Abstract: Background: Acquired aplastic anemia (AA), the prototypical bone marrow failure syndrome, is inferred to result from immune-mediated destruction of hematopoietic progenitors, as most patients respond to immunosuppressive therapies. Clonal hematopoiesis in AA is evident in the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells in as many as half of patients and by identification of uniparental disomies involving 6p (6pUPD) chromosome in 13% of cases. In addition, "clonal transformation", as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) is a serious long-term complication in 10-15% AA patients. Methods: We performed targeted deep sequencing and SNP array-based copy number (CN) analysis of peripheral blood- or granulocyte-derived DNA from 439 patients with AA (280 from US and 159 from Japanese cohorts) for a panel of 103 candidate genes, chosen because they are known to be frequently mutated in myeloid neoplasms. Germline DNA was available for 288 out of 439 patients and was used to confirm the somatic origin of mutations. Whole exome sequencing (WES) was performed in 52 cases. Where serial samples were available, the chronology of detected mutations was also investigated. Results: Targeted deep sequencing provided highly concordant results between the US and Japanese cohorts; approximately one third of AA patients had mutations in genes commonly affected in myeloid neoplasms, and about one third of patients in whom mutations were identified had multiple mutations. Multi-lineage involvement of mutations was confirmed in 6 cases using flow-sorted bone marrow samples. However, compared to myeloid neoplasms, mutations in AA were at much lower variant allele frequencies (VAFs) ( 〈 10% on average) and most frequently involved 5 genes: PIGA, BCOR/BCORL1, DNMT3A and ASXL1 (Fig.1). Although CN abnormalities were rare, about 13% of AA patients in both cohorts showed 6pUPD. Combined, clonal hematopoiesis was detected in as many as 46.5% and 47.8% of US and Japanese patients, respectively. We focused efforts on the large NIH cohort, due to accessible serial samples and well characterized clinical phenotypes at many time points. For 46 cases for which diagnostic samples were available, mutations were detected from at the time of diagnosis but at very low VAFs. The size of DNMT3A or ASXL1 mutated clones tended to increase over time, regardless of the emergence of chromosomal anomalies or blasts, whereas that of BCOR or PIGA mutated clones was more likely to decrease or remain stable. In both patient cohorts, presence of an acquired mutation was associated with older age, but did not correlate with response to immunosuppressive therapy (IST) or overall survival (OS). Mutations in PIGA and BCOR/BCORL1 were more common in AA than in MDS/AML and when combined, were associated with favorable OS (favorable mutations) (P = 0.044). Conversely, 17 high-risk mutations were extracted to predict poor OS (Fig. 2), which combined with favorable mutations, could be used to stratify AA patients with regard to OS (P = 0.0025). WES allowed capture of more mutations and better characterization of clonal hematopoiesis: more than 60% of AA patients had somatic mutations by combined targeted and whole exome sequencing. In 36 cases, WES was performed for all available serial samples, which enabled comprehensive monitoring of the dynamic chronological behavior of hematopoietic clones for as long as a decade after diagnosis. In many cases, clonal hematopoiesis developed gradually and was unrelated to the severity of cytopenias or to clinical evolution to abnormal cytogenetics, marrow dysplasia, and leukemia. Acquisition of new mutations within founder clones and subsequent selection shaped highly complex clonal structures in some cases (Fig. 3). The emergence of clonal hematopoiesis predated the development of MDS or leukemic transformation, with clones often detectable at time of diagnosis. Conclusions: Clonal hematopoiesis in AA was prevalent, associated in about half of cases with mutations in genes recurrently mutated in myeloid neoplasms. The highly biased set of mutated genes associated with clonal hematopoiesis in AA is evidence for Darwinian selection of particular cell clones under in the bone marrow failure environment. Mutations could be used to better predict prognosis of AA patients. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Abstract: 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 〈 0.001). Other independent prognostic factors included the International Prognostic Scoring System (IPSS) score and the mutational category (i.e., Group-I, -II, and -III) for PFS, while the presence of complex karyotypes, together with IPSS score, Group-I, -7/del(7q), age, and del(20q) were among the negative predictors of OS. 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.

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

Abstract: Background: DNA hypomethylating agents, such as 5-azacitidine (5-aza) and decitabine, comprise the current standard in therapy for patients with high-risk myelodysplastic syndromes (MDS), with dramatic responses in some patients. However, the responses are poorly predictable and their impact on clonal dynamics has not been fully elucidated. Patients and Methods: We enrolled a total of 119 patients with high-risk MDS who were treated with 5-aza . Bone marrow samples were collected before (n = 71) and both before and after (n = 48) treatment and analyzed by targeted-capture sequencing using RNA baits designed for 67 known or putative driver genes in myeloid neoplasms and 1,674 single nucleotide polymorphisms, which enabled detection of both mutations and copy number alterations on the same platform. In 9 of the 48 patients, pre- and post-therapy samples were further analyzed by whole exome sequencing (WES). Results: Average number of driver mutations before 5-aza was 2.8 per patient and 107 (90%) patients had multiple mutations. Most frequently mutated were TP53 (27%), followed by RUNX1, TET2, DNMT3A, and ASXL1. Reflecting high-risk disease subtypes of the subjects, splicing factor mutations were relatively rare (29 %) in the current cohort. Chromosomal abnormalities were identified in 65 (55%) patients, where 7q- and /or 5q- were the most frequent. Among 48 patients with serially collected samples, 46 had one or more mutations, enabling an evaluation of clone dynamics. In total 163 and 146 mutations were detected before and after treatment, respectively. About two thirds (110/163) of the mutations before 5-aza remained detectable after treatment. By contrast, the remaining one third showed a dynamic clonal behavior; 36 mutations in 22 cases were newly acquired, whereas 53 in 28 cases disappeared. Among those newly acquired, most frequently observed were mutations in STAG2 and EP300 (n = 3), of which STAG2 (7 cases) also represented the most frequent targets of disappeared mutations after treatment. In WES in 9 patients, a total of 112 mutations were identified either before or after 5-aza treatment with a mean of 10.4 or 8.9 mutations per sample, respectively. Among these, 63 were found at both pre- and post-therapy samples, whereas 17 and 32 mutations were newly acquired or disappeared during treatment, Given that only 4 newly acquired and 8 lost mutations had been detected by targeted-capture sequencing, respectively, WES enabled more sensitive detection of alternation of clones during 5-aza treatment, which were demonstrated in 8 (89%) subjects, rather than 5 (56%) in targeted-capture sequencing. Clinical outcomes have been reported for 22 patients as of the time of abstract submission; 5 achieved complete remission (CR), 9 stable disease (SD), and 5 progressive disease (PD). Alteration in clone size was frequently associated with clinical response. The size of dominant clones significantly decreased in 4 of 5 cases with CR, whereas stable or increased in 12 of 14 patients with SD or PD. In patients with SD or PD, acquisition of new mutations was common (10/14) during 5-aza treatment and potentially implicated in the resistance to 5-aza-treatment. Of interest, newly acquired mutations were also found in 2 CR samples, albeit at low allele frequency, even though the clone size of dominant clones was substantially reduced, suggesting the evolution of alternative MDS subclones or expansion of preexisting non-leukemic hematopoietic clone. Although CR was achieved in 3 of 6 patients with TP53 mutations, the TP53-mutationsdid not totally disappeared but were still detectable in CR samples in 2 cases, suggesting that TP53 mutated clones have not been completely eradicated by 5-aza treatment. Conclusion: Our study successfully depicted the structure of clones and their dynamics in high-risk MDS on 5-aza treatment. Alteration in the size of the dominant clones was frequently associated with a clinical response. Clonal evolution was common even in patients who achieved CR. Tracking the mutations in MDS patients during 5-aza treatment provides the opportunity to detect clones resistant to 5-aza and might be used to guide 5-aza therapy. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria. Kiyoi:Celgene Corporation: Consultancy; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding. Naoe:Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Astellas Pharma Inc.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Amgen Astellas BioPharma K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene K.K.: Honoraria, Research Funding; CMIC Co., Ltd.: Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Pfizer Inc.: Research Funding. Makishima:The Yasuda Medical Foundation: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

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

Abstract: Acute myeloid leukaemia ( AML ) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT , NPM 1 , CEBPA and FLT 3 in both adult and paediatric AML . In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH 1/2 and DNMT 3A ) in adult AML . In this study, whole‐exome sequencing ( WES ) of 22 paediatric AML patients revealed mutations in components of the cohesin complex ( RAD 21 and SMC 3 ), BCORL 1 and ASXL 2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML . Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [ STAG 2 , RAD 21 and SMC 3 in 17 patients (8·3%)], epigenetic regulators [ ASXL 1/ ASXL 2 in 17 patients (8·3%), BCOR / BCORL 1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML . Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.

Abstract: Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602