Abstract: TP53 and RAS-pathway mutations predict very poor survival, when seen with CK and MDS/MPNs, respectively. For patients with mutated TP53 or CK alone, long-term survival could be obtained with stem cell transplantation.

Abstract: 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.

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

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

Abstract: 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.