Mononuclear cells were enriched through Ficoll-Hypaque gradient centrifugation and cryopreserved until use. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). The mutational status of 81 protein-coding genes was determined centrally at The Ohio State University by targeted amplicon sequencing using two different gene panels on the MiSeq platform (Illumina, San Diego, CA; see Additional file 1 for details). MuCor was used for integrative data analysis [32]. Details about the variant calling are outlined in the Additional file 1. In addition to the 81 genes assessed by HTS, testing for CEBPA mutations was performed as previously described, thus resulting in mutational status of 82 genes being assessed in our study [33]. Only patients with biallelic CEBPA mutations were considered as mutated [2]. The presence or absence of FLT3-ITD, as well as quantification of the FLT3-ITD to FLT3 wild-type allelic ratio, was determined as previously described [34].

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