Hierarchical relationships of FLT3-ITD and D835 clones. (A) In all 6 cases, the predominant FLT3-ITD clones in blasts_naive and blasts_resist were detectable in the LIC (the engrafted population). (B) Quantitative analysis of FLT3-ITD allelic burden in the engrafted LIC clone showed heterozygosity in 4 samples and homozygosity in 2 samples. (C) Allele specific enzyme digestion for D835 mutation was performed in both transplanted and engrafted blasts_naive and blasts_resist. D835Y mutation was observed in the LIC population but not in the input blasts_naive in AML1, AML4, and AML10. The D835H mutation was not detectable in either transplanted or engrafted blasts_naive, in AML3 and AML7. In AML8, it was detectable in the input and engrafted blasts_resist. In the blasts_naive, it was only 3% (Table 1). (D) When mice engrafted with blasts_naive from AML8 were treated with sorafenib, D835H mutation emerged. (E) Quantitative analysis of FLT3-D835 allelic burden confirmed the enrichment of the D835Y clone in the LIC population of blasts_naive in patients AML1, AML4, and AML10. No significant difference before and after transplantation was observed for blasts_resist.