Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel
Nupur Das1, Devasis Panda1, Smeeta Gajendra1
1Department of Laboratory Oncology, Dr. BRAIRCH, AIIMS, New Delhi, India.
Insights
This study identifies leukaemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs) in acute myeloid leukemia (AML) patients. A 10-color flow cytometry panel effectively distinguishes LSCs from HSCs, revealing LSC heterogeneity.
Area of Science:
- Hematology
- Cancer Biology
- Immunophenotyping
Background:
- Comprehensive characterization of leukaemic stem cells (LSCs) and their immunophenotypic (IPT) differences from normal hematopoietic stem cells (HSCs) remain limited.
- Understanding LSC heterogeneity is crucial for developing targeted therapies in acute myeloid leukemia (AML).
Purpose of the Study:
- To unravel the IPT characteristics and heterogeneity of LSCs in AML.
- To establish a reliable method for distinguishing LSCs from HSCs.
Main Methods:
- Multiparametric flow cytometry (MFC) using a single 10-color tube panel with LSC-associated and backbone markers.
- Single-cell whole transcriptome sequencing of bone marrow samples.
Main Results:
- LSCs and HSCs were identified in 88.2% and 71.6% of samples, respectively.
- Significantly higher expression of COMPOSITE, CD45RA, CD123, CD33, and CD44 was observed in LSCs compared to HSCs (p < 0.0001).
- No significant difference in LSC-specific antigen expression was found between CD34+ and CD34- LSCs. LSC subpopulations were identified in 4.4% of cases with high concordance between MFC and single-cell sequencing.
Conclusions:
- A single 10-color MFC panel is proposed as an easy and reproducible tool for identifying and discriminating LSCs from HSCs.
- LSCs exhibit inter- and intra-sample heterogeneity in antigen expression, warranting further single-cell molecular analysis to understand their role in AML progression.
Introduction:
Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing.
Materials And Methods:
Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample.
Results:
LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods.
Conclusion:
A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.
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