Recapitulation of the biologic differences between blasts_naive and blasts_resist in NOD/SCID mice. (A) When transplanted at equal cell doses, blasts_resist exhibited superior engraftment than blasts_naive, which was significant at 9 and 12 weeks after transplantation. The engrafting cells were all CD33⁺, consistent with the immunophenotype expected of AML. (B) Limiting dilution assays were performed, showing that the LIC frequencies in blasts_resist were 12.0-, 12.2-, and 13.3-fold higher than those of blasts_naive in AML1, AML3, and AML10, respectively. (C) NOD/SCID mice transplanted with blasts_resist exhibited inferior survivals than those of blasts_naive. All mice that died after 6 weeks after transplantation were confirmed to have > 90% human engraftment in the BM. Twenty-eight mice were transplanted with blasts_naive and another 28 with blasts_resist. All mice were killed at 12 weeks after transplantation. (D) Two of 28 mice transplanted with blasts_resist developed myeloid sarcoma in the body and the eye, with tumor cells being CD45⁺/CD33⁺/CD19⁻. (E) Sorafenib significantly reduced leukemia engraftment by blasts_naive but not blasts_resist in NOD/SCID mice. Each point represented the result from one mouse (5 different experiments from paired samples from 3 patients AML4, 7, 8). (F-G) Sorafenib but not vehicle (DMSO in carboxymethylcellulose) treatment induced differentiation of engrafting cells, as indicated immunophenotypically (F) and morphologically (G). Nikon Eclipse E800M; eyepiece 10×; objective 40×; numerical aperture of objective lens 0.95; camera Nikon DS cooled camera head DS-5MC and DC camera control unit DS-L2; magnification of picture 400×; software for imaging processing Adobe Photoshop CS5 Extended Version 12.0.4x32. (H) Sorafenib had no effect on CD11b expression in mice engrafted with blasts_resist. (I) PCR for FLT3-ITD in marrow cells from mice engrafted with blasts_naive. In all 3 cases, despite a reduced level of engraftment as shown in panel B after sorafenib treatment, the predominant FLT3-ITD clone persisted, which was also confirmed by quantitative analysis (supplemental Figure 4). Patient AML14 was not included in the clinical trial and has not been treated with sorafenib.