A Leukemia-Associated CD34/CD123/CD25/CD99+ Immunophenotype Identifies FLT3-Mutated Clones in Acute Myeloid Leukemia

Purpose We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in AML. Experimental Design: Bone marrow samples from 132 patients with acute myeloid leukemia (AML) were analyzed by 9-color multiparametric flow cytometry (MPFC). We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Results: Within the CD34+ve cell fraction we identified a discrete population expressing high levels of the IL-3 receptor alpha-chain (CD123) and MIC-2 (CD99) in combination with the IL-2 receptor alpha-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics (ROC) showed that, within the CD34+ve cell fraction a percentage of CD123/CD99/CD25+ve cells ≥ 11.7% predicted FLT3-ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25+ve clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1+ve AML who relapsed with FLT3-ITD/NPM1+ve AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. Conclusion: Our results suggest that the CD34/25/123/99+ve LAIP is strictly associated with FLT3-ITD positive cells.