DNMT3A R882 Mutations Research Articles

A rare case of acute myeloid leukemia with ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) fusion gene in an elderly patient

Abstract Introduction. We report one elderly patient diagnosed with a rare subtype of acute myeloid leukemia (AML) and also with a very rare fusion gene involving ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) genes. Material and methods. Clinical examination and routine analysis were performed including peripheral blood smear, immunophenotyping of the peripheral blood by flow cytometry and several molecular analyses. Results. Peripheral blood smear showed 80% blasts with round and some with convoluted nuclei, with basophilic cytoplasm, identified as monoblast and the majority of cells as promonocytes. Peripheral blood immunophenotyping was consistent with monocytic differentiation. Molecular analysis was negative for FLT3 ITD, FLT3 D835, NPM1, and DNMT3A R882 mutations. Multiplex ligation-dependent probe amplification revealed no copy number aberration. Ligation-dependent reverse transcription polymerase chain reaction (LD-RT-PCR) analysis identified the presence of one gene fusion between ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) genes. The patient had no significant comorbidities, the renal function was normal and Eastern Cooperative Oncology Group performance status was 2 at diagnosis and 1 after treatment. She was treated with decitabine. She became transfusion independent and a reduction of the number of blasts was obtained. Conclusions. The outcome of our AML patient was favorable but other patients with fusion genes involving ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) should be reported, contributing to a better characterization of the disease, to monitor the minimal residual disease and in the end to more targeted treatment options. LD-RT-PCR represent a valuable multiplex technique for fusion gene analysis.

Dissociation of the DNMT3A-HDAC1 Repressor Complex Induces PD-L1 Expression

Mutations of DNMT3A are one the most frequently observed alterations in AML patients. The DNMT3A R882 mutation appears to confer a dominant-negative loss-of function effect and changes the DNA binding preference according to recent studies. DNMT3A R882 mutations are also found at increasing frequency with age in healthy elderly populations and are one of the earliest pre-malignant alterations in the clonal evolution progression to leukemia. Studies have shown that DNMT3A mutations decrease overall DNA methylation and through less clear mechanisms, also impact the epigenetic landscape by effecting changes in histone acetylation. Here we investigated potential mechanisms by which loss of DNMT3A activity changes histone acetylation. DNMT3A binds with many proteins that regulate chromatin biology and gene transcription. Among those interacting proteins, we focused on the DNMT3A-HDAC interaction and their regulation of target gene suppression. To investigate whether there are differences in binding of wild-type vs. mutant DNMT3A to HDACs, we performed immunoprecipitation and Western blotting assays using Myc- tagged wild-type and R882 mutated DNMT3A. We found that DNMT3A R882 mutants showed reduced interaction with HDAC1 and 2. In addition, upon treatment with HDAC inhibitors (HDACi), DNMT3A mutant protein was more easily dissociated from HDAC1/2 than was wild-type DNMT3A. Intriguingly, covalent modification of DNMT3A R882 by SUMO1 protein was significantly enhanced relative to wild type DNMT3A. Together, we suggest that the weak complex formation between mutant DNMT3A and HDACs results from augmented SUMOylation of the R882 mutant. Because the DNMT3A R882 mutation reduces its methyl transferase activity, we investigated which genes would be upregulated from the DNMT3A repressor complex. To do this, we established isogenic TF-1 cell lines that harbor haploid DNMT3A knockout (DNMT3A+/-) using the Cripsr-Cas9 system. We also treated cells with HDACi and 5-azacytidine (5-aza) which inhibit HDAC and DNMT, respectively. Interestingly, we discovered that PD-L1 expression is induced by HDACi and 5-aza treatment. Chemical inhibition by 5-aza or genetic inhibition by knockout reduces DNMT3A activity and synergized with HDACi to increase PD-L1 expression. Flow cytometry analysis also demonstrated increased membrane PD-L1 expression in response to HDACi. We also found out that DNMT3A+/- resulted in higher Histone H3K27 acetylation, which is known as a gene activation mark. Higher H3K27 acetylation in DNMT3A+/- cell confirms the findings by other groups but the mechanisms by which this occurs are unknown. We suggest that haploinsufficiency of DNMT3A results in a reduced DNMT3A-HDAC interaction leading to higher H3K27 acetylation and increased PD-L1 expression. Our results also revealed that HDACi treatment induced cell cycle arrest, DNA damage and apoptosis at increasing levels in DNMT3A+/- cell. Even though the DNMT3A+/- TF-1 showed increased sensitivity to HDACi treatment, we observed a correlation of higher phosphor- ERK1/2 and PD-L1 levels in the surviving cells. The enhanced expression of PD-L1 and activation of ERK1/2 may explain in part how mutated DNMT3A contributes to drug resistance and immune checkpoint avoidance. Many oncology clinical trials are underway utilizing HDACi. However, the questions of which mutational backgrounds might be most sensitive to these agents and how to best combine them with other agents remain to be answered. To test whether reduced DNMT3A activity increases PD-L1 expression in vivo, we crossed floxed DNMT3A mice with Mx1-Cre mice. After 4 weeks of induction of Cre recombinase by injecting pIpC in the progeny carrying both genetically engineered changes, lineage depleted mouse BM cells were analyzed for PD-L1 expression using quantitative RT-PCR. BM cells derived from DNMT3A knockout mice showed increased expression of PD-L1 compared to wild-type mice. Treatment of these BM cells with an HDACi and/or 5-aza resulted in a synergistic induction of PD-L1 expression for the combination. Taken together, we suggest that mutant DNMT3A induces higher H3K27 acetylation along with PD-L1 expression due to a looser complex between HDAC1 and mutant DNMT3A. Therefore, we suggest that combined treatment with an HDACi and an immune checkpoint inhibitor targeting the PD-L1/PD-1 axis might be a promising strategy for treating DNMT3A mutant AML patients. Disclosures Small: InSilico Medicine: Membership on an entity's Board of Directors or advisory committees; Pharos I, B & T: Consultancy, Research Funding.

DNMT3AR882 Alters the Epigenome of Hematopoietic Cells

The gene that encodes DNA methyltransferase 3A (DNMT3A) is mutated in nearly 40% of normal karyotype acute myeloid leukemia (AML) patients. More importantly, DNMT3A mutations are almost invariably the initiating event for the disease; however, the mechanisms by which they contribute to AML initiation are not yet clear [1, 2]. Regardless, ancestral clones containing the DNMT3A mutation are hard to eradicate, and often persist in clinical remission [3, 4]. We have recently shown that the most common DNMT3A mutation in AML patients (R882H; R878H in mice) is a dominant negative mutation that causes a focal, canonical pattern of DNA hypomethylation that is present in pre-leukemic hematopoietic cells, and appears to evolve further in fully transformed AML cells [5]. However, it is not yet clear how the DNMT3AR882H mutation can cause the clonal expansion of HSPCs, or how it makes HSPCs more susceptible to transformation by secondary mutations. We have utilized the conditional Dnmt3aR878H knock-in model described by Guryanova et. al. [6] and established an hematopoietic-specific model of clonal hematopoiesis driven by DNMT3AR882 mutations. With this model, we set out to investigate the epigenetic and functional consequences of Dnmt3aR878H expression in murine hematopoietic cells. The bone marrow cells of 6-week-old Dnmt3aR878H x Vav1-CREmice are >95% floxed; although these mice have a distinct focal hypomethylation phenotype, it is not as striking as that of Dnmt3a deficient bone marrow cells (this is expected, since the dominant negative R882H mutation reduces DNMT3A activity by 80%, not 100%). Importantly, Dnmt3aR878H and WT RNA are expressed at a precise 50:50 ratio, mimicking the ratio found in human patients with this mutation [7]. To establish the "baseline" methylation status of Dnmt3a+/+ vs. Dnmt3aR878H/+ bone marrow cells, we harvested marrow from 6-week-old littermates and performed whole genome bisulfite sequencing on 7 independent mice from each genotype. The R878H bone marrow cells have focal, canonical regions of DNA hypomethylation that strongly resemble those seen in human patients with DNMT3AR882 mutations. 2,621 differentially methylated regions (DMRs) exist in the Dnmt3aR878H/+ marrow samples, and >99% are hypomethylated. Dnmt3a-/- bone marrow cells have > 8,000 DMRs compared to Dnmt3a+/+ bone marrow cells, highlighting the intermediate methylation phenotype in Dnmt3aR878H marrow (Figure 1A). Further, the hypomethylation phenotype is stable with aging, and does not progress between weeks 2 and 52 of life (data not shown). Previous studies utilizing bulk RNA sequencing demonstrated very few differentially expressed genes (DEGs) in primary human AML samples with DNMT3AR882H. Therefore, we utilized single cell RNA sequencing to identify DEGs in the progenitor populations of Dnmt3aR878H hematopoietic cells. In addition to the methylation phenotype outlined above, we have identified a set of 117 DEGs (30 genes up and 87 genes down) in lineage negative, c-KIT positive cells from Dnmt3aR878H bone marrow, including Hspa1a and Hspa1b, Cxcl2 and Cxcl12, Fosb and C1q complement genes, with pathway analyses suggesting dysregulation of the proteasome, chemokine and inflammatory signaling, and apoptotic and proliferative pathways (Figure 1B). These pathways have all been implicated in cancer, and may provide a rationale for the Dnmt3aR878H -driven pre-leukemic phenotype. Extensive hematopoietic immunophenotyping has revealed subtle population changes in progenitor and stem populations of 6-week old Dnmt3aR878H mice; we identified significantly lower granulocyte-monocyte progenitors (GMP) and multipotent progenitors (MPP), and a significant increase in SLAM cells. Although these differences are significant, they do not result in any gross lineage population skewing in the bone marrow or peripheral blood. In sum, these data define some of the epigenetic consequences of the Dnmt3aR878H mutation in hematopoietic cells, and establish this mouse model as a valid one for the study of this mutation. This is the first report of the pre-leukemic state mediated by Dnmt3aR878H and these data have important implications in the design of future pharmacological agents targeting this mutation in patients with AML. Disclosures No relevant conflicts of interest to declare.

Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (>10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P < .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q<.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q<.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

PPM1D and DNMT3A Mutations in Myelodysplasia and Clonal Hematopoiesis

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.

Genetic Vulnerabilities of DNMT3AR882H in Myeloid Malignancies

Mutations affecting the gene for the de novo DNA Methyltransferase 3A (DNMT3A) are the most common drivers of clonal hematopoiesis (CH) and amongst the most common somatic events in acute myeloid leukaemia (AML). Approximately two thirds of AML-associated DNMT3A mutations are heterozygous substitutions affecting codon R882, located within the methytransferase domain and are correlated with global hypomethylation. DNMT3A mutations are initiating events in leukemogenesis and leukemic progression relies on the acquisition of additional mutations in genes such as NPM1, TET2, IDH1/2, FLT3 andNRAS. AMLs harboring DNMT3A-R882 mutations display increased resistance to chemotherapy and carry a worse prognosis for patients, but the molecular basis of this is not well understood. To improve our understanding of the molecular effects of mutant DNMT3A, we developed a conditional model of DNMT3A-R882H. Our conditional Dnmt3a-flox-R882H allele retains the native locus intact, but generates the Dnmt3a-R882H mutant upon Cre-loxP recombination (Mx1-Cre). We observed that Dnmt3aR882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential in serial re-plating assays and increased BM repopulation ability in competitive transplants. Approximately 30% of Dnmt3aR882H/+ mice developed leukaemia after a long latency (median of 532 days). Introduction of Flt3-ITD mutation significantly accelerated spontaneous disease, in agreement with other published reports, with majority of mice succumbing to AML after a median of 192 days. Due to the frequent co-occurrence and strong cooperation between Dnmt3aR882H/+ with Flt3ITD/+, we sought to identify potential genetic vulnerabilities of this combination, to facilitate patient treatment. We therefore generated Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ mice, induced Dnmt3aR882H mutation with pIpC and isolated hematopoietic stem and progenitor cells (HSPCs). We cultured HSPCs in vitro and performed whole-genome CRISPR dropout screens to identify genetic vulnerabilities. This identified 526 dropout genes at a genome-wide FDR of 20%, including pan-essential genes such as components of the ribosome, proteasome or spliceosome. To identify essential genes specific to the Dnmt3aR882H/+/Flt3ITD/+ genotype, we performed analogous screens in the non-leukemic mouse hematopoietic precursor cell-7 (HPC-7) and used published data indicating essential genes for normal human CD34 cells. Overlap of these screens revealed 196 dropouts present only in Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ cells. Among these were genes coding for "druggable" candidates such as kinases and histone modifying enzymes, including SET Domain Containing 1B (Setd1b), one of the six mammalian proteins (also Setd1a and Mll1-4) that can catalyze methylation of H3K4. Each of these six proteins can interact with obligate proteins as a part of the methyltransferase complex, but also form distinct complexes with non-redundant function. We firstly validated that the knockout of Setd1b induced by gRNA significantly reduced the growth of pre-leukemic and leukemic HSPCs from both Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ and Dnmt3aR882H/+/Mx1-Cre+/Rosa-Cas9+ mice. Importantly, Setd1b deletion in normal HSPC had no major effect on cell growth. To investigate the molecular role of Setd1b in Dnmt3aR882H context, we asked if Setd1b catalytic activity and linked transcriptional programs were responsible for the observed phenotypes. For this, we performed histone profiling and RNA-sequencing in Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+HSPC upon Setd1b KO vs control and observed a striking reduction of H3K4me3 marks at particular loci in association with loss of expression of corresponding genes. These included several known to be required for cellular proliferation and transformation, some of which were themselves dropouts in our original screen. Importantly the majority of genes were also significantly upregulated when gene expression from Dnmt3aR882H/+/Flt3ITD/+ HSPC were compared with Flt3ITD/+ HSPC. Our data propose that the catalytic activity of Setd1b plays a mechanistic role in driving the growth/fitness-promoting effects of Dnmt3aR882H/+. Blocking the catalytic activity of SETD1B may therefore represent a plausible therapeutic option for the treatment of AML of even CH patients harboring mutations inDNMT3A. Disclosures Mazan: Selvita S.A.: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding.

Influence of DNMT3A R882 mutations on AML prognosis determined by the allele ratio in Chinese patients

BackgroundThe influence of DNMT3A R882 mutations on adult acute myeloid leukemia (AML) prognosis is still controversial presently. The influence of R882 allele ratio on drug response and prognosis of AML is unknown yet. Besides, it is obscure whether anthracyclines are involved in chemoresistance resulted from R882 mutations.MethodsDNMT3A R882 mutations in 870 adult AML patients receiving standard induction therapy were detected by pyrosequencing. Associations of the mutants with responses to induction therapy and disease prognosis were analyzed.ResultsDNMT3A R882 mutations were detected in 74 (8.51%) patients and allele ratio of the mutations ranged from 6 to 50% in the cohort. After the first and second courses of induction therapy including aclarubicin, complete remission rates were significantly lower in carriers of the DNMT3A R882 mutants as compared with R882 wildtype patients (P = 0.022 and P = 0.038, respectively). Compared with R882 wild-type patients, those with the R882 mutations showed significantly shorter overall survival (OS) and disease-free survival (DFS) (P = 1.92 × 10−4 and P = 0.004, respectively). Patients with higher allele ratio of R882 mutations showed a significantly shorter OS as compared with the lower allele ratio group (P = 0.035).ConclusionOur results indicate that the impact of DNMT3A R882 mutations on AML prognosis was determined by the mutant-allele ratio and higher allele ratio could predict a worse prognosis, which might improve AML risk stratification. In addition, DNMT3A R882 mutations were associated with an inferior response to induction therapy with aclarubicin in Chinese AML patients.

PS1060 CO‐OCCURRENCE OF DNMT3A‐R882, FLT3 AND NPM1 MUTATIONS IDENTIFIES A SUBSET OF AML PATIENTS WITH ADVERSE PROGNOSIS

Background:DNMT3A mutations are found in approximately 22% of adult acute myeloid leukemia (AML) cases, occurring more commonly in a hotspot at codon 882. These mutations are an early event in leukemogenesis, causing impaired DNA methylation and aberrant gene expression. Previous studies have shown that isolated DNMT3A mutations may cause limited clonal hematopoiesis in healthy individuals, however, additional mutations in genes such as FLT3 or NPM1 are required for leukemic transformation. Despite its well‐recognized role in AML development, little is known about the prognostic repercussions of the interaction between these three genes.Aims:Here, we aimed to investigate i) the clinical and biological differences between #R882 (hotspot) and non‐R882 mutations in AML and ii) the impact of the synergism between DNMT3a, FLT3 and NPM1 mutations on patient survival.Methods:218 non‐selected adult AML patients (non‐AML M3) followed in a reference center in northeast Brazil were enrolled. Additionally, 180 patients from The Cancer Genome Atlas (TCGA) databank were evaluated as an independent cohort. Brazilian patients were treated according to the 7+3 chemotherapy protocol followed by consolidation with high doses of cytarabine, while patients from the TCGA cohort were treated according to the National Comprehensive Cancer Network recommendations. Screening for DNMT3a mutations was performed by direct Sanger Sequencing.Results:DNMT3A R882 mutations were associated with FAB monocytic AML subtypes (M4 and M5), higher leukocyte counts, normal karyotype and FLT3 and NPM1 mutations (more evident for FLT3/NPM1 double‐positivity), while non‐R882 mutations were associated with NRAS and IDH2 mutations. Analysis of TCGA patient's transcriptome data revealed that patients harboring concomitant DNMT3a, FLT3 and NPM1 mutations had different patterns of gene expression compared to their counterparts with isolated mutations. Next, to address whether the association between DNMT3a R882, FLT3 and NPM1 mutations is clinically relevant, we created a composite variable grouping patients harboring DNMT3a R882 mutations with at least one mutation in FLT3 or NPM1 (here referred to as R882/FLT3/NPM1). Strikingly, patients assigned to the R882/FLT3/NPM1 group had significantly poorer overall (Brazilian cohort: HR: 2.44, 95% CI: 1.35–4.44; P = 0.002 and TCGA cohort: HR: 1.83, 95% CI: 1.09–3.06; P = 0.022) and disease‐free survival rates (Brazilian cohort: HR: 1.94, 95% CI: 1.08–5.92; P = 0.027 and TCGA cohort: HR: 2.06, 95% CI: 1.11–3.81; P = 0.021) in both cohorts. Similar results were observed when analysis was restricted to the normal‐karyotype group. On the other hand, individual analysis of DNMT3a (or #R882 only) mutations showed only a tendency for shorter survival that did not reached statistical significance in both cohorts. Proportional hazards modeling showed that R882/FLT3/NPM1, but not single mutational status, remained independently predictive of poor outcomes (Brazilian cohort: HR: 1.97, 95% CI: 1.06–3.84; P = 0.048 and TCGA cohort: HR: 2.14, 95% CI: 1.24–3.68; P = 0.006).Summary/Conclusion:AML patients with R882 mutations have distinct clinical and molecular signatures from those with non‐R882. At the transcriptome level, triple positive‐mutated patients (DNMT3a‐R882, FLT3 and NPM1) cluster differently from the single positives ones, suggesting functional interaction between these mutations. By transposing these findings into a composite variable, we show that the synergism between DNMT3a, FLT3 and NPM1 mutations identifies a subset of AML with a particularly adverse prognosis.

Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis.

Background: According to the recent National Comprehensive Cancer Network (NCCN) guidelines, the risk level in acute myeloid leukemia (AML) patients with FLT3-ITD and NPM1 double mutation (AMLFLT3-ITD+/NPM1+) depends on the allelic ratio of FLT3-ITD. But despite a low or high allelic ratio of FLT3-ITD, AMLFLT3-ITD+/NPM1+ patients belong to the favorable or intermediate risk, for whom allogeneic stem cell transplantation is not obligated. However, some latest studies pointing out that NPM1 and FLT3-ITD double mutation patients showed an inferior prognosis, which have raised concern about the risk categorization and more effective treatment of AMLFLT3-ITD+/NPM1+ patients.Methods: A total of 76 patients were selected for coexisting FLT3 and NPM1 mutations with normal cytogenetics. The prognostic risk factors were analyzed, and treatment strategies including allogeneic stem cell transplantati1on and chemotherapy were compared.Results: In 76 AMLFLT3-ITD+/NPM1+ patients, 36.8% of patients had hyperleukocytosis (HL) and DNMT3A R882 mutation was the most common concomitant gene (23.7%). For 53 patients in the complete remission (CR), 22 had received allogeneic hematopoietic stem cell transplantation (allo-HSCT) on first complete remission (CR1). Patients in transplantation group had better overall survival (OS) and disease-free survival (DFS) than chemotherapy only (P=0.002 and 0.001, respectively). In multivariable Cox model analyses, HL and DNMT3A R882 mutation were independent adverse prognostic factors (all P<0.05) for AMLFLT3-ITD+/NPM1+ patients. Nevertheless, allo-HSCT was an independent good factor of OS and DFS (P=0.001 and 0.000; HR =0.173 and 0.138; 95% CI were 0.062–0.483 and 0.049–0.389). And allo-HSCT could moderately improve the poor prognosis of AML FLT3-ITD+/NPM1+/DNMT3A R882+.Conclusion: Although, AMLFLT3-ITD+/NPM1+ patients are categorized as favorable or intermediate risk levels according to recent NCCN and ELN guidelines, these patients should receive allo-HSCT in CR1 for a longer survival. AMLFLT3-ITD+/NPM1+ patients with DNMT3A R882 mutation had a very poor prognosis, and allo-HSCT could moderately improve their survival.

Delayed Hematologic Recovery in AML Patients after Induction Chemotherapy Is Associated with Inferior Relapse-Free Survival and Persistence of Preleukemic Mutations

Introduction:Induction chemotherapy debulks the leukemic burden in AML patients. Blood count recovery usually occurs during the fourth week of starting chemotherapy in patients who achieve a morphologic remission in bone marrow. However, a subset of patients experience significantly delayed recovery. The relevance of delayed recovery on long-term clinical outcomes and its contributing factors have not been well studied. Specifically, the association between recurrent mutations in AML and hematologic recovery is unknown.Methods:We studied a total of 262 newly diagnosed adult AML patients treated between September 2014 and December 2017 at Princess Margaret Cancer Centre who achieved a complete remission (CR) or CR with incomplete count recovery (CRi) after one cycle of induction chemotherapy. The regimens consisted of 3+7 (N=194) and FLAG-IDA (N=68). We collected information on disease characteristics and blood count results at baseline and during chemotherapy. Mutation profiling of diagnostic samples was performed using a 54-gene next generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina). Detection of persistent mutations in remission samples was performed using a custom 37-gene duplex sequencing platform with a lower detection limit of ~0.05% variant allele frequency (VAF).Results:Of the cohort of 262 patients, 256 patients (97.7%) achieved neutrophil recovery (defined as > 1x109/L), with time to recovery ranging from 17 to 84 days. Two hundred forty-four (93.1%) patients achieved platelet recovery (defined as > 100x109/L); time to recovery ranged from 17 to 117 days. The percentage of patients who achieved neutrophil and platelet count recovery before day 35 was 82.4% and 84.0% respectively (Fig. 1).To evaluate the prognostic significance of delayed recovery, we categorized patients who achieved CR into two groups, “normal” or “delayed” recovery, according to whether they achieved recovery before or after day 35, respectively. Relapse-free survival (RFS) of patients with delayed recovery was significantly worse than those with normal recovery and only marginally better than those with CRi (P=0.02; Fig. 2). Analysis restricted to 3+7 treated patients showed the same trend (P=0.02), excluding the possibility that the inferior outcome was due to treatment of higher risk patients with more intensive regimens.To study the factors associated with delayed recovery, we performed multivariable Cox regression analysis that included clinical factors and mutations identified at the time of diagnosis as covariates. Four factors were found to be independently correlated with delayed recovery: treatment with FLAG-IDA, truncating ASXL1mutations, SRSF2mutations, and DNMT3AR882 mutations (Table 1). Because FLAG-IDA is the preferred frontline regimen for higher risk patients at our institution, we performed a secondary analysis restricted to patients treated with 3+7 to exclude chemotherapy regimen as a potential confounding variable. This analysis identified six independent factors: AML with myelodysplasia-related changes, lower hemoglobin levels at presentation, truncating ASXL1mutations, TET2mutations, CEBPAmutations, and DNMT3AR882 mutations (Table 1).Somatic mutations in DNMT3A, TET2, ASXL1, and SRSF2(DTAS) mutations are associated with preleukemic conditions, such as myelodysplastic syndrome and age-related clonal hematopoiesis, and frequently persist in remission. These mutations are acquired in hematopoietic stem cells resulting in their propagation to progenitors and terminally differentiated blood cells. We hypothesized that the persistence of DTAS mutations in progenitors might compromise their capacity for reconstitution of normal hematopoiesis resulting in delayed recovery. To test this hypothesis, we performed duplex sequencing on peripheral blood DNA samples collected from a random subset of 43 patients during remission. The detection of DTAS mutations in remission above a VAF of 2% was strongly associated with delayed recovery (P=0.0004; Fig. 3).Conclusion:Delayed hematologic recovery in AML patients after induction chemotherapy is associated with inferior RFS and persistence of preleukemic mutations (i.e., DTAS mutations). Our results support a model in which progenitors harboring DTAS mutations have reduced repopulation capacity leading to delayed hematologic recovery after induction chemotherapy. DisclosuresGupta:Incyte: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schimmer:Otsuka Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding. Yee:Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding; Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees. Maze:Novartis: Consultancy, Honoraria. Bratman:Roche: Other: SVB is a co-inventor on a patent describing methods for circulating tumor DNA analysis, which has been licensed to Roche Molecular Diagnostics.. Schuh:Shire: Consultancy; Jazz: Consultancy; Novartis: Consultancy; Otsuka: Consultancy; Teva: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen Inc.: Consultancy.

Invariant Patterns of Clonal Succession Determines Specific Phenotypic and Clinical Features of Myelodysplastic Syndromes (MDS)

MDS arises through stepwise acquisitions of multiple mutations. Mutation configuration (bi-allelic vs. mono-allelic), specific distributions (hot spot), timing of acquisitions/ranks within clonal hierarchies, and combinatorial spectra, can all affect the pathogenesis and clinical phenotype of this disease. We performed integrative analyses of these processes to determine which relationships are deterministic vs. random. We analyzed 1,809 clinically annotated MDS patients. Deep targeted NGS was applied to a panel of the 36 genes frequently mutated in myeloid neoplasms. Copy number alterations (CNA) were evaluated by combining karyotyping, microarray, and digital CNA analysis. With a mean coverage of 862x, after removing SNPs and errors, we identified 3,971 somatic mutations.Frequently mutated genes/CNAs were TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%), each present in >10% of cases. DNMT3AMT affected the canonical site (R882) in 17% (35/203) of DNMT3AMT cases, were truncating in 39% (79/203) and were other missense mutations in 44% (89/203). Bi-allelic alterations of EZH2 and TP53 most commonly involved a mutation paired with a copy number deletion or UPD. In contrast, RUNX1 and TET2 commonly involved multiple mutations of the gene. Examining intragenetic relationships, we assumed that the impact of individual mutations depends on their clonal hierarchical position. To discriminate 1st hits (“dominant”) from subsequent “secondary mutations”, we used a stringent binominal distribution algorithm to compare the expected vs. observed VAFs using read counts (Figure 1). A mutation with the largest VAF in a sample was defined as “dominant”. Mutations with overlapping 95% CI of expected VAFs were “co-dominant”, and those with non-overlapping 95% CIs were “secondary”. These assumptions were validated by Pyclone (concordance rate > 95%). Accordingly, 1,474 (36%) and 1,372 (35%) mutations were dominant and secondary, respectively. SF3B1, DNMT3A, U2AF1, and TP53 were more likely to be dominant, ASXL1, CBL, ETV6, and KRAS were more likely to be secondary. For example, SF3B1 mutations were dominant in 17% (303/1809) of patients and secondary in 2% (39/1,809), p<.0001, q<.01.78 common combinations were identified among these different types of mutations. For instance, compared to U2AF1S34 mutations, U2AF1Q157 mutations were more associated with ASXL1 mutations and del(7q), and less with DNMT3A and BCOR mutations (p<.01). Compared to TET2 mono-allelic mutations, TET2 bi-allelic mutations co-existed more with ZRSR2 and SRSF2 mutations, but less with del(5q). DNMT3AR882 mutations at 77% (27/35) were more likely to be dominant than truncating or other missense DNMT3A mutations at 51% (40/79) and 45% (47/98), p=.0046, q=.07. We also evaluated the impact of different types of mutations and their combinations on disease phenotypes and survival. Many relationships were identified between mutations in different genes and bi-allelic vs. mono allelic hits. Among frequently correlated dominant/secondary pairs, pairs of dominant EZH2 mutations and secondary RUNX1 or ASXL1 mutations were associated with higher-risk subtypes (q=.08) (Figure 2), whereas patients with dominant SF3B1 mutations and secondary JAK2 mutations had myeloproliferative features and lower-risk subtypes (q<.001; q=.09). DNMT3AR882 mutations associated with worse overall survival than did truncating and other missense DNMT3A mutations. Five pairs of dominant and secondary mutations significantly affected survival (p<.01, q<.1). We hypothesized that if a fraction of MDS originates from clonal hematopoiesis of indeterminate potential (CHIP), the compositions of dominant mutations in MDS should be similar to those in CHIP. MDS patients with dominant mutations of TET2, DNMT3A, and ASXL1 were defined as “CHIP-derived MDS” and had more secondary TET2 and ZRSR2 mutations than “De novo MDS”. In conclusion, we report a comprehensive analysis of various mutational scenarios and the resultant clinical features. Most significantly, our results demonstrate how invariant patterns of evolution evolve with certain dominant hits dictating high probabilities of specific secondary events. Such patterns coincide with unique combinations of clinical properties that arise with hits along specific pathways. [Display omitted] DisclosuresNazha:MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Epigenetic Modifier Mutations in the LL-100 Panel

The NCI-60 human cell line panel, developed for use in drug development comprises sixty human cancer cell lines derived from nine different tissues. Only six cell lines of the NCI-60 were derived from blood cancers. Therefore, most forms and subtypes of leukemia and lymphoma are not represented in the NCI-60 panel. To respond to this apparent gap, we suggest the novel LL-100 panel, 100 leukemia and lymphoma cell lines representing the major leukemia/lymphoma entities, for basic research and drug development.Whole exome sequencing and RNA sequencing were performed to identify mutations in 100 cell lines. Here we list the 100 cell lines, ordered by subtype and show mutations in epigenetic modifier genes.We found cell lines with mutations in ASXL1, EZH2, IDH1, TET2 and in DNMT3A. Hitherto, cell line OCI-AML3 was the only human cell line described with a DNMT3A mutation. Twenty-two percent of patients with acute myeloid leukemia contain DNMT3A mutations and the median overall survival with DNMT3A mutations is shorter than without. Most DNMT3A mutations are heterozygous and alter amino acid R882, R882H being the most common DNMT3A mutation in AML. Exogenously mutant murine R878H (equivalent to human R882H) inhibits DNMT3A activity in a dominant negative manner. We describe here that the AML cell line SET-2 carries a heterozygous G to A transition at chr2_25234373 (hg38) which leads to the DNMT3A R882H amino acid substitution. Chip-based methylation analysis revealed that the described DNMT3A targets IRF8, KLF2, HOXA11 and HOXB2 are hypomethylated in cell lines OCI-AML3 (DNMT3A R882C) and in SET-2 (DNMT3A R882H). These data suggest that SET-2 is a novel model cell line for functional analysis of the DNMT3A R882 mutation and a first gain in knowledge through data mining the LL-100 panel. DisclosuresNo relevant conflicts of interest to declare.

Characteristics of DNMT3A-R882 Mutation in AML

IntroductionGenetic mutations play an important role in the development and progression of acute myeloid leukemia (AML). One of the common aberration in AML is mutation in the epigenetic modifying gene, DNA methyltransferase 3α (DNMT3A). Despite the active investigations, the exact impact of mutation on the development of AML is not completely known. The occurrence of mutation in pre-leukemic cells explains a particular attention to DNMT3A. The most common mutation is located in codon R882 (DNMT3AR882mut). The objective of this study is to compare clinical and prognostic characteristics of AML patients in relation to presence of DNMT3AR882mut. The quantification of the mutation burden in follow up samples was performed both after standard therapy and after allogeneic stem cell transplantation (alloSCT). In addition, it was investigated whether the quantification of the mutational burden of DNMT3AR882mut is significant to the progression of disease.MethodsSamples of 580 AML were retrospective analyzed using HRM-PCR, capillare electrophorese, and Sanger Sequencing. The median observation period was 495 days. Of 580, 69 have DNMT3AR882mut. Mutation burden was evaluated in follow-up samples by quantitative PCR. The statistical methods were selected according to sample distribution and evaluated with SPSS (significance level p <0.05).ResultsDNMT3AR882mut were associated with a higher level of leukocytes and blasts at diagnosis, with M4-M5 variant of AML, and with normal karyotype. It was found that NPM1 and FLT3-ITD are more frequent co-mutations that have a significant effect on the prognosis of disease. Analysis of mutation burden of DNMT3AR882mut at diagnosis showed a large spread (0.02 - 66.9 %). At the time of diagnosis, DNMT3AR882mut transcript levels did not correlate with clinical and prognostic characteristics. The mutation burden decreased after therapy, but was always visible in CR after standard therapy. In CR after allogeneic stem cell transplantation (alloSCT) with complete donor chimerism mutation was not detected. In relapse of AML, an increasing of the mutation burden were found in all patients, both after therapy, and after alloSCT. In relapse samples, the same mutant clone was found.ConclusionThe DNMT3A mutation is a common genetic aberration in AML patients, which is associated with specific clinical and prognostic data. The presence of co-mutations, especially NPM1 and FLT3-ITD, has a significant effect on the prognosis of patients.Quantitative detection of DNMT3AR882mut at different time points of disease revealed the persistence of mutated clone after standard therapy and disappearance of DNMT3AR882mut after alloSCT. It is suggest that alloSCT is the optimal treatment option for the eradication of DNMT3AR882mut in AML patients. DisclosuresBullinger:Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Bayer Oncology: Research Funding.

FLT3-ITD compared with DNMT3A R882 mutation is a more powerful independent inferior prognostic factor in adult AML patients after Allogeneic Hematopoietic Stem Cell Transplantation: A retrospective cohort study

ObjectiveThis study aimed to evaluate DNMT3A exon 23 mutations and their prognostic impacts in the presence of NPM1 and FLT3 mutations in acute myeloid leukemia (AML) patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT).Materials and MethodsThis study comprised 128 adult AML patients referred to the Hematology-Oncology and Stem Cell Research Center of Shariati Hospital. NPM1 and FLT3-ITD mutations were detected by fragment analysis. For DNMT3A exon 23 mutation analysis, we used Sanger sequencing. Overall survival (OS) and relapse-free survival (RFS) curves were estimated by the Kaplan-Meier method and the log-rank test was used to calculate differences between groups.ResultsThe prevalence of DNMT3A exon 23 mutations was 15.6% and hotspot region R882 mutations were prominent. RFS and OS were compared in patients with and without DNMT3A exon 23 mutations using univariate analysis and there was no significant difference between these groups of patients. On the contrary, the FLT3-ITD mutation significantly reduced the OS (p=0.009) and RFS (p=0.006) in AML patients after allogeneic HSCT. In the next step, patients with AML were divided into four groups regarding FLT3-ITD and DNMT3A mutations. Patients with DNMT3A R882mut/FLT3-ITDpos had the worst OS and RFS. These results indicate that DNMT3A mutations alone do not affect the clinical outcomes of AML patients undergoing allogeneic HSCT, but when accompanied by FLT3-ITD mutations, the OS was significantly reduced (5-year OS 0% for DNMT3A R882mut/FLT3-ITDpos patients vs. 62% DNMT3A R882wt/FLT3-ITDneg, p=0.025) and the relapse rate increased.ConclusionIt can be deduced that DNMT3A R882mut/FLT3-ITDpos is an unfavorable prognostic factor in AML patients even after allogeneic HSCT.

FLT3-ITD with DNMT3A R882 double mutation is a poor prognostic factor in Chinese patients with acute myeloid leukemia after chemotherapy or allogeneic hematopoietic stem cell transplantation.

To investigate clinical characteristics and outcomes of transplantation in AML patients with FLT3-ITD/DNMT3A double mutation, we retrospectively analyzed 206 Chinese patients with AML after Sanger sequencing. Our analysis showed that AML patients with FLT3-ITD and DNMT3A R882 mutations had a higher white blood cell count and a lower complete remission (CR) rate after first induction chemotherapy. All 206 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in status of CR. These results indicate that AML patients with FLT3-ITD and DNMT3A R882 double mutation show a higher 2-year cumulative incidence of relapse (CIR), lower 2-year overall survival (OS) rate, and lower 2-year leukocyte-free survival (LFS) after allo-HSCT. The univariate and multivariate analyses confirmed that disease status prior to transplantation, FLT3-ITD, FLT3-ITD, and DNMT3A R882 double mutation were independent factors for poor prognosis after allo-HSCT. In summary, the present cohort study demonstrated that FLT3-ITD and DNMT3A R882 double mutation predicts poor prognosis in Chinese AML patients receiving chemotherapy or allo-HSCT treatment.

CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% invitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patientswith or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Ourfindings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

Analysis of DNA methyltransferase 3A gene mutations in patients with Philadelphia-negative myeloproliferative neoplasms

Context: Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) from a group of disorders characterized by dysregulated JAK-STAT functionality, abnormal hematopoiesis, as well as increased production of proliferative cytokines. In addition to JAK2V617F mutation, additional gene alterations that are involved in epigenetic mechanisms, particularly de novo DNA methyltransferase 3A (DNMT3A), have been described in Ph-negative MPN biology. Aims: The aim of this study is to evaluate the H/C/S/P mutations in codon R882 of DNMT3A gene among patients with Ph-negative MPNs. Subjects and Methods: This study was conducted on 64 newly diagnosed patients with PV, ET, and PMF who referred to Shafa Hospital, Ahvaz, Iran. In the beginning, 5 mL whole blood was drawn from each patient, and the DNMT3A R882 codon mutations were investigated following the isolation of peripheral blood mononuclear cells by DNA amplification protocol using polymerase chain reaction and DNA sequencing techniques. Results: The R882H G > A mutation, which results in an amino acid substitution at position 882 of DNMT3A gene from arginine (R) to histidine (H), was observed in two patients (3.1%) with JAK2V617F positive PV and JAK2V617F negative PMFs. Conclusions: Based on the results, DNMT3A-R882 mutations occur at a low frequency in patients with Ph-negative MPNs. To the best of our knowledge, this is the first study to specifically estimate the prevalence of DNMT3A mutations among Ph-negative MPN patients living in the Middle East. It is recommended to investigate these mutants as a secondary defect along with common major complications in such patients.

Impact of Distinct Genetic and Epigenetic Aberrations on Survival and Response in Acute Myeloid Leukemia Patients Receiving Epigenetic Therapy

Background: Treatment with hypomethylating agents such as decitabine, which results in complete and partial remission rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are no candidates for intensive chemotherapy. While clinical parameters such as reduced performance status, high leukocyte counts, elevated lactate dehydrogenase (LDH) levels as well as poor-risk cytogenetics are associated with lower response rates and shorter overall survival, there exists only limited data on molecular biomarkers that enables for selection of AML patients who are likely to benefit from epigenetic therapy. Shen et al. (JCO 2010) could show that distinct DNA methylation changes are prognostic for overall survival in myelodysplastic syndrome patients, however, response to decitabine therapy could not be predicted. Additionally, mutations in the epigenetic-modifier gene DNMT3A were recently reported to be associated with response and survival in AML patients treated with hypomethylating agents, however, while some of these studies revealed a positive association with outcome, other studies showed no association with response and overall survival (Metzeler et al., Leukemia 2012, DiNardo et al., Leuk Lymph 2014, Coombs et al., Blood 2015).Results: In order to contribute further knowledge regarding prognosis and response to hypomethylating therapy in AML patients, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331, Lübbert et al. Haematologica 2012) to identify potential molecular biomarkers. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, in our cohort patients harboring DNMT3A R882 mutations showed shorter overall survival (hazard ratio (HR): 2.15, 95%-confidence interval (CI): 0.91-5.12, p=0.08). Promoter DNA methylation analyses using pyrosequencing revealed shorter overall survival of patients with higher levels of methylation of ERα (HR: 1.50, CI: 0.97-2.32, p=0.07) and OLIG2 CpG4 (HR: 1.52, CI: 0.96-2.41, p=0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR: 1.74, CI: 1.03-2.96, p=0.04 and HR: 1.61, CI: 0.96-2.71, p=0.07, respectively) whereas the effect of DNMT3A R882 mutations was reduced to HR: 1.94 (CI: 0.79-4.73, p=0.15). In contrast, none of the investigated molecular markers was associated with response to treatment.Conclusion: In addition to the well established adverse prognostic clinical parameters such as patients’ age, reduced performance status and elevated LDH levels, we provide further evidence that pretreatment genetic and especially epigenetic analyses including DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status may be potential molecular biomarkers in AML patients undergoing epigenetic therapy. DisclosuresLübbert:Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

Prognostic Impact of FLT3-ITD and DNMT3A R882 Double Mutations in Patients with Acute Myeloid Leukemia

Background: FLT3-ITD and DNMT3A R882 mutations are both independent factors for poor prognosis of acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been confirmed to improve the outcome of AML with either of gene mutations. However, the clinical characteristics of AML with both of FLT3-ITD and DNMT3A R882 mutations and their outcome post allo-HSCT had been rarely evaluated.Objective: To investigate the clinical characteristics and transplantation outcomes of AML patients with the double mutations.Methods: A total of 241 patients were included in this retrospective analysis. Molecular genetic mutation sets in bone marrow (including FLT3-ITD, DNMT3A, C-kit, CEBPA, FLT3-TKD, and NMP1) of 241 AML patients were detected at diagnosis using direct sequencing method. And the clinical data of these patients were analyzed retrospectively.Results: (1)According to the genetic mutation types, the 241 AML patients were divided into four groups: FLT3-ITD+DNMT3A R882+, FLT3-ITD+DNMT3A R882-, FLT3-ITD-DNMT3A R882+ and FLT3-ITD-DNMT3A R882- groups, which consisted of 19, 38, 21 and 163 patients, respectively. (2) Patients with FLT3-ITD+DNMT3A R882+ have high white blood cell count and a low complete remission rate after first induction chemotherapy (52.6%), which are common in M5 M2 M4 with normal chromosome karyotype, higher early cumulative incidence of relapse(6m-CIR 36.8±1.3% 12m-CIR 49.7±1.6%) and cumulative mortality rate (6m-CMR 26.3±10.1% 12m-CMR 48.5±11.8%) . (3) 2-year CIR of the FLT3-ITD+DNMT3A R882+ was (72.2±2.7%), which was significantly higher than that of the FLT3-ITD+DNMT3A R882- group (38.6±0.7%) and FLT3-ITD-DNMT3A R882+ group (36.8±1.6%). While the 2-year overall survival (OS) rate and 2-year leukemia-free survival (LFS) rate of double mutation group were (30.9±13.3% ) and (11.3±10.2%), respectively, which were considerably lower than those of the FLT3-ITD+DNMT3A R882- and FLT3-ITD-DNMT3A R882+ groups (2y-OS: 67.5±7.8%, 61.4±12.4%: 2y-LFS: 47.9±8.4%, 56.8±12.5%). Meanwhile, the 2-year CIR rates of the FLT3-ITD+DNMT3A R882- and FLT3-ITD-DNMT3A R882+ groups were significantly higher than that of the FLT3-ITD-DNMT3A R882- group (16.3±0.1%), while their 2-year OS rate and 2-year LFS rate were significantly lower than those of the FLT3-ITD-DNMT3A R882- group (2y-OS: 82.5±3.1% 2y-LFS: 80.9±3.2%). (3) Univariate and multivariate analyses revealed that disease status prior to transplantation, grade III-IV aGVHD, FLT3-ITD, DNMT3A R882 and FLT3-ITD+DNMT3A R882+ were independent factors for poor prognosis post transplantation.Conclusion: AML patients with FLT3-ITD and DNMT3A R882 double mutations still present a very poor prognosis after transplantation with low OS and LFSas well as a high CIR rate. Therefore, it is necessary to explore prevention strategy for relapse post transplantation. DisclosuresNo relevant conflicts of interest to declare.

DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling.

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3AR882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.