Abstract
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 acute myeloid leukemia (AML). Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Within the CD34(+) cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-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 showed that, within the CD34(+) cell fraction a percentage of CD123/CD99/CD25(+) cells ≥11.7% predicted FLT3-ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25(+) clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1(+) AML who relapsed with FLT3-ITD/NPM1(+) 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. Our results suggest that the CD34/CD25/CD123/CD99(+) LAIP is strictly associated with FLT3-ITD-positive cells.
Highlights
Immunophenotyping by multiparametric flow cytometry (MPFC) is a valuable and effective tool for diagnostic characterization, classification, and minimal residual disease (MRD) monitoring in acute myeloid leukemia (AML)
The main demographic characteristics and karyotype of patients according to NPM1/fms-like tyrosine kinase 3 (FLT3) gene status are reported in Supplementary Table S2
CD34þ cells derived from the FLT3 and NPM1-mutated group expressed the lowest levels of CD38 (Fig. 1B)
Summary
Immunophenotyping by multiparametric flow cytometry (MPFC) is a valuable and effective tool for diagnostic characterization, classification, and minimal residual disease (MRD) monitoring in acute myeloid leukemia (AML). Several studies have defined surface and cytoplasmic markers that are aberrantly expressed on AML blasts at diagnosis [1, 2]; these combinations may identify leukemia-associated immunophenotypes (LAIP), which allow sensitive monitoring of MRD during follow-up. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Compared with molecular evaluation of MRD through PCR amplification of genetic AML lesions, MPFC offers the advantage of being applicable to the vast majority (i.e., >90%) of cases [6, 7]
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