Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity
M A van der Pol1, J M Pater, N Feller
1Department of Hematology, Medical Center Vrije Universiteit, Amsterdam, The Netherlands.
Abstract:
Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.
Insights
Minimal residual disease (MRD) in acute myeloid leukemia (AML) can be detected using leukemia-associated phenotypes (LAP). A new assay simultaneously detects LAP and drug resistance markers, enabling better MRD monitoring.
Area of Science:
- Hematology
- Cancer Biology
- Immunophenotyping
Background:
- Relapse in acute myeloid leukemia (AML) is often driven by minimal residual disease (MRD), which are leukemia cells persisting after treatment.
- Leukemia-associated phenotypes (LAP) are cell surface markers unique to leukemia cells, enabling MRD detection.
- Understanding drug resistance mechanisms, such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity, is crucial for effective AML treatment.
Purpose of the Study:
- To develop and validate a novel assay for simultaneous detection of LAP and drug transporter activity (Pgp/MRP) in AML.
- To enable functional characterization of MRD cells for Pgp and MRP activity.
- To facilitate longitudinal and multicenter studies for improved MRD detection and quantification in AML.
Main Methods:
- Development of a fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid to measure Pgp and MRP activity.
- Simultaneous labeling of cells with monoclonal antibodies for LAP detection.
- Validation of the assay on newly diagnosed AML samples and MRD situations, including fresh and cryopreserved cells.
Main Results:
- The assay successfully detected LAP in 80% of diagnosed AML patients.
- Simultaneous detection of LAP and Pgp/MRP activity in MRD cells was achieved.
- No significant differences in LAP expression or Pgp/MRP function were observed between fresh and cryopreserved AML samples.
Conclusions:
- The developed assay provides a robust method for simultaneous detection and functional characterization of MRD in AML.
- This approach can enhance the understanding of multidrug resistance in AML and improve patient monitoring.
- The assay's applicability to both fresh and cryopreserved samples supports its use in multicenter and longitudinal studies.
