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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.

Context Pheochromocytoma and paraganglioma (PPGL) may appear as a complication of cyanotic congenital heart disease (CCHD-PPGL) with frequent EPAS1 mutations, suggesting a close link between EPAS1 mutations and tissue hypoxia in CCHD-PPGL pathogenesis. Objective Our aim is to further investigate the role of EPAS1 mutations in the hypoxia-driven mechanism of CCHD-PPGL pathogenesis, particularly focusing on metachronous and/or multifocal CCHD-PPGL tumors. Methods We performed whole-exome sequencing (WES) for somatic and germline mutations in 15 PPGL samples from 7 CCHD patients, including 3 patients with metachronous and/or multifocal tumors, together with an adrenal medullary hyperplasia (AMH) sample. Results We detected EPAS1 mutations in 15 out of 16 PPGL/AMH samples from 7 cases. Conspicuously, all EPAS1 mutations in each of 3 cases with multifocal or metachronous tumors were mutually independent and typical examples of parallel evolution, which is suggestive of strong positive selection of EPAS1-mutated clones. Compared to 165 The Cancer Genome Atlas non–CCHD-PPGL samples, CCHD-PPGL/AMH samples were enriched for 11p deletions (13/16) and 2p amplifications (4/16). Of particular note, the multiple metachronous PPGL tumors with additional copy number abnormalities developed 18 to 23 years after the resolution of hypoxemia, suggesting that CCHD-induced hypoxic environments are critical for positive selection of EPAS1 mutants in early life, but may no longer be required for development of PPGL in later life. Conclusion Our results highlight a key role of activated hypoxia-inducible factor 2α due to mutated EPAS1 in positive selection under hypoxic environments, although hypoxemia itself may not necessarily be required for the EPAS1-mutated clones to progress to PPGL.

Intravascular large B-cell lymphoma (IVLBCL) is a unique type of extranodal lymphoma characterized by selective growth of tumor cells in small vessels without lymphadenopathy. Greater understanding of the molecular pathogenesis of IVLBCL is hampered by the paucity of lymphoma cells in biopsy specimens, creating a limitation in obtaining sufficient tumor materials. To uncover the genetic landscape of IVLBCL, we performed whole-exome sequencing (WES) of 21 patients with IVLBCL using plasma-derived cell-free DNA (cfDNA) (n = 18), patient-derived xenograft tumors (n = 4), and tumor DNA from bone marrow (BM) mononuclear cells (n = 2). The concentration of cfDNA in IVLBCL was significantly higher than that in diffuse large B-cell lymphoma (DLBCL) (P < .0001) and healthy donors (P = .0053), allowing us to perform WES; most mutations detected in BM tumor DNA were successfully captured in cfDNA and xenograft. IVLBCL showed a high frequency of genetic lesions characteristic of activated B-cell–type DLBCL, with the former showing conspicuously higher frequencies (compared with nodal DLBCL) of mutations in MYD88 (57%), CD79B (67%), SETD1B (57%), and HLA-B (57%). We also found that 8 IVLBCL (38%) harbored rearrangements of programmed cell death 1 ligand 1 and 2 (PD-L1/PD-L2) involving the 3′ untranslated region; such rearrangements are implicated in immune evasion via PD-L1/PD-L2 overexpression. Our data demonstrate the utility of cfDNA and imply important roles for immune evasion in IVLBCL pathogenesis and PD-1/PD-L1/PD-L2 blockade in therapeutics for IVLBCL.

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.

Introduction Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment (TIE) has become a focus of intense research; however, there are few reports on the dynamics of the TIE in leukemia, especially in bone marrow (BM), the primary site of leukemia. Mass cytometry, which allows high-dimensional analysis with single-cell resolution, is a powerful tool for the characterization of the TIE, and enables us to examine pediatric BM samples containing only a few numbers of immune cells. Methods Primary and recurrent BM samples were collected serially from pediatric patients with BCP-ALL at Kyoto University Hospital from 2006 to 2013. Mononuclear cells were isolated by density centrifugation and viably preserved until they were used. We examined the TIE of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) by analyzing serial BM samples from patients in primary and recurrent disease phases by mass cytometry, using 39 immunophenotype markers, and transcriptome analysis. Results and Discussion The proportion of BCP-ALL cells in the samples was 87.3-97.8% (mean: 94.0%) at onset, and 81.3-98.8% (mean: 92.2%) at relapse. High-dimensional single-cell analysis by mass cytometry elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that the TIE during relapse comprised a T helper 1 (Th1)-polarized immune profile, together with the increase of effector regulatory T cells (Tregs). Th1 cells are typically known as supporters of cytotoxic T lymphocytes which would eliminate leukemia cells and work against relapse, but recently, it is reported that Th1 cells directly support ALL proliferation in vitro (Traxel et al. Oncogene. 2019; 38:2420-2431), and that the concentrations of pro-inflammatory cytokines and Th1 cytokines (IFN-γ and IL-12) were elevated in patients with ALL, which could create favorable conditions for ALL (Perez-Figueroa et al. Oncol Rep. 2016; 35: 2699-2706, Vilchis-Ordonez et al. Biomed Res Int. 2015; 2015: 386165). Therefore, there is a possibility that the Th1-polarized immune profile would work in favor of leukemia survival for relapse. Furthermore, Gene set enrichment analysis based on RNA expression identified the enrichment of six immune-related pathways were enriched at the time of relapse; chemokine activity, chemokine production, complement activation, positive regulation of cytokine production involved in immune response, positive regulation of lymphocyte chemotaxis, and positive regulation of lymphocyte migration. These expression signatures suggest that BCP-ALL cells attract lymphocytes, and upregulate immune activities at relapse. Conclusion In summary, a TIE characterized by a Th1-polarized immune profile, with the increase of effector Tregs, may be involved in the pathophysiology of recurrent ALL, and BCP-ALL cells at relapse were enriched with gene expressions related to lymphocyte attraction and activities. This information could contribute to the development of effective immunotherapeutic approaches against BCP-ALL relapse. Disclosures Ogawa: Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

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.

MLL(KMT2A) rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML). MLL fusion patterns are associated with patient prognosis; however, little is known about the distribution and prognostic significance of driver mutations according to specific MLL fusion partner genes. In this study, we conducted sequence analysis of 338 genes in pediatric patients with MLL-rearranged (MLL-r) AML (n = 56; JPLSG AML-05 study) alongside data from TARGET cohort pediatric MLL-r AML (n = 104) and non-MLL-r AML (n = 581). In addition, mutation data and clinical information from adult MLL-r AML patients (n=81), collected at the MLL Munich Leukemia Laboratory, were also analyzed. The frequencies of driver mutations differed according to MLL fusion partner genes in pediatric MLL-r AML patients (JPLSG AML-05 study + TARGET cohort, n = 160). FLT3 mutations or internal tandem duplications were more frequent in patients with MLL-MLLT3 (27/63, 42.9%) and MLL-MLLT1 (4/11, 36.4%). RAS pathway genes were more frequently mutated in specific groups, such as KRAS in those with MLL-MLLT10 (17/37, 45.9%), MLL-MLLT4(AFDN) (5/12, 41.7%), MLL-MLLT1 (3/11, 27.3%), and other (4/14, 28.6%) fusions, and NRAS in those with MLL-ELL (10/23, 43.5%) and MLL-MLLT4 (4/12, 33.3%) fusions. Other pathway mutations also coexisted with specific partner genes; SETD2 mutations were frequent in patients with MLL-MLLT4 fusions (4/12, 33.3%), and STAG2 mutations were frequent in those with MLL-ELL (6/23, 26.1%). Next, we examined the prognostic significance of each driver mutation. In pediatric MLL-r AML patients (n = 160), among eight frequently mutated genes (≥ 5%: ≥ 8 patients with mutations) and one copy number change (trisomy 8), only KRAS mutations were significantly associated with adverse prognoses, in terms of both event-free survival (EFS) and overall survival (OS). Compared with patients without KRAS mutations (n = 118, KRAS-WT), those with KRAS mutations (n = 42, KRAS-MT) had significantly inferior prognoses (KRAS-WT vs. KRAS-MT; 5 year (5y)-EFS: 51.8% vs 18.3%, P < 0.0001; 5y-OS: 67.3% vs 44.3%, P = 0.003). The adverse prognostic impact of KRAS mutation was confirmed in adult MLL-r AML. By contrast, there were no significant differences in EFS or OS (KRAS-WT vs. KRAS-MT; 5y-EFS: 50.6% vs 54.5%, P = 0.55; 5y-OS: 67.1% vs 64.9%, P = 0.84) between patients with KRAS-WT (n = 533) and KRAS-MT (n = 48) non-MLL-r AML. We also analyzed the prognostic significance of KRAS mutations according to MLL fusion partner. First, the prognoses of patients were compared for each fusion partner gene. Consistent with a previous report, patients with MLL-MLLT10 (n = 37), MLL-MLLT4 (n = 12), and MLL-MLLT1 (n = 11) had poor prognoses compared to those with other fusion types. Therefore, we dichotomized patients into high- (MLLT10 + MLLT4 + MLLT1, n = 60) and intermediate/low- (MLLT3 + ELL + Others, n = 100) risk groups. The frequency of KRAS mutation was significantly elevated in the high-risk group (26/60, 43.3%) relative to the intermediate/low-risk group (16/100, 16.0%; P = 0.0002). Further, in the high-risk group, KRAS-MT patients (n = 26) had significantly adverse prognoses compared with KRAS-WT patients (n = 34; KRAS-WT vs. KRAS-MT; 5y-EFS: 31.5% vs 10.3%, P = 0.007; 5y-OS: 52.4% vs 40.5%, P = 0.16). Moreover, KRAS-MT patients in the intermediate/low-risk group (n = 16) had significantly adverse prognoses compared with KRAS-WT patients (n = 84; KRAS-WT vs. KRAS-MT; 5y-EFS: 60.3% vs 31.3%, P = 0.02; 5y-OS: 73.4% vs 50.0%, P = 0.04). Finally, we investigated whether KRAS mutation is an independent prognostic factor in patients with pediatric MLL-r AML. Multivariate Cox regression analysis was performed including the following variables: age, WBC, MLL fusion gene, driver mutations, and trisomy 8. The results indicated that KRAS mutation was the only prognostic factor predicting both poor EFS (hazard ratio (HR), 2.21; 95% confidence interval (CI), 1.35-3.59; P = 0.002) and poor OS (HR, 1.85; 95% CI, 1.01-3.31; P = 0.045), whereas there were no statistically significant findings for MLL fusions. These results suggest that mutations in MLL-rearranged AML are associated with MLL fusion partners, and KRAS mutations frequently coexist with high-risk MLL fusions. KRAS mutations are distinct adverse prognostic factors in MLL-r AML, regardless of risk subgroup, therefore, potentially useful for accurate treatment stratification. Disclosures Ogawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene. Despite the use of tyrosine kinase inhibitors (TKIs), a minority of chronic phase (CP) CML patients fail TKI therapies and progress to blast crisis (BC), showing dismal outcomes. Although genetic studies have revealed that CML-BC frequently carries not only ABL1 mutations but other driver mutations, our knowledge about the mechanism of TKI resistance and progression to BC is still limited by a relatively small number of patients and/or genes analyzed in each study. Moreover, it remains elusive whether mutations can predict clinical outcomes of BC patients, in which few biomarkers are known. Here, we investigated a large cohort of CML patients to reveal the landscape of genetic lesions and those predicting clinical outcomes in CML. Methods We performed whole-exome sequencing (WES) of paired CP and BC samples from 52 patients and targeted-capture sequencing that covered myeloid driver genes in 32 BC and 19 CP samples. Combined with public WES data for 24 BC and 77 CP, we analyzed a total of 108 BC and 148 CP samples. Results In WES analysis of paired CP and BC samples, an average of 5.3 nonsynonymous mutations were acquired during disease progression from CP to BC. Notably, a Poisson regression model revealed that the number of acquired mutations was positively correlated with time to progression from CP to BC (P < 0.001) and negatively with TKI therapy after CP diagnosis (P = 0.0093), although the correlation of the number of driver mutations in CML-BC with time to progression was not clear. These results suggest that the use of TKI effectively reduces the size of tumor populations at risk for clonal evolution by acquiring random mutations, by which prevents BC. In CML-BC, we found frequent mutations not only in known mutational targets in other hematological malignancies, such as RUNX1, ABL1, ASXL1, BCOR/BCORL1, TP53, and WT1, but also in other genes recently reported in BC (UBE2A and SETD1B) and previously unreported mutational targets in cancer (KLC2 and NBEAL2). Deep amplicon-sequencing revealed that ASXL1 mutations were already present at the time of CP diagnosis in most cases, whereas others such as RUNX1, ABL1, and TP53 mutations were absent in CP and newly emerged during progression to BC. Some abnormalities, such as +21, +8, and ASXL1 mutations, were more enriched in myeloid than lymphoid crisis, while others, including CDKN2A/B and IKZF1 deletions, -7/del(7p), -9/del(9p), and ABL1 mutations, vice versa. By contrast, abnormalities such as RUNX1 mutations and double Ph were almost equally observed in both crises. In univariate analysis of clinical factors for overall survival (OS) in 77 CML-BC cases for whom survival information was available, TKI-containing therapy for BC was significantly associated with a better OS, whereas genetic lesions including ASXL1 and TP53 mutations, del(17p), amp(17q), +19, and +21 had a negative impact on OS. Conspicuously, patients with TP53 mutations, del(17p), and amp(17q) showed an especially dismal outcome. We then performed a multivariate analysis using a Cox proportional hazard regression model, focusing on 36 TKI-treated patients, because TKI-containing therapy has been shown to improve OS and therefore, is a standard choice of therapy. We found that ASXL1 and BCOR mutations, complex copy-number alterations, amp(17q), and +21 were independent predictors for worse prognosis. Based on the number of these unfavorable factors, patients were classified into three subgroups showing distinct prognosis, where the 2-year OS rate was 71.8%, 15.6%, and 0% for patients with 0, 1, and ≥2 risk factors, respectively (P < 0.001). Finally, we explored the genetic abnormalities and clinical outcomes in CML-CP. In CP, only ASXL1 was mutated at a frequency comparable to that in BC, while others, including TET2, KMT2D, PTPN11, RUNX1, and WT1, were mutated at much lower frequencies. Of interest, patients who later developed BC more frequently had at least one genetic abnormality, suggesting that mutations found at the time of CP might play a role in driving CML cells to BC under the pressure of TKI treatment. Conclusion Our study clarified a comprehensive registry of genetic lesions in BC in a large cohort of CML patients and their prognostic impact, which should provide a clue to the development of better therapy/management for patients with CML. Disclosures Takaori-Kondo: Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; Eisai: Research Funding; Nippon Shinyaku: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; MSD: Honoraria. Mitani:CHUGAI: Research Funding; Takeda: Research Funding; KYOWA KIRIN: Consultancy, Research Funding. Ogawa:Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: 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.

Background DNA hypomethylating agents (HMAs), including azacytidine (AZA) have been established as key drugs for higher-risk myelodysplastic syndromes (MDS). We and others have explored the role of mutation profile before AZA administration on predicting outcomes. Actually, we have previously identified mutated-TP53 as a marker associated with higher rate of achieving complete remission (CR). In addition, mutations in TP53 and DDX41 predicted reduced and prolonged survival after treatment, respectively. However, the clinical significance of evaluating clone size changes early after treatment has not been determined. In this study, we explored the role of post-treatment clone size in predicting outcomes of AZA treatment for MDS and related diseases. Methods We enrolled 290 AZA-treated cases, including 88 from a Japanese prospective study (JALSG MDS-212 trial), 149 from Karolinska Institute, and 53 from a retrospectively collected Japanese cases. The diagnoses were MDS (n=242), MDS/MPN (n=25), and AML-MRC (n=23). For all patients, tumor samples were collected both before and after AZA administration and were analyzed for mutations in 66 genes implicated in myeloid neoplasms using targeted-capture sequencing. The median cycles of AZA treatment before sampling was 4 (range 1-7). Clone size was calculated from variant allele frequency adjusted for ploidy or allelic imbalances.Survival was calculated with a Cox regression model. Results In post-treatment samples, we identified 870 mutations in 51 genes in 255 (88%) patients with a median of 3 mutations per sample, while 943 mutations were seen in 279 (96%) patients in the pre-treatment samples. Most frequently detected mutations in post-treatment samples were seen in TET2, TP53, RUNX1, and ASXL1. Germline DDX41 mutations were excluded from clone size evaluation. Median clone sizes were 0.63 and 0.54 for pre-treatment and post-treatment samples (P=.011), respectively. The largest clone sizes (max(VAF)) in post-treatment samples had a strong negative correlation with hematological response according to IWG criteria (P < .0001). We next explored whether max(VAF) in post-treatment samples provides a more precise estimation of long-term survival than IPSS-R. Max(VAF) further stratified each IPSS-R risk group in subgroups with discrete OS (P < .0001 for IPSS-R very high and P = .0004 for high risk group). Incorporating pre-treatment mutation data (mutations in TP53 and DDX41) and max(VAF) values in addition to IPSS-R scores and clinical response, we constructed a multivariate model and found that all these factors had an independent and significant impact on OS (Figure 1A). Next, we examined whether max(VAF) combined with IPSS-R and clinical response can improve the model. For this purpose, we randomly split the cohort into 75% training and 25% validation subsets and for each split, we constructed different models using the training set, performance of which was evaluated by calculating the concordance index (c-index) using the validation set. The mean c-index in 10,000 simulation sets increased by 0.025 by adding response data to IPSS-R score (I versus IR in Fig 1B). Further improvements were obtained by adding gene mutation and max(VAF), in which the c-index increased by 0.034 (IR versus IGR in Fig 1B) and 0.010 (IGR versus IGRP in Fig 1B), respectively. For the 53 patients who received allogeneic stem cell transplantation, the median post-transplant OS was 82.6 months (range, 36.3 to not reached). Notably, max(VAF) significantly stratified OS after allo-SCT (HR, 3.3; 95%CI, 1.3 to 8.3; P = .014). Conclusions Our study revealed that post-treatment clone size significantly correlated with clinical response and the evaluation of post-treatment clone size allows for more precise prognostication after AZA treatment compared with IPSS-R and clinical response alone. Table Disclosures Naoe: NIPPON SHINYAKU CO.,LTD.: Speakers Bureau; Sysmex co.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau. Miyazaki:Celgene: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Papaemmanuil:Kyowa Hakko Kirin: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; MSKCC: Patents & Royalties; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Ogawa: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; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

Introduction: Myelodysplastic syndromes (MDS) is thought to develop and progress as a result of accumulation of genetic mutations. This multicenter, open-label, phase II study examined impact of gene mutations on the effect of darbepoetin alfa (DA) for anemia. Death within 1 year and progression to acute myeloid leukemia (AML) were also examined. Methods: DA ≤240 μg was administered once weekly for 16 weeks to DA-naive, low-risk MDS (low or Intermediate-1 [Int-1] risk defined by the International Prognostic Scoring System [IPSS] risk classification) patients with anemia. DNA was extracted from the peripheral blood of patients, and presence of previously reported high-frequency gene mutations in MDS (SF3B1, TET2, SFRS2, ASXL1, DNMT3A, etc.) was analyzed by next-generation sequencing. Primary endpoint was association between frequently observed mutated genes and therapeutic effect of DA (IWG criteria 2006 (HIE)) at 16 weeks. A secondary endpoint was survival analysis results for death and progression to AML within one year after 16 weeks of treatment, with the date of first treatment as the starting date. For AML, progression to AML and subsequent death were stated as events, and the patients without confirmed progression to AML were censored at the date of last known survival. For overall survival, death from any cause was considered an event, and for the survival cases, the study was terminated at the date of last known survival. For the primary endpoint, relative risk and 95% confidence intervals (CI) were calculated using Wilson's score method; statistical significance was assessed using Pearson's chi-square test. Multivariate analysis was performed by adding baseline erythropoietin (EPO) levels (low: Results: Of the 85 patients enrolled between August 2016 and May 2019, 4 patients who were judged ineligible and 2 patients who discontinued the study prior to the start of treatment were excluded, and 79 subjects were included in the analysis (full analysis set). Of 79 subjects, 52 (65.8%) were male (median age 77.0 [29-90] years). IPSS risk was low in 27 (36.7%) and Int-1 in 50 (63.3%) subjects. The frequently mutated genes (³10%) were SF3B1 (24, 30.4%), TET2 (20, 25.3%), SRSF2 (10 cases, 12.7%), ASXL1 (9, 11.4%), and DNMT3A (8, 10.1%). Overall response rate was 70.9%. Univariate logistic regression analaysis did not show any significant association between these mutations and therapeutic efficacy of DA. The same results were obtained when the analysis was limited to red blood cell transfusion-dependent subjects (n=15). After adjusting against baseline EPO values, mutations of ASXL1 gene were associated with significantly worse response (odds ratio 0.180 [0.035-0.928], p = 0.040). Six (7.6%) confirmed cases of AML and 17 (21.5%) deaths (death before confirmed AML) were observed. Overall, 21 deaths were observed including 4 deaths after progression to AML. The median survival time (95% confidence interval) from the start of treatment to confirmed AML or death was 41.3 months (30.6 months - Not reached). Conclusions: The results of this exploratory study suggest that the presence of ASXL1 gene mutations may result in poor response to the anemic treatment with DA in low-risk MDS. The current analysis of progression to AML and death included subjects whose observation period did not reach the prespecified number of days. For such cases, the outcome research will be conducted around October 2020 and updated results will be presented at the ASH 2020 conference. Disclosures Ichikawa: Novartis, Takeda: Honoraria. Shibayama:Taiho: Research Funding; Shionogi: Research Funding; Ono: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Takeda: Honoraria, Research Funding; Merck Sharp & Dohme: Research Funding; Sumitomo Dainippon: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Kirin: Honoraria; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Fujimoto: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Teijin: Research Funding; Mundi Pharma: Honoraria. Maeda:Kyowa Kirin Co.Ltd.: Honoraria, Research Funding. Kawabata:Celgene Corporation: Consultancy; Celgene Corporation: Honoraria; Sanofi K. K: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co., Ltd: Honoraria. Matsumura:Shionogi & Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Janssen Pharmaceutical K.K: Speakers Bureau; Amgen K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED.: Speakers Bureau; Pfizer Japan Inc.: Speakers Bureau; Novartis Pharma KK: Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding. Mitani:CHUGAI: Research Funding; Takeda: Research Funding; KYOWA KIRIN: Consultancy, Research Funding.

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.

To the editor: Chronic myeloid leukemia (CML) represents a distinct subtype of myeloproliferative neoplasm originated from a deregulated hematopoietic stem cell, in which a constitutively activated breakpoint cluster region-Abelson (BCR-ABL) fusion kinase plays a crucial role.[1][1] Although its

Biallelic mutations in the neuroblastoma amplified sequence (NBAS) gene have been reported to cause two different clinical spectra: short stature with optic nerve atrophy and Pelger-Huet anomaly (SOPH) syndrome and infantile liver failure syndrome 2 (ILFS2). Here, we describe a case of a 3-year-old Japanese boy who presented with fever-triggered recurrent acute liver failure (ALF). The clinical characteristics were considerable elevation of liver enzymes, severe coagulopathy, and acute renal failure. In addition to the liver phenotype, he had short stature and Pelger-Huet anomaly in the peripheral granulocytes. Whole-exome and Sanger sequencing of the patient and his parents revealed that he carried novel compound heterozygous missense mutations in NBAS, c.1018G>C (p.Gly340Arg) and c.2674 G>T (p.Val892Phe). Both mutations affect evolutionarily conserved amino acid residues and are predicted to be highly damaging. Immunoblot analysis of the patient’s skin fibroblasts showed a normal NBAS protein level but a reduced protein level of its interaction partner, p31, involved in Golgi-to-endoplasmic reticulum retrograde vesicular trafficking. We recommend NBAS gene analysis in children with unexplained fever-triggered recurrent ALF or liver dysfunction. Early antipyretic therapy may prevent further episodes of ALF. Novel mutations in a gene called NBAS have been identified in a Japanese boy with recurrent acute liver failure. Researchers led by Junko Matsuda from Kawasaki Medical School, Okayama, Japan, searched for the genetic cause of a young boy’s recurrent episodes of fever-triggered liver dysfunction. They sequenced the entire protein-coding portion of his genome and that of his parents. They found that the boy had inherited two defective copies of the NBAS (neuroblastoma amplified sequence) gene, one from each parent. Laboratory experiments indicated that these mutations impaired the ability of the protein encoded by NBAS to function correctly. The authors recommend testing for NBAS mutations in any children with unexplained liver problems, and then treating with fever-reducing therapies to prevent future life-threatening episodes of liver failure.

Excessive soluble fms-like tyrosine kinase-1 (sFlt-1) has been strongly implicated in preeclampsia. An increase in the serum sFlt-1 level occurs before the onset of preeclampsia, and the sFlt-1 level is already higher in women who are predisposed to preeclampsia than in normotensive pregnant women. This study aimed to investigate the relation between arginase and sFlt-1 in the plasma of preeclamptic women and normotensive pregnant women. We suggested that a regulatory mechanism exists that suppresses the level of sFlt-1. The relationship between arginase, one of the nitric oxide (NO) modulators, and sFlt-1 was examined. First, the pregnant women were divided into 4 groups: group 1, sFlt-16000 pg/ml and arginase activity110 U/L; group 2, sFlt-1 ≥6000 pg/ml and arginase activity110 U/L; group 3, sFlt-1 ≥6000 pg/ml and arginase activity ≥110 U/L; and group 4, sFlt-16000 pg/ml and arginase activity ≥110 U/L. Groups 2 and 3 comprised preeclamptic women. The preeclampsia/normotensive ratio increased from groups 1 to 3. Under the higher sFlt-1 condition, lower arginase activity was associated with lower occurrence of preeclampsia. Next, in human umbilical endothelial vein cells (HUVECs), a slightly higher concentration of sFlt-1, as in group 2, reduced arginase expression and arginase activity, and S-(2-boronoethyl)-l-cysteine (BEC; arginase inhibitor) impaired sFlt-1 secretion. In contrast, a higher level of sFlt-1 increased arginase expression and activity in HUVECs, as in group 3. These results showed that arginase controlled sFlt-1 elevation to some extent. In conclusion, our results suggest the existence of a mechanism to maintain the level of sFlt-1. Soluble Flt-1 negatively regulated itself against increasing serum sFlt-1 in preeclampsia. Moreover, this study revealed that arginase inhibitors are a potential treatment option for preeclampsia.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.

Introduction: Age-related clonal hematopoiesis (CH) has been implicated in an increased risk of myeloid neoplasms. While common driver genes mutated in CH largely overlap to those in myeloid neoplasms, a notable exception is protein phosphatase Mg2+/Mn2+dependent 1D gene (PPM1D), encoding a p53-targeting phosphatase. Although it is known to be involved in DNA damage response pathways and more frequently mutated in therapy-related myeloid neoplasms than in primary ones, its role in CH and myeloid neoplasms has not been fully understood. Aim: To identify genetic features associated with PPM1D mutations, we examined genetic profiles in the large cohorts of healthy elderly individuals and patients with myelodysplasia. Methods: We enrolled 10,826 healthy individuals (>60y) and 1,213 cases with myelodysplasia, including myelodysplastic syndromes (MDSs), myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) (n=1,080), and secondary acute myeloid leukemia (sAML) (n=133), of which 567 cases were treated by hematopoietic stem cell transplantation (HSCT) through the Japan Marrow Donor Program just after sampling, and 332 of them underwent any therapy before sampling. Samples from healthy individuals were subjected to multiplex-amplicon sequencing for 22 genes, including PPM1D and other genes, related to CH or myeloid neoplasms. Myelodysplasia samples had previously been sequenced for major myeloid drivers, except for PPM1D, which was newly sequenced in this study. Results: Frequency of PPM1D mutations in myelodysplasia and healthy individuals was 3.1% and 0.42%, with a median variant allele frequency (VAF) of 0.043 and 0.056, respectively. PPM1D mutations were more frequent in cases with previous treatment (4.8%) than in those without known history of therapy (2.3%) (P=0.038). In MDS and MDS/MPN cases, 59.5% of PPM1D mutations had accompanying mutations, in which DNMT3A mutations were the most frequently identified (16.2%, n=6). These 6 cases were diagnosed with RCUD (n=1), RCMD (n=2), RAEB-2 (n=2), or CMML (n=1). The association between PPM1D and DNMT3A mutations was also seen in 7 of 45 healthy individuals with PPM1D mutations, of which one had a DNMT3A-R882 mutation. In the HSCT cohort, 192 cases harbored ≥2 mutations of the 22 CH-related genes, and the relative temporal order of these mutations was investigated using Bradley-Terry model relying on their tumor cell fractions. The estimate of PPM1D mutations tended to be smaller than that of DNMT3A mutations. To further confirm chronological order of these mutations, VAF values were compared between them in the individuals with concurrent PPM1D and DNMT3A mutations (n=13; 6 myeloid neoplasms and 7 healthy donors). In the combined cohort, the VAFs of PPM1D and DNMT3A mutations were correlated (Spearman; correlation coefficient=0.87, P=1.2x10e-5). In both neoplastic and healthy cohort, the VAFs of DNMT3A-R882 mutations were larger than those of accompanying PPM1D mutations. These findings suggest that these mutations should be acquired in the same cell populations and that DNMT3A mutations might occur prior to PPM1D mutations. With regard to copy number alterations associated with PPM1D-mutated myelodysplasia, del(5q) (16.7%) and complex(-like) karyotypes (13.9%) were among the most frequent chromosomal abnormalities. Approximately 65% of PPM1D-mutated tumor samples had normal karyotype, which was similar to PPM1D-unmutated cases. PPM1D mutations did not significantly influence overall survival, although PPM1D mutations tended to negatively affect clinical outcome among patients who were treated with HSCT (Hazard ratio, 1.61; 95% confidence interval, 0.95 to 2.70). Conclusion: PPM1D mutations were more enriched in myelodysplasia than in CH, and the median value of VAF in PPM1D mutations in CH was not significantly different from that in myelodysplasia. The size of PPM1D-mutated clones tended to be relatively smaller compared with that of clones with other mutations in myelodysplasia. PPM1D and DNMT3A mutations might be cooperatively associated in the pathogenesis of myelodysplasia and CH. Disclosures Baer: 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. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership.

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.

Background DNA hypomethylating agents (HMAs), including azacytidine (AZA) and decitabine, have been established as key drugs for higher-risk myelodysplastic syndromes (MDS). Recently, a marked efficacy of decitabine has been reported in TP53-mutated myeloid malignancies, although it is not approved for MDS in many countries. Thus it is of an urgent interest whether the excellent response to TP53-mutated myeloid neoplasms is recapitulated with AZA and if so, what is the biomarkers reliably predicting response to AZA. Methods In this study we enrolled a total of 384 AZA-treated MDS patients, including 179 from a Japanese prospective study (JALSG MDS-212 trial), 163 from Karolinska Institute, and 42 from a Japanese multi-institutional registry, and analyzed mutations in 66 genes commonly mutated in myeloid neoplasms together with copy number abnormalities, using targeted-capture sequencing. For all patients, sequencing was performed on pre-AZA samples. Post-AZA samples were also analyzed for the majority (279/394, 73%) of patients. Results Analysis of pre-AZA specimens identified 1,085 driver mutations in 53 genes in 361 (94%) patients with a mean of 3.0 mutations/sample. CNAs were detected in 258 (67%) cases. Most frequently observed were mutations in TP53, RUNX1, TET2, SRSF2, and ASXL1, and other high-risk CNAs, such as -7/7q LOH, 5q LOH, and 17p LOH. TP53 lesions were found in 102 (27%) cases, of which 87 cases (85%) had biallelic lesions caused by either multiple mutations (N=28) or 17p LOH involving the TP53 locus (N=59). Sixty-seven cases (17%) achieved complete remission (CR) with a median observation period of 7.5 months. TP53 mutation, found in 48% of the cases, was the only lesion that was significantly associated with CR (OR=3.4, p72 years old, hazard ratio: HR 1.36, p=0.03) and high karyotype risks (Very High/High according to IPSS-R-based classification system, HR 1.31, p=0.04) were associated with a shorter OS, while mutations in DDX41 predicted a significantly better OS (HR 0.62, p Conclusions Our study revealed distinct response profile to AZA treatment according to TP53 mutation status, underscoring the need of different tools to estimate treatment outcomes for them. High remission rate in TP53-mutated cases does not lead to long-term outcomes due to short remission duration, if ever obtained. Successive transplantation to AZA treatment during remission phase might rescue patients from this intractable disease. Disclosures Takaori: Novartis: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Papaemmanuil:Celgene: Research Funding. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Qiagen Corporation: Patents & Royalties; RegCell Corporation: 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.

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Genetic alterations, including mutations and copy-number alterations, are central to the pathogenesis of myelodysplastic syndromes and related diseases (myelodysplasia), but their roles in allogeneic stem cell transplantation have not fully been studied in a large cohort of patients. We enrolled 797 patients who had been diagnosed with myelodysplasia at initial presentation and received transplantation via the Japan Marrow Donor Program. Targeted-capture sequencing was performed to identify mutations in 69 genes, together with copy-number alterations, whose effects on transplantation outcomes were investigated. We identified 1776 mutations and 927 abnormal copy segments among 617 patients (77.4%). In multivariate modeling using Cox proportional-hazards regression, genetic factors explained 30% of the total hazards for overall survival; clinical characteristics accounted for 70% of risk. TP53 and RAS-pathway mutations, together with complex karyotype (CK) as detected by conventional cytogenetics and/or sequencing-based analysis, negatively affected posttransplant survival independently of clinical factors. Regardless of disease subtype, TP53-mutated patients with CK were characterized by unique genetic features and associated with an extremely poor survival with frequent early relapse, whereas outcomes were substantially better in TP53-mutated patients without CK. By contrast, the effects of RAS-pathway mutations depended on disease subtype and were confined to myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). Our results suggest that TP53 and RAS-pathway mutations predicted a dismal prognosis, when associated with CK and MDS/MPNs, respectively. However, for patients with mutated TP53 or CK alone, long-term survival could be obtained with transplantation. Clinical sequencing provides vital information for accurate prognostication in transplantation.

While searching for vitamin D(3) analogues which inhibit neutrophil recruitment in the lung without elevating plasma calcium level, we found that (5Z,7E)-(1S,3R)-20(R)-[(5E)-(2S)-2-hydroxy-2-methyl-cyclopentanone-5-ylidene]methyl-9,10-secopregna-5,7,10(19)-triene-1,3-diol (TEI-A00114) had the best efficacy and calcemic action. TEI-A00114 has a vitamin D receptor affinity 2.5-fold weaker and a vitamin D binding protein affinity 330.9-fold weaker than those of 1α,25(OH)(2)D(3). The estimated effective doses for 40% inhibition (ED(40)) via peroral and intratracheal administration are 7.6 and 0.4 μg/kg, respectively. TEI-A00114 was also tested by inhaled administration, and its ED(40) was calculated as 0.2 μg/kg. The estimated 40% inhibitory concentration (IC(40)) of TEI-A00114 on interleukin (IL)-8 production induced by lipopolysaccharide and on IL-1β in human whole blood cells in vitro were 9.8 × 10(-8) or 1.8 × 10(-9)M, respectively. These levels of TEI-A00114's activities are equal to those of 1α,25(OH)(2)D(3). On the other hand, the calcemic action of TEI-A00114, which was evaluated at day 14 after sequential peroral quaque die administration, was 89-fold weaker (molar ratio) than that of 1α,25(OH)(2)D(3). These results indicate that TEI-A00114 has a dissociated profile between inhibition of neutrophil recruitment in the lung and calcemic action, suggesting its suitability over 1α,25(OH)(2)D(3) as a candidate for the treatment of acute lung injury.

Background: Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease, characterized by expansion of undifferentiated myeloid precursor cells. The outcome for AML has improved through optimal treatment protocols, new drugs, and better supportive care; however, relapse remains common and patients with relapsed AML have poor prognosis. Recent genome-wide analyses revealed several recurrently mutated genes in AML, however, few of these driver mutations have been developed as therapeutic targets to date. In AML, t(8;21) and MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape is limited. Patients and Methods: The AML-05 study is a Japanese nationwide multi-institutional study of children (age < 18 years old) with de novo AML, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). The trial was registered with the UMIN Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm; number UMIN000000511) and conducted in accordance with the principles set down in the Declaration of Helsinki, and approved by the Ethics Committees of all participating institutions. All patients, or their parents / guardians, provided written informed consent. For whole-exome sequencing (WES), whole-exome capture was accomplished by liquid phase hybridization of sonicated genomic DNA using a bait cRNA library (SureSelect Human ALL Exon V5 or V5 Inc RNA 5 kit), following the manufacturer's protocol. Massively parallel sequencing of the captured targets was performed using a HiSeq 2000/2500 (Illumina) with the paired-end 126-133 bp read option. For targeted sequencing, target enrichment was performed using a SureSelect custom kit (Agilent) designed to capture all coding exons of the 338 genes. Similarly, CCND1, CCND2, and CCND3 were also captured and sequenced in t(8;21) AML samples. For cell cycle analysis, cell lines were treated with DMSO, palbociclib (500 nM), or abemaciclib (500 nM) for 24 h. Then, cells were stained with propidium iodide and analyzed using a FACS Canto II flow cytometer (BD Biosciences). Results and Discussion: First, we analyzed paired AML tumor and germline samples from nine pediatric MLL-rearranged AML patients by WES. In total, 52 mutations (mean, 5.8 mutations/patient) were identified, including known mutational targets in AML, such as FLT3, BRAF, SETD2, BCORL1, and WT1. Moreover, novel CCND3 mutation was detected in one patient. Next, we analyzed 56 samples from patients with pediatric MLL-rearranged AML enrolled in the JPLSG AML-05 study, using targeted sequencing. We selected 338 genes, among which were previously reported and putative driver genes, including CCND3, for targeted sequencing. We identified eight mutations in CCND3 in five pediatric MLL-rearranged AML patients (8.9%). All mutations were clustered in the PEST domain. Four of the eight mutations were R271fs, which is a known hot-spot mutation in lymphoid malignancies. Mutations of the other D-type cyclins (CCND1, CCND2) have been reported in t(8;21) AML (Leukemia, 2017); therefore, we also searched for mutations in CCND1, CCND2, and CCND3 by targeted sequencing of samples from pediatric t(8;21) AML patients (n=105). CCND1 (n=3, 2.9%) and CCND2 (n=8, 7.6%) mutations were detected; however, no mutations of CCND3 were detected. By contrast, there were no mutations of CCND1 or CCND2 in MLL-rearranged AML (n=56), suggesting that mutations of D-type cyclins exhibit a subtype-specific pattern in AML. A recent study demonstrated that genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4/6 inhibitor (Cancer Cell, 2017), therefore, we also examined the effects of CDK4/6 inhibitors (abemaciclib and palbociclib) on two t(8;21) AML cell lines (Kasumi-1 and SKNO-1) and five MLL-rearrangement AML cell lines (ML-2, MV4-11, MOLM-13, THP-1, and NOMO-1) were analyzed. All cell lines described above exhibited impaired proliferation after treatment with CDK4/6 inhibitors. Furthermore, treatment of these cell lines with CDK4/6 inhibitors resulted in detection of lower frequencies of S/G2/M phase cells by flow cytometry, suggesting that cells were arrested in G1 phase via CDK4/6 inhibition. These data provide further insights into the genetic basis of, and potential therapeutic strategies in t(8;21) and MLL-rearranged acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Introduction Epstein-Barr virus (EBV) is a double-stranded DNA virus that infects >95% of the human population and is associated with a substantial risk of cancer development. Most infections in children and adolescents are asymptomatic or result in infectious mononucleosis; however, in some patients, EBV is associated with various hematological malignancies including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), and extranodal NK/T-cell lymphoma. EBV infection is also present in a portion of epithelial cell neoplasms such as gastric cancer and nasopharyngeal carcinoma. Despite the large population risk of cancer associated with EBV, it is poorly understood why only a small subset of EBV-infected individuals develop neoplasms, while others do not. Patients and Methods We designed a target enrichment system to capture several EBV strains including the Akata strain, which is responsible for the majority of EBV infections in Japan. We analyzed the genomes of EBV strains in 139 patients with various EBV-associated diseases and 17 EBV-positive cell lines. Next-generation sequencing reads were aligned to the Akata reference genome to analyze nucleotide variations, copy number alterations, and structural variations including sequence insertions in the human genome. The institutional review board of Nagoya University Graduate School of Medicine approved this study. Results We identified a median of 645 single nucleotide variants (SNVs) in the EBV genomes, 78% of which affected coding sequences. SNVs in coding sequences were significantly biased toward synonymous variants, suggesting negative selection pressure. The SNVs detected in noncoding sequences were enriched in two evolutionarily conserved viral noncoding RNAs (EBER1 and EBER2), particularly in the PAX5-binding domain of EBER2. However, most SNVs identified in the EBV genome do not seem to affect the development of neoplasms, as hierarchical clustering of EBV genomes from neoplastic and non-neoplastic diseases based on SNVs revealed no significant association between the EBV strain and disease type. In addition to SNVs, we identified frequent intragenic deletions in the EBV genomes of patients with EBV-positive DLBCL (10/14, 71%), extranodal NK/T-cell lymphoma (10/23, 43%), chronic active EBV infection (27/77, 35%), and other EBV-associated neoplasms (2/7). Such deletions were also identified in several EBV-associated cell lines (6/17), but not in non-neoplastic diseases such as infectious mononucleosis (0/4) and post-transplant lymphoproliferative disorders (0/14), suggesting a unique role of these mutations in the neoplastic proliferation of EBV-infected cells. Frequent deletions were detected in BamHI A rightward transcripts microRNA clusters (31/156), which suppress viral transcription factors (BZLF1 and BRLF1) required for the lytic reactivation of EBV. Deletions also were associated with several genes essential for virus production (20/156). These observed deletions are thought to upregulate lytic cycle-associated genes, some of which benefit neoplasms by inducing genomic instability and immune escape and mitigate cell damage caused by the production of viral particles. In fact, deletion of one essential gene, BALF5, resulted in upregulation of the lytic cycle and promotion of lymphomagenesis in a xenograft model. Discussion Although the essential roles of several latency-associated genes, such as LMP-1 and EBNA-2, in EBV-mediated immortalization and transformation of human lymphocytes have long been discussed, our finding raises the possibility that lytic cycle-associated genes also contribute to lymphomagenesis. This agrees with reports that lytic cycle-associated genes are expressed in Burkitt lymphoma, DLBCL, and chronic active EBV infection, and that BZLF1-deficient lymphoblastoid cells exhibit significantly impaired tumorigenicity in mice. In addition, essential gene deletions lead to the protection of EBV-infected cells from lysis. Further studies are warranted to exploit these findings for the design of novel therapeutics for EBV-associated neoplasms. Disclosures Kiyoi: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Nakamura:Roche/Chugai,: Research Funding; Kyowa-Kirin: Research Funding.

Introduction Immunoglobulin (Ig)/ T-cell receptor (TCR) gene rearrangements are the most widely used clonal marker to detect residual leukemic cells in patients with Acute Lymphoblastic Leukemia (ALL). Ig/TCR gene rearrangements based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with ALL. Although the standard method of real-time quantitative PCR (RQ-PCR) provides very good sensitivity in MRD measurement, the workflow is very complicated and time-consuming, requiring expert technique and much work for operation, which limits the number of patients to be examined for MRD monitoring and the number of testable markers per patient. Material and methods To reveal clonal architecture and detect appropriate MRD marker, we designed capture probes covering the coding and recognition signal sequences of V, D, J genes of the Ig/TCR loci. We performed high-throughput target-capture sequencing in 208 pediatric cases with BCP-ALL and 35 pediatric cases with T-cell ALL, including 20 relapsed cases and 14 MRD marker negative cases. Extracted DNA samples were enriched with about 420 capture probes (Agilent Technology) and sequenced by HiSeq2500 platform (Illumina) in order to obtain enough sequence coverage (> 500 mean depth). Sequenced data were analyzed with Ig/TCR recombination analysis tool Vidjil (Giraud et al, 2014) and V(D)J recombination clones were listed according to a number of detected read for each clone. Results Total 2379 clonal Ig/TCR gene rearrangements (median 9 per patient, range 0-82) were detected by capture sequencing among 236 (97%) cases. A clonal IGH sequence with V(D)J recombination was identified in 91% of BCP-ALL cases, followed by TRG (68 %), IGK (67 %), TRA+D (66%), TRD (59 %), TRB (49%), and IGL (15 %), respectively. On the other hand, clonal TRG V(D)J recombination was detected in 74% of T-ALL cases, followed by TRB (69%), TRD (57%), IGH (26%), and TRA+D (6%), respectively. About half of BCP-ALL cases were identified two independent IGH rearrangements. These frequencies agree with previous reports obtained by PCR based experiments. In the cases in this study with well-characterized clonal Ig/TCR gene rearrangements by PCR and Sanger sequencing, our capture sequencing was able to detect all rearrangements used in MRD measurements. Although 8 BCP-ALL cases in this study were marker-negative in standard PCR-MRD diagnostics, clonal Ig/TCR gene rearrangements were identified for 5 out of 8 cases by capture sequencing. Some of the hidden clonal rearrangements showed specific and good quantitative amplification by RQ-PCR and can be used as sensitive PCR-MRD targets. On the other hand, all the MRD marker negative 6 T-ALL cases were not detected clonal Ig/TCR gene rearrangements. Finally, we compared the clonal architecture based on Ig/TCR gene rearrangements between diagnosis and relapse in relapsed B-ALL patients. Changes in the clonal architecture were associated with remission duration. In very early relapse cases, detected Ig/TCR rearrangements and their proportion at relapse are very similar to those at diagnosis. In early to late relapse cases, some major Ig/TCR gene rearrangements were lost at relapse and other minor rearrangements expanded at relapse. Most of the identified Ig/TCR gene rearrangements were different between at diagnosis and at relapse in a case relapsed after more than 10 years. Loss of rearrangements were commonly seen in TRA, TRB, and IgL, while most of the IgK and TRD rearrangements were steady during disease course. Conclusion Introducing target capture sequencing enables to high throughput sample preparation and automated data analysis. Capture sequencing is a useful method for comprehensive detection of Ig/TCR gene rearrangements and contributes to better understanding clonal architecture and detecting appropriate MRD markers in ALL patients. Disclosures No relevant conflicts of interest to declare.

MDS arises through stepwise acquisitions of multiple mutations. Mutation configuration (bi-allelic vs. mono-allelic), specific distributions (hot spot), timing of acquisitions/ranks within clonal hierarchies, and combinatorial spectra, can all affect the pathogenesis and clinical phenotype of this disease. We performed integrative analyses of these processes to determine which relationships are deterministic vs. random. We analyzed 1,809 clinically annotated MDS patients. Deep targeted NGS was applied to a panel of the 36 genes frequently mutated in myeloid neoplasms. Copy number alterations (CNA) were evaluated by combining karyotyping, microarray, and digital CNA analysis. With a mean coverage of 862x, after removing SNPs and errors, we identified 3,971 somatic mutations. Frequently mutated genes/CNAs were TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%), each present in >10% of cases. DNMT3AMT affected the canonical site (R882) in 17% (35/203) of DNMT3AMT cases, were truncating in 39% (79/203) and were other missense mutations in 44% (89/203). Bi-allelic alterations of EZH2 and TP53 most commonly involved a mutation paired with a copy number deletion or UPD. In contrast, RUNX1 and TET2 commonly involved multiple mutations of the gene. Examining intragenetic relationships, we assumed that the impact of individual mutations depends on their clonal hierarchical position. To discriminate 1st hits ("dominant") from subsequent "secondary mutations", we used a stringent binominal distribution algorithm to compare the expected vs. observed VAFs using read counts (Figure 1). A mutation with the largest VAF in a sample was defined as "dominant". Mutations with overlapping 95% CI of expected VAFs were "co-dominant", and those with non-overlapping 95% CIs were "secondary". These assumptions were validated by Pyclone (concordance rate > 95%). Accordingly, 1,474 (36%) and 1,372 (35%) mutations were dominant and secondary, respectively. SF3B1, DNMT3A, U2AF1, and TP53 were more likely to be dominant, ASXL1, CBL, ETV6, and KRAS were more likely to be secondary. For example, SF3B1 mutations were dominant in 17% (303/1809) of patients and secondary in 2% (39/1,809), p 78 common combinations were identified among these different types of mutations. For instance, compared to U2AF1S34 mutations, U2AF1Q157 mutations were more associated with ASXL1 mutations and del(7q), and less with DNMT3A and BCOR mutations (p Disclosures Nazha: MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing < 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P Conclusion TP53 mutations at diagnosis are common in Hypodiploid ALL and also found in a substantial fraction of MLL rearrangement and IGH-DUX4 ALL, where the mutations predict a poor prognosis. TP53 mutation is also found in NCI-SR cases but may not be associated with poor prognosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

MDS is a heterogeneous group of myeloid neoplasms caused by genetic and epigenetic alterations. During the past decade, the major driver mutations in MDS have been fully investigated. However, the role of epigenetic alterations, particularly those of DNA methylation, has less intensively been studied, even though abnormal DNA methylation has long been implicated in the pathogenesis of MDS. In this study, we analyzed DNA methylation status of bone marrow mononuclear cells from 320 cases with MDS-SLD (n = 7), MDS-RS (n = 63), MDS-MLD (n = 51), MDS-EB (n = 186), MDS-U (n = 1), and MDS with isolated del(5q) (n = 12), using Illumina 450K methylation array. Mutations in major driver genes (51 genes) and abnormal genomic copy numbers were also interrogated using targeted-capture sequencing. Using unsupervised consensus clustering, we identified 3 subgroups showing unique DNA methylation profiles. Subsequently, we assessed differentially methylated positions (DMPs) associated with each subgroup. Differentially hypermethylated positions (hyper-DMPs) were significantly more enriched in Group 3 (n = 82) (P < 0.001), while differentially hypomethylated positions (hypo-DMPs) were more prominent in Group 1 (n = 125). Group 1 was significantly enriched for SF3B1 (46%) mutations (q < 0.01), while Group 2 (n = 131) was characterized by the enrichment of ASXL1 (38%), RUNX1 (30%), TP53 (26%), STAG2 (15%), and SETBP1 (6.7%) mutations (q < 0.01). In contrast, Group 3 (n = 64) was significantly enriched for TET2 (67%) and IDH1/2 (12% and 15%, respectively) mutations (q < 0.01), suggesting a strong association between DNA methylation and gene mutations. To further elucidate mutation-specific DNA methylation patterns, supervised analysis was performed for each mutation. As expected from their enrichment in Group 3 (q < 0.01), TET2 and IDH1/2 mutations were significantly associated with hyper-DMPs (P < 0.001) involving 1891 and 8330 promotor sites, respectively. Conspicuously, among these hypermethylated promoter sites, >1616 were commonly hypermethylated, strongly supporting the common impact of TET2 and IDH1/2 mutations on deregulated DNA methylation. To clarify prognostic impact of abnormal DNA methylation, we first interrogated the correlation between unique methylation subgroups and revised IPSS. Patients with very low or low risk were significantly dominant (74%) in Group 1 (q < 0.01), and very high or high risk cases were significantly enriched (68%) in Group 2 (q < 0.01). In accordance with this finding, patients in Group 3 showed significantly shorter overall survival (OS) compared to Group 1 (HR: 1.94, 95%CI: 1.11-3.4, P < 0.05) and OS was even worse in Group 2 patients (vs. Group 1: HR: 5.18, 95%CI: 3.21-8.36, P < 0.001). Strong correlations between epigenetic and genetic profiles were further interrogated using a Bayesian statistical model; on the basis of DNA methylation and gene mutations, the original 3 clusters were re-classified into 5 discrete clusters, clusters A, B, C, D, and E (n = 124, 17, 74, 46, and 59, respectively); patients in Group 1 and 3 largely clustered into Cluster A and E, respectively, while Group 2 was further subclassified into clusters B, C, and D. Clusters B and D were characterized by a conspicuos enrichment of DNMT3A (88%) and TP53 (69%) mutations (q < 0.001), while Cluster C was characterized by higher frequency of ASXL1 (71%), RUNX1 (54%), STAG2 (27%), and EZH2 (21%) mutations (q < 0.001). In contrast to significant associations between epigenetic regulators and unique methylation clusters, splice factor mutations tended to be clustered into multiple clusters, depending on type of co-occurring mutations. For example, combined SF3B1 and TET2 mutations (n = 20) were enriched in Cluster A, where highly associated with MDS-RS, while patients with SF3B1 and RUNX1 mutations (n = 9) were more grouped in Cluster C, mostly showing MDS-EB phenotype (89%). Similarly SRSF2 mutations with RUNX1 and/or ASXL1 mutations (n = 36) were enriched in Cluster C, largely associated with MDS-EB phenotype (80%), while those with TET2 or IDH1/2 (n = 39) were mainly grouped into Cluster C, many of which showed MDS-EB phenotype (74%). These findings highlight differential roles of mutated epigenetic regulators and splicing factors in abnormal DNA methylation. In conclusion, we elucidated the collaborative impact of DNA methylation profiles and mutation status on heterogeneous pathogenesis and prognosis in MDS. Figure. Figure. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene. Despite the dramatic improvement of its prognosis in recent years by the development of tyrosine kinase inhibitors (TKIs), a minority of chronic phase (CP) CML patients fail to respond to TKI therapies and progress to blast crisis (BC), showing dismal clinical outcomes. While acquired mutations in ABL1 kinase have been identified as a common mechanism for TKI resistance, recent genetic studies have revealed that patients with BC frequently harbor one or more genetic alterations implicated in myeloid malignancies, suggesting additional mutations other than ABL1 mutations might drive disease progression. However, our knowledge about the mechanism of TKI resistance and progression to BC is largely limited by the scarcity of matched CP and BC samples, which were investigated for genetic alterations in relatively small number of genes. Here, we performed comprehensive genomic studies of CML-BC using paired CP and BC samples to investigate the mutation profiles associated with BC. Method We performed whole-exome sequencing of 53 patients with CML-BC, including 40 myeloid and 13 lymphoid crisis cases, as well as corresponding CP controls to investigate acquired mutations during disease progression from CP to BC. We also performed targeted-capture sequencing of known and putative driver genes in an additional 15 CML-BC samples. Combined, a total of 68 CML-BC samples were analyzed for somatic mutations, copy number abnormalities, and structural variations. Results Commonly affecting ASXL1, GATA2, and IKZF1, mutations were found only in a minority of CP cases (10/53 [19%]). However, most cases acquired somatic mutations during disease evolution from CP to BC; in whole-exome sequencing, an average of 17 additional non-synonymous mutations were newly acquired per case during evolution from CP to BC. Mutations in CML-BC frequently involved known driver genes, such as ASXL1, RUNX1, ABL1, TP53, BCOR/BCORL1, and WT1. In addition, we identified novel targets of recurrent mutations, including UBE2A, NBEAL2 and KLC2. Of note, most these driver mutations were not detected in corresponding CP samples and newly acquired, whereas ASXL1 mutations were often found in corresponding CP samples in a minor population, suggesting that ASXL1 mutations at CP might play an important role in the disease progression to BC. Mutational profiles were similar between cases with and without a history of TKI therapy before BC, except for frequent ABL1 mutations among TKI-treated cases, mostly affecting the kinase domain. Compared with lymphoid BC, myeloid BC showed a higher number of somatic mutations, which was more prominent for ASXL1, TP53, and WT1 mutations. Copy number abnormalities were rarely found in CML-CP cases (8/53), but were common and newly acquired in 29 (55%) cases with CML-BC, 18 of which showed complex karyotype-like (≥3) abnormalities. Amplification of chromosome 6 and/or 8 were characteristics of myeloid BC, while deletion of chromosome 7 was more characteristic of lymphoid BC. In some cases, structural variations other than BCR-ABL1 translocation were newly acquired in CML-BC, which frequently involved genes implicated in myeloid malignancies such as RUNX1, CBFB, and MECOM. When mutations, copy number abnormalities, and structural variants were combined, most BC cases had at least one driver alterations, which might be involved in CML-BC. Conclusion Through a comprehensive sequencing analysis using paired samples of CP and BC, we demonstrate a role of additional driver events during the clonal evolution to BC. Additional mutations were common even in CML-CP, some of which might contribute to the progression to BC. Disclosures Takaori-Kondo: Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria.

Subclinical clonal expansions, referred to as clonal hematopoiesis of indeterminate potential (CHIP), are present in blood of otherwise healthy individuals and their frequency increases with age. While many CHIP-associated mutations are present in MDS, only a small proportion of asymptomatic individuals with CHIP progress to MDS. We assumed that: i) a proportion of CHIP mutations will eventually serve as ancestral hits that manifest as MDS upon acquisitions of additional genetic alterations, and ii) MDS from antecedent CHIP may be a MDS disease subtype that is distinct from de novo MDS characterized by more penetrant primary hits. Separating ancestral vs. secondary hits in MDS patients and comparing by meta-analyses their frequencies to those in CHIP may enable molecular and clinical characterizations of CHIP-related MDS. Our study cohort consisted of 1,809 clinically annotated MDS patients. Deep targeted NGS was conducted for a panel of the 36 most frequently mutated "myeloid" genes, which revealed 3,971 somatic mutations in MDS patients after removing SNPs and errors. To discriminate between dominant and subsequent rising secondary mutations, we used a stringent binominal distribution algorithm to define VAFs confidence intervals via loci read counts. Sample maximal VAF mutations were defined as "dominant" and those with overlapping VAF 95% CI were defined as "co-dominant"; mutations with lower non-overlapping 95% CIs were "secondary". These definitions are consistent with those of other methods such as Pyclone, with 95% concordance (1,253/1,317). They yield 1,474 (36%) dominant and 1,372 (35%) secondary mutations. We compared a meta-analysis of frequently mutated genes in 1,693 healthy CHIP individuals with somatic mutations from the CHIP meta-analysis to dominant mutations in MDS patients. Mutations in DNMT3A, TET2, ASXL1, and JAK2 were more frequent in CHIP than MDS [e.g.,DNMT3A; 52% (888/1,693) vs. 6% (110/1,809), p We set out to compare clinical, molecular and demographic features of C- vs. NC-MDS. There were 459 (25%) C-MDS and 498 (28%) NC-MDS cases out of 1809 MDS patients. 95% of patients with C-MDS (437/459) had at least one TET2 (51%, 234/459), DNMT3A (24%, 110/459) or ASXL1 (20%, 93/459) dominant mutation. The top 5 dominant mutations in patients with de novo MDS were U2AF1 (15%, 75/498), ZRSR2 (10%, 52/498), RUNX1 (9%, 47/498), STAG2 (9%, 43/498), and EZH2 (8%, 40/498). 52% (257/498) of patients with de novo MDS had at least one of these mutations. Focusing on secondary mutations, patients with C-MDS had a significantly higher frequency of secondary TET2 and ZRSR2 mutations than those with de novo [e.g., TET2, 17% (77/459) vs. 8% (41/498), p Prospective sequencing of serial samples of CHIP to development of MDS may be needed to fully reveal the landscape of C-MDS. This may not be practical, as over a million healthy donors would need to be followed for years to obtain adequate numbers of C-MDS cases. If our hypothesis that there exist clinical and molecular characteristics of C-MDS is correct, alternative methods that begin with MDS cases could then be employed. Regardless, detecting and monitoring CHIP is warranted, as this will lead to biomarkers that predict risks and thus preventative interventions. Disclosures Nazha: MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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.

While germline predisposition to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has long been recognized mainly through rare familial and pediatric cases, it has been drawing an increasing attention, on the basis of the recent discovery of novel risk alleles for MDS/AML through studies relying on revolutionized sequencing technologies; according to these studies, it suggest that more numbers of MDS/AML cases than expected might have germline predisposition. Moreover, it is suggested that germline variations may also confer predisposition to age-related clonal hematopoiesis or "CHIP", which has been implicated in the development of MDS/AML. In this study, we explored germline predisposition to MDS and CHIP through intensive sequencing of blood samples from large cohorts of AML/MDS patients and 'hematologically' healthy individuals (HHIs), in which germline variants in 21 genes implicated in sporadic or familial MDS/AML or CHIP were interrogated among patients with MDS/AML from the Japan Marrow Donor Program (n=797) and HHIs aged >60 years from Biobank Japan (n=10,852). Germline variants were referred to NCBI dbSNP Build 151 database, excluding the entries in COSMIC ver.7 and in-house database, followed by manual curations. Somatic mutations and CHIP in the 21 genes were also analyzed for MDS/AML and HHIs, respectively. In total, 30,286 germline variants, including both synonymous and non-synonymous changes, were detected in 21 genes in the entire cohort. By comparing their frequencies between in MDS/AML and HHIs, we identified 6 germline variants in showing a significant enrichment in MDS/AML. Among these most frequently observed was variants in DDX41, for which a total of 3,721 variants were detected in 3,688 HHIs. Among these, 3 variants were significantly enriched in MDS/AML, including p.A500fs (OR=13.1 [6.6-25.9] (95%CI) (n=15), p.S363del (OR=41.0, [4.3-349.5]) (n=3), and p.Y259C (OR=34.2, [6.6-176.8]) (n=5). Of interest, 14 of 23 MDS patients with one of these alleles carried somatic DDX41 mutations, typically p.R525H, which were not found in any of HHIs, further supporting the relevance of these DDX41 risk alleles. Also including an additional 2 nonsense/splicing variants, 5 DDX41 alleles found in 25 MDS/AML patients were thought to represent germline predisposition to MDS/AML. Similarly, RUNX1 p.H85N (OR=9.10, [1.52-54.52]) (n=2), CBL p.P782L (OR=4.27, [1.56-11.70]) (n=5), and GNAS p.H69N (OR = 2.90, [1.28-6.59]) (n=7) showed a significant enrichment in MDS/AML. Combined, these putative risk alleles accounted for 4.6% (37/797) of sporadic MDS and sAML. None of these alleles were observed in the Caucasian population of Exome Aggregation Consortium dataset, suggesting Asian origins of these variants. We next evaluated the effects of germline variants on CHIP. CHIP mutations were detected in 929 HHIs, where DNMT3A mutations (n=290) were most prevalent, followed by TET2 (n=124) and ASXL1 (n=68) mutations. By comparing allele frequency of each of 1,276 germline variants between healthy donors with and without CHIP, we identified two haplotypes at the JAK2 and TET2 loci, defined by T/A at c.C489T/c.G2490A (JAK2) and G/G/T at c.G652A/c.G3117A/c.T4140C (TET2), which were significantly enriched in the cases carrying CHIP with the JAK2 (p.V617F) and TET2 mutations, respectively (T/A vs. C/G; OR=3.36, [1.41-8.01] for JAK2 and G/G/T vs. A/A/C; OR=1.85, [1.19-2.86] for TET2). Intriguingly, the JAK2 risk haplotype (C/G) were also enriched in MDS cases with JAK2 p.V617F mutations (T/A vs. C/G; OR=3.06, [1.26-7.60]). Similarly, the TET2 risk haplotype (G/G/T) tended to be enriched in MDS cases with TET2 mutations, although not statistically significant. Finally, variant allele frequency of JAK2 p.V617F mutations in CHIP exceeded 0.5 in 4 out of 26 JAK2 CHIP-positive patients (15%), suggesting the presence of loss of heterozygosity (LOH) in chromosome 9p. In conclusion, through a large-scale detection of germline variants in 21 common drivers of MDS/AML as well as CHIP, we identified multiple novel germline variants or haplotypes that showed a significant predisposition to the development of adult-onset MDS or CHIP, respectively. Our findings provide novel insights into the genetic basis of myeloid leukemogenesis and the development of CHIP. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Kanda:Otsuka: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Pfizer: Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

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.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.

N , N ′-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the σ factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.

Two synthetic derivatives of the flavonol kaempferol were prepared in order to probe the biological mechanism of action of this natural product. Efficient synthetic routes to both a benzophenone-containing derivative and an amine-containing derivative are reported herein.

In a study of the rat intestinal P(i) transport system, an activator protein for rat Na/P(i) co-transport system (PiUS) was isolated and characterized. We also investigated the effects of restriction of vitamin D and P(i) (two of the most important physiological and pathophysiological regulators of P(i) absorption in the small intestine) on intestinal P(i) transport activity and the expression of Na/P(i) co-transporters that are expressed in rat small intestine. Rat PiUS encodes a 424-residue protein with a calculated molecular mass of 51463 Da. The microinjection of rat PiUS into Xenopus oocytes markedly stimulated Na(+)-dependent P(i) co-transport activity. In rats fed with a low-P(i) diet, Na(+)-dependent P(i) co-transport activity was increased approx. 2-fold compared with that of rats fed a normal P(i) diet. Kinetic studies demonstrated that this increased activity was due to an elevation of V(max) but not K(m). The PiUS mRNA levels showed an approximate doubling in the rats fed with the low-P(i) diet compared with those fed with the normal P(i) diet. In addition, after the administration of 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] to vitamin D-deficient animals, the P(i) uptake was significantly increased in the Na(+)-dependent component in the brush border membrane vesicle (BBMV) at 24 and 48 h. In addition, we found a further high-affinity Na/P(i) co-transport system in the BBMV isolated from the vitamin D-replete animals. The levels of type III Na/P(i) co-transporter PiT-2 mRNA were increased 24 and 48 h after 1,25-(OH)(2)D(3) administration to vitamin D-deficient animals, whereas PiUS and the type IIb Na/P(i) co-transporter mRNA levels were unchanged. In conclusion, we first cloned a rat activator protein, PiUS, and then studied its role along with that of other type III Na/P(i) co-transporters. PiUS and PiT-2 might be important components in the regulation of the intestinal P(i) transport system by P(i) restriction and 1,25-(OH)(2)D(3).

Three types (typeI, II, andIII) of sodium-dependent phosphate cotransporters have recently been isolated and shown to be expressed in the mammalian kidney. Understanding of the functional roles and regulation of each transporter is still fragmented, however. We analyzed the functional roles of each transporter by using antisense oligonucleotides in theXenopus oocytes expression system, and by localization in the proximal tubules of rat kidney using immunohistochemistry. Phosphate uptake in brush border membranes was increased by about 2 times in rats fed a low-phosphate, as compared with a high-phosphate, diet. Expression of typeI, II, andIII transporter mRNAs was observed in renal poly(A)+RNA, isolated from the rats fed a low-phosphate diet. Phosphate uptake increased about 2.5-fold inXenopus oocytes injected with the poly(A)+RNA, compared with those given RNA from rats fed a high-phosphate diet. Hybrid depletion of the typeII sodium-dependent phosphate transporter (NaPi-2), but not of the typeI (rNaPi-1) or typeIII transporters (PiT-1 and PiT-2), significantly decreased phosphate transport activity in oocytes injected with the poly(A)+RNA from each experimental group rat kidney. In rats fed the lowphosphate diet, NaPi-2 immunoreactivity increased markedly in the brush border membranes of renal proximal tubular cells, whereas rNaPi-1 protein was not changed. This study suggests that the typeII transporter functions mainly as a sodium-dependent phosphate cotransporter, and is regulated by dietary phosphate in the rat kidney.

Fanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach., 「アルデヒド分解酵素遺伝子」ALDH2が解き明かす難病の謎. 京都大学プレスリリース. 2013-09-13

To improve the intestinal absorption of human calcitonin (hCT), novel lipophilic derivatives of hCT were synthesized by chemical modification with short-chain length fatty acids such as acetic acid and caproic acid, and the intestinal absorption and stability of these derivatives were examined in rats. The intestinal absorption properties of hCT and its acyl derivatives, acetylated hCT (Ac-hCT) and caproylated hCT (Cap-hCT), were estimated by measuring its hypocalcemic effects. Their intestinal absorption and stability in the intestinal mucosal homogenates were improved by increasing the carbon number of the fatty acid. Furthermore, we investigated the effects of co-administered absorption enhancers, sodium glycocholate (Na-GC), N-lauryl-β-D-maltoside (LM), sodium deoxycholate (Na-DC) and sodium salicylate (Na-Sal), on the intestinal absorption of hCT and its acyl derivatives. Their intestinal absorption was slightly enhanced by the addition of various absorption enhancers except for Na-Sal. However, the extent of the promoting effect was a little higher for a native hCT rather than for acyl-hCT derivatives. These results suggested that it may be possible to achieve the further absorption enhancement of hCT by using a combination of acylation and absorption enhancers.

1. 1. In medium containing 10 or 20 mM KCI, NH 4 Cl (10 mM) addition significantly diminished tetraphenylphosphonium bromide (TPP + ) uptake in A7r5 vascular smooth muscle cells. 2. 2. Addition of 10 mM NH 4 Cl significantly potentiated the 45 Ca 2+ uptake of A7r5 cells in medium containing 20 mM KCI; this increased uptake could be reduced to the basal level by pretreatment with 0.1 μM verapamil. 3. 3. In rat aortic strips, KCI at 10 mM induced a slight contraction, that was greatly potentiated by the simultaneous addition of 10 mM NH 4 C1. In this case, also, pretreatment with verapamil (1 μM) completely abolished the NH 4 C1-potentiating effect. 4. 4. The results suggest that ammonium ion amplifies the K + -induced contraction of rat aorta by facilitating transmembraneous Ca 2+ influx through the voltage-dependent calcium channel, which may be due to potentiation of KC1-induced depolarization of the plasma membrane.

Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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.