Related Experiment Video
Updated: Apr 12, 2026
Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up
Published on: March 26, 2018
A Leukemia-Associated CD34/CD123/CD25/CD99+ Immunophenotype Identifies FLT3-Mutated Clones in Acute Myeloid Leukemia
Daniela F Angelini1, Tiziana Ottone2, Gisella Guerrera1
1Neuroimmunology and Flow Cytometry Units, Fondazione Santa Lucia-I.R.C.C.S., Rome, Italy.
Insights
A specific leukemia-associated immunophenotype (LAIP) involving CD34/CD25/CD123/CD99+ cells can identify acute myeloid leukemia patients with FLT3-ITD mutations, aiding in relapse risk assessment.
Area of Science:
- Hematology
- Immunophenotyping
- Molecular Diagnostics
Background:
- Acute myeloid leukemia (AML) is a heterogeneous disease.
- Accurate risk stratification is crucial for effective AML patient management.
- Identifying specific biomarkers for high-risk patients is an ongoing challenge.
Purpose of the Study:
- To evaluate leukemia-associated immunophenotypes (LAIP) in AML.
- To correlate LAIP with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status.
- To improve the identification of AML patients at high risk of relapse.
Main Methods:
- Analysis of bone marrow samples from 132 AML patients using nine-color multiparametric flow cytometry.
- Confirmation of FLT3 and NPM1 mutations via RT-PCR and RQ-PCR.
- Identification and quantification of specific cell populations within the CD34+ fraction.
Main Results:
- A distinct CD34+ cell population expressing CD123, CD99, and CD25 was identified.
- The presence of this CD123/CD99/CD25+ population strongly correlated with FLT3-ITD mutations (r=0.71).
- A threshold of ≥11.7% for these cells predicted FLT3-ITD with >90% sensitivity and specificity.
- These LAIP clones were detected in patients who relapsed with FLT3-ITD mutations, with low copy numbers present at diagnosis.
Conclusions:
- The CD34/CD25/CD123/CD99+ LAIP is a reliable indicator of FLT3-ITD positivity in AML.
- This immunophenotypic marker can aid in identifying patients with a higher risk of relapse.
- Further investigation into LAIP for AML risk stratification is warranted.
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 acute myeloid leukemia (AML).
Experimental Design:
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.
Results:
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.
Conclusions:
Our results suggest that the CD34/CD25/CD123/CD99(+) LAIP is strictly associated with FLT3-ITD-positive cells.
More Related Videos
05:24Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia
Published on: February 21, 2021
07:38Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants
Published on: June 6, 2025