Triple-mutated leukemic subclones can be distinguished from double-mutated clones based on GPR56 expression. (A) Schematic overview of the sorting strategy of 10 triple-mutated AML samples. ^+/+, CD34⁺GPR56⁺; ^−/+, CD34⁻GPR56⁺; −/−, CD34⁻GPR56⁻. (B) FLT3-ITD mutant allele frequencies determined by kmer approach in RNA-Seq data obtained from CD34⁺GPR56⁺ and CD34⁻GPR56⁻ fractions. Divergent FLT3-ITD frequencies were found in the sorted fractions of samples marked in red, whereas no difference was found in samples marked in blue. Of note, in the 5 samples, in which FLT3-ITD ratios were not divergent in the sorted fractions, FLT3-ITD was close to 50% allele frequency indicating that it was not subclonal in 12H007, 10H166, 09H043, whereas in 2 samples (14H007, 10H101) FLT3-ITD was below 50% in all fractions. (C) Confirmation of divergent FLT3 mutant/wild-type ratios in samples 07H042 and 07H062 by PCR using genomic DNA isolated from CD34⁺GPR56⁺ (^+/+), CD34⁻GPR56⁺ (^−/+), and CD34⁻GPR56⁻ (^−/−) sorted fractions (upper, bars indicating FLT3 mutant/wild-type ratios; lower, agarose gel showing FLT3 wild-type band at 325 bp, +93 bp ITD in sample 07H062, and +54 bp ITD in 07H042). (D) Variant allele frequencies (VAF) of mutations with known leukemogenic potential in GPR56 positive and negative fractions shown for 5 AML samples with divergent FLT3-ITD load. Mutations with VAF close to the diagonal line indicate no difference between the sorted fractions. See supplemental Table 5 for information on CD34⁻GPR56⁺ fractions. (E) Detailed VAF analysis in GPR56 positive and negative sorted fractions for sample 09H083. (F, left) Comparison of VAF in unsorted sample 09H083 compared with the corresponding unsorted relapse sample 10H068. (Right) Fluorescence-activated cell sorter (FACS) plots showing CD34 and GPR56 protein expression in sample 09H083 at diagnosis and in the corresponding relapse sample. The loss of the PTPN11 clone at relapse is accompanied by the loss of the GPR56 negative population.