A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid
A van Stijn1, A Kok, M A van der Pol
1Department of Haematology, VU University Medical Center, Amsterdam, The Netherlands.
Abstract:
Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-x(L), Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.
Insights
A new flow cytometry method allows measurement of apoptosis proteins in minimal residual disease (MRD) cells from acute myeloid leukemia (AML) patients. This technique helps understand apoptosis resistance in AML relapse.
Area of Science:
- Hematology
- Immunology
- Molecular Biology
Background:
- Minimal residual disease (MRD) cells are implicated in acute myeloid leukemia (AML) persistence and relapse.
- Flow cytometry for leukemia-associated phenotype (LAP) detection is feasible in 80% of AML patients.
- Apoptosis resistance is a key mechanism in chemotherapy failure.
Purpose of the Study:
- To develop and validate a flow cytometry method for assessing apoptosis-related protein expression in AML MRD.
- To investigate the role of apoptosis resistance in the emergence of MRD in AML.
Main Methods:
- Developed a four-color flow cytometry assay to measure apoptosis proteins (Bcl-2, Bcl-x(L), Mcl-1, Bax).
- Validated the assay using Western blot analysis in leukemia cell lines.
- Assessed the impact of permeabilization on LAP expression in diagnostic and MRD AML samples.
Main Results:
- Western blot validation showed significant correlation (R=0.70, P<0.0001).
- Permeabilization procedure did not significantly affect LAP expression in diagnostic (P=0.57) or MRD (P=0.43) samples.
- The developed protocol allows analysis of apoptosis proteins in diagnostic and MRD samples.
Conclusions:
- The validated flow cytometry method enables analysis of apoptosis-related proteins in AML at diagnosis and during MRD monitoring.
- This approach will enhance understanding of apoptosis resistance's role in AML MRD emergence and relapse.
- The findings support the use of this method for improved characterization of AML MRD.
