Background Recent genetic studies have identified frequent mutations affecting a number of genes encoding RNA splicing factors (SDs) in myelodysplastic syndromes (MDS) and related myeloid neoplasms (MNs). Among these, most frequently mutation are SF3B1, SRSF2, U2AF1, and ZRSR2, whose functions have been intensively studied. By contrast, U2AF2, another SF, is also recurrently mutated in MNs. U2AF2 is a component of the U2 auxiliary factor that forms a heterodimer with U2AF1 for the recognition of the 3’ splice site (3'SS). U2AF2 contains a sequence-specific RNA-binding region with two RNA recognition motifs and identifies polypyrimidine (Py) tract signals of nascent transcripts. However, compared to other splicing factor mutations, U2AF2 mutations are much rare, preventing the detailed analysis of their role in leukemogenesis. Methods To characterize the role of U2AF2 mutations, we systematically analyzed mutation spectrum in 6,369 with different MNs using targeted-capture sequencing. We also performed RNA sequencing of cKit(+) bone marrow cells from 6 U2AF2 mutated MDS cases along with 52 MDS patients without common splicing factor mutations and 25 healthy individuals. THP1 and HL60 leukemia cells with exogenous expression of wildtype or mutant U2AF2 were established to evaluate the splicing response. We also used clustered regularly interspaced short palindromic repeats /CRISPR-associated protein-9 nuclease (CRISPR/cas9) to introduce the p.190_195del mutation to U2AF2 in K562 and MOLM-13 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant U2AF2. Results In total, U2AF2 mutations were found in 34 (0.53%) of 6,369 MN cases, of which 29 had the recurrent p.190_195del mutation, the median age of U2AF2 mutations was 55 years (range 31-79) and 55% were diagnosed with MDS, 31% with CMML and 7% with AML. U2AF2-mutated patients showed a male predominance (90%). The most frequently co-mutated gene was ASXL1 with 52%, followed by SETBP1 (35%) STAG2 (21%),RUNX1 (21%), CSF3R, NRAS, DNMT3A, NF1, PTPN11, ETV6, and STAT3. To investigate the effects of the U2AF2 p.190_195del mutation on RNA splicing, we analyzed transcriptome sequencing, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events . Various types of AS events were identified, including skipped exons (SEs), alternative 5’ ss exons (A5SSs), alternative 3’ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs). Among these, the skipping exons are the most frequent in both U2AF2-mutant primary MDS cases and cell lines. To explore these alternative splicing results in more detail, we next examined differentially skipped exons. Unsupervised uniform manifold approximation and projection (UMAP) analysis based on the standardized splicing ratio of skipping exons segregated U2AF2-mutated patients from other MDS cases without common splicing factor mutations and healthy individuals. To prioritize mutant U2AF2-induced alterative splicing events, we intersected significant skipping exons across 2 datasets: MDS patient samples with and without U2AF2 p.190_195del mutations, and MDS patient samples with U2AF2 p.190_195del mutations and healthy individuals. The differential skipping exon usages and differential gene expression profiles enables both consensus sequence analysis and pathway/gene-set enrichment analysis. Conclusions Our study revealed that the U2AF2 p.190_195del regulated aberrant alternative splicing facilitated MDS progression through perturbations in splice isoforms. A better understanding of the U2AF2 p.190_195del and its critical specific changes will provide novel insights into disease pathophysiology and potentially inform future treatment decisions.

Background Acute myeloid leukemia (AML) is a heterogeneous disease and is primarily defined by genetic abnormalities. Although accumulating evidence suggests the role of epigenetics in the pathogenesis of AML, it has not fully been investigated in a large cohort of patients. Methods We enrolled 1,563 primary AML cases from Swedish (n=1,040) and Japanese (n=523) cohorts and performed ATAC-seq (n=1,563) as well as multi-omics analysis, including targeted-capture sequencing (n=1,563), RNA-seq (n=1,398), whole-genome sequencing (n=207), ChIP-seq (n=120), drug screening (n=112), and single-cell RNA/ATAC-seq (n=31). Results ATAC-seq analysis identified 185K recurrent peaks, most of which were found within intergenic/intronic regions. An unbiased clustering analysis based on ATAC-seq identified 16 unique subgroups with distinct genetic drivers, transcriptome profiles, differentiation states, key transcription factors, and clinical features. Among these, three groups were well-known entities defined by t(8;21), inv(16), and t(15;17). In contrast, the remaining 13 subtypes represent a novel classification framework not defined by single genomic abnormalities. Patients with HOX-related gene abnormalities, such as NPM1 mutation as well as KMT2A and NUP98 rearrangement, converged into four subgroups (D-G). Associated with global chromatin changes in the HOXA locus from repressive to active state, these ATAC subgroups were characterized in common by an elevated expression of the entire HOXA cluster genes, exhibiting unique clinical and molecular features. For example, subgroup D is characterized by co-occurring NPM1 and TET2/IDH1/IDH2 mutations, older age, and high WBC/blast counts, while another HOX-subgroup (E) had monocytic nature and frequent RAS pathway mutations. By contrast, subgroups I and J were enriched for bi-allelic CEBPA mutations with and without frequent bZIP domain inframe mutations, respectively. GATA2 and WT1 mutations were common in subgroup I, while subgroup J was enriched for myelodysplasia-related mutations. TP53 mutations were largely clustered into subgroups (N, O and P) characterized by erythroid, immature progenitor, and tumor microenvironment cells, respectively. Additional ATAC subgroups included those having frequent RUNX1 (K), IDH1/IDH2 (L), and DDX41 (M) mutations, or showed a CMML-like AML phenotype (H). We next analyzed gene regulatory networks by combining RNA-seq and ATAC-seq, revealing key transcription factors (TFs) in each ATAC-subgroup. HOXA members played central roles in the HOX-related subgroups, while IRF members, including IRF4, 7, 8 and 9, were key TFs in the subgroup enriched for RUNX1 mutations (K), leading to the upregulation of the interferon pathway. ATAC subgroups also impacted patients' survival and significantly improved the risk prediction of ELN, enabling further stratification of each ELN risk group. Next, we performed an in vitro drug screening for 112 samples against 250 compounds and obtained a drug sensitivity profile for each ATAC-subgroup. As expected, the subgroup with frequent FLT3-ITD mutations showed a high sensitivity to a FLT3 inhibitor (quizartinib), while other subgroups (C, F, H) with monocytic differentiation and common RAS pathway mutations were sensitive to MEK inhibitors. Furthermore, we noted an unexpected sensitivity of subgroup K samples to multiple ABL inhibitors, even though they had no known ABL-related kinase mutations. To validate these findings, we predicted ATAC-subgroups for four external AML cohorts based on gene expression and successfully reproduced an equivalent ATAC-subgroups with similar clinical, genetic, and transcriptomic features as well as drug sensitivities. Finally, we performed single-cell RNA/ATAC-seq and profiled a total of 233,000 mononuclear cells from 31 patients. We observed that leukemic cells were separately clustered from normal cells, while cells from the same ATAC subgroups were co-clustered, supporting that leukemic cells had their own epigenetic profiles unique to each cluster. Conclusion Through a large-scale multi-omics analysis of AML, we revealed a comprehensive landscape of chromatin accessibility of AML, highlighting the role of epigenetic profiling as a powerful tool for deciphering heterogeneity of AML, which could be used for a better stratification of patients and therapeutics.

IntroductionDespite the significant impact of clonal hematopoiesis (CH) on leukemogenesis, the pathogenesis of CH is still not fully understood.MethodsUtilizing a novel single-cell sequencing platform that allows for simultaneous detection of mutations and gene expression, we examined the gene expression profiles of hematopoietic stem and progenitor cells (HSPCs) harboring CH-related mutations from CH(+) cases, which was compared with that of wild-type (WT) cells from both CH(+) and CH(−) cases. Age-related changes in the bone marrow (BM) environment were also assessed using CH(−) cases.ResultsIn 12 patients with CH, genes associated with cell proliferation were upregulated in mutant cells. Significantly, mutant cells showed decreased expression of genes related to inflammatory responses, which were enhanced in BM cells from aged CH(−) cases, indicating the potential contribution of aged BM environment to the positive selection of mutant cells. Unexpectedly, WT cells from 3 TET2-CH(+) cases demonstrated significant upregulation of genes related to interferon response and cell proliferation, compared with those from age-matched CH(−) cases, suggesting the altered BM environments. Notably, when competitively transplanted with Tet2-knockout (KO) cells, WT HSPCs displayed enhanced expression of genes associated with cell proliferation and interferon signalling, compared with those transplanted with WT cells, implying non-cell autonomous effects of mutant cells.ConclusionsThese results suggest that mutant cells in CH(+) BM may exert non-cell autonomous effects on WT cells. Alongside aged BM environments, these effects may contribute to the positive selection of CH clones, playing a pivotal role in the pathogenesis of CH.

Age-related clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) frequently harbor mutations in genes encoding epigenetic regulators contributing to epigenetic dysregulation. However, it is not fully understood how CH/CCUS clones are positively selected and undergo phenotypic changes through these epigenetic alterations. In this regard, the analysis of the impact of these mutations on epigenesis at a single cell resolution would help understand the mechanism of clonal selection of CH and CCUS. Unfortunately, however, because of the frequent allele dropout in detecting mutations, few studies have addressed this with sufficient sensitivity to date. In the current study, we have developed a droplet-based simultaneous single cell gene mutations and DNA accessibility (ATAC-seq) profiling platform utilizing the Mission Bio Tapestri, which has achieved high sensitivity and accuracy in single cell genotyping and providing novel insight into the role of epigenetic mutations on chromatin accessibility. First, we evaluated the performance of our platform through the analysis of a sample in which two cell lines, HL-60 and SKM-1, having a distinct variant in RUNX1 (heterozygous) and BCOR (hemizygous), respectively, were intentionally mixed. A total of 3,579 cells were clustered into two groups based on DNA accessibility data alone and annotated for identities based on gene accessibility profiles. We next assessed genotyping accuracy for the BCOR and RUNX1 variant. Our platform resulted in detection of only 3.7 % of doublets and genotype assignment of 74 % of cells with 99 % accuracy for the BCOR variant genotyping and 80 % of cells with 97 % accuracy for genotyping of the RUNX1 variant. These results suggest that our single cell platform can successfully integrate single cell ATAC-seq and genotyping of targeted loci, allowing for the simultaneous genotyping and ATAC with high accuracy and sensitivity for thousands of cells. Then, to elucidate the epigenetic consequences by epigenetic mutations in primary CH/CCUS samples, we applied our platform to the analysis of whole bone marrow (WBM) cells from patients with CH (n = 3) and CCUS (n = 3) carrying single TET2 mutation. A total of 20,565 cells were clustered based on chromatin accessibility data and annotated by gene accessibility profiles. TET2 targeted sites were reliably genotyped in 81 (74-89) % of cells. TET2 mutations were highly enriched in myeloid cells, plasmacytoid dendritic cells (pDCs), and B cell progenitors. Within the hematopoietic stem and progenitor cell (HSPC) fraction, TET2-mutated HSPCs showed a higher accessibility to the binding motifs for transcription factors associated with myeloid differentiation (C/EBP family), lymphoid lineage priming (SPI1), and pDC differentiation (SPIB) compared with wild-type HSPCs. Compared with wild-type B cells, TET2-mutated B cells had a lower accessibility to the AP-1 family motif associated with B cell differentiation, including the BACH2 binding motif. To characterize the chromatin accessibility profiles of clones comprising multiple mutations, we next analyzed WBM cells from a CCUS patient with double TET2 mutations (TET2-D390fs and TET2-A1512V) and a ZRSR2 mutation. The ATAC-seq successfully profiled 3,725 cells, of which 2,468 (66.3 %) were analyzed for all three mutated loci, allowing for the reconstruction of clonal hierarchy. Linearly acquired, ZRSR2/TET2-D390fs double mutant and triple mutant clones were enriched in myeloid cells, pDCs and B cell progenitors, whereas they were depleted in T and plasma cells. In the ATAC-based pseudotime analysis, the fraction of ZRSR2/TET2-mutant and triple mutant clones decreased along B cell differentiation, suggesting the impaired B cell differentiation of these clones. Compared with wild-type B cells, ZRSR2/TET2-mutant B cells had lower ETS and POU family motif activity and the triple mutant B cells further lost the AP-1 family motif. In summary, we have developed a novel single cell analysis platform enabling high-throughput simultaneous genotyping and ATAC analysis with high accuracy for primary human samples. Applying this to the analysis of CH/CCUS samples, we demonstrated how epigenetic mutations disrupt the transcriptional mechanism, contributing to the development of CH/CCUS.

Azacitidine is a mainstay of therapy for myelodysplastic syndrome (MDS)-related diseases. The purpose of our study is to elucidate the effect of gene mutations on hematological response and overall survival (OS), particularly focusing on their posttreatment clone size. We enrolled a total of 449 patients with MDS or related myeloid neoplasms. They were analyzed for gene mutations in pretreatment (n = 449) and posttreatment (n = 289) bone marrow samples using targeted-capture sequencing to assess the impact of gene mutations and their posttreatment clone size on treatment outcomes. In Cox proportional hazard modeling, multihit TP53 mutation (hazard ratio [HR], 2.03; 95% confidence interval [CI], 1.42-2.91; P < .001), EZH2 mutation (HR, 1.71; 95% CI, 1.14-2.54; P = .009), and DDX41 mutation (HR, 0.33; 95% CI, 0.17-0.62; P < .001), together with age, high-risk karyotypes, low platelets, and high blast counts, independently predicted OS. Posttreatment clone size accounting for all drivers significantly correlated with International Working Group (IWG) response (P < .001, using trend test), except for that of DDX41-mutated clones, which did not predict IWG response. Combined, IWG response and posttreatment clone size further improved the prediction of the original model and even that of a recently proposed molecular prediction model, the molecular International Prognostic Scoring System (IPSS-M; c-index, 0.653 vs 0.688; P < .001, using likelihood ratio test). In conclusion, evaluation of posttreatment clone size, together with the pretreatment mutational profile as well as the IWG response play a role in better prognostication of azacitidine-treated patients with myelodysplasia.

Supplementary Figure from Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia

Abstract Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome, whole-exome, and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains and amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains and amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL. Significance: This study reveals the major role of gains, amplifications, and mutations of EPOR and JAK2 in the pathogenesis of pure erythroleukemia. Their frequent response to ruxolitinib in patient-derived xenograft and cell culture models highlights a possible therapeutic role of JAK2 inhibition for erythroleukemia with EPOR/JAK2-involving lesions. This article is highlighted in the In This Issue feature, p. 369

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML.

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background

Through intensive efforts of genome sequencing of myeloid malignancies, a comprehensive registry of driver mutations has been revealed, virtually providing us with a complete spectrum of driver mutations in these diseases. Importantly, there have been significant correlations between driver mutations, which suggests that some combinations of genetic events confer strong selective advantage on mutated stem cells. Next-generation sequencing technology have also revealed that clonal hematopoiesis is a common, age-related process in which a somatically mutated hematopoietic precursor gives rise to a genetically distinct subpopulation in the blood. Furthermore, novel germline mutations were identified, indicating that mutated stem cells appear long before myelodysplastic syndrome (MDS) presentation. Such founding mutations are thought to be acquired and positively selected in a well-organized manner to allow for expansion of the initiating clone to compromise normal hematopoiesis, ultimately giving rise to MDS and subsequent transformation to acute myeloid leukemia (AML) in many patients.

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.

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, p<0.0001) together with hematological improvements (HI-E: OR=2.32, p=0.011, HI-N: OR=2.29, p=0.038, HI-P: OR=1.88, p=0.039). Marrow CR (mCR), PR, SD with HI were also achieved in additional 71 (18%), 11 (3%), and 37 (9.6%) cases, respectively. However, 51% of the cases that achieved any response had disease progression with a median duration of only 313 days. Despite favorable initial response, TP53-mutation was still associated with dismal long-term outcomes (median OS 374 days versus 687 days in TP53-unmutated cases, p<0.0001), which is in compatible with a short duration of remission period (median 199 days for CR cases and median 182 days for mCR/PR cases with TP53 mutation) in the former group. Given distinct characteristics in both genetic and clinical features in TP53 mutated cases, we subsequently inquired for additional biomarkers predicting outcomes according to the mutation status of TP53. In the TP53-mutated cases, platelet counts larger than median value of 7.1×104/μL was the only factor that predicted better survival (P=0.02). In the TP53-unmutated cases, higher age (>72 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<0.001). Notably, those who received transplantation following AZA therapy (N=14) had a significantly prolonged survival with a median OS of 758 days for TP53-mutated cases, where TP53 mutations had disappeared after AZA treatment in six cases and decreased to <10% in VAF in other 4 patients. Clonal dynamics was evaluated by measuring change of copy number-adjusted variant allele frequency (aVAF) between pre- and post-AZA samples. Both post-AZA mutational burden (p<0.0001) and the size of reduction in aVAF during treatment (p<0.0001) significantly correlated with clinical response (trend test). 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.

Background Leukemic cell populations are highly heterogeneous in terms of both gene mutations and gene expression, which is shaped by acquisition of multiple mutations and expansion of adapted clone. This evolutional process is clinically important because it is observed in the contexts of treatment resistance and relapse as well as leukemic transformation, and molecular mechanisms involved in clonal selection can be exploited as a therapeutic target. Nevertheless, direct analysis of such mechanisms in patients' cells is hampered by technical difficulties to characterize both clonal structure and gene expression at a single-cell resolution. On this issue, we have recently developed a new method which enables simultaneously detection of mutations and whole transcriptome information at single-cell level by extensively modifying an existing single cell RNA-seq (Nakagawa et al. ASH abstract 2018). The aim of this study is to understand heterogeneity of clones and to clarify mechanisms behind clonal expansion in AML by longitudinal analysis using our novel single-cell sequencing platform. Results In order to estimate clone frequencies and select samples to be analyzed by single-cell sequencing, we first sequenced bulk bone marrow cells from patients with AML. Of interest, we found that AML samples frequently harbored multiple clones having different Ras pathway mutations, most frequently involving NRAS, which exhibited dynamic change in their clone size during the course of AML. These are interesting targets of the analysis of mechanism of clonal evolution of AML. Thus, three patients having multiple (n=3-5) Ras pathway mutations were investigated by sequencing their bone marrow Lin-, CD34+ cells using the newly established single-cell method, which successfully separated distinct clones having distinct mutations, where all of detected Ras pathway mutations were present in independent clones as expected. In order to examine these independent clones with Ras pathway mutations might show equal or heterogenous cellular phenotypes, proliferation or differentiation statuses as determined from transcriptome data was analyzed for all detected NRAS mutated clones. Among the NRAS mutated clones, some showed significant increase in proliferation-associated gene expression signature (calculated as proliferation score) compared with NRAS wild type clones, and no NRAS mutated clones showed decrease of the score, which is consistent with pro-proliferative function of Ras pathway. Interestingly, such increase in proliferation showed considerable heterogeneity among clones, where some NRAS mutated clones showed greatly increased proliferation scores compared to other NRAS mutated clones. Differentiation statuses of NRAS clones also showed heterogeneity among clones. In order to examine whether this inter-clone proliferation difference correlates with clone dynamics, we then analyzed longitudinal bone marrow samples for a patient who showed different proliferation between clones. The NRAS mutated clone with highly increased proliferation compared with wild type clone (NRAS p.G12S) had undergone rapid expansion in 3 months (cell frequency 0.08 to 0.74) in spite of continuous azacitidine treatment, while the NRAS mutated clone with less increase in proliferation (NRAS p.G12D) had showed regression (cell frequency 0.72 to 0.14). To investigate the mechanism of this therapy-resistant clonal expansion, we compared transcriptome data of these clones. Unlike the regressed clone, the expanded clone uniquely exhibited increase in expression of genes in PI3K/AKT pathway and unfolded protein response (UPR) pathway, one of cellular stress response pathway. UPR is recently reported to responsible for the promoted survival and competitive advantage in mouse hematopoietic stem cells with Nras mutations (Liu et al. Nat. Cell Biol. 2019). Our data suggest that the enhanced UPR pathway contributes to clonal expansion also in human AML with Ras pathway mutations. Conclusions Using a newly developed single-cell sequencing platform, we have successfully characterized gene expression profiles associated with clonal evolution of AML with Ras pathway mutations. Simultaneous measurement of both mutations and transcriptomes at a single-cell level will help understand the mechanism of clonal evolution of AML. Disclosures Inagaki: Sumitomo Dainippon Pharma Co., Ltd.: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Yoda:Chordia Therapeutics Inc.: Research Funding. Ogawa:RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy.

Recent advances in single-cell sequencing (sc-Seq) technologies have enabled high-throughput transcriptome analysis in thousands of cells to understand the heterogeneity among cancer populations in terms of genome-wide gene expression. However, its application to the analysis of clonal evolution of cancer populations is largely limited by the lack of an efficient sc-Seq platform that allows for accurate detection of gene mutations at the same time with transcriptome analysis. The major challenge here is a frequent allele dropout of just two copies per single cell, which results in an inaccurate genotype assignment for many cells, preventing identification of relevant genotype-phenotype correlations. To overcome this, we developed a novel sc-Seq platform (scMutSeq) that allows for precise determination of both genotype and genome-wide gene expression simultaneously with negligible allele dropouts, on the basis of the Fluidigm C1 Single-Cell mRNA Seq HT system and applied it to the analysis of clonal evolution and intratumor heterogeneity of myelodysplastic syndromes (MDS) characterized by frequent clonal evolution to acute amyloid leukemia (AML).

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. 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.

Background The genetic architecture of coronary artery disease has not been fully elucidated, especially in Asian countries. Moyamoya disease is a progressive cerebrovascular disease that is reported to be complicated by coronary artery disease. Because most Japanese patients with moyamoya disease carry the p.R4810K variant of the ring finger 213 gene (RNF213), this may also be a risk factor for coronary artery disease; however, this possibility has never been tested. Methods and results We genotyped the RNF213 p.R4810K variant in 956 coronary artery disease patients and 716 controls and tested the association between p.R4810K and coronary artery disease. We also validated the association in an independent population of 311 coronary artery disease patients and 494 controls. In the replication study, the p.R4810K genotypes were imputed from genome-wide genotyping data based on the 1000 Genomes Project. We used multivariate logistic regression analyses to adjust for well-known risk factors such as dyslipidemia and smoking habits. In the primary study population, the frequency of the minor variant allele was significantly higher in patients with coronary artery disease than in controls (2.04% vs. 0.98%), with an odds ratio of 2.11 (p = 0.017). Under a dominant model, after adjustment for risk factors, the association remained significant, with an odds ratio of 2.90 (95% confidence interval: 1.37–6.61; p = 0.005). In the replication study, the association was significant after adjustment for age and sex (odds ratio = 4.99; 95% confidence interval: 1.16–21.53; p = 0.031), although it did not reach statistical significance when further adjusted for risk factors (odds ratio = 3.82; 95% confidence interval: 0.87–16.77; p = 0.076). Conclusions The RNF213 p.R4810K variant appears to be significantly associated with coronary artery disease in the Japanese population.