Abstract: DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the 〉 480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with 〉 10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing 〉 20% difference in mean methylation levels between responders and non-responders (q 〈 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

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

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.

Abstract: DDX41 was identified as a causative gene for late-onset familial myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While DDX41 is thought to be one of the most frequent targets of germline mutations responsible for sporadic cases with AML/MDS and other myeloid neoplasms, the entire spectrum of pathogenic DDX41 variants and their effect size therein are still to be elucidated, and so was the clinical picture of DDX41-mutated myeloid neoplasms. In this study, through an international collaboration, we investigated DDX41 variants in a total of 5,609 sporadic cases with different myeloid neoplasms from different ethnicities, using next generation sequencing. Mutations in the major driver genes commonly mutated in AML/MDS were also examined. Frequencies of germline DDX41 variants were compared between sporadic cases with myeloid neoplasms and healthy individuals (n=13,906). We also characterized genetic/clinical features of DDX41-mutated myeloid neoplasms. We identified a total of 208 (3.6%) patients with DDX41 variants, of whom approximately 50% had both germline and somatic mutations, whereas 37% and 13% had either germline or somatic mutations alone, respectively. Somatic mutations were found in 58% of patients with germline mutation, which was significantly higher than those without (0.21%) (P & lt;0.0001). No somatic mutation was identified in healthy individuals. Among 174 germline variants, truncating and missense mutations were found in 93 and 81 cases, respectively, whereas only 1.9% of somatic mutations were truncating (P & lt;0.0001). Among 21 cases with somatic mutations alone, 4 had multiple somatic mutations and an additional 4 had loss-of-heterozygosity of the DDX41 locus (5q35.3), including 3 with uniparental disomy and 1 with deletion. Thus, 8 out of 21 cases with somatic mutation alone were suspected to have biallelic DDX41 mutations. Germline DDX41 variants showed a conspicuous ethnic diversity; the most frequent germline variants were A500fs in Japan, D140fs in USA, Q41* in Germany, G218D in Italy, M1I in Sweden, S21fs in Thailand. The M1I variant was also seen in other European countries, but not in Japan or Thailand, while no A500fs mutation was found in Europe. Among the Japanese population, significant enrichment in myeloid neoplasms was observed not only for truncating variants, such as A500fs (odds ratio (OR)=12.1) and E7X (OR=11.0) but also for missense variants, including Y259C (OR 14.3) and E256K (OR 7.81), frequently accompanied by a somatic DDX41 mutation (Figure 1). Patients with germline and/or somatic DDX41 variants were significantly older than those without (P=0.00076) and more prevalent in male than female (OR=3.14; P & lt;0.0001). DDX41 variants were significantly more frequent in MDS (4.7%) and AML (2.9%), compared with other myeloid neoplasms (0.58%). Among AML, mutations were more frequent in AML with myelodysplasia-related changes (P & lt;0.00001). Patients with MDS having both germline and somatic mutations were more likely to classified in refractory anemia with excess blasts (RAEB), compared with those with germline or somatic alone (P=0.029). DDX41 variants were significantly associated with lower WBC and granulocyte counts. Most frequently co-occurring mutations included those in ASXL1, SRSF2, TET2, CUX1, and DNMT3A, of which only CUX1 mutations were statistically significant. Overall, no difference was observed in overall survival (OS) between DDX41-mutated and unmutated cases. However, among RAEB cases, DDX41 variants were associated with a significantly longer OS (P=0.0039). In summary, the majority of DDX41-mutated cases had a germline variant, although a minority had somatic mutations alone. Pathogenic DDX41 alleles have a large ethnic diversity, where not only truncating variants but also missense variants are associated with an increased risk of the development of myeloid neoplasms. Disclosures Kanda: Chugai Pharma: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mundipharma: Honoraria; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria; Alexion Pharmaceuticals: Honoraria; Shire: Honoraria; Mochida Pharmaceutical: Honoraria; Daiichi Sankyo: Honoraria; Shionogi: Research Funding; Meiji Seika Kaisha: Honoraria; Sanofi: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding. Miyazaki:NIPPON SHINYAKU CO.,LTD.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Ogawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: 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.