Kurzfassung: Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for patients with myelodysplastic syndromes (MDS), whose benefit, however, is frequently offset by accompanying mortality and morbidity, underscoring the importance of accurate prognostication before the therapeutic choice. For this purpose, several systems, such as the International Prognostic Scoring System (IPSS), are being successfully applied to clinics, and recent genome profiling studies indicate that molecular diagnostics can further improve the prediction. Nevertheless, existing systems are based on the observation from those patients who were untreated or only supportively treated and therefore, may not successfully be applied to the prognostication of the patients who are actually treated by HSCT. Methods: We analyzed patients with MDS (N = 719) from a cohort of Japan Marrow Donor Program (JMDP) who were treated by unrelated HSCT between 2006 and 2013. Peripheral blood DNA was subjected to targeted deep sequencing in 68 major driver genes for the detection of both somatic mutations and copy number variations (CNVs) with accurate determination of their allelic burdens. Results: The median age at HSCT and observation period were 53 years old (20-66) and 372 days (2-3001), respectively. At the diagnosis, 63, 203, 163 and 65 patients have low, intermediate-1, intermediate-2 and high risk classified on the basis of IPSS, respectively (IPSS data was not available for 250 patients). The median time from diagnosis to HSCT was 274 (9-10900) days. Mutations were observed in 75% of the patients, of which TP53 was most frequently mutated (14.3%), followed by U2AF1 (13.2%), RUNX1 (12.2%), ASXL1 (11.0%) and DNMT3A (9.3%). The mean number of mutations was 2.1 per patient and the mean allelic burden was 23.4%. To evaluate karyotyping we combined metaphase cytogenetics and copy number variations using targeted sequencing data. Complex karyotype, chromosome 7 anomaly, deletion 5q, and deletion 20q were observed in 174 (24.4%), 173 (24.3%), 91 (12.8%), and 50 (7.0%) of the patients, respectively. Combined, 86.6% of the patients had one or more genetic lesions. Patients with one or more mutations or CNVs showed unfavorable overall survival (Hazard Ratio (HR) 2.46, P = 2.12 x 10-5). Univariate analysis for each gene identified mutations in TP53 (HR 2.85, P 〈 2.0 x 10-16), NRAS (HR 1.90, P = 5.4 x 10-4), ETV6 (HR 1.54, P = 0.029), CBL (HR 2.25, P = 5.3 x10-5), EZH2 (HR 1.74, P = 0.014), KRAS (HR 2.01, P = 2.0 x 10-3), U2AF2 (HR 1.97, P = 0.027), JARID2 (HR 2.09, P = 0.039), and RIT1 (HR 2.16, P = 0.023) as the unfavorable factors for the overall survival. Besides, mutations in PRPF8 had a favorable effect on overall survival (HR 0.50, P = 0.029). Then, we performed multivariate analysis with stepwise model selection of these significant mutations and clinical parameters. Mutations in TP53 (HR 2.31, P=0.015), and ETV6 (HR 2.57, P=0.015) remained significant together with complex karyotype (HR 2.15, P = 0.0063), grade of acute graft versus host disease (GVHD) (Grade I or II: HR 1.95, P = 0.011, Grade III or IV: HR 4.18, P = 7.94 x 10-5), and the number of red blood cell transfusion received before HSCT ( 〉 =10 times: HR 2.64, P = 0.027). Next, we analyzed the impact of mutations on relapse in cases who achieved complete response after HSCT (N = 423 (58.8%)). Patients with mutations in one or more genetic lesions showed unfavorable relapse free survival (HR 2.27, P = 1.65 x 10-4). Univariate analysis for each gene revealed mutations in TP53 (HR 3.09, P = 9.5 x 10-16), NRAS (HR 2.21, P = 0.0019), ETV6 (HR 1.90, P = 0.012), PRPF8 (HR 0.40, P = 0.046), and WT1 (HR 2.24, P = 0.013) were significant for the relapse free survival. Multivariate analysis and stepwise model selection identified ETV6 (HR 2.98, P = 0.011), WT1 (HR 4.01, P = 0.014), complex karyotype (HR 2.39, P = 0.0083), IPSS High (HR 6.22, P = 0.0053), and Grade III or IV acute GVHD (HR 2.91, P = 0.0071) as unfavorable factors. Conclusions: This large study of MDS cases treated by unrelated HSCT demonstrated that somatic mutations of several driver genes were novel prognostic factors for overall and relapse free survival. These genetic factors were independent of well-known prognostic makers, and therefore could be used to better guide therapy for MDS patients. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Introduction Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin's lymphoma. Although most cases (~95%) show histology of diffuse large B-cell lymphomas (DLBCLs), PCNSL shows very different biological and clinical characteristics from systemic DLBCL. Nevertheless, our knowledge about the molecular pathogenesis of PCNSL and genetic differences between both lymphomas are still incomplete. Method To obtain a comprehensive view of the genetic alterations, including mutations in non-coding regions as well as structural variants (SVs), we performed whole-genome sequencing (WGS) of 22 PCNSL cases. Subsequently, to unravel the genetic differences between PCNSL and systemic DLBCL, we re-analyzed WGS data from systemic DLBCL cases (N = 47) generated by the Cancer Genome Atlas Network (TCGA) and Cancer Genome Characterization Initiative (CGCI) using our in-house pipeline. The mean depth of WGS for tumor samples were 49X and 37X for PCNSL and DLBCL cases, respectively. Whole-exome sequencing (WES) was also performed for an additional 37 PCNSL cases to reliably capture driver alterations and also to analyze mutational signatures in PCNSL, which were compared to those obtained from the WES data for DLBCL from TCGA (N = 49). Results WGS identified 10.5 and 5.6 mutations per mega-base on average in PCNSL and DLBCL, respectively. We first explored the density of somatic mutations and identified 64 and 33 genomic loci showing significantly high mutation densities in PCNSL and DLBCL, respectively. In PCNSL, most of these loci corresponded to known targets of somatic hypermutations (SHMs) induced by activation-induced cytidine deaminase (AID), including those for IG genes (IGK, IGH and IGL), BCL6, and PIM1, as well as those for known driver genes, such as MYD88 and CD79B. Although most of the hypermutated regions were overlapped between PCNSL and DLBCL, some regions were differentially affected by hypermutations between both lymphoma types. For example, BCL2 and SGK1 loci were frequently affected by SHMs in germinal center B-cell (GCB) DLBCL, while not in PCNSL. In terms of non-coding driver mutations, we identified frequent mutations in a PAX5 enhancer region in 8/22 (36%) of PCNSL and 18/47 (38%) of DLBCL cases. SVs were common in both lymphoma types, where 104 (PCNSL) and 57 (DLBCL) SVs were detected per sample. SV clusters were identified in 34 (PCNSL) and 13 (DLBCL) regions, of which several clusters were commonly seen in both PCNSL and DLBCL, and included IG loci, BCL6, FHIT, TOX and CDKN2A. In PCNSL, SVs were clustered within the loci for known targets of SHMs, such as BCL6, BTG2 and PIM1. As was the case with somatic mutations, the SV cluster corresponding to BCL2 was only seen in DLBCL. We then analyzed these clustered breakpoints for their proximity to known sequence motifs targeted by AID (CpG and WGCW). Breakpoints of SVs found in the targets of SHMs, including PIM1, BCL6, BTG2 and BCL2, showed an enrichment at or near the CpG, supporting the involvement of AID in the generation of these SVs. By analyzing these SV clusters, we identified several novel driver genes in PCNSL. For example, WGS and WES identified an enrichment of breakpoints of deletions (7/22) and loss-of-function mutations (6/37) in GRB2, strongly indicating its tumor suppressor role in PCNSL. We also analyzed pentanucleotide signatures of mutations in coding sequences detected by WES of PCNSL and DLBCL, taking into consideration the two adjacent bases 3' and 5' of the substitutions as well as transcription strand biases. Two predominant mutational signatures were identified in PCNSL: the AID signature characterized by C 〉 T mutations within the WRCY motif targeted by SHMs and the age-related signature involving C 〉 T transition at CpG dinucleotides. For DLBCL, an additional signature (signature 17 according to Alexandrov et al.) was detected as well, which had been reported in DLBCL with an unknown mechanistic basis. Conclusions Comprehensive genomic analyses of a large cohort of PCNSL and DLBCL cases have revealed the major targets of somatic mutations and SVs, including novel driver genes. In both PCNSL and systemic DLBCL, an enhanced AID activity is thought to be associated with generation of both SHMs and SVs, although the activity and targets of AID seem to substantially differ between both lymphoma types, suggesting distinct pathogenesis therein. Disclosures Kataoka: Boehringer Ingelheim: Honoraria; Yakult: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Kurzfassung: 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.

Kurzfassung: 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.

Kurzfassung: Introduction Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma, of which approximately 95% are diffuse large B-cell lymphomas (DLBCLs). Despite the substantial development of intensive chemotherapy during the past two decades, overall clinical outcome of PCNSL has been poorly improved especially in elderly and so has been our knowledge about the molecular pathogenesis of PCNSL, in terms of driver alterations that are relevant to the development of PCNSL. Method To delineate the genetic basis of PCNSL pathogenesis, we performed a comprehensive genetic study. We first analyzed paired tumor/normal DNA from 35 PCNSL cases by whole-exome sequencing (WES). Significantly mutated genes identified by WES and previously known mutational targets in PCNSL and systemic DLBCL were further screened for mutations using SureSelect-based targeted deep sequencing (Agilent) in an extended cohort of PCNSL cases (N = 90). Copy number alterations (CNAs) have been also investigated using SNP array-karyotyping (N =54). We also analyzed WES and SNP array data of systemic DLBCL cases (N = 49) generated by the Cancer Genome Atlas Network (TCGA) to unravel the genetic difference between PCNSL and systemic DLBCL. Results The mean number of nonsynonymous mutations identified by WES was 183 per sample, which was comparable to the figure in systemic DLBCL and characterized by frequent somatic hypermutations (SHMs) involving non-Ig genes. A higher representation of C 〉 T transition involving CpG dinucleotides and hotspot mutations within the WRCY motif targeted by SHM further suggested the involvement of activation-induced cytidine deaminase (AID) in the pathogenesis of PCNSL. We found 12 genes significantly mutated in PCNSL (q 〈 0.1), including MYD88, PIM1, HLA-A, TMEM30A, B2M, PRDM1, UBE2A, HIST1H1C, as well as several previously unreported mutational targets in systemic DLBCL or PCNSL, such as SETD1B, GRB2, ITPKB, EIF4A2. Copy number analysis identified recurrent genomic segments affected by focal deletions (N = 27) and amplifications (N = 10), most of which included driver genes targeted by recurrent somatic mutations or known targets of focal CNAs such as CDKN2A and FHIT. Subsequent targeted sequencing finally identified a total of 107 significantly mutated genes, of which 43 were thought to be targeted by SHM according to their mutational signature and genomic distribution. Most cases with PCNSL (98%) had mutations and CNAs involving genes that are relevant to constitutive NF-KB/Toll-like receptor (TLR)/BCR activity, including those in MYD88 (80%), CD79B/A (60%), CARD11 (18%), TNFAIP3 (26%), GRB2 (24%) and ITPKB (23%). Genetic alterations implicated in escape from immunosurveillance were also frequently identified in as many as 76% of cases. Mutations of HLA-B (64%), HLA-A (36%), HLA-C (28%), B2M (14%) and CD58 (12%) were commonly detected in addition to CNAs in 6p21.32 (HLA class II), 1p13.1 (CD58) and 15q15.2 (B2M), suggesting the importance of immune escape in the pathogenesis of PCNSL. SHMs were also seen in most cases (98%), which affected not only known targets of AID including PIM1, IGLL5 and BTG2 but also previously unreported genes involved in cell proliferation, apoptosis, or B cell development. The pattern of frequently mutated genes in PNCSL was more uniform compared with that in systemic DLBCL, and similar to that found in the activated B cell subtype of DLBCL (ABC-DLBCL), which was in accordance with the previous report of immunophenotypic analysis of PCNSL. On the other hand, mutations of HLA class I genes (HLA-B, HLA-A) were more frequently mutated in PCNSL compared with ABC-type DLBCL. Conclusion WES, SNP array karyotyping and follow-up targeted sequencing of a large cohort of PCNSL cases revealed the genetic landscape of PCNSL, which were more homogeneous than that of systemic DLBCL, and thought to be involved in activation of constitutive NF-KB/TLR/BCR signaling, escape from immunosurveillance, as well as highly frequent SHMs. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Background Copy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms. Materials and Methods We enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%. Results To obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions. Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy. Conclusion We obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Kurzfassung: Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders of hematopoietic stem cells, characterized by defective hematopoiesis and dysplasia of multiple blood lineages. Patients with MDS could achieve complete remission only by allogeneic hematopoietic stem cell transplantation (HSCT). However, because of its high mortality and morbidity, long-term survival is accomplished only in the half of the patients, underscoring the importance of accurate prognostication before the therapeutic choice. For this purpose, several systems, such as the International Prognostic Scoring System, are being successfully applied to predicting patients' clinical outcomes. Advanced molecular diagnostics of recent years might further improve the prediction. Nevertheless, originally established based on the data from patients who are untreated or only supportively treated, existing systems may not always be applied properly to the prediction of outcomes of the patients who are treated by HSCT. Methods: We enrolled 790 patients with MDS who were treated by unrelated bone marrow transplantation between 2006 and 2013 through the Japan Marrow Donor Program. Oncogenic variants and copy number alterations were identified by targeted-capture sequencing of peripheral blood-derived DNA using RNA baits designed for 69 known or putative driver genes in myeloid neoplasms and 1,674 single nucleotide polymorphisms. Results: The median age at HSCT and observation period were 51 years old (16-66) and 1106 days (48-6018), respectively. At the time of transplant, 29%, 34%, 24%, and 5.2% of the cases were diagnosed as low-risk MDS, high-risk MDS, secondary acute myeloid leukemia, and myelodysplastic / myeloproliferative neoplasms, respectively, while the disease subtype was unknown for the remaining 8.2% of the cases. Mutations were observed in 73% of the patients, where U2AF1 was most frequently mutated (13.8%), followed by RUNX1 (12.9%), ASXL1 (12.8%), TP53 (12.4%), and NRAS (7.1%). The mean number of mutations was 2.0 per patient with a mean allelic burden of 43.8%. Sequencing data were successfully used for sensitive detection of CNVs and copy-neutral loss-of-heterozygosity (LOH) (or uniparental disomy; UPD). Among the most frequent lesions were 7q LOH, complex karyotype-like CNVs as defined by 3 or more CNVs excluding UPD in targeted sequencing), 5q LOH, trisomy 8, and 17p LOH, observed in 15.5%, 12.9%, 10.1%, 7.0%, and 6.8% of the patients, respectively. Mutations in TP53, CBL, and EZH2 significantly co-occurred with LOH in 17p, 11q, and 7q, with odds ratios of 190, 75.5, and 11.7, respectively. On the basis of these findings, we combined frequently identified LOH lesions with associated mutations for further analyses of survival. In univariate analysis of overall survival (OS), 9 lesions were significantly associated with shorter OS; TP53 mutation and/or 17p LOH (TP53 / 17p LOH) (HR 2.76, P = 1.6 x 10-14), CSNK1A1 / 5q LOH (HR 2.66, P = 2.13 x 10-12), CBL / 11q LOH (HR 2.42, P = 3.88 x 10-7), EZH2 / 7q LOH (HR 2.29, P = 1.46 x 10-12), NRAS mutations (HR 1.86, P = 2.0 x10-4), ETV6 / 12p LOH (HR 1.83, P = 2.84 x 10-5), 1q gain (HR 1.73, P = 0.0037), 20p LOH (HR 1.57, P = 0.030), and FLT3 mutations (HR 1.53, P = 0.027). The number of these unfavorable lesions significantly correlated with OS (P=8.9 x 10-16). Specifically, those with at least one mutation showed a significantly shorter OS, compared to those with none of these mutations (HR 2.54, P 〈 2.0 x 10-16). TP53 / 17p LOH was the most unfavorable among the 9 lesions by multivariate analysis (HR 1.97, P=1.6 x 10-14). Multivariate analysis with clinical factors revealed that the presence of at least 1 of the 9 lesions was independently associated with poor OS (HR 2.05, P 〈 2.0 x 10-16), together with well-known clinical factors negatively affecting OS, including red blood cell transfusion before HSCT (HR 1.93, P=0.0036), 3 or more grade of the performance status at HSCT (HR 1.92, P=1.6 x 10-4), and older age (HR 1.69, P=2.0 x 10-4). The presence of at least one lesion negatively affected OS irrespective of the presence of complex karyotype-like CNVs. Conclusions: The present study highlights the clinical significance of somatic mutations and CNVs in MDS cases treated by HSCT. Our findings suggest that the novel set of lesions identified in this study could be successfully used for the prediction of outcome in MDS in the setting of stem cell transplantation. Disclosures Kataoka: Yakult: Honoraria; Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria. Kanda:Otsuka Pharmaceutical: Honoraria, Research Funding. Makishima:The Yasuda Medical Foundation: Research Funding. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Kurzfassung: ATL subtypes are further classified into molecularly distinct subsets with different prognosis by genetic profiling. PD-L1 amplifications are a strong genetic predictor for worse outcome in both aggressive and indolent ATL.

Kurzfassung: 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.