Acquisition of mutations in the evolution of SCN to AML. (A) The 12 somatic nonsynonymous mutations identified in the leukemic blasts were analyzed in the SCN phase using amplicon-based deep sequencing. The percentage of mutated amplicons per mutation is shown. Based on their frequencies in the AML population, all mutations are considered to be heterozygous, implying that the number of cells carrying the mutations is estimated to be twice the number of mutated amplicons. (B) Single myeloid colonies grown from the BM sample obtained 15 years before leukemia development were analyzed for the presence of mutations in CSF3R, LLGL2, and ZC3H18 (see supplemental Table 5 for more information). (C) The presence of different CSF3R mutations in the BM obtained 15 and 9 years before leukemia development and in the leukemic phase was investigated by amplicon-based deep sequencing. The percentage of mutated amplicons per mutation is shown. T595I indicates the CSF3R mutation T595I; d715-d730, CSF3Rδ mutations at amino acid positions 715-730.