Abstract: 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.

Abstract: der(1;7)(q10;p10) is a recurrent chromosomal abnormality found in a wide variety of myeloid neoplasms observed in as high as 6% of myelodysplastic syndromes (MDS) in Asian populations, while rarely observed in Caucasian populations. It is thought to be generated by a recombination between two highly homologous centromere alphoid sequences which lead to an unbalanced abnormality of monosomy of 7q and trisomy of 1q. However, despite the presence of -7q, der(1;7) has been associated with a better prognosis compared to monosomy 7 or other del(7q) (-7/del(7q)). In addition to its association with +8 and del(20q), frequent RUNX1 mutations and a paucity of mutated TP53 have been reported in der(1;7) tumors, but otherwise, the molecular features of this abnormality have been poorly characterized in the literature. This is most likely because it is very rare in Caucasians, even though it represents one of the most prevalent lesions among Asian populations. The purpose of our study is to clarify the frequency and mutational landscape of der(1;7) in myeloid neoplasms on the basis of targeted-capture sequencing. A total of 1,707 MDS cases, including 944 German and 763 Japanese cases, were enrolled, from which we identified 73 (4.0%) cases with der(1;7). The prevalence was 〉 20 times higher in Japanese (9.0%) than German (0.43%) cohorts (p 〈 0.0001). We also identified a strong male predominance in der(1;7)-positive cases (90.4%) compared to negative cases. Also including an additional 22 cases, somatic mutations and copy number abnormalities in der(1;7) were interrogated in a total of 95 cases, which included 84 (88.4%) with MDS, 9 (9.5%) with AML, and 2 (2.1%) with MPN. Among MDS patients, 29 were low-risk, 47 were high-risk, and the rest were not specified. In mutation analysis, at least one mutation was detected in 98% of der(1;7) cases, most frequently affecting RUNX1 (42%), followed by EZH2 (26%), and ETNK1 (25%). Copy number analysis showed a high frequency of del(20q) and trisomy 8 in der(1;7) cases: 27.4% and 18.9% respectively. On the basis of mutant cell fractions, most of these mutations were present in subclones acquired within the major population harboring der(1;7). In particular, most of the EZH2 (7q35-q36) mutations were thought to be secondary events in der(1;7)-positive cases, while representing initial events acquired before UPD(7q) or -7/del(7q) in der(1;7)-negative cases. Of interest, der(1;7) was associated with a low frequency of TP53 mutations, which were seen only in 3% of cases with der(1;7), whereas highly prevalent in non-der(1;7) cases with -7/del(7q) (52%), which is concordant with a better clinical outcome was observed in der(1;7) cases compared with non-der(1;7) cases with monosomy 7 or other del(7q). Another unique feature of der(1;7) positive MDS was an extremely high frequency of RUNX1 mutations. However, the most prominent finding with secondary mutations in der(1;7) cases is the frequent hot spot mutation in ETNK1, which were originally reported in 8.8% of myeloid neoplasms with MPN features, like SETBP1 mutations. ENTK1 mutations were found in as many as 25% (23/95) of der(1;7) cases, while rarely seen in -7/del(7q) (1/89) (p 〈 0.0001) or amp(1q) (2/68) (p=0.0001). Despite the high frequency of trisomy 8 observed in der(1;7) cases, none were associated with ETNK1 mutations. In addition, all of the RAS pathway mutations (positive in 16 cases) were observed in der(1;7) cases with wild-type ETNK1, while none were in ETNK1-mutant cases. Morphologically, these ETNK1-mutated der(1;7) cases presented with an increased eosinophil count in peripheral blood (760.9/ul vs. 78.1/ul) (p 〈 0.001), compared to those without EKNK1 mutations, suggesting that ENTK1-mutated der(1;7) cases represent a novel disease entity within der(1;7), characterized by unique genetic features and increased eosinophils. In conclusion, der(1;7) is a genetically and clinically distinct subset of myeloid neoplasms, which showed unique features that are distinct from MDS cases in -7 and other del(7q). Especially, ETNK1 mutations subdivided cases with der(1;7) into two groups of genetically distinct subsets as shown in Figure 1. In the future, inhibition of the kinase activity in ETNK1 could be a novel therapeutic strategy in such a previously unrecognized subset as characterized by der(1;7) and eosinophilia. Figure 1 Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Handa:Ono: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Qiagen Corporation: Patents & Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership.

Abstract: 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.

Abstract: 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 ( & lt;50%) of erythroid cells in bone marrow were enrolled. Publicly available information on clinical and genetic findings was collected from previous publications, including The Cancer Genome Atlas and BeatAML datasets. In total, 1,012 cases with AML were enrolled in this study. The genetic profile in PEL and MEL were also compared with that of NEL. Finally, we recognized biological significance of the molecular defects by functional analysis of xenograft model, and showed novel therapeutic possibilities targeting pathogenic pathways. 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 & lt;0.05). Compared to STAG2-mutated cases in NEL, those in MEL showed significantly higher frequency of KMT2A-PTD. Group C (n=16, 15%) was defined by NPM1 mutations frequently accompanied by PTPN11 mutations (50% in Group C). This combination was also distinct in MEL and was significantly more frequent than in NPM1-mutated NEL cohort (P & lt;0.05). In Group D (n=29, 28%) without any TP53, STAG2, or NPM1 mutations (triple-negative), RUNX1, ASXL1, BCOR, PHF6, and U2AF1 mutations were enriched compared to triple-negative NEL. In addition, novel loss-of-function USP9X mutations were exclusively identified in Group D AEL (P & lt;0.05, compared to triple-negative NEL), which has been implicated in erythroid leukemogenesis through the TGF beta pathway. Serial sample sequencing analysis revealed that JAK2 mutation positive myeloproliferative neoplasms evolved into PEL. Clinically, patients in Group A showed shorter overall survival time compared to those in any other group. To further search for a novel therapeutic target to the most severe type of AEL, JAK2 inhibitor was administered to xenograft models (from 2 cases) having JAK2 and EPOR amplifications and TP53 mutations. Engrafted cells showed in vitro activation of STAT5 and proliferative capacity enhanced with erythropoietin. In vivo analysis showed that inhibition of JAK/STAT pathway significantly suppressed cell growth and prolonged overall survival. 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.

Abstract: 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.

Abstract: Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

Abstract: Background In patients with Myelodysplastic Syndromes (MDS), TP53 mutations associate with high-risk presentation, complex karyotype, acute myeloid leukemia (AML) progression and poor response to hematopoietic stem cell transplantation. These associations highlight the relevance of TP53 as a prognostic and predictive biomarker. Consistent with its role as a tumor suppressor, bi-allelic targeting of the TP53 locus is a frequent but not an obligatory event. Despite the central role of TP53 in MDS, the clinical implications of TP53 mutations in the context of allelic state have not been extensively studied. Methods Under the auspices of the International Working Group for Prognosis in MDS, we sequenced a representative cohort of 3,324 peri-diagnosis MDS patients on a next generation sequencing (NGS) panel optimized for myeloid disease. Conventional G-banding analysis (CBA) was available for 2,931 patients. Focal (~3MB) gains and deletions and regions of NGS-derived copy-neutral loss of heterozygosity (cnLOH) were assessed using an in-house algorithm CNACS. Putative oncogenic mutations in TP53 were characterized by consideration of normal controls and established population databases. A validation cohort of 1,120 samples with independent but comparable molecular and clinical annotation was sourced from a compendium of Japanese MDS data to include JALSG-MDS212, JMDP registry, and regional registries. Results NGS-derived ploidy alterations and CBA show a high genome-wide concordance. From NGS profiles, 11% of patients (n=360) are subject to cnLOH, of which 80 target the TP53 locus. We characterize 490 TP53 mutations in 380 patients, representing 11% of the cohort. Amongst those patients, 22% (n=85) and 21% (n=78) have a deletion or a cnLOH involving the TP53 locus, respectively. Taken together, these segregate patients into two TP53 states: a mono-allelic state where one wild type allele remains (33% of TP53 mutated patients, n=126); and a multi-hit state where TP53 is altered multiple times by either mutations, deletions or cnLOH (67% of TP53 mutated patients, n=254). We find that TP53 state shapes clinical presentation and outcomes. Mono-allelic TP53 patients present with more favorable disease than multi-hit TP53 patients: they are less cytopenic, have lower bone marrow blasts (median 4 vs. 9%, p & lt;0.0001) and are enriched in low risk WHO subtypes. We show that the established association between mutated TP53 and complex karyotype is specific to the multi-hit TP53 state (OR=66, CI: 33-141, p & lt;0.0001). Critically, we show that multi-hit TP53 associates with worse overall survival as compared to mono-allelic TP53 (HR=3.7, CI: 2.7-5.1, p & lt;0.0001; Figure 1a) and more pronounced AML transformation (HR=5.3, CI: 3.1-8.9, p & lt;0.0001; Figure 1b). Patients with mono-allelic TP53 mutations have a similar survival to that of wild type TP53 patients and track overall IPSS-R, whereas multi-hit TP53 stratifies adverse prognostic subgroups independent of the IPSS-R. We formally test this using multivariate models that consider age, peripheral blood counts, blasts and IPSS-R cytogenetic score and show that multi-hit TP53 state is an independent prognostic factor for overall survival and AML transformation, whilst mono-allelic TP53 state is not significant. We also observe a significant difference in overall survival between TP53 states in the context of therapy-related MDS (HR=3.1, CI: 1.2-7.9, p=0.03). Last, analyses of 12 serial samples identify multi-hit targeting of the TP53 locus as a critical driver of AML transformation in the context of TP53-mutated MDS. These findings are replicated in the validation cohort. Conclusions TP53 is a natural candidate for incorporation in molecularly informed risk stratification schemas (molecular IPSS-R). We show that TP53 state rather than mutation alone is an independent diagnostic and prognostic biomarker in MDS. We propose that ascertainment of TP53 state is critical in prospective clinical sequencing for risk estimation, disease monitoring and future correlative research into predictors of response to established and investigational therapies. Disclosures Bernard: Celgene: Research Funding. Hasserjian:Jazz Pharmaceuticals: Consultancy; Promedior, Inc.: Consultancy. Germing:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria. Cargo:Celgene: Research Funding. Santini:Acceleron: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kotsianidis:Celgene: Research Funding. 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. Savona:Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding. Ades:Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Stevenson:Celgene: Research Funding. Fenaux:Jazz: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Aprea: Research Funding; Celgene Corporation: Honoraria, Research Funding. Platzbecker:Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Heuser:Synimmune: Research Funding; Bayer Pharma AG, Berlin: Research Funding. Valent:Blueprint: Research Funding; Pfizer: Honoraria; Celgene: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Deciphera: Honoraria, Research Funding. Miyazaki:Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Otsuka: Honoraria; Chugai: Research Funding; Novartis: Honoraria; Kyowa-Kirin: Honoraria. Finelli:Novartis: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Gattermann:Novartis: Honoraria; Takeda: Research Funding; Alexion: Research Funding. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Bejar:Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Greenberg:Notable Labs: Research Funding; Celgene: Research Funding; Genentech: Research Funding; H3 Biotech: Research Funding; Aprea: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Ogawa:Qiagen Corporation: Patents & Royalties; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; RegCell Corporation: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership. Papaemmanuil:Celgene: Research Funding.

Abstract: Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.