Prospective analyses of FLT3-ITD and FLT3-D835 mutant clones at different stages of diseases in 6 patients treated with sorafenib. (A) Amplification of FLT3-ITD by PCR. The 329 bp band corresponded to wild-type (WT) FLT3 and the 344-416 bp bands corresponded to ITD. The DNA sequences of these bands were all confirmed by sequencing (supplemental Figure 2). AML3 relapsed after HSCT and achieved 100% donor chimerism after sorafenib treatment, so that the ITD clone was undetectable at CRi. (B) ITD allelic burden after sorafenib treatment persisted but decreased at CRi/nCRi, but resurged when the response was lost. (C) In 4 patients with FLT3-ITD⁺ AML treated outside this study, chemotherapy reduced the ITD clone to below the detection limit at CR. (D) In 6 sorafenib-treated patients, allele-specific EcoRV digestion showed that the D835 mutation (indicated by arrow) was not detectable before treatment. When the response was lost, however, the D835 mutation emerged in 4 patients (AML1, AML4, AML8, and AML10). In AML8, the mutation was detectable even at CRi. (E) DNA sequencing demonstrated the D835Y mutation in AML1, AML4 and AML10, and D835H mutation in AML8. The EcoRV cut site was underlined in black and point mutation site (G→T/C mutation) was indicated by an asterisk. (F) Quantitative analysis showed that the TKD D835Y/H mutation had emerged when the response to sorafenib was lost.