neoplasms (MPN) (n=31), secondary AML derived from these myeloid neoplasms (sAML) (n=47), and de novo AML (n=410), as well as Japanese healthy controls (n=17,186) using targeted deep sequencing, whole exome sequencing, and/or whole genome sequencing, though which pathogenic germline DDX41 variants and their clinical, pathological, and genetic features were investigated. Risk of detected germline variant for myeloid neoplasms was evaluated by comparing their frequencies between patients and healthy individuals. The germline origin of candidate risk alleles was confirmed using buccal mucosa samples. We identified 4 germline variants that were significantly enriched in the patient cohort (n=58, 3.6 %) compared to healthy controls (n=42, 0.2%) (OR=15; 95%CI: 10.2-22.8), including two truncating variants, p.A500fs (OR=14; 95%CI: 8.7-24.4), p.E7X (OR=12.6; 95%CI: 2.1-86), and two missense alleles, p.Y259C (OR=15; 95%CI: 4.1-60), and p.S363del (OR=11; 95%CI: 3.4-35). Four additional truncating and splice-site variants, Y279X (n=1), R124fs (n=1), c.571+2T>G (n=2), and c.298+1G>T (n=1), were also considered as risk alleles, although a small number of each variant precluded an accurate estimation of enrichment in the patient cohort. Overall, as many as 63 (3.9%) patients harbored one of these variants, where each variant allele was invariably heterozygous. Of note, none of these 8 risk variants have been reported in the Caucasian population. To further evaluate the pathogenic role of these DDX41 germline variants, clinical features and somatic genetic events were investigated. The median age at diagnosis did not significantly differ between patients with and without DDX41 variants (60 and 56 years old in variant carrier and in non-carrier, respectively), suggesting that DDX41-mediated myeloid leukemogenesis shows an age-dependence similar to that in other sporadic cases. Compared with DDX41-unmutated cases, DDX41-mutated cases showed a higher male predominance (52/11 and 987/559, respectively; OR=2.7; 95%CI: 1.4-5.1) and were more likely to have an initial diagnosis of MDS rather than de novo AML (55/8 and 1,094/402, respectively; OR=2.5; 95%CI: 1.2-5.4). Patients with germline DDX41 mutations had a median of 1 (0-5) somatic mutations. Somatic DDX41 mutations were found in 43 (2.6%) cases, a majority of which harbored a germline DDX41 risk variants (32/63 (52%) and 11/1,549 (0.7%), respectively; OR=143; 95%CI: 67-311). Other targets of somatic mutations in risk allele-positive cases included ASXL1 (n=9), DNMT3A (n=6), TP53 (n=5), and JAK2 (n=4), for which no significant correlation was observed between risk allele-positive and negative cases. Abnormal cytogenetics and copy number abnormalities were detected in 30 (48%) of the patients with DDX41 risk alleles, none of which were significantly in the risk allele-positive cases. In conclusion, we identified DDX41 germline risk variants among the Japanese population. Germline DDX41 variants were seen in a substantial fraction of Japanese patients with sporadic myeloid neoplasms. Found in the general Japanese population at very low frequencies, these risk alleles account for the largest germline risk for myeloid neoplasms. Somatic DDX41 mutations were common with a prominent mutational hotspot, almost exclusive found in patients with DDX41 risk alleles. Given the high prevalence of DDX41 germline variants and the late onset of associated MDS and sAML, their detection may help better manage patients and carriers who carried DDX41 risk alleles, even when no family history is known. Figure. Figure. Disclosures Ishiyama: Alexion Pharmaceuticals, Inc.: Honoraria. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Asou:Chugai Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau. Naoe:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding. Usuki:Otsuka Pharmaceutical Co., Ltd.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Boehringer-Ingelheim Japan: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Novartis: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Miyawaki:Novartis Pharma KK: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Consultancy; Astellas Pharma Inc.: Consultancy.

Background Intensive efforts of genome sequencing studies during the past decade identified >100 driver genes recurrently mutated in one or more subtypes of myeloid neoplasms, which collectively account for the pathogenesis of >90% of the cases. However, approximately 10% of the cases have no alterations in known drivers and their pathogenesis is still unclear. A possible explanation might be the presence of alterations in non-coding regions that are not detected by conventional exome/panel sequencing; mutations and complex structural variations (SVs) affecting these regions have been shown to deregulate expression of relevant genes in a variety of solid cancers. Unfortunately, however, no large studies have ever been performed, in which a large cohort of myeloid malignancies were analyzed using whole genome sequencing (WGS) in an attempt to identify a full spectrum of non-coding alterations, even though its efficacy have been demonstrated in many solid cancers. In this study, we performed WGS in a large cohort of pan-myeloid cancers, in which both coding and non-coding lesions were comprehensively analyzed. Patients and methods A total of 338 cases of myeloid malignancies, including 212 with MDS, 70 with AML, 17 with MDS/MPN, 23 with t-AML/MDS, and 16 with MPN were analyzed with WGS, of which 173 were also analyzed by transcriptome sequencing. Tumor samples were obtained from patients' bone marrow (N=269) or peripheral blood (N=69), while normal controls were derived from buccal smear (N=263) or peripheral T cells (N=75). Sequencing of target panel of 86 genes were performed for all samples. Sequencing data were processed using in-house pipelines, which were optimized for detection of complex structural variations (SVs) and abnormalities in non-coding sequences. Results WGS identified a median of 586,612 single nucleotide variants (SNVs) and 124,863 short indels per genome. NMF-based decomposition of the variants disclosed three major mutational signatures, which were characterized by age-related C>T transitions at CpG sites (Sig. A), C>T transitions at CpT sites (Sig. B), and T>C transitions at ApTpN context (Sig. C). Among these, Sig. C showed a prominent strand bias and corresponds to COSMIC signature 16, which has recently been implicated in alcohol drinking. Significant clustering of SNVs and short indels were interrogated across the genome divided into different window sizes (1Kbp, 10Kbp, 100Kbp) or confining the targets to coding exons and known regulatory regions, such as promoters, enhancers/super enhances, and DNase I hypersensitive sites. Recapitulating previous findings, SNVs in the coding exons were significantly enriched in known drivers, including TP53, TET2, ASXL1, DNMT3A, SF3B1, RUNX1, EZH2, and STAG2. We detected significant enrichment of SNVs in CpG islands, and promoters/enhancers. We also detected a total of 8,242 SVs with a median of 15 SVs/sample, which is more prevalent than expected from conventional karyotype analysis. Focal clusters of complex rearrangements compatible with chromothripsis were found in 8 cases, of which 7 carried biallelic TP53 alterations. NMF-based signature analysis of SVs revealed that large (>1Mb) deletions, inversions, and tandem duplications and translocations are clustered together and were strongly associated with TP53 mutations, while smaller deletions and tandem duplications, but not inversions, constitute another cluster. As expected, FLT3-ITD (N=15) and MLL-PTD (N=12) were among the most frequent SVs. Unexpectedly, in addition to known SVs associated with t(8;21) (RUNX1-RUNX1T1) (N=6) and t(3;21) (RUNX1-MECOM) (n=1) as well as non-synonymous SNVs within the coding exons (N=30), we detected frequent non-coding alterations affecting RUNX1, including SVs (N=15) and SNVs around splicing acceptor sites (N=5), suggesting that RUNX1 was affected by multiple mechanism, where as many as 38% of RUNX1 lesions were explained by non-coding alterations. Other recurrent targets of non-coding lesions included ASXL1, NF1, and ETV6. Conclusions WGS was successfully used to reveal a comprehensive registry of genetic alterations in pan-myeloid cancers. Non-coding alterations affecting known driver genes were more common than expected, suggesting the importance of detecting non-coding abnormalities in diagnostic sequencing. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Mochida Pharmaceutical: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Shire Japan: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Miyawaki:Otsuka Pharmaceutical Co., Ltd.: Consultancy; Novartis Pharma KK: Consultancy; Astellas Pharma Inc.: Consultancy.

Recently, somatic mutations of the nucleophosmin gene (NPM1), which alter the subcellular localization of the product, have been reported in acute myeloid leukemia (AML). We analyzed the clinical significance of NPM1 mutations in comparison with cytogenetics, FLT3, NRAS, and TP53 mutations, and a partial tandem duplication of the MLL gene (MLL-TD) in 257 patients with AML. We found NPM1 mutations, including 4 novel sequence variants, in 64 of 257 (24.9%) patients. NPM1 mutations were associated with normal karyotype and with internal tandem duplication (ITD) and D835 mutations in FLT3, but not with other mutations. In 190 patients without the M3 French-American-British (FAB) subtype who were treated with the protocol of the Japan Adult Leukemia Study Group, multivariate analyses showed that the NPM1 mutation was a favorable factor for achieving complete remission but was associated with a high relapse rate. Sequential analysis using 39 paired samples obtained at diagnosis and relapse showed that NPM1 mutations were lost at relapse in 2 of the 17 patients who had NPM1 mutations at diagnosis. These results suggest that the NPM1 mutation is not necessarily an early event during leukemogenesis or that leukemia clones with NPM1 mutations are sensitive to chemotherapy.

The outcome for adult patients with BCR-ABL-positive acute lymphoblastic leukemia (ALL) remains dismal and long-term survival can hardly be achieved except by allogeneic hematopoietic stem cell transplantation (HSCT). The Japan Adult Leukemia Study Group (JALSG) has recently started a phase 2 trial with intensive chemotherapy and imatinib for newly diagnosed BCR-AB-positive ALL patients, and we present here the interim results for the first 24 patients. All patients except one case of early death (96%) attained complete remission (CR) after a single course of remission induction therapy. Polymerase chain reaction (PCR) negativity was achieved in 28% of the patients on day 28, in 50% on day 63, and in up to 78% during the follow-up period. The toxicity profile was almost similar to that with chemotherapy alone. As a result, 15 patients (63%) could receive an allogeneic HSC transplant during their first CR. Although the number of patients is small and the observation period is too short, the combination therapy is very promising and produces high-quality CR for most newly diagnosed patients with BCR-ABL-positive ALL. This is especially useful because it provides the patients with a better chance to receive an allogeneic HSC transplant.

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human malignancy. An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene (FLT3/ITD) is found in 20% of patients with acute myeloid leukemia (AML) and is strongly associated with leukocytosis and a poor prognosis. On the other hand, mutations of the c-KIT gene, which have been found in mast cell leukemia and AML, are clustered in 2 distinct regions, the JM domain and D816 within the activation loop. This study was designed to analyze the mutation of D835 of FLT3, which corresponds to D816 of c-KIT, in a large series of human hematologic malignancies. Several kinds of missense mutations were found in 30 of the 429 (7.0%) AML cases, 1 of the 29 (3.4%) myelodysplastic syndrome (MDS) cases, and 1 of the 36 (2.8%) acute lymphocytic leukemia patients. The D835Y mutation was most frequently found (22 of the 32 D835 mutations), followed by the D835V (5), and D835H (1), D835E (1), and D835N (1) mutations. Of note is that D835 mutations occurred independently of FLT3/ITD. An analysis in the 201 patients newly diagnosed with AML (excluding M3) revealed that, in contrast to the FLT3/ITD mutation (n = 46), D835 mutations (n = 8) were not significantly related to the leukocytosis, but tended to worsen disease-free survival. All D835-mutant FLT3 were constitutively tyrosine-phosphorylated and transformed 32D cells, suggesting these mutations were constitutively active. These results demonstrate that the FLT3 gene is the target most frequently mutated to become constitutively active in AML.

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic myeloid neoplasms, in which disease progression is quite common, eventually terminating in secondary acute myeloid leukemia (sAML). To elucidate differential roles of mutations in MDS progression and sAML evolution, we investigated clonal dynamics of somatic mutations using targeted sequencing of 699 MDS patients, of which 122 were analyzed for longitudinally collected samples. Combining publicly available data, mutational data in a total of 2,250 MDS cases were assessed for their enrichment in specific disease subtypes. All samples were obtained after informed consent. Genotyping data from samples with low- (n=1,207) and high-risk (n=683) MDS as well as sAML (n=360) were available for most prevalently mutated 25 driver genes. In univariate comparison between low- and high-risk MDS, the majority of differentially mutated genes were enriched in high-risk MDS, except for SF3B1, which was more frequently mutated in low-risk MDS. Multivariate analysis was performed using a least absolute shrinkage and selection operator model. As a result, mutations in 7 genes (FLT3, PTPN11, WT1, IDH1, NPM1, IDH2,and NRAS) designated as 'Type-1' mutations, were significantly enriched in sAML compared to high-risk MDS. When comparison was made between high- and low-risk MDS, mutations in 10 genes, including GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2, were enriched in high-risk MDS. The latter mutations are designated as 'Type-2' mutations, excluding NRAS and IDH2 mutations, which were already assigned to the Type-1 category. To characterize the chronological behavior of Type-1 and Type-2 mutations, we performed longitudinal analyses of 122 cases, of which 90 progressed to sAML. Overall, driver mutations tended to increase their clone sizes between two time points. In accordance with their significant enrichment in sAML, Type-1 mutations were more likely to be newly acquired at the second time points, compared to Type-2 and other mutations (P=0.0001). By contrast, in patients with high-risk MDS at the second time point, Type-2 mutations were more dominant than Type-1 mutations, and most of the Type-2 mutations (88%) increased their clone sizes at the second sampling. Similarly, Type-2 mutations found in high-risk MDS or sAML evolving from low-risk MDS increased their clone sizes more frequently (30 out of 38 mutations (79%)) than Type-2 mutations in stable low-risk MDS without disease progression over time (4 out of 11 (36%)) (P=0.02). These findings suggest that Type-1 and Type-2 mutations might be associated with progression from high-risk MDS to sAML and low- to high-risk MDS, respectively. To further clarify the effects of the different classes of mutations on progression to sAML, 429 patients with MDS were analyzed for progression free survival (or PFS). Patients with Type-1 mutations (Group-I) had a significantly shorter PFS, compared to those who had Type-2 mutations but lacked Type-1 mutations (Group-II) (HR=1.82, 95% CI:1.08−3.05; P=0.025). Nevertheless, PFS in Group-II cases was still significantly shorter than that in other cases (HR=2.46, 95% CI:1.43−4.23; P=0.001). Of note, some Group-II cases subsequently acquired Type-I mutations during progression to sAML. By contrast, SF3B1-mutated patients tended to show slower progression to sAML, unless they carried either of Type-1 or 2 mutations (Group-III). Finally, the effects of these mutations on overall survival (OS) were assessed in a larger cohort of patients with MDS (n=1,347). Group-I cases were shown to have a significantly shorter OS than Group-II cases (HR=1.50, 95% CI:1.20−1.86; P In conclusion, our study has elucidated clonal dynamics associated with MDS progression and sAML evolution. Close monitoring of these sets of distinct mutations in the prospective fashion may help in the prediction of the clinical outcome in MDS. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Background Copy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms. Materials and Methods We enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%. Results To obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions. Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy. Conclusion We obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Introduction Acute erythroid leukemias (AELs) are infrequent acute leukemias characterized by a predominant erythroid population, dysplasticity and ring sideroblasts. The 2016 revision of the World Health Organization (WHO) Classification re-classified previous AELs as myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) not otherwise specified (non erythroid subtype, or pure erythroid type), according to blast percentage in all nucleated bone marrow cells. Recent studies have offered the evidence that genetic profiles are associated with pathophysiology and clinical outcomes in myeloid malignancies. However, characteristic mutation profiles of AEL and its genetic differences or similarities with other-types of AML and MDS have not been fully elucidated due to the lack of a sufficient number of samples, although frequent TP53 mutations have been reported (35-43.5%). Method We performed a comprehensive genetic study, in which paired tumor/normal DNA from 24 AEL cases were analyzed by whole-exome sequencing (WES). Subsequently, in total 41 cases were screened for mutations in 69 driver genes associated with myeloid malignancies using targeted-capture sequencing. Genome-wide copy number analysis was also performed based on allele frequency of heterozygous single nucleotide polymorphisms and sequencing depth. Frequencies of driver mutations in AEL were compared to those in other-types of AML analyzed by the Cancer Genome Atlas Network (TCGA) (N = 197). Results The mean number of nonsynonymous mutations identified by WES was 10 per sample (range, 0-21), which was similar to that in other-types of AML or MDS. Most frequently mutated genes were STAG2, TP53, RUNX1 and TET2 (17%, respectively), followed by WT1 and BCOR (13%, respectively). All TP53 mutated cases harbored complex karyotypes. Subsequent targeted sequencing identified 3.4 mutations per sample on average (range, 0-9). TP53 was mutated in 8 cases (20%), and lower frequency of TP53 mutations in our cohort was attributed to the relatively lower proportion (21%) of cases with complex karyotype compared to those in the previous reports. Commonly mutated genes in other-types of AML, such as NPM1, FLT3, and DNMT3A, were relatively infrequent in AEL and affected 7 (17%), 4 (9.8%) and 3 (7.3%) cases, respectively. Genes associated with splicing machinery were mutated in 5 cases (12%), including SRSF2 (9.8%) and U2AF1 (2.4%). Of note, mutations in genes implicated in cohesin complex were observed in as many as 32% of cases, including those in STAG2 (24%), SMC1A (4.9%) and RAD21 (2.4%). Mutations in epigenetic regulators were also frequently identified in AEL, including those in WT1 (20%), TET2 (15%) and IDH1/2 (7%). Mutations in TP53 were mutually exclusive with those in cohesin complex (p = 0.04) and epigenetic regulators (p = 0.05). When we compared the frequencies of recurrent mutations in AEL to those in other-types of AML analyzed by TCGA, mutations in STAG2 (p = 6*105), SRSF2 (p = 7*104), WT1 (p = 0.01), GATA2 (p = 0.008), and BCOR (p = 0.02) were significantly more frequent in AEL, whereas those in FLT3 (p = 0.01) and DNMT3A (p = 0.007) were significantly less frequent in AEL. Mutations in cohesin complex and splicing factors were frequently observed both in AEL and MDS, and higher frequency of these genetic lesion in AEL rather than other-types of AML suggested that AEL and MDS are similar disease entities. Recently, genomic subgroups of AML with mutations in cohesin complex, spliceosome and TP53 have been associated with poor prognosis in AML, and poor prognosis of AEL was thought to be due to higher frequency of these genetic lesions. Conclusion WES and follow-up targeted sequencing revealed the genetic landscape of AEL. Frequent mutations in TP53, splicing factors and cohesin complex were characteristic to AEL, and thought to be involved in its pathophysiology. Our data showed the genetic similarities between AEL and MDS, which were compatible with the partial integration of AEL into MDS in the recent revision of WHO classifications. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Heuser:Pfizer: Research Funding; BerGenBio: Research Funding; Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; Celgene: Honoraria; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding.

Background The percentage of blasts in the bone marrow in the early stage of remission induction therapy for acute myeloid leukemia (AML) has been reported to affect not only remission rate, but also long-term prognosis. If early post-treatment response to therapy was determined to affect prognosis, this finding could be a basis for selection of a proactive treatment such as hematopoietic stem cell transplantation; however, the proper assessment period and the percentage of blast cell survival vary among reports. Therefore, we retrospectively analyzed the relationship between prognosis and the percentage of blasts in the bone marrow in the early post-treatment stage in cases registered for the AML201 study conducted by the Japan Adult Leukemia Study Group (JALSG). Patients and Methods The JALSG AML201 study was a multicenter randomized study in which 1064 patients with newly diagnosed AML patients were registered between December 2001 and December 2005. For remission induction therapy, we compared standard-dose idarubicin or high-dose daunorubicin in combination with cytarabine. For consolidation therapy, we compared standard-dose multiagent chemotherapy or high-dose cytarabine alone, and no apparent differences were observed in overall survival (OS) between both treatment groups. The percentage of blasts in the bone marrow on day 15 was strongly associated with remission; using the receiver operating characteristic curve as a reference, we divided patients into a ≥5% and a Results Among the 1064 patients registered for the JALSG AML201 study, 600 patients for whom bone marrow examination was performed on Day 15 were selected as subjects for the present study. The patients consisted of 375 men and 225 women with a median age of 47 years (15-64 years). A total of 377 patients (62.8%) had Conclusion As an early assessment of bone marrow following remission induction therapy, having Disclosures Fujita: Chugai Pharmaceutical CO.,LTD.: Honoraria. Kiyoi:Novartis Pharma K.K.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Celgene Corporation: Consultancy; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding. Naoe:Amgen Astellas BioPharma K.K.: Honoraria; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Pfizer Inc.: Research Funding; CMIC Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Bristol-Myers Squibb: Honoraria; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding.

Introduction: Acute myeloid leukemia (AML) with hyperleukocytosis is considered to have a poor prognosis due to a high early death rate secondary to complications such as disseminated intravascular coagulation (DIC), tumor lysis syndrome (TLS), and leukostasis. The usefulness of pre-treatment reduction of leukemic cells is controversial. We reviewed the clinical characteristics and outcome of adult patients with newly diagnosed AML with or without hyperleukocytosis registered to the Japan Adult Leukemia Study Group (JALSG) AML201study and examined whether early death in induction therapy for this AML subtype influenced the outcome. Methods: We retrospectively reviewed the records of 1,022 patients enrolled in the JALSG AML201 study, which was a phase 3 clinical trial for AML newly diagnosed from December 2001 to December 2005. The JALSG AML201 study administered cytarabine from day 1 to 7 as the induction therapy combined with idarubicin or daunorubicin (Ohtake S, et al. Blood. 2011, 24;117:2358-65.). None of the patients received pre-treatment with oral hydroxyurea or leukapheresis. Patients with performance status (PS) of 4 or severe comorbidities such as septic shock were excluded. Patients' characteristics and outcomes such as rate of early death (defined as death occurring within the first thirty days of treatment), achievement of complete remission (CR), overall survival (OS) rate and cumulative incidence of recurrence (CIR) were compared between patients with hyperleukocytosis and those without hyperleukocytosis. Result: We analyzed retrospectively the records of 1,057 patients with AML registered with the JALSG AML201 study and excluded 35 patients whose leukocyte count or outcome was not recorded. We found 113 AML patients with an initial leukocyte count greater than 100 × 109L−1 (HiLC group). There were 66 males and 47 females, and the median age was 45 years (range: 16-64). Median of leukocyte count before treatment was 160 × 109L−1 (range: 102-382 × 109L−1). According to the French-American-British (FAB) classification, there were four as M0, thirty-four as M1, fifty-six as M2, thirty as M4, and nineteen as M5. Cytogenetic analysis was performed in 107 patients. Eight patients had favorable karyotype, 90 had intermediate karyotype, and nine had unfavorable karyotype. Fifteen percent of patients with hyperleukocytosis had PS of two or greater. We compared the outcome of this AML subset with 909 patients with a leukocyte count lower than 100 × 109L−1 (median count: 10,900, range: 0.77-99.5× 109L−1) who received the same induction chemotherapy as the JALSG AML201 protocol (LoLC group). There was no significant difference between the two groups for age and sex; median age of the patients in LoLC group was 47 years (range: 15-64) with 542 males and 367 females. The frequency of the patients with a favorable karyotype or PS of 0 or 1 was lower in HiLC group than in LoLC group, whereas the rate of FAB M4 or M5 was higher in HiLC as compared to LoLC group. Eighty-two patients (72.5%) in HiLC group and 714 patients (78.5%) in LoLC group achieved CR. Sixty (53%) and twenty-two (19.5%) achieved CR after one and two cycles of treatment, respectively. In contrast, 577 (63%) and 137 (15%) in LoLC group achieved CR after one and two cycles of chemotherapy, respectively. The rate of CR was not significantly different between two groups (p=0.118). However, 5 year OS was significantly lower in HiLC as compared to LoLC group (26.9% vs. 49.4%, p < 0.001). Interestingly, the rate of early death was not different between the two groups (1.65% vs. 1.77%, p = 1.0), although the rate of severe complications, such as DIC, was higher in HiLC compared to LoLC group (32% vs. 10%, p < 0.001). The frequency of brain hemorrhage (1% vs. 1%, p = 1.0) and gastrointestinal hemorrhage (4% VS. 3%, p = 0.294) was not different between two groups. One patient died of brain hemorrhage and one of DIC in HiLC group, whereas five patients died of brain hemorrhage, four of sepsis, and three of pneumonia in LoLC within the first thirty days of treatment. CIR was higher in HiLC (64.7%) as compared to LoLC group (51.6%) (p = 0.0048). Conclusion: We conclude that hyperleukocytosis on presentation did not significantly affect the rate of early mortality during the induction phase of the treatment. It is considered that intensive chemotherapy without pre-treatment would be possible if appropriate supportive cares were given to manage fatal complications such as DIC. Disclosures Kiyoi: MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; AlexionpharmaLLC.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Phizer Japan Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Celgene Corporation: Consultancy; Chugai Pharmaceutical Co. LTD.: Research Funding. Naoe:Amgen Astellas BioPharma K.K.: Honoraria; CMIC Co., Ltd.: Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties.

Acute myeloid leukemia (AML) is the most common leukemia in older adults. Chemotherapy is the standard treatment, however, treatment efficacy and tolerability deteriorates with age, and patients are susceptible to relapse after complete remission (CR). Therefore, new therapeutic strategies for preventing relapse after consolidation therapy are urgently needed. Wilms' tumor 1 (WT1) is a promising target of new immunotherapies for acute myeloid leukemia (AML) as well as other cancers. OCV-501 is a helper peptide derived from the WT1 protein. Preclinical studies have shown that it induced OCV-501-specific Th1 responses dose-dependently and stimulated helper activity of the specific Th1 cells in peripheral blood mononuclear cells from healthy donors. OCV-501 also enhanced the increase in WT1-killer peptide-specific cytotoxic T lymphocytes (CTLs). The stimulated OCV-501-specific Th1 clones in an HLA class-II restricted manner formed a complex with HLA class-II protein and showed significant OCV-501-specific cytolytic activity against B-lymphoblastoid cell lines (B-LCL). These results suggested that OCV-501 activates both direct and indirect antitumor (anti-leukemic) cellular immunity, including specific cytotoxic Th1 cells and WT1-peptide-specific CTLs. Based on this, a phase 1 trial was carried out to assess the safety and tolerability as well as efficacy of OCV-501 in elderly AML patients in CR. This was an open label, multi-center trial conducted in Japan. The study involved AML patients who have achieved their first CR with an induction regimen and completed standard consolidation therapy, and have been identified as WT1 mRNA positive, with one of the following HLA class II types: HLA-DRB1*01:01, *04:05, *15:01, *15:02, *08:03, or *09:01. The trial consisted of 3 cohorts at a dose of 0.3 mg in cohort 1, 1 mg in cohort 2, and 3 mg in cohort 3, whereby administration commenced with cohort 1 and progressed to subsequent cohorts, depending on the assessment for dose limiting toxicity (DLT) in the preceding cohort. OCV-501 was administered subcutaneously weekly for 4 weeks, followed by end of treatment and post-treatment examinations after 1 and 4 weeks from the last administration, respectively. Safety and tolerability which included any occurrence of treatment-emergent adverse events (TEAEs) and DLT were assessed, and the maximum tolerated dose (MTD) was determined. Efficacy was evaluated by relapse of AML, WT1 mRNA level and immune response to OCV-501 using delayed-type hypersensitivity (DTH) test. Nine patients were enrolled and all completed the study. No DLT was observed in any of the 3 cohorts. Injection site reactions, including erythema, induration, mass, pain, and pruritus were observed in all patients. However, these drug-related TEAEs were Grade 1 or 2 in severity. There were neither deaths nor serious TEAEs during the treatment period and none of the patients discontinued OCV-501 administration due to TEAE. The result of this trial suggested that OCV-501 once a week for 4 weeks at doses of 0.3, 1, and 3 mg was safe and well-tolerated in AML patients in CR and the MTD was considered to be ≥ 3 mg. In the efficacy analysis, neither relapse nor blast cells were observed in any patients and a reduction in WT1 mRNA level was found in 5 patients. Immunological response was observed in all patients in the OCV-501-specific DTH test. In conclusion, OCV-501, which was suggested to activate both direct and indirect anti-leukemic cellular immunity, showed a favorable safety and tolerability profile in elderly patients who have AML in CR, and the MTD was shown to be ≥ 3 mg. The results suggested the clinical beneficial of this vaccine and further clinical studies are highly warranted. This trial was registered at www.clinicaltrials.gov as NCT 01440920. Disclosures Sakura: Otsuka Pharmaceutical: Research Funding. Kobayashi:Celgene: Research Funding; Pfizer: Research Funding; Ohtsuka Pharmaceutical: Research Funding; Ariad: Research Funding; SymBio Pharmaceuticals: Research Funding. Toga:Otsuka Pharmaceutical: Employment. Sogo:Otsuka Pharmaceutical: Employment. Heike:Sumitomo Dainippon Pharma: Consultancy; Chugai Pharma: Consultancy; Otsuka Pharma: Consultancy.

Chk2 is a novel checkpoint kinase isolated as a human homologue of yeast Cds1/Rad53.[1][1] Recent analyses have revealed that it is among key molecules signaling DNA damage via the ATM protein kinase to p53.[1][1] [2][2] Of great interest is the report that germ line mutations of the Chk2 gene are

We conducted a multi-institutional randomized study to determine whether high-dose daunorubicin would be as effective as standard-dose idarubicin in remission-induction therapy for newly diagnosed adult patients younger than 65 years of age with acute myeloid leukemia. Of 1064 patients registered, 1057 were evaluable. They were randomly assigned to receive either daunorubicin (50 mg/m2 daily for 5 days) or idarubicin (12 mg/m2 daily for 3 days) in combination with 100 mg/m2 of cytarabine by continuous infusion daily for 7 days as induction therapy. Complete remission was achieved in 407 (77.5%) of 525 patients in the daunorubicin group and 416 (78.2%) of 532 in the idarubicin group (P = .79). Patients achieving complete remission received intensive postremission therapy that consisted of either 3 courses of high-dose cytarabine or 4 courses of standard-dose therapy. Overall survival rates at 5 years were 48% for the daunorubicin group and 48% for the idarubicin group (P = .54), and relapse-free survival rates at 5 years were 41% and 41% (P = .97), respectively. Thus, high-dose daunorubicin and standard-dose idarubicin were equally effective for the treatment of adult acute myeloid leukemia, achieving a high rate of complete remission and good long-term efficacy. This study is registered at http://www.umin.ac.jp/ctrj/ as C000000157.

MDS and related disorders, including MDS/MPN and sAML that evolved from these conditions constitute disease continuum characterized by a wide spectrum of molecular lesions which often overlap. Here, we defined general mutational spectrum and clonal architecture in a large cohort (n=718) of MDS studied by whole exome sequencing (WES) and target deep sequencing. Within this cohort 97 cases were studied at multiple time points to clarify the clinical impact of clonal dynamics on phenotype commitment or outcomes. All samples were obtained after informed consent, according to protocols approved by the respective ethics boards of the participating institutions. When mean and maximum variant allele frequency (VAF) for whole mutations were at one time-point evaluated in disease phenotypes, significantly higher averaged values suggested their larger clones in sAML and CMML compared to MDS. Clustering analysis of multiple mutational events by Pyclone software discriminated the cases with multiple mutational clones (positive heterogeneity) and those with a single expansion of MDS clone (no heterogeneity detected). Over 80% of low-risk MDS and all the sAML harbored multiple clusters of mutations. These results suggest that intra-tumor heterogeneity of MDS is most likely due to various sizes of clonal and subclonal mutations, likely impacting clinical behavior. To delineate clonal dynamics in MDS, we assessed mutational burden and their temporal changes in serially collected samples (n=97). Among these, Pyclone analysis was applied to exome sequencing at two time points (n=11 pairs). All cases showed various mutational clusters with individual expansions and declines, including initially present, newly acquired or disappearing during clinical course. Initial subclones were identified at disease presentation in 55% of cases, of which in 86% the subclones expanded to occupy whole MDS population with clonal sweep. New subclones acquired during clinical course were identified in 91%, in which 60% cases harbored clonal sweep. Disappearing clones were observed in 55% of cases. Next, we applied clustering analysis on clonal size of driver mutations evaluated at multiple time points (n=97 cases) to categorize the most frequently mutated genes into 3 subtypes. Mutational burden of PTPN11 most frequently increased and were associated with leukemic evolution (an example of type I gene). Similarly, CBL, NRAS, STAG2, RUNX1, and IDH1 were categorized into the type I genes, demonstrating increased clonal size resulting in the evolutions into high-risk phenotypes. Although JAK2 mutations were related to the stable clinical course when the mutational burden decreased, cases with highly expanded JAK2 mutations resulted in leukemic evolution (occasional evolution or expansions; type II gene). DNMT3A, SRSF2, TP53, U2AF1, and ASXL1 mutations were also categorized into such type II consequences with occasional progression. The last category (type III) included clonal/founder genes EZH2, TET2, SF3B1 and PRPF8, demonstrating random shifts of clonal size and lack of association with leukemic evolution. The proposed hierarchical categorization correlates with clinical parameters. Cases with the increasing burden of type I gene mutations showed most significant increases in myeloblasts. Overall survival measured from second sampling time points in the cases with increasing type I mutations was significantly shorter in the whole cohort (HR=2.05, 95%CI; 1.14-3.79, P=0.016) and in the cases solely with IPSS INT-1 (HR=2.37, 95%CI; 1.01-5.97, P=0.048). Subcohorts classified according to the presence or absence of increasing type I mutations did not differ with regard to the IPSS categories. In contrast, increased mutational burden of type II and III genes did not correlated with any of the clinical parameters examined, even though some gene mutations including TP53, EZH2, and U2AF1 represented poor prognostic factors at disease presentation. In conclusion, this work demonstrates that detailed understanding of clonal dynamics allows for new insights into clinical significance of somatic mutations, made possible only by serial sample sequencing at multiple time points. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in more uniform and larger cohort of MDS. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Shih:Novartis: Research Funding.

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 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 ( 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.

Adult T-cell leukemia/lymphoma (ATL) is a distinct subtype of peripheral T-cell neoplasms associated with human T-cell leukemia virus type-1 retrovirus. ATL includes a heterogeneous group of patients in terms of pathological and clinical features as well as prognosis, suggesting the presence of underlying molecular pathogenesis that could explain such heterogeneity among patients. Recently, we performed an integrated molecular analysis of a large number of ATL cases and delineated a comprehensive registry of gene mutations and other genetic/epigenetic lesions in ATL. In this study, we investigated possible correlations between these genetic/epigenetic lesions and clinical/pathological phenotypes in a large set of ATL patients, with a special focus on the impact of mutations and copy number alterations (CNAs) on clinical outcome. We analyzed a total of 361 ATL samples, including acute (n = 192), lymphoma (n = 66), chronic (n = 89), and smoldering (n = 14) subtypes, for recurrent mutations and CNAs. Each subtype had characteristic genetic/epigenetic features, suggesting a distinct molecular pathogenesis therein. Aggressive (acute and lymphoma) subtypes were characterized by a higher number of mutations and CNAs including focal amplifications/deletions, hyperploid status, and CIMP phenotype, compared with indolent (chronic and smoldering) tumors. Two mutations (TP53 and IRF4) and eight focal deletions involving 1p13 (CD58), 6p21 (HLA-B), 9p21 (CDKN2A), 10p11 (CCDC7), 13q32 (GPR183), 16q23 (WWOX), 17p13 (TP53), and 19q13 (CEBPA), were more common in aggressive ATL than in indolent ATL. In contrast, showing a similar mutational distribution to those found in large granular lymphocytic leukemia, STAT3 mutations were characteristic of the indolent diseases. Gene set enrichment analysis of RNA-seq data showed a significant enrichment of MYC pathway and genes regulating cell cycle and DNA repair in upregulated genes in aggressive ATL. Next, we assessed the impact of mutations and CNVs on prognosis among 215 ATL cases, for which survival data were available. In the entire cohort, mutation in CCR4 and IRF4, focal amplification in 9p24 (CD274) and 14q32 (BCL11B), and focal deletion in 9p21 (CDKN2A) were found to be significant predictors of poor overall survival, after adjustment for disease subtype and age. Multivariate analysis revealed that disease subtype (aggressive vs. indolent) was the most significant predictor of clinical outcome in ATL. Subsequent multivariate analysis according to disease subtype showed that within the patients with aggressive ATL, older age (≥ 70 years), CCR4 mutations, and 9p24 amplification were independently associated with an adverse outcome. Based on the number of the risk factors they owned, patients with aggressive ATL were classified into three categories showing marked difference in 3-year overall survival (OS) (P < 0.001): those with no risk factors (OS, 32%), with one risk factor (18%), and with two or more (0%). Among the patients with indolent ATL, we found IRF4 and TP53 mutations, 9p24 amplification, and deletions in 9p21 and 10p11 were independently associated with reduced survival. Interestingly, these alterations, except for 9p24 amplification, were also identified as genes more frequent in aggressive ATL. More importantly, based on these risk factors, the patients with indolent ATL can be classified into two categories showing very different prognostic profiles: patients with no risk factors (OS, 89%) and those with one or more risk factors (21%) (P < 0.001, HR = 16.8, 95% CI:5.4-52.5), suggesting that patients with indolent ATL having a genetic feature of the aggressive subtypes might genetically and biologically represent a distinct subset, which should be better managed as having an aggressive disease. Among these poor prognostic factors, 9p24 amplification and CCR4 mutation are especially interesting, because these lesions might be plausible targets of available agents, including anti-PD1/PD-L1 and anti-CCR4 antibodies. In conclusion, based on the comprehensive genetic profiling, we demonstrated that the known subtypes of ATL can be further classified into genetically and biologically distinct subsets of tumors characterized by discrete sets of genetic lesions and substantially different prognosis. Our results suggest that molecular profiling can improve the prediction of prognosis in ATL patients and better guide therapy. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Shin-bio: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Background: FMS-like tyrosine kinase 3 (FLT3) mutations in patients with acute myeloid leukemia (AML) are strongly associated with poor prognosis and leukocytosis. Approximately 15-35% of AML patients have internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence and 5-10% have a missense mutation of D835 within the tyrosine kinase domain. ASP2215 potently inhibits FLT3 with established preclinical activity against both ITD and D835 mutations. Preliminary results from a Phase 1/2, first-in-human study suggest ASP2215 is generally well tolerated and associated with antileukemic activity in FLT3 mutation-positive (FLT3+) subjects with relapsed or refractory AML (R/R AML). The safety and antileukemic activity of ASP2215 is currently being evaluated in an ongoing, Phase 1 study in Japanese subjects with relapsed or refractory (R/R) AML. Methods: This open-label, dose-escalation study, conducted in adult Japanese subjects (≥18 years) with R/R AML, consisted of a single- and a repeated-dose periods. ASP2215 (20-300 mg) was orally administered to subjects as a single dose (Cycle 0) and then once daily (Cycle 1 and subsequent cycles, each cycle was 28 days) until one of the discontinuation criteria were met. Dose escalation proceeded in a step-wise manner, based on the onset of dose-limiting toxicity (DLT) during Cycles 0 and 1, with the next dose level determined sequentially using the Bayesian Continual Reassessment Method (CRM). The study design allowed that at least one subject would receive the 20 mg dose and at least three subjects would receive higher doses (40-300 mg) of ASP2215. Primary endpoints included ASP2215 tolerability and determination of maximum tolerated dose (MTD, defined as the dose where posterior mean DLT incidence is ~33% when calculated using Bayesian CRM). Secondary endpoints included clinical response (based on Cheson's criteria) and pharmacokinetic profile. Results: As of June 24, 2015, 18 subjects with a median age of 71.5 years (range: 60-81 years) and 94% with ECOG 0-1 have received ASP2215 at dosages of 20 mg (n=1), 40 mg (n=4), 80 mg (n=4), 120 mg (n=4), and 200 mg (n=5). Three subjects were FLT3-ITD mutation positive, eight were FLT3-ITD negative, and the FLT3-ITD status for the other seven was unknown; FLT3 point mutation status for the majority (94%) of these subjects is not yet available. Only one DLT (tumor lysis syndrome), experienced at the 120 mg dose, has been reported suggesting MTD has not yet been reached. Abnormal hepatic function (n=5) and stomatitis (n=3) were the only treatment-related AEs occurring in ≥3 subjects. Grade ≥3 AEs were thrombocytopenia (n=2), febrile neutropenia (n=1), device-related infection (n=1), tumor lysis syndrome (n=1) and pneumonia (n=1). Five serious AEs deemed to be possibly or probably treatment related by the investigators (febrile neutropenia, edema, pneumonia, device-related infection, subdural hematoma) occurred in three subjects. ASP2215 plasma concentrations increased with doses from 20 mg to 200 mg with median time to maximal concentration of 2-6 hr. A total of 13 subjects had evaluable bone marrow to assess antileukemic activity; six of whom achieved at least partial response (Table). Conclusions: These preliminary findings in Japanese patients with R/R AML suggest ASP2215 is a well-tolerated FLT3 inhibitor with dose dependent increase in PK and has shown antileukemic activity in Japanese patients. ASP2215 tolerability, PK profile, and antileukemic activity are consistent with the ongoing dose-escalation/dose-expansion study being conducted in the US and Europe. Based on the positive findings, randomized Phase 3 trials have been planned. Table 1. Best Clinical Response by ASP2215 Dose and FLT3 Mutation Status Dose (mg) FLT3-ITD mutation status Best response Assessment day with first report of best response 20 Unknown CRi Day 28 40 Negative NR Day 29 Negative PR Day 29 Negative NR Day 29 80 Negative CR Day 28 Negative NR Day 11 Positive PR Day 28 Unknown NR Day 30 120 Negative NR Day 29 Positive CRi Day 57 200 Negative CRp Day 39 Positive NR Day 21 Unknown NR Day 27 CR, Complete remission; CRi, complete remission with incomplete hematologic recovery; CRp, complete remission with incomplete platelet recovery; NR, no response; PR, partial response. Disclosures Naoe: Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding. Off Label Use: ASP2215 is an investigational product for the treatment of AML. Sakura:Astellas: Other: Principal Investigator of the clinical research; study fee. Kobayashi:Gilead Sciences: Research Funding. Usuki:Kyowa Hakko Kirin: Other: personal fees, Research Funding; Shire: Research Funding; Takeda Pharmaceutical: Research Funding; Fujimoto Pharmaceutical: Research Funding; Shionogi: Other: personal fees; SymBio Pharmaceutical: Other: personal fees, Research Funding; Eisai: Research Funding; Otsuka Pharmaceutical: Research Funding; MSD: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; Novartis: Other: personal fees, Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; GlaxoSmithKline: Other: personal fees, Research Funding; Taiho Pharmaceutical: Other: personal fees, Research Funding; Fuji Film RI Pharma: Other: personal fees; Chugai Pharmaceutical: Other: personal fees; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Boehringer Ingelheim: Other: personal fees, Research Funding; Bristol-Myers Squibb: Other; Astellas: Research Funding. Iida:Astellas: Other: Principal Investigator of the clinical research; study fee. Morita:Astellas: Honoraria, Other: Personal fees. Bahceci:Astellas Pharma Global Development: Employment. Kaneko:Astellas Pharma, Inc.: Employment. Yamada:Astellas Pharma, Inc.: Employment. Takeshita:Astellas Pharma, Inc.: Employment. Miyawaki:Astellas Pharma Inc.: Consultancy, Other: personal fees; Ohtsuka Pharma Co, LTD.: Other: Safety Data Committee.

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples. A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H). The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers. Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed. RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator. Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

The association of ethnicity with the incidence of graft-versus-host disease (GVHD) and other clinical outcomes after transplantation is controversial. We compared the results of HLA-identical sibling bone marrow transplantations for leukemia, performed between 1990 and 1999, among different ethnic populations, including 562 Japanese, 829 white Americans, 71 African Americans, 195 Scandinavians, and 95 Irish. Results for adults and children were analyzed separately. Multivariate analyses of adult patients showed that white Americans, African Americans, and Irish cohorts were at significantly higher risk for acute GVHD than Japanese or Scandinavian cohorts (relative risk [RR] = 1.77, P < .001; RR = 1.84, P < .006; RR = 2.22, P < .001, respectively). White Americans, African Americans, and Irish, but not Scandinavians, were at significantly higher risk for early (within 3 months of transplantation) transplant-related mortality (TRM) compared with Japanese (RR = 2.99, P < .001; RR = 5.88, P < .001; RR = 2.66, P < .009, respectively). No differences in the risk for chronic GVHD, relapse, and overall survival were noted. In the pediatric cohort (limited to Japanese and white Americans), white Americans were at significantly higher risk for acute (RR = 1.93; P = .04) and chronic (RR = 3.16; P = .002) GVHD. No differences in other clinical outcomes were noted. Our findings suggest that ethnicity may influence the risk for GVHD, though overall survival rates after transplantation remain similar.

Although FLT3 mutations are essentially found in myeloid-lineage leukemia cells, a high level of FLT3 expression was recently observed in MLL gene-rearranged acute lymphoblastic leukemia without FLT3 mutations. Here, we analyzed the biologic and clinical significance of the FLT3 transcript level in comparison with several gene alterations in 181 de novo acute myeloid leukemia (AML) cases. The mean expression level in AML was higher than that in normal mononuclear cells, whereas the range varied widely. A high level of FLT3 is related to internal tandem duplication of the FLT3 gene (FLT3/ITD), the mutations within the activation loop of FLT3 (FLT3/D835Mt), and tandem duplication of the MLL gene (MLL-TD) but not to p53 or N-RAS gene mutations. Furthermore, a high expression level in AML cases with FLT3 mutations was not related to MLL-TD. Overexpressed FLT3 revealed autophosphorylation and had the same sensitivity to the FLT3 inhibitor as FLT3/ITD. Overexpression of FLT3 (more than 200,000 copies/microgRNA) was an unfavorable prognostic factor for overall survival in 91 AML cases without FLT3/ITD. These results indicated that FLT3 overexpression may distinguish a novel disease entity in AML without FLT3 mutations and serve as a therapeutic target for FLT3 inhibitors.

By retrospectively analyzing 288 patients with de novo myelodysplastic syndrome (MDS), we sought to determine the prevalence and clinical characteristics of bone marrow eosinophilia and basophilia that were detected at presentation. Bone marrow eosinophilia and basophilia were defined as a differential count of each cell type exceeding 5.0% and 1.0%, respectively. Of 288 patients with MDS, 36 (12.5%) fulfilled this criterion for bone marrow eosinophilia (MDS-Eos); 34 patients (11.8%) showed basophilia (MDS-Bas), and 11 (3.8%) satisfied both criteria (MDS-EosBas). The remaining 229 patients had neither eosinophilia nor basophilia in their bone marrow (MDS−/−) at presentation. Cytogenetic analysis was carried out on unstimulated bone marrow cells obtained from 264 patients. When the cytogenetic categorization of the IPSS (International Prognostic Scoring System) for MDS was applied, significantly higher numbers of MDS-Eos and MDS-Bas patients had chromosomal abnormalities carrying intermediate or poor prognosis, compared with the MDS−/− patients. Specific chromosomal abnormalities and complex karyotypes were associated with MDS-Eos and/or MDS-Bas. In accordance with these results, the overall survival rate was significantly lower, and the evolution to acute myelogenous leukemia (AML) occurred more frequently in the MDS-Eos and MDS-Bas than in the MDS−/− patients. Multivariate analysis demonstrated that bone marrow basophilia was an independent risk factor for evolution to AML. Our study indicates that bone marrow eosinophilia and basophilia in patients with MDS predict a poorer prognosis.

Background: A combination of all-trans retinoic acid (ATRA) and chemotherapy (CT) has dramatically improved the prognosis of acute promyelocytic leukemia (APL). Nevertheless, considerable number of patients are either refractory to the treatment or relapse after an initial complete remission (CR). Although prognostic factors for APL have been studied, the influence of chromosomal variations in addition to t(15;17) remains controversial. One of the reasons is due to the numbers of cases studied were relatively small (47 to 513 cases). Here, we analyzed clinical features and outcomes of 775 APL caseswith or without additional chromosome abnormalities (ACAs) who were treated with ATRA and CT in the JALSG-APL studies including a large number of cases analyzed for karyotype. Methods: 1,024 cases aged between 15 and 70 yrs with newly diagnosed APL were enrolled in the JALSG APL92, 95, 97 and 204 studies and 775 patients were assessable for karyopypes. All protocols included induction therapy with ATRA and CT, following several courses of post-remission chemotherapy including anthracyclines. Arsenic trioxide (ATO) was not included. Clinical and biological characteristics such as age, gender, initial leukocyte count, platelet count, number of APL cells, DIC score, lack of Auer-rod and incidence of variant type were analyzed in relation to chromosomal abnormalities in 766 cases. CR rate, relapse rate (RR), overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were assessed and compared between patients with or without ACAs. Each variation of ACAs was also analyzed with clinical and biological features. This work was supported in part by the National Cancer Center Research and Development Fund (26-A-24), Grants-in-Aid from the Cancer Research from the Japanese Ministry of Health, Labor and Welfare (#23-004 and #25100501). These studies were approved by our IRB. Results: ACAswere noted in 235 patients (30%). Sanz score and the initial leukocyte count were significantly lower in patients with ACAs (p=0.027 and p=0.027, respectively). No other clinical or biological differences were found between patients with and without ACAs. The subgroups of ACAs were shown in Figure 1. Trisomy 8 was found in 76 cases (32%). Other ACAs were found involving chromosome 15 in 37 cases (16%), both chromosomes 15 and 17 in 31 cases (13%), chromosome 7 in 19 cases (8%), chromosome 9 in 12 cases (5%), chromosome 6 in 8 cases (3%), chromosome 21 in 7 cases (3%) and alternative ACAs in 43 cases (18%). A low initial leukocyte count ( Conclusions: The present study is the largest to date to focus on the influence of ACAs on clinical outcomes of patients with APL treated with ATRA and CT. The analysis revealed exact variation and frequency of ACAs. We found that patients with ACAs were associated with the lower initial leukocyte count and the lower survival outcomes in elderly patents, suggesting a possible link to age and post-remission chemotherapy. Some promising agents, such as ATO, tamibarotene and gemtuzumab ozogamicin might change the prognostic factors, including ACAs. Careful chromosomal analyses, especially ACAs related to chromosome 15 and/or 17, need to be analyzed by molecular methods and performed in future prospective studies with alarge number of cases. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Kiyoi: Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Kobayashi:Ohtsuka: Research Funding; Behringer: Research Funding; Simic: Research Funding. Asou:Chugai Pharmaceutical Co., Ltd.: Research Funding. Miyazaki:Nippon-Shinyaku: Honoraria.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner. In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL. RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants. In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. Disclosures No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in > 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.

Somatic mutations constitute key pathogenetic elements in MDS. Unbiased whole exome sequencing (WES) and deep NGS led to discovery of new somatic mutations and also to the recognition of i) tremendous diversity of mutations and their combinations; ii) individual intra-tumor heterogeneity and clonal hierarchy. Chromosomal lesions further increase the complexity of molecular defects. While in MDS molecular defects are acquired in order, observations made in AML highlight the importance of ancestral events; e.g., t(8;21), inv16 or t(15;17) and other lesions that are used as the basis for nosological sub-classification. Thus, it is the identity of individual ancestral events or their classes rather than the spectrum of secondary events or the distribution of mutations, that will allow for molecular, functionally-relevant and diagnostically useful classification within MDS. This would explain why only a few somatic mutations have been found to be prognostically important, as their position in the clonal hierarchy has not been accounted for. With this in mind, we applied WES (N=206) and targeted deep NGS (N=836) and studied 100 samples serially with analyses focused on ancestral events. Globally, through WES we identified and validated 2386 mutational events in 1458 genes. Of these, 112 genes were mutated at significant frequencies (q Using cross-sectional analyses, the highest mean VAF could be interpreted as consistent with the ancestral nature of the mutations, as seen for instance in a proportion of TET2 and SF3B1 mutant cases. In contrast, the lowest mean VAF indicated secondary events, as occur in NPM1 and RAS pathway mutations. Similar conclusions were made based on cross-sectional analyses showing a similar distribution of ancestral but not secondary events in MDS and sAML. All gene mutations were categorized into those that are predominantly ancestral and those that are facultatively secondary. The most frequent founder mutations were identified (TET2, DNMT3A, SF3B1, ASXL1, TP53, U2AF1, RUNX1, SRSF2) and used to sub-classify approximately 80% of patients, with the remainder containing more infrequent ancestral mutations. While in a combined fashion (as both founder and secondary events) many of these mutations were not predictive of prognosis, they gained relevance when only cases affected by ancestral mutations were used for prognostication. Thus some of the mutations, when present as secondary events may not be predictive. Founding mutations may determine subsequent clinical and molecular features. While other frequently affected genes, SF3B1 or ASXL1, are not associated with a significant increase in the number of concomitant mutations, cases with TET2 mutations showed significantly more frequent mutations per case than those with wild-type TET2 (14.6 vs. 9.1; p=0.001). Moreover, ancestral TET2 mutations were associated with concomitant mutations due to high C-to-T transitions, possibly because reduced 5-hydroxymethylcytosine might create the specific mutator milieu. Most important is the association not of any type, but of ancestral mutations with certain pathomorphologic features and outcomes. Founding TET2 mutations are associated with MPN/MDS while secondary TET2 mutations are present in MDS. Ancestral DNMT3A mutations determine a rapid progression to AML, whereas subclonal DNMT3A mutations are also found in high-risk MDS. RAS pathway mutations are ancestral in CMML and also secondarily positive in the late stage of MDS (sAML). Specific ancestral events may determine subsequent mutational events, and while both types of mutation may affect the clinical phenotype, the initial events are less diverse and more subtype-specific. In conclusion, WES clarified the distinct landscape and ordering of the somatic mutational spectrum in MDS. Disclosures No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive form of peripheral T-cell lymphoma (PTCL), which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone may not be sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL. The purpose of this study is to understand the genetic basis of ATL, 32 cases with different ATL subtypes, including acute (N=15), chronic (N=6), lymphoma (N=10), smoldering (N=1) types were analyzed by whole exome sequencing as well as copy number analysis. With a mean coverage of 119, 94% of the target sequences were analyzed at more than 20 depth on average. A total of 2,862 somatic changes were detected in 32 cases with a true positive rate of 99% (329 of the 334 tested were confirmed by PCR-based deep sequencing). These consisted of 2,512 missense mutations, 174 nonsense mutations, 65 splice-site mutations, and 111 indels. The mutation rate of 89 (44-227) per sample was significantly higher than that in acute myeloid leukemia (7.3–13), myelodysplastic syndromes (9.2) and chronic lymphocytic leukemia (11.5). Recurrent mutations were observed in 350 genes, of which 192 were considered to be significantly mutated (q < 0.05) compared to background mutation rates (1.79 mutations per megabase). To investigate significantly mutated pathways, each pathway registered in the Kyoto Encyclopedia of Genes and Genomes, BioCarta, Reactome, Sigma-Aldrich and Signaling Transduction KE was tested on the basis of the background mutation rate observed in whole-exome sequencing data, which revealed a number of significantly mutated functional pathways, including pathways involeved in T cell receptor signaling, leukocyte trans-endothelial migration, VEGF and WNT signalings and other signaling pathways. Genes for epigenetic regulations were also among the frequent targets of gene mutations. We performed targeted deep sequencing of TET2, IDH1/2 and DNMT3A in 182 ATL cases. In total, 19 TET2 mutations were identified in 16 cases (8.7%). Different subtypes of ATL were almost evenly affected with 9 out of 67 acute, 3 out of 42 chronic and 4 out of 56 lymphoma types having TET2 mutations. Less frequent mutations of IDH2 and DNMT3A (both 1%) were also identified. Our findings on genetic alterations provide a novel insight into the pathogenesis of ATL. Disclosures: No relevant conflicts of interest to declare.

Background MPAL is a rare subtype of acute leukemia that accounts for 2-5% of all acute leukemia cases. It has been reported that patients with MPAL tend to have worse prognosis compared with those with AML or ALL. However, the efficacy and safety of allogeneic stem cell transplantation (allo-SCT) for adult MPAL patients has yet to be elucidated. Objective The aim of this retrospective study was to assess the outcomes of allo-SCT for adult MPAL and compared with those for AML/ALL using a matched pair analysis method. Patients and Methods This study included patients over 15 years of age who were diagnosed with MPAL according to the WHO classification and underwent allo-SCT for the first time between January 2001 and December 2010. Their clinical data were collected from the database of the Kanto Study Group for Cell Therapy. The clinical features and transplant outcomes of MPAL patients were then compared with those of adult patients with AML, ALL, and Philadelphia chromosome-positive acute leukemia (Ph+AL). We selected control cohorts at the rate of one to five using an optimal matching method with the following seven matching factors: age, sex, disease status at the time of transplant, the intensity of conditioning, human leukocyte antigen disparity between donor and recipient, donor type (related or not), and time of transplant (2001-2005 or 2006-2010). Overall survival (OS) and relapse-free survival (RFS) rates was estimated by the Kaplan-Meier method and compared using the log-rank test. Cumulative incidences (CI) of relapse and non-relapse mortality (NRM) were compared using the stratified Gray test. P Results Eighteen MPAL patients (9 men, 9 women) with a median age of 40 years (range, 16–61 years) were identified. Immunophenotypic analysis showed B+M phenotype in 12 patients, B+T phenotype in five, and B+T+M phenotype in one. Seven patients possessed Ph. Fourteen patients were in remission at the time of transplant. Sixteen patients were conditioned with myeloablative regimens. Stem cell sources were related donor bone marrow in four patients, unrelated donor bone marrow in 13, and cord blood in one. Among 18 MPAL patients, 5-year OS and RFS rates were 48.1% and 39.7%, respectively, and 5-year CI of relapse and NRM were 43.3% and 17.1%, respectively. Patients in remission at the time of transplant showed significantly better outcomes than those not in remission (5-year OS: 71.8% vs. 0%, p = 0.001; 5-year RFS: 58.7% vs. 0%, p = 0.004). By contrast, no significant difference was seen in OS and RFS when stratifying patients according to immunophenotype and cytogenetic abnormalities. In matched pair analysis, 90 AML, 90 ALL, and 35 Ph+AL patients were extracted from the database as control. The 5-year OS rate of MPAL patients was similar to those of AML patients (48.1% vs. 48.1%; p = 0.855) and ALL patients (48.1% vs. 37.8%; p = 0.426). Similarly, the 5-year OS rate of Ph+MPAL patients was not significantly different from that of Ph+AL patients (68.6% vs. 39.9%; p = 0.234). No significant difference was observed in RFS, CI of relapse, and CI of NRM when comparing MPAL patients with AML and ALL patients, and Ph+MPAL patients with Ph+AL patients. Conclusion Transplant outcomes of adult MPAL patients were comparable to those of both AML and ALL patients, although the statistical power was possibly insufficient due to the relatively small cohort. The existing transplant procedures were not satisfactory for MPAL patients who were not in remission at the time of transplant. On the other hand, MPAL patients who underwent allo-SCT while in remission showed excellent outcomes. Therefore, transplant early in the disease course may lead to improved outcomes for patients with this serious disease. Disclosures: No relevant conflicts of interest to declare.

Chromosomal aberrations and somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a clonal hematologic malignancy characterized by cytopenias, a dysplastic bone marrow and propensity to clonal evolution. Next generation sequencing (NGS) enables definition of somatic mutational patterns and clonal architecture as a discovery platform, and for clinical applications. We systematically applied NGS to 707 cases of MDS and MDS-related disorders. 205 cases (low-risk MDS: N=78, high-risk MDS: N=42, MDS/MPN: N=48 and sAML: N=37) were tested by whole exome sequencing (WES). For validation in an additional 502 patients (low-risk MDS: N=192, high-risk MDS: N=104, MDS/MPN: N=111 and sAML: N=95), targeted deep NGS was applied for 60 index genes which were most commonly affected in the cohort analyzed by WES. For NGS data analysis a statistical pipeline was developed to focus on: i) identification of the most relevant somatic mutations, and ii) minimization of false positive results. We studied serial samples from 21 exemplary informative patients. We also compared somatic mutational patterns to those seen in primary AML TCGA cohort (N=201). Given the size of the cohort, there was, for example, a 87% chance of seeing mutations at a frequency of 1% and a 98% of seeing those with a frequency of 2%. While focusing on the most common events, we observed 1117 somatic mutations in 199 genes. The 88 genes mutated mutated in >1% of cases with MDS carried 388 mutations in MDS+sAML (2.5/case), 128 in MDS/MPN (2.7/case) and 398 in pAML (2.0/case). The average number of mutations per case increased during progression (2.2 in lower-risk, 2.8 in higher-risk MDS, 3.4 in sAML). In MDS, the 30 most frequently affected genes were present at least once in 70% of patients. The 30 most frequently mutated genes in MDS/MPN were mutated in 82% of patients. Individual mutations were also sub-grouped according to their function. When we compared three MDS subcategories (lower-risk, higher-risk MDS and sAML) in a cross-sectional view, RTK family, RAS family, IDH family and cohesin family mutations were more frequently detected in the sAML group than in the MDS group. In contrast, the frequency of the DNMT family, TET2 and ASXL family gene mutations did not increase in frequency in the sAML cohort. In addition to better definition of mutational patterns of known genes, we have also defined new mutations, including in the RNA helicase family and the BRCC3pathway. Clonal architecture analysis indicates that mutations of TET2, DNMT3A, ASXL1, and U2AF1 most likely represent ancestral/originator events, while those of the IDH family, RTK family and cohesin family are typical secondary events. Establishment of mutational patterns may improve the precision of morphologically-based diagnosis. The comparison between MDS-related diseases (MDS+sAML) and pAML revealed a notably different mutational pattern suggestive of a distinct molecular derivation of these two disease groups. While RTK, IDH family and NPM1 mutations were more frequently observed in the pAML cohort, mutations of SF3B1 and SRSF2, were more common in MDS+sAML. With regard to the connections between individual mutation combinations, RTK mutations were strongly associated with DNMT, but not with RAS family mutations in the pAML cohort, while the mutual association between TET2 and PRC2 family, cohesin family and RUNX1were encountered in the MDS+sAML cohort. Individual mutations may have prognostic significance, including having an impact on survival, either within the entire cohort or within specific subgroups. In the combined MDS cohort, TP53 family mutations were associated with a poor prognosis (HR; 3.65, 95%CI; 1.90-7.01, P In conclusion, our study continues to indicate the power of NGS in the molecular analysis of MDS. MDS and related disorders show a great deal of pathogenetic molecular overlap, consistent with their morphologic and clinical pictures, but also distinct molecular differences in mutational patterns. Some of the specific mutations are pathognomonic for specific subtypes while some may convey a prognostic rather than discriminatory value. Disclosures: Makishima: Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Polprasert:MDS foundation: Research Funding.

Background The current guidelines for acute myeloid leukemia (AML) issued by the Japan Society for Hematopoietic Cell Transplantation were updated in 2009 to reflect that AML in intermediate-risk or unfavorable-risk patients younger than 55 years of age should be treated with allogeneic stem cell transplantation (allo-SCT) at the first complete remission (1CR). However, most of the eevidence' for this recommendation was based on the results of either prospective studies with randomization according to donor availability or retrospective studies. Therefore, the influence of eselection bias' is inevitable. On the other hands, physicians sometimes have difficulty deciding whether a patient should receive upfront allo-SCT or final treatment with chemotherapy, as the outcomes of patients with 1CR at allo-SCT and 2nd CR (2CR) at allo-SCT are comparable. Aims We conducted a subgroup analysis to verify the validity of allo-SCT in AML patients in 1CR using the dataset of patients registered in prospective studies. [Methods] We examined the disease status and outcomes of the AML patients registered in the AML97 and AML201 studies conducted by the Japan Adult Leukemia Study Group (823 patients and 1,064 patients, respectively) who underwent allo-SCT. Results A total of 752 patients with AML were registered in the JALSG study and underwent allo-SCT. Their estimated 5-year overall survival (OS) was 44%. The number of patients according to the disease status at allo-SCT was 262 in 1CR, 130 in 1st relapse (1rel), 192 in 2CR and 98 with primary induction failure to CR (PIF). The estimated 5-year OS of the patients with intermediate-risk or unfavorable-risk cytogenetics in 1CR was significantly superior to that of the patients with 1rel or 2CR (57% vs. 41%, p Conclusions The use of upfront allo-SCT in AML patients with 1CR is recommended, as the allo-SCT outcomes of patients with 1CR are superior to those observed after relapse, although the 5-year OS of patients with 2CR is not inferior to that of patients with 1CR. Disclosures: No relevant conflicts of interest to declare.

Background Immunophenotyping is a standard diagnostic procedure in leukemia, but unlike cytogenetic classifications, which are reliable indicators for prognostic interpretation, immunophenotyping has, thus far, yielded inconsistent results. Recently, the presence of CEBPA, NPM1, or FLT3 mutations, as a molecular characteristic, was identified as an important prognostic factor for normal karyotype (NK)-acute myeloid leukemia (AML). Because AML with the CEBPA mutation is closely associated with specific surface antigen expression, we investigated the prognostic implications and characteristics of a certain immunophenotype in NK-AML. Design and Methods The frequency of CEBPA mutation according to cytogenetic classification in 318 AML patients was investigated, and then the surface antigen expression profile in NK-AML was assessed. Thereafter, the prognosis and patient characteristics of a specific immunophenotype was investigated in 329 NK-AML patients from the Japan Adult Leukemia Study Group (JALSG) AML97 study. Factor differences between groups of patients with and without this immunophenotype were compared by Fisher’s exact and Student’s t-tests. Complete remission (CR), disease-free survival (DFS), event-free survival (EFS) and overall survival (OS) was assessed using Fisher’s exact and log-rank tests. We used a multivariate Cox proportional hazard regression model to assess factors that could possibly affect the clinical outcome. Results The CEBPA mutation, including 12 monoallelic and 29 biallelic mutations, was detected in 41 of the 318 AML patients. AML with the CEBPA mutation was frequently observed in NK-AML and in cytogenetically intermediate-risk AML, but rare in favorable- or adverse-risk AML. NK-AML with the CEBPA mutation was closely associated with CD4 negativity as well as CD7 and CD34 positivity in 133 NK-AML cases. Furthermore, CD15 positivity is known to be associated with the CEBPA mutation. Therefore, we investigated the prognostic implications and characteristics of the CD4- CD7+ CD15+ CD34+ immunophenotype in NK-AML in the JALSG AML97 study. NK-AML with the CD4- CD7+ CD15+ CD34+ immunophenotype was classified as the CEBPA type, and NK-AML without this immunophenotype was considered as the non-CEBPA type. Of the 329 NK-AML patients analyzed, 39 and 243 were classified as having CEBPA and non-CEBPA type NK-AML, respectively. NK-AML patients with CEBPA type tended to have better EFS and OS than those with non-CEBPA type (5-year EFS, 48.4% vs 30.6%, P = 0.039; 5-year OS, 67.3% vs 35.7%, P = 0.0019). Multivariate analysis showed that the CEBPA type and white blood cell (WBC) counts of >20 × 109/L were independent prognostic factors for better EFS and OS. Compared to non-CEBPA type NK-AML, CEBPA type was associated with higher MPO-positive rates, frequent presentation with Auer rods, and a FAB classification of M1 or M2. Conclusions NK-AML with the CD4- CD7+ CD15+ CD34+ immunophenotype is a clinically discrete entity, indicating the presence of the CEBPA mutation, which may have a possible role in risk stratification. Disclosures: Taniwaki: Novartis: Honoraria.

It is now evident that the cell cycle machinery has a variety of elements negatively regulating cell cycle progression. However, among these negative regulators in cell cycle control, only 4 have been shown to be consistently involved in the development of human cancers as tumor suppressors: Rb (Retinoblastoma susceptibility protein), p53, and two recently identified cyclin-dependent kinase inhibitors, p16INK4A/MTS1 and p15INK4B/MTS2. Because there are functional interrelations among these negative regulators in the cell cycle machinery, it is particularly interesting to investigate the multiplicity of inactivations of these tumor suppressors in human cancers, including leukemias/lymphomas. To address this point, we examined inactivations of these four genes in primary lymphoid malignancies by Southern blot and polymerase chain reaction-single- strand conformation polymorphism analyses. We also analyzed Rb protein expression by Western blot analysis. The p16INK4A and p15INK4B genes were homozygously deleted in 45 and 42 of 230 lymphoid tumor specimens, respectively. Inactivations of the Rb and p53 genes were 27 of 91 and 9 of 173 specimens, respectively. Forty-one (45.1%) of 91 samples examined for inactivations of all four tumor suppressors had one or more abnormalities of these four tumor-suppressor genes, indicating that dysregulation of cell cycle control is important for tumor development. Statistical analysis of interrelations among impairments of these four genes indicated that inactivations of the individual tumor-suppressor genes might occur almost independently. In some patients, disruptions of multiple tumor-suppressor genes occurred; 4 cases with p16INK4A, p15INK4B, and Rb inactivations; 2 cases with p16INK4A, p15INK4B, and p53 inactivations; and 1 case with Rb and p53 inactivations. It is suggested that disruptions of multiple tumor suppressors in a tumor cell confer an additional growth advantage on the tumor.

The cyclin-dependent kinase 4-inhibitor (CDK41; p16; or MTS1) gene has been proposed as a candidate for a tumor-suppressor gene located in chromosome 9p21, a frequently deleted region in a wide spectrum of human cancers, including leukemias. Recent studies disclosed that it was frequently deleted or mutated in a variety of primary human cancers, including acute lymphoblastic leukemia. The purpose of this study is to figure out the precise manners and frequencies of p16 gene inactivation in diverse hematopoietic tumor types and thus to clarify its significance in development of human hematopoietic malignancies. A total of 410 tumor specimens from patients with primary hematopoietic malignancies were examined for deletions of the p16 gene as well as the neighboring p15 gene and the nearby interferon alpha gene by Southern blot analysis. Tumor-specific mutations or small deletions of the p16 gene were also studied in 74 patients using single-strand conformation polymorphism analysis and direct sequencing. Loss of the p16 gene was most frequently observed among the three genes examined and was found in 59 of the 410 patients: 2 of 134 with acute myelocytic leukemia, 41 of 105 with acute lymphocytic leukemia, 2 of 15 with chronic lymphocytic leukemia, 5 of 14 with adult T-cell leukemia, 4 of 33 with non-Hodgkin's lymphoma, 3 of 8 with mixed-lineage leukemia, and 2 of 61 with chronic myelocytic leukemia. In 16 of the 59 patients, the p16 deletions occurred due to rearrangements within the small region between the p15 exon 2 and the p16 exon 2. Tumor-specific mutations or small deletions of the p16 gene were not detected in the 74 patients examined, including 12 of 14 patients with hemizygous deletions of the gene. Loss of the p16 gene is frequent in and highly specific to lymphoid malignancies (54 of 183 [30%] in lymphoid tumor v2 of 219 [1%] in myeloid tumors; P < .0001). The deletion analyses strongly suggest that the p16 gene is a tumor-suppressor gene located in chromosome 9p21 that is involved in development of human lymphoid tumors. Gene deletions but not minute mutations should be the predominant mechanism of p16 gene inactivation in these types of tumors.

Abstract 2584 Background: Core binding factor (CBF) acute myeloid leukemia (AML) is a specific subgroup of AML, which is commonly related with rearrangements of genes encoding subunits of CBF, AML1 /RUNX1 (AML1-ETO) in t(8;21)(q22;q22) or CBFB (CBFB-MYH11) in inv(16)(p13q22)/t(16;16)(p13;q22), and both clinical outcomes are usually superior in de novo AML. According to literatures, the frequency of patients with t(8;21)(q22;q22) among AML is higher in Japan and Korea than Western countries. However, the accurate incidence and clinical features of CBF-AML are unclear in a large cohort. Patients and methods: We have analyzed 1,536 de novo AML patients from 2 clinical trials of Japan Adult Leukemia Study Group (JALSG, AML97 and AML201 studies, registered from December 1997 to December 2005) and 876 from 3 institutes in Korea (diagnosed from January 2007 to December 2011). Patients from 15 to 64 years old were included in this study, but those with FAB-M3/t(15;17)(q22;q12–21) were excluded. Results: The incidence of CBF-AML was similar in both Japan and Korea (23.3% and 21.2%, P=0.2), however, that of t(8;21) and inv(16) each was a little different between both countries. t(8;21) and inv(16) were observed in 18.6% and 4.9% in Japan, and in 15.0% and 6.3% in Korea, respectively. Of these, the incidence of t(8;21) in Korea was lower than that in Japan (P=0.02), but higher than those reported from Western countries [4.9–8.6% in MRC, CALGB, and SWOG/ECOG except t(15;17), P The prognoses of patients with t(8;21) and inv(16) were similar in complete remission (CR) rate between both countries, but different in 5-year overall survival (OS) and disease-free survival (DFS) rates. CR rate was 91.3% (Japan) and 93.9% (Korea) in t(8;21) (p=0.3), and 98.7% and 100% in inv(16) (P=0.6), respectively. In t(8;21), OS was relatively better in Japan than in Korea (64.6% versus 54.9%, P=0.055), however, DFS tended to be worse in Japan than in Korea (47.6% versus 56.1%, P=0.47). In inv(16), almost the same results with t(8;21) were observed (72.6% versus 65.7% in OS, P=0.2, and 47.7% versus 62.2% in DFS, P=0.06). The white blood cell (WBC) counts and WBC index were prognostic factors in patients with t(8;21), and significantly related to clinical outcome in Japanese patients, however, no significant differences were observed in Korean patients. Patients with t(8;21) having high WBC count (>=10×109/L) were found to have a poor outcome in Japan (44.8% versus 74.5% in OS and 32.4% versus 57.5% in DFS; P Additional chromosome abnormalities found in t(8;21) and inv(16) were similar in both countries. In t(8;21), a sole t(8;21) was observed in 25.5% and 25.2%, a loss of a sex chromosome (-X/-Y) in 60.8% and 60.3%, del(9q) in 8.4% and 6.1%, and other abnormalities including 7q- in 23.8% and 29.0% in Japan and Korea, respectively. However, correlations of additional chromosome abnormalities and clinical outcome were different in each country. In Japan, patients with -X/-Y had relatively favorable OS than those with sole t(8;21) [69.1% versus 57.5%, P=0.08]. However, opposite correlation was observed in Korea [48.1% in -X/-Y versus 59.6% in sole t(8;21), P=0.03], although sole t(8;21) had identical OS in both counties [57.5% versus 59.6%, P=0.4] Conclusions: High incidence of t(8;21) in AML was observed in both countries, however, prognostic significance of various factors, such as WBC counts, WBC index, and additional chromosome abnormalities, were different in each country. One of the reasons for these differences may be dependent on the different treatment protocols in each country, and further analysis of factors associated with prognosis in each country is needed. Disclosures: No relevant conflicts of interest to declare.

Abstract 2574 Background: Mixed phenotype acute leukemia (MPAL) has historically been known as biphenotypic acute leukemia (BAL), and novel diagnostic criteria for this disease entity are described in the World Health Organization (WHO) classification 4th edition. As the most common recurrent genetic abnormality observed in MPAL is the bcr-abl fusion gene, Philadelphia chromosome-positive MPAL (Ph+MPAL) has been recognized as one distinctive disease entity. The prognosis of Ph+B-cell acute lymphoblastic leukemia (Ph+B-ALL) has been dramatically improved with the introduction of imatinib, and the goal of this study was to determine whether imatinib results in a survival benefit in the context of Ph+MPAL. Patients and Methods: We retrospectively analyzed 42 consecutive adult patients who were diagnosed with Ph+AL between January 2001 and March 2012 at Gunma University Hospital and Saiseikai Maebashi Hospital in Gunma, Japan. Ph+AL was diagnosed based on detection of the bcr-abl fusion gene with the polymerase chain reaction method and the presence of more than 20% of blasts in the peripheral blood and/or bone marrow. Patients with a previous history of chronic myelogenous leukemia were excluded. The lineage of leukemia cells was defined according to the WHO classification 4th edition. All patients received intensive chemotherapy and concurrent administration of imatinib. The c2-test was used for comparison of binary variables. The Mann-Whitney U test was used for comparison of continuous variables. Overall survival (OS) rate was estimated by the Kaplan-Meier method and were compared using the log-rank test. P < 0.05 was considered as statistically significant. Results: According to the WHO classification 4th edition, 13 (31%) patients were categorized as Ph+MPAL (positive for both myeloid and B-cell lineage), 27 (64%) patients were categorized as B-cell lineage acute lymphoblastic leukemia (Ph+B-ALL), and two (5%) patients were categorized as acute myeloid leukemia (Ph+AML). Patients with Ph+AML were excluded from this study, as the number of patients was relatively small. Of the 40 Ph+AL patients, 23 patients were men, and 17 were women, and the median age was 53 years (range, 16–75 years). Age, sex, white blood cell counts, lactate dehydrogenase levels, and the prevalence of additional cytogenetic abnormalities at diagnosis were not significantly different when comparing the groups, although patients with Ph+MPAL showed significantly higher frequency of major bcr-abl gene than those with Ph+B-ALL (69% and 19%, respectively; p < 0.01). Immunophenotypic analysis revealed that Ph+MPAL patients expressed CD10 and CD34 with significantly lower frequency than Ph+B-ALL patients. Notably, positivity of myeloid antigens (CD13 and 33) was similar between both groups. The complete response (CR) rates after the initial induction therapy were not significantly different when comparing Ph+MPAL and Ph+B-ALL (100% vs. 85%, respectively, p = 0.14). Likewise, the 5-year-OS rate was similar when comparing patients with Ph+MPAL and Ph+B-ALL (55% vs. 53%, respectively, p = 0.87). Of the 13 patients with Ph+MPAL, six patients received AML-type chemotherapy, and seven patients received ALL-type chemotherapy as the initial induction therapy. All patients achieved CR after the initial induction therapy, and there was no significant difference in 5-year OS according to the therapeutic strategy (AML-type vs. ALL-type), (50% vs. 63%, respectively, p = 0.71). Among 12 patients younger than 65 years old who were alive at >3 months after the diagnosis, eight patients underwent allogeneic hematopoietic stem cell transplantation (allo SCT). The 5-year OS was significantly better for patients who underwent allo-SCT than for those who received chemotherapy alone (58% vs. 20%, respectively, p = 0.05). Conclusion: Among adult Ph+AL patients, mixed phenotype was more frequently observed than expected, and Ph+MPAL patients showed unique clinical features, including immunophenotype and the type of bcr-abl fusion gene. Although BAL has been considered as a negative prognostic factor, Ph+MPAL patients showed comparable prognosis to those with Ph+B-ALL who received imatinib-containing intensive chemotherapy. Therefore, the established theory that mixed phenotype is associated with poor outcomes should be revisited among these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 1464 Background: Retrospective studies have shown that AYA with ALL treated with pediatric protocols have better outcomes than similarly aged patients with adult protocols, but prospective studies comparing AYA with pediatric schedules are scarce. So, we conducted a phase II multicenter study (JALSG ALL202-U) in collaboration with the Japan Association of Childhood Leukemia Study (JACLS) to determine the efficacy and safety of pediatric type therapy (JACLS ALL-02-HR). Methods: From June 2002 to September 2009, patients (age range 16–24 years) with previously untreated ALL (excluding Philadelphia-positive ALL and mature B-cell ALL) were consecutively registered in this study. For patients with t (4; 11), allogeneic stem cell transplantation (HSCT) was recommended during their first complete remission (CR), if donors were available; whereas for patients without t (4; 11), there were no criteria for choosing HSCT. This protocol consisted of induction therapy (pre-phase with oral corticosteroids and 5-drug induction), consolidation phase including 1) consolidation therapy, 2) sanctuary therapy with two cycles of high dose MTX, 3) reinduction therapy, 4) reconsolidation therapy and 2-year maintenance therapy. Intrathecal chemotherapy was administered during each cycle. In comparison with the former JALSG ALL-97 adult protocol, the pediatric-type therapy showed an increase in cumulative dose of CPM (1.5-fold), VCR (1.2-fold), L-asp (18-fold) and MTX (3.7-fold), respectively. As for the intrathecal chemotherapy, the duration was longer (ALL97; 4 to 21 week, ALL202; 1 to 94 week) and the frequency was increased from 8 to 15 times. Results: There were 138 eligible patients: median age was 19 years old and 56% were male. Of the eligible patients, 134 patients (97.1%) achieved CR. The estimated the 4-year OS rate was 74% and 4-year DFS rate was 71%, respectively. Compared with previous JALSG ALL-97 study, CR rate (97.1% ALL202, 84% ALL97; p=0.01), 4-year DFS rate (fig 1, 71% ALL202, 46% ALL97; p=0.0001) and 4-year OS rate (fig 2, 74% ALL202, 46% ALL97; p=0.0002) were significantly higher in this study. During induction therapy, major grade III-IV adverse events (AEs) were FN, DIC, pancreatitis and AST/ALT. The frequency of FN was higher with ALL202-U protocol (43%) than with ALL-97 protocol (12%). During consolidation phase and maintenance therapy, the most common toxicity was FN. The treatment duration during induction therapy and consolidation phase was longer than planned. In comparison with the outcome of JACLS ALL-02-HR protocol, grade 3 to 4 AEs were similar. Conclusion: This pediatric-type therapy was very efficient, yielding a 97.1% CR rate, 71% 4-year DFS and 74% 4-year OS in AYA patients. The protocol was tolerable, but FN and treatment prolongation were observed. Grade 3 to 4 AEs were similar to pediatric group. The data reported here were collected at the time of interim analysis. The latest data including AEs are currently being reviewed and will be disclosed at the presentation. Disclosures: No relevant conflicts of interest to declare.

Abstract 410 Background: ATRA with anthracycline-based chemotherapy markedly improved the prognosis of APL, leading to disease free survival rates 70–80%. But continuous relapse in post-remission period has been a major problem. The role of maintenance therapy for patients in molecular remission after consolidation therapy is uncertain and previous study showed ATRA with or without 6-MP/MTX for 1 to 2 years would be the choice. JALSG APL204 study was intended to decrease the relapse rate in post remission period and applied new synthetic retinoid Tamibarotene (Am80) for maintenance therapy. Am80 is an oral synthetic retinoid with benzoic acid and RARα specific agonist invented in Japan in 1984. High differentiation potential and low affinity to cytoplasmic retinoic acid binding protein may contribute to a superior anti-APL cell effect than ATRA. Early phase II study showed CR rate was 58% for relapsed APL previously treated with ATRA (Blood 90:967–973, 1997). Furthermore Am80 has lower incidence of muco-cutaneous side effects because of no binding affinity to RARγ. Methods: APL204 study was designed as prospective randomized phase III trial comparing Am80 with ATRA in maintenance therapy. Patients (pts) with untreated PML/RARA positive APL between the age of 15 to 69 are eligible. Induction therapy was stratified according to the initial WBC counts: WBC Results: Between April 2004 to December 2010, 347 pts were enrolled and 345 pts were evaluable. Median age was 48 (range 16–68) years old, male : female = 183 : 162 and M3 : M3v = 322 : 23. Median WBC count at diagnosis was 700 (70–127,000)/μl and number of pts in each induction group were A=133, B=56, C=70, D=86 pts. In induction therapy, overall CR rate was 94.5% (326/345) and 95.5%(A), 92.9%(B), 88.6%(C), 98.8%(D) in each group. During consolidation therapy 11 pts (3.4%) were dead, including 9 pts (2.8%) for therapy related toxicity and 2 pts (0.6%) for resistant disease. Two hundred and seventy pts (85.7%) were evaluable for maintenance randomization, 134 pts in Am80 and 136 pts in ATRA. With median follow up of 51 (15–97) months, 5-year RFS was 90.9% (Am80) and 83.2% (ATRA) (P=0.1284), OS 92.4% and 98.1% (P=0.3543) in each group. In group C (WBC ≧10,000/μl), 5-year RFS was 87.7% (Am80) and 59.9% (ATRA) (P=0.0297), it was statistically significant, but in group A and B there was no significant differences. Overall relapse rate was 13.7% (37 pts) after randomization. The most frequent adverse effect of Am80 was a transient hyperglycemia. For all patients in APL204 study, 5-year EFS were 76.6%, RFS 82.9%, OS 87.5%. Conclusions: Maintenance therapy with Am80 has a moderate effect to decrease the relapse rate compared with ATRA but the difference was not statistically significant at 5 years. Both ATRA and Am80 maintenance seem to be effective compared to our previous APL97 study. Notably Am80 is significantly effective in high risk group as WBC ≧10,000/μl (Group C). These results may lead to new strategy for high risk APL. In the future study applying ATO in consolidation therapy and Am80 in maintenance therapy may be a promising strategy to improve the curability and decrease the toxicity of therapy for newly diagnosed APL. This trial was registered at University Hospital Medical Information Network (UMIN)-CTR ID C000000154 in Japan. Disclosures: No relevant conflicts of interest to declare.

Abstract 782 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 both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, 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, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 2511 Background: Although prognosis of acute myeloid leukemia (AML) with t(8;21) is better than other types of AML, outcome of the patients has not been satisfied. Previously, aberrant antigen expression has been reported as risk factor for AML with t(8;21). However, in the reported series, number of cases was not large enough and chemotherapy regimens were variable. We investigated the association of prognosis and several biomarkers including immunophenotype, WBC count, age, and performance status for large number of AML patients with t(8;21) uniformly treated in JALSG AML97 regimen. Patients and Methods: Seven hundred eighty-nine eligible AML patients were evaluated for the multicenter JALSG AML97 study. Adult patients with de novo AML except for APL, ages 15–64 years, were registered consecutively from 103 institutions that participated in JALSG from December 1997 to July 2001. One hundred forty-four patients with AML with t(8;21) were analyzed in this study with a median 1205 days of observation term from diagnosis. Complete remission (CR), relapse-free survival (RFS), and overall survival (OS) rates were analyzed by Fisher's exact test and log-rank test. Factors that would affect clinical outcome were analyzed by multivariate Cox proportional hazard regression model. Results: AML with t(8;21) frequently expressed CD19, CD34, and CD56 compared to other subtypes of AML. CD11b was rarely expressed. Expression of CD19 favorably affected on CR rate (96% in CD19 positive and 87% in negative patients, p20×109/L, CD19 negativity, and CD56 positivity were adverse factors for RFS. CD56 expression was the only independent adverse factor for RFS by multivariate analysis (73.7% in CD56 negative and 48.2% in CD56 positive patients at 3 yrs) although its expression did not affect on OS. There was no difference of age, sex, WBC count, presence or absence of Auer rod, performance status, or CD15 expression between CD56 positive and negative cases. Expression of CD19 was more common in CD56 negative patients (50% in CD56 negative and 30.6% in CD56 positive patients, p Conclusions: We demonstrated that the expression of CD56 was a distinctive adverse factor in a large number of AML patients with t(8;21) treated with JALSG AML97 regimen. CD56 positive AML patients with t(8;21) are possible candidates for hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2697 Adult T cell leukemia (ATL) is an aggressive peripheral T-cell neoplasm highly resistant to conventional therapies. The development of ATL is thought to be initiated by immortalization of T cells by human T cell leukemia virus type I (HTLV-I) infection in early childhood, followed by accumulating genetic hits during a long latency period, which eventually cause neoplastic transformation of T-cells. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL, except for the role of tax protein in T-cell immortalization and alterations of other genes, including TP53, p16 and TCF8. So, in order to understand the genetic basis of ATL, we performed whole genome sequencing of paired-normal DNA from an ATL patient. In total 77 non-silent somatic mutations were detected, among which a TET2 mutation (R1261C) immediately drew our attention. TET2 mutations are found in a wide variety of myeloid malignancies in high frequency and implicated in their pathogenesis. The TET families of proteins are thought to be involved in the epigenetic regulation of gene expression through catalyzing conversion of 5'-methyl cytosine to 5'-hydroxymethyl cytosine, which are supposed to be further converted to unmethylated cytosine. One of the recent interests in TET2 mutations was the recent report of frequent TET2 mutations in peripheral T cell neoplasms, including angioimmunoblastic T-cell lymphomas, peripheral T-cell lymphomas not otherwise specified, as well as other B cell neoplasms, which is in agreement with the observation in TET2-deficient mice, which showed an expansion of bone marrow progenitor pools involving immature lymphoid populations, indicating that deregulated epigenetic machineries could be also involved in the development of mature lymphoid neoplasms. To explore this hypothesis, 144 samples of ATL were screened for mutations in TET2 and other epigenetic regulators commonly mutated in myeloid malignancies, including DNMT3A, IDH1/2 and spliceosome genes, using target deep sequencing. We found 16 TET2 mutations in 13 out of the 144 ATL cases (9%) together with other mutations, which was similar to the frequencies reported in other peripheral T cell neoplasms. Deep sequencing allowed for accurate estimation of allelic burden of these mutations. In most cases, TET2 mutations were found in the major tumor populations, indicating their early origin during clonal evolution. In addition, DNMT3A (2%), IDH1/2 (1%), SF3B1 (2%), ZRSR2 (1%) were also mutated, although at lower frequencies. In conclusion, we found TET2 mutation through whole genome sequencing. TET2 mutations were relatively common in ATL. Together with other mutations frequently found in myeloid malignancies, our finding provided an intriguing insight into the role of deregulated DNA methylation in the pathogenesis of ATL and also suggested that a common mechanism may underlie between the two very different blood cancers. Disclosures: No relevant conflicts of interest to declare.

Abstract 2036 BACKGROUND: There are limited numbers of studies focusing on the role of hematopoietic stem cell transplantation (HSCT) as a salvage treatment for acute promyelocytic leukemia (APL) after relapse in the all-trans-retinoic acid (ATRA) era. We retrospectively analyzed the outcomes of APL patients who underwent allogeneic or autologous HSCT during second complete remission (CR2) and compared them with those in APL patients who did not receive HSCT. PATIENTS & METHODS: A total of 302 adult patients with previously untreated de novo APL were registered in the Japan Adult Leukemia Study Group (JALSG) APL97 study between 1997 and 2002, and treated with ATRA and chemotherapy (Asou et al., 2007). Of 283 assessable patients with t(15;17) and/or PML-RARA, 267 (94.3%) achieved CR and 67 (23.7%) relapsed. Except for 2 patients unable to complete the follow-up survey, 65 relapsed patients received salvage treatment with ATRA alone (n=17), ATRA plus chemotherapy (n=33), chemotherapy alone (n=6), tamibarotene alone (n=7), allogeneic HSCT (n=1), unknown (n=1), and 58 of these patients (89%) achieved CR2. Of the patients who achieved CR2, 27 received HSCT (21 allogeneic and 6 autologous) during CR2, 30 did not receive HSCT, and 1 was not assessable. All 6 patients who underwent autologous HSCT were confirmed of having achieved molecular CR. We compared the survival rates of patients with CR2 achievement who received HSCT (HSCT group) or did not receive HSCT (no-HSCT group). Survival was calculated from the date of CR2 to death or last visit. Probabilities of survival were estimated using the Kaplan-Meier method and compared by the log-rank test. RESULTS: Median age at first relapse was significantly lower in the HSCT group (36 years; range: 22 – 60) than in the no-HSCT group (53 years; range: 16 – 72) (P = 0.006). Median first CR duration was 21.6 months (range: 5.5 – 80.8) in the HSCT group and 17.6 months (range: 5.9 – 90.5) in the no-HSCT group (P = 0.75). Among patients in the no-HSCT group, 6 underwent allogeneic (n = 4) or autologous (n = 2) HSCT during CR3 or more. Five-year survival rate in the HSCT and no-HSCT groups were 70.4% and 77.4%, respectively (P = 0.86). Six patients died within 5 years of CR2 in the HSCT group. The causes of death included complications of allogeneic HSCT (n = 4) and relapse after HSCT (n = 2; 1 autologous, 1 allogeneic). In contrast, 7 patients in the no-HSCT group died of complications of allogeneic HSCT after second relapse or later (n = 4), disease progression (n = 2), and ischemic heart disease (n = 1). Among younger patients (aged ≤39 years), there was no statistically significant difference in 5-year survival between the HSCT group (100.0%) and the no-HSCT group (82.5%) (P = 0.10). In contrast, among older patients (aged ≥40 years), 5-year survival rate was significantly higher in the no-HSCT group (78.0%) than in the HSCT group (40.5%) (P = 0.04) (using the time-dependent covariates). Within the HSCT group, 5-year survival rate was significantly better in younger patients (n = 15) (100.0%) than in older patients (n = 12) (50.0%) (P = 0.006). Among the 27 patients in the HSCT group, there was no significant difference in 5-year survival between patients who underwent allogeneic HSCT (76.2%) and those who underwent autologous HSCT (83.3%) (P = 0.69). However, the 5-year cumulative incidence of relapse was significantly higher in patients who underwent autologous HSCT (58.3%) than those who underwent allogeneic HSCT (11.3%) (P = 0.01). After second relapse, 3 of 4 patients who underwent autologous HSCT at CR2 achieved CR3 through treatment with arsenic trioxide (ATO), tamibarotene or high-dose cytarabine. CONCLUSIONS: This study indicates that allogeneic HSCT is recommended in younger APL patients (aged ≤39 years) during CR2, while autologous HSCT is accompanied by frequent relapses. Our retrospective study also revealed that outcomes were significantly better in elderly patients who did not receive HSCT than those who did during CR2. This may result from a high HSCT-related mortality rate among elderly patients, and suggests a need for appropriate salvage treatment after relapse. We can now use ATO for salvage therapy routinely, allowing for further improvement of relapsing APL outcomes, even for patients not eligible for HSCT. Disclosures: Takeshita: Takeda: Research Funding; Novaltis: Research Funding. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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 ( 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.