SummaryMutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1-50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B-cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.

BackgroundThe availability of larger-scale SNP data sets in the chicken genome allows to achieve a higher resolution of the pattern of linkage disequilibrium (LD). In this study, 36 k and 57 k genotypes from two independent genotyping chips were used to systematically characterize genome-wide extent and structure of LD in the genome of four chicken populations. In total, we analyzed genotypes of 454 animals from two commercial and two experimental populations of white and brown layers which allows to some extent a generalization of the results.ResultsThe number of usable SNPs in this study was 19 k to 37 k in brown layers and 8 k to 19 k in white layers. Our analyzes showed a large difference of LD between the lines of white and brown layers. A mean value of r2 = 0.73 ± 0.36 was observed in pair-wise distances of < 25 Kb for commercial white layers, and it dropped to 0.60 ± 0.38 with distances of 75 to 120 Kb, the interval which includes the average inter-marker space in this line. In contrast, an overall mean value of r2= 0.32 ± 0.33 was observed for SNPs less than 25 Kb apart from each other and dropped to 0.21 ± 0.26 at a distance of 100 kb in commercial brown layers. There was a remarkable similarity of the LD patterns among the two populations of white layers. The same was true for the two populations of brown layers, while the LD pattern between white and brown layers was clearly different. Inferring the population demographic history from LD data resulted in a larger effective population size in brown than white populations, reflecting less inbreeding among brown compared to white egg layers.ConclusionsWe report comprehensive LD map statistics for the genome of egg laying chickens with an up to 3 times higher resolution compared to the maps available so far. The results were found to be consistent between analyzes based on the parallel SNP chips and across different populations (commercial vs. experimental) within the brown and the white layers. It is concluded that the current density of usable markers in this study is sufficient for association mapping and the implementation of genomic selection in these populations to achieve a similar accuracy as in implementations of association mapping and genomic selection in mammalian farm animals.

Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention. Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G). Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation. Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse. We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. Disclosures No relevant conflicts of interest to declare.

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