Kurzfassung: Background: Recent advance in genetic analysis has revealed that many mutations are associated with the development, progression and/or prognosis of core-binding factor acute myeloid leukemia (CBF-AML). Although KIT mutation is the most frequently identified in CBF-AML, its prognostic relevance remains controversial. We conducted the prospective, multicenter cooperative study (JALSG CBF-AML209-KIT, UMIN Clinical Trials Registry UMIN000003434, http://www.umin.ac.jp/ctr/) to evaluate the prognostic impact of KIT mutation, the incidence and clinical relevance of the other gene mutations and prognostic impact of the minimal residual disease (MRD) in CBF-AML. Methods: A total of 199 patients 16 to 64 years of age with newly diagnosed de novo AML were enrolled in this study if they had a RUNX1-RUNX1T1 or CBFB-MYH11 chimeric transcript and achieved complete remission within 2 courses of the standard induction therapies consisting of cytarabine and either daunorubicin or idarubicin. All patients were to be received 3 courses of high-dose cytarabine therapy (2 g/m2 by 3-hour infusion every 12 hours for 5 days) and no further chemotherapy until relapse. MRD level was evaluated in BM after the completion of the 3-course of consolidation therapy by the quantitation of RUNX1-RUNX1T or CBFB-MYH11 transcript in 112 patients. Target sequencing of 56 genes frequently identified in myeloid malignancies including exons 8, 10, 11 and 17 of the KIT gene were analyzed using the preserved DNA extracted from AML cells at diagnosis. Results: A total of 68 KIT mutations were identified in 63 of 199 patients (31.7%); 42 of 132 (31.8%) and 21 of 67 (31.3%) patients with RUNX1-RUNX1T1 and CBFB-MYH11, respectively. Mutation in exon 17 was the most frequently identified (73.5%), followed by in exon 8 (20.6%) and in exon 10-11 (5.9%). Mutation in exon 8 was more frequent in AML with CBFB-MYH11 (37.5%) than that with RUNX1-RUNX1T1 (11.4%, P=0.014). Although mutation at N822 residue in exon 17 was identified in 13/44 (29.5%) KIT mutations of the patients with RUNX1-RUNX1T1, no patient with CBFB-MYH11 had this mutation (P=0.008); however, mutation at the D816 residue was equally identified in patients with RUNX1-RUNX1T1 (21/44, 47.7%) and CBFB-MYH11 (13/24, 54.1%). The median BM blast percentage of the KIT mutation positive-patients (73.5%) was significantly higher than that of negative-patients (53.8%, P 〈 0.0001). The median follow-up time was 1556 days (range, 356 - 2453 days). There was no significant difference in RFS and OS between the patients with RUNX1-RUNX1T1 and CBFB-MYH11. The 2-year RFS rate was 67.1% in KIT mutation negative-patients and 48.6% in positive-patients (P=0.0033). The 2-year OS rate was 91.0% in KIT mutation negative-patients and 74.6% in positive-patients (P=0.0005). In the patients with CBFB-MYH11, KIT mutations did not affect either RFS or OS. However, in the patients with RUNX1-RUNX1T1, KIT mutations had a strong impact both on 2-year RFS (72.8% vs. 39.5%, P 〈 0.0001) and OS rates (89.7% vs. 63.5%, P 〈 0.0001) in KIT mutation negative- and positive-patients, respectively. Among 3-types of KIT mutations, only the mutation in exon 17 had a worse prognostic impact on CBF-AML, which was also observed in the patients with RUNX1-RUNX1T1 (P 〈 0.001), but not in those with CBFB-MYH11 (P=0.724). Furthermore, mutations at the D816 and N822 residues showed a worse prognostic impact (P 〈 0.0001). In addition to KIT mutations, NRAS (21.7%), FLT3 (12.1%) and ASXL2 (12.0%) genes were frequently identified in CBF-AML. ASXL1 mutation was a favorable factor for RFS in RUNX1-RUNX1T1 and NRAS mutation was a poor prognostic factor for RFS in CBFB-MYH11. The 2-year RFS rate of the patients without MRD was significantly higher than that of those with MRD (81.3% vs. 55.4%, P=0.0075). Notably, the presence of the MRD was associated with worse RFS in the patients with CBFB-MYH11 (P=0.0144), but not in those with RUNX1-RUNX1T1 (P=0.1018). Conclusions: This large-scale prospective study demonstrated that KIT mutation has an adverse effect for OS and RFS only on AML with RUNX1-RUNX1T1 but not on AML with CBFB-MYH11. Notably, we demonstrated that only mutations in the exon 17 of KIT gene had an adverse effect both on the RFS and OS of the patients with RUNX1-RUNX1T1, and the presence of MRD was a poor factor for RFS in AML with CBFB-MYH11. Figure 1 Figure 1. Disclosures Sawa: Celgene Corporation: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis International AG: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Mundipharma K.K.: Honoraria. Dobashi:Pfizer Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Celgene Co.: Research Funding; Sysmex Co.: Research Funding. Asou:Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Yakult Honsha Co., Ltd.: Speakers Bureau; SRL Inc.: Consultancy; Eisai Co., Ltd.: Research Funding; Asahi Kasei Pharma Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Pfizer Japan Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding. Kiyoi:Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Bristol-Myers Squibb: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; FUJIFILM Corporation: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Kurzfassung: KIT exon 17 mutation is a poor prognostic factor in AML patients with RUNX1-RUNX1T1, but not in those with CBFB-MYH11. NRAS mutation is a poor prognostic factor in AML patients with CBFB-MYH11.

Kurzfassung: 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.

Kurzfassung: DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the 〉 480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with 〉 10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing 〉 20% difference in mean methylation levels between responders and non-responders (q 〈 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.