Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells

Acute myeloid leukemia (AML) withFLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts forFLT3-ITD-positivity. The aim of this study was to characterize the distribution ofFLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture ofFLT3-ITD(mut)AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients withFLT3-ITD(mut)AML (n = 12). A higherFLT3-ITD(mut)load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/-,CD25-,CD99low/-) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higherFLT3-ITD(mut)burden was also observed in LPCs of AML patients with a smallFLT3-ITD(mut)clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows thatFLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target inFLT3-ITD(mut)AML.