A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia.
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.
van Stijn
A
A
Department of Haematology, VU University Medical Center, Amsterdam, The Netherlands.
Kok
A
A
van der Pol
M A
MA
Feller
N
N
Roemen
G M J M
GM
Westra
A H
AH
Ossenkoppele
G J
GJ
Schuurhuis
G J
GJ
D000208:Acute Disease; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D017209:Apoptosis; D015153:Blotting, Western; D001853:Bone Marrow / Q000473:pathology; D002463:Cell Membrane Permeability / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D015973:Gene Expression Regulation, Leukemic; D019254:Genes, bcl-2; D006801:Humans; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D018365:Neoplasm, Residual; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012503:Saponins / Q000494:pharmacology; D013048:Specimen Handling; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012503:Saponins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein