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Bio-Technical Methods Section (BTS)

A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia

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-xL, 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.

Introduction

Complete remission rates as high as 70–80% have been reported in adult patients with acute myeloid leukemia (AML),1,2,3 but approximately 60–70% of the patients will eventually relapse because of the persistence of residual leukemia cells. These minimal residual disease (MRD) cells are undetectable by conventional morphological techniques. Detection of MRD at different stages of the disease, that is, after chemotherapy or in stem cell transplants, can provide useful information to tailor therapy, such as early intervention or purging of the stem cell graft. Several molecular and immunological methods have been developed to detect MRD cells, among which PCR and flow cytometry are the most suitable. Detection by flow cytometry (FC) is based on the idea that AML cells frequently show aberrant leukemia-associated phenotypes (LAPs) characterized by asynchronous antigen expression, crosslineage antigen expression and antigen overexpression.4,5,6,7,8,9,10,11,12 Although the use of FC has some drawbacks such as interpatient heterogeneity in antigen expression that requires a large panel of monoclonal antibodies (MoAbs) to cover all potential aberrancies, or the occurrence of phenotypic shifts,13,14,15 the FC method is very quick, sensitive and quantitative. Moreover, it is the only method that allows functional analysis on viable MRD cells thereby enabling to gain insight into the mechanisms by which the leukemia cells survive. These mechanisms include apoptosis resistance. A great deal of apoptosis research in AML has been focused on apoptosis-related proteins in de novo AML cells, and showed that AML patients with high Bcl-2 levels or high Bcl-2/Bax ratios at diagnosis have a poor clinical outcome.16,17,18,19,20 Analysis of apoptosis-related proteins in MRD cells that survived chemotherapy would offer insight into the role of apoptosis resistance in treatment failure in AML. For example, it might reveal characteristics present only in subpopulations at presentation of the disease. The aim of the present study was therefore to design a technique that enables detection and quantification of apoptosis-related proteins such as Bcl-2, Bcl-xL, Bax, Mcl-1 and p53 in MRD cells. With the conventional Western blot method, this would not be possible because of the limited number of MRD cells available. We have recently developed a technique that allows the detection of ABC transporter activity in MRD (van der Pol et al,21 Haematologica, 2003, 88, in press). The present approach was aimed to characterize the role of apoptosis-related proteins in MRD in AML. We have set up a four-color FC method and determined in five cell lines whether this method could reliably replace Western blot analysis and whether the permeabilization procedure necessary for the detection of intracellular apoptosis-related proteins would influence the profiles of cell surface antigen expression required for the identification of MRD cells.

Materials and methods

Cell lines

Human AML cell lines HL60, THP-1, KG1, the chronic myeloid leukemia blast crisis cell line K562 and the human histiocytic lymphoma cell line U937 were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). All cell lines, except KG-1 that was cultured in DMEM supplemented with 20% FCS, were cultured in RPMI supplemented with 10% FCS at 37°C in a humid atmosphere with 5% CO2. The cells were maintained at 0.1–1 × 106 cells/ml and were adjusted to a concentration of 0.3 × 106 cells/ml 24 h before experimental use.

Patients

Following informed consent, AML bone marrow samples were obtained at the time of diagnosis, at follow-up (after first, second and third cycle of chemotherapy, peripheral blood stem cell transplants, at regular time points after completion of treatment) and at relapse. FAB classification was as follows: M0 (n=5), M1 (n=5), M2 (n=8), M4 (n=3), M4eo (n=3), M5 (n=8), M6 (n=1), RAEB-t (n=2), unknown (n=3).

Antibodies

Unlabeled p53 (DO-7), unlabeled Bcl-2 (clone 124) mouse monoclonal antibody, FITC-conjugated mouse anti-human Bcl-2 (clone 124) and FITC-conjugated anti-rabbit were from Dako Diagnostics BV, The Netherlands. The rabbit polyclonal antibodies Bax (P-19), Bcl-xL (S-18) and Mcl-1 (S-19) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The MRD monoclonal antibody panel consisted of the following FITC-conjugated MoAbs: CD2 (T11, Coulter, Coultronics, Mergency, France), CD5 (DK23, Dako), CD7 (CLB-T-3A1/1,7, CLB, Amsterdam, The Netherlands), CD11c (KB90, Dako), CD13 (WM-47, Dako), CD15 (MMA, BD, Becton Dickinson, San Jose, CA, USA), CD19 (J4.119, Immunotech, Marseille, France), CD33 (CD33-4D3, Caltag, Caltag Laboratories, San Francisco, CA, USA), CD34 (8G12, BD), CD38 (T16, Immunotech), CD45 (J33, Immunotech), CD61 (RUUPL7F12, BD), CD65 (VIM2/GM-4102, AnderGrub, Kaumberg, Austria). PE-conjugated MoAbs were: CD2 (T11, Coulter), CD7 (8h8.1, Immunotech), CD11b (D12, BD), CD13 (My7, Coulter), CD15 (80H5, Immunotech), CD33 (My9, Coulter), CD34 (8G12, BD), CD38 (HB7, BD), CD45 (T29/33, Dako), CD56 (My31, BD), CD117 (104D2, BD), CD133 (CD133/1, Miltenyi Biotech, Bergisch Gladbach, Germany). PerCP-conjugated MoAbs were: CD4 (2D1, BD), CD19 (SJ25C1, BD), CD34 (8G12, BD), CD45 (2D1, BD), HLA-DR (L243, BD). APC-conjugated MoAbs were: CD2 (G11, Imgen, Antwerpen, Belgium), CD13 (22A5, Imgen), CD14 (MOP9, BD), CD19 (SJ25C1, BD), CD33 (P67.6, BD), CD34 (8G12, BD), CD38 (HB7, BD), CD45 (J33/2D1, Immunotech/BD), CD117 (104D2, BD), HLA-DR (T436, Caltag).

Reagents

NaVO3, NaCl, Tris-HCl, EDTA, DTT, SDS, bromophenol blue, saponin and Triton X-100 were purchased from Sigma-Aldrich Chemie (Steinheim, Germany) and protease inhibitors (complete mini protease inhibitors) from Boehringer Mannheim, Mannheim, Germany. Molecular weight marker was the Bio-Rad rainbow marker (Bio-Rad Laboratories Ltd, Hertfordshire, UK).

Western blot analysis

Cell pellets of 1–5 × 106 cells were immediately frozen in liquid nitrogen and stored at –80°C until analysis. Frozen–thawed cell pellets were lysed in 50 μl/2 × 106 cells of lysing solution containing 0.06 mM NaVO3, 176 mM NaCl, 11.8 mM Tris-HCl pH 7.8, 5.9 mM EDTA, 1.2% Triton X-100 and protease inhibitors at 4°C for 30 min. To estimate the total protein level, a commercially available kit that employs the Bradford method22 was used. Sample buffer containing 0.19 M Tris-HCl pH 6.8, 30 mM DTT, 2% SDS, 0.1% bromophenol blue and 30% glycerol were added to the lysate in a ratio of lysate:sample buffer of 2:1. The samples were incubated at 100°C for 5 min. HL60 cell line was used as a positive control for Bcl-2, Bax, Bcl-xL and Mcl-1 expression and KG1 cell line was used as a positive control for p53 expression. After lysis, the cells were appropriately diluted in sample buffer so as to give a five-point standard curve for each blot by loading 20 μl containing 5, 10, 15, 20 and 25 μg (HL60) or 1, 3, 5, 7 and 10 μg (KG1) of total cellular protein. Each sample was subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli23 using the mini-protean II system supplied by Bio-Rad Laboratories Ltd (Hertfordshire, UK). Acrylamide gels (15%) were used for the analysis of all proteins. After electrophoresis, proteins were transferred to nitrocellulose membranes (Hybond-C; Amersham, Little Chalfont, UK) using the mini-protean II system (Bio-Rad Laboratories Ltd). After transfer, the blots were immersed for 1 h in a blocking buffer of 2% BSA in PBS containing 0.1% Tween (PBS-T). Primary antibody was diluted in blocking buffer and incubated with the blots for 1 h as follows: Bcl-2, Bax, Bcl-xL and Mcl-1 at 1:500 and p53 at 1:2000. The blots were washed thoroughly with PBS-T and incubated with secondary antibody (horseradish peroxidase (HRP) labeled anti-mouse or HRP labeled anti-rabbit, diluted 1:1000 in PBS-T) for 1 h. After a final wash in PBS-T, HRP was detected using Lumi-Light reagent (Boehringer Mannheim, Mannheim, Germany) as described in the manufacturer's instructions. Chemiluminescence was detected by autoradiography using X-ray film. The integrated optical density (IOD) of the resulting bands was determined by densitometry using a Bio-Rad densitometer. The identity of the target protein was determined on the basis of a molecular weight marker. A standard curve of the positive reference cell line was produced for each blot by plotting the IOD of each standard band against the amount (μg) of total protein cell lysate loaded. For each cell line, the IOD was compared with the standard curve of the positive control cell lines. The amount of specific protein in the cell line was then presented as a ratio relative to the control cell line. This way of quantification, instead of using a house-keeping protein, was chosen to avoid that differences in size and volume, as in primary AML samples, contribute to a quantification error.

Establishment of LAPs and MRD detection

The phenotypical analysis of de novo AML was performed on whole bone marrow or mobilized peripheral blood samples upon staining with FITC-, PE-, PerCP- and APC-conjugated MoAbs. A detailed description of the establishment of a LAP and MRD detection has previously been published.21,24

Apoptosis-related protein detection in cell lines, de novo AML and in MRD by flow cytometry

For a detailed description of the procedure and quantification of apoptosis-related protein expression, see the appendix, ‘Method in focus’.

Statistics

The Spearman ρ correlation test was used to show the correlation between the FC and Western blot analysis. The Wilcoxon signed ranks test was performed to compare MRD percentages with and without the permeabilization procedure. All statistical analyses were performed using the SPSS software program. A P-value <0.05 was considered as significant.

Results and discussion

Comparison of apoptosis-related protein detection by flow cytometry and Western blot analysis

To prove that the flow cytometric method described here can be reliably used in AML samples at diagnosis and in MRD in AML, flow cytometric detection of apoptosis-related proteins was first compared with Western blot analysis, which we considered to be the golden standard. The methods showed to correlate well in a linear fashion for Bcl-2, Bcl-xL, Mcl-1, Bax and p53 (R=0.7; P<0.0001). We conclude that Bcl-2, Bcl-xL, Mcl-1, Bax and p53 can be reliably detected by flow cytometry, which is in accordance with a recent study in AML only investigating Bcl-2.25

Morphological and phenotypical preservation after permeabilization procedure

If simultaneous analysis of apoptosis-related proteins and MRD by FC is to be performed accurately, the membrane permeabilization step required for intracellular protein detection should not modify the cell surface labeling. Although the FSC/SSC characteristics undergo changes by the permeabilization procedure, it is possible to exclude debris characterized by a low FSC/SSC (Figures 1a and b). After the permeabilization procedure, the blast population, characterized by a low CD45 expression and a low SSC (Lacombe et al,26 see Figures 1c and d), is comparable to the fresh sample as shown in Figure 1g. Furthermore, Figures 1c and d illustrate the usefulness of the CD45 staining in combination with SSC to exclude neutrophils, monocytes and lymphocytes from MRD phenotyping. This gating is of particular importance since it has been described that neutrophils differ considerably of normal and leukemia progenitor cells in Bcl-2 and Bcl-xL mRNA content.27

Figure 1
figure1

Effect of permeabilization on scatter properties and antigen expression. (a–d) AML patient BM sample after the first cycle of chemotherapy, (e, f) AML patient bone marrow sample at diagnosis. The samples were stained for extracellular antigens alone (a, c and e) or were followed by a permeabilization procedure (b, d and f) to obtain access to intracellular antigens as described in Materials and methods. FSC/SSC was used to exclude debris from the analysis (a, b). CD45 in combination with SSC (in c and d) has been used to discriminate distinct cell populations as described elsewhere:26 blast cells (R2), lymphocytes (R3), monocytes (R4), neutrophils (R5). When necessary, we confirmed the position of the population with a transformed-SSC using Paint-A-gate software. The aberrant antigen combination CD117+ CD7+ (R7) being the LAP of the blasts of this patient has been established at diagnosis with a panel of 15 quadruple MoAbs combinations (see under Materials and methods). CD117+ CD7− (R6) and CD117− CD7low (R9) cells are AML cells that do not bear the aberrant antigen combination, which thus cannot be traced as MRD. CD117− CD7+ (R8) are CD7+ lymphocytes. Gate settings were changed in some cases to fit a distinct population within the region. (g) Quantitative comparison (percentage of gated cells) of the populations within the regions indicated is shown. In this figure, the actual antigen and intracellular staining is not shown.

Since the identification of MRD relies on the detection of LAPs,21,24 the effect of the permeabilization procedure on the phenotypical analysis of diagnosis and follow-up bone marrow AML material was studied. Figures 1e and f illustrate the approach using an AML with blasts expressing CD117 with coexpression of CD7 (LAP: CD117+ CD7+) at diagnosis. A small influence of the permeabilization procedure on the expression of the surface antigens CD117 and CD7 can be seen, although the percentages within the selected gates did not change (Figure 1e–g). To determine the preservation of all surface antigens involved in the study during the permeabilization procedure, we tested 38 diagnosis (17 cases with a CD34+ LAP, 13 cases with a CD117+ LAP, three cases with a CD133+ LAP and five cases with a phenotype including both CD34 and CD117 as a progenitor marker) and 42 MRD samples. Results are presented as LAP+ cells as a percentage of the progenitor cell population (CD34+ or CD117+ or CD133+). As can be seen in Figure 2, percentages of LAP+ cells at diagnosis and in follow-up samples with and without the permeabilization procedure correlated well, showing a close to y=x relation. In this cohort, none of the antigens that were used (as described in Figure 2) were affected by the permeabilization procedure. From these results we conclude that the permeabilization procedure can be applied to the phenotypical analysis of both de novo and MRD cells without loss of cells of interest.

Figure 2
figure2

Effect of the permeabilization procedure on phenotypic assessment of LAP combinations of AML blasts at diagnosis and in follow-up. The frequency of cells bearing a LAP determined by phenotyping alone or after the permeabilization procedure. The most common LAP in this group of patients was either CD7+ CD34+ (26%: n=10) or CD7+ CD117+ (16%: n=6). Other LAPs included: CD2+ CD34+ (n=1), CD5+ CD34+ (n=1), CD4+ CD34+ (n=1), CD11b+ CD34+ (n=2), CD19+ CD34+ (n=1), CD22+ CD34+ (n=1), CD56+ CD34+ (n=1), CD15+ CD117+ (n=3), CD19+ CD117+ (n=1), CD56+ CD117+ (n=2), CD133+ CD34− (n=3), CD34+ CD117+ (overexpression) (n=5). The percentage of LAP+ cells has been determined relative to the total number of CD34+, CD117+ or CD133+ blasts at diagnosis and in follow-up samples. Results from 38 de novo (closed circles) and 42 follow-up (open and gray circles) AML samples are shown. Follow-up samples were divided into samples with (open circles) and without (gray circles) complete remission. The percentage of LAP+ blasts correlated significantly between the measurements with and without the permeabilization procedure (diagnosis: R=0.990; P<0.0001; n=38 and follow-up: R=0.974; P<0.0001; n=42, respectively) in a close to y=x relation. An enlargement of the graph from 0 up to 20% is shown in the inset to better visualize the lower percentages.

Detection of intracellular apoptosis-related proteins in de novo AML and MRD

An example of an AML at diagnosis (Figure 3, left column) and a sample from the same patient after the first cycle of chemotherapy (Figure 3, middle and right columns) is demonstrated. At diagnosis, the AML presented with blasts (R2, Figure 3b) expressing CD34 and with coexpression of CD2 (CD34+ CD2+ cells; see R5, Figure 3c) MRD cells, present in the BM sample after first cycle of chemotherapy, show up in R5 in Figure 3d. Gating on the LAP+ MRD cells now offers the possibility to analyze the protein expression at different time points of the disease. Accurate gating on leukemia CD34+ cells is of extreme importance since the presence of normal progenitor cells (CD34+ CD2−, R6 in Figure 3d) might easily affect the results. Such would be especially important with differences between normal and leukemia progenitors as described, for example, for Bcl-2 and Bcl-xL mRNA levels.27 Indeed, the MRD cells in R5 (Figure 3d) have a different Bcl-2 expression than the CD34+ CD2− normal cells in R6 (11.5 vs 8.5, respectively, not shown).

Figure 3
figure3

Apoptosis-related protein expression in de novo and follow-up AML. An example of Bcl-2, Bcl-xL, Mcl-1 and Bax expression in BM from de novo AML (left column) and from the same patient after first course of chemotherapy (middle and right column) is shown. The surface antigen staining of the cells made it possible to gate on specific subpopulations, while the permeabilization procedure after the surface staining allowed staining cells for Bcl-2, Bcl-xL, Mcl-1 and Bax (see Materials and methods). Blast cells were specified by gating on the CD45low/SSC low population (R2). Within the CD45low/SSC low gate, regions were set around the LAP+ cells: CD34+ CD2+ (R5) and LAP− cells: CD34+ CD2− (R6) population in (c) and (d). At presentation of disease, the percentage of CD34+ blasts expressing CD2 is 94%. After the first cycle of chemotherapy, part of the CD34+ blasts expressed CD2. Apoptosis-related protein (solid lines) and isotype control (dotted lines) of the CD34+ CD2+ cells within region R5 are shown for de novo and follow-up. Bcl-2 (e, f), Bcl-xL (g, h), Mcl-1 (i, j) and Bax (k, l). Right column of figures show the Bax expression after the first cycle of chemotherapy in (m) blast cells (from R2 in b), (n) lymphocytes (from R3 in b), (o) MRD cells (from R5 in d), (p) normal progenitor cells (from R6 in d). Ratios calculated relative to the isotype control were indicated in each plot.

This method can not only be extended to other cell populations that can be phenotypically discriminated from each other, for instance blast cells (CD45 low), lymphocytes (CD45 high cells) or normal progenitor cells (CD34+ CD2− cells) but also to other apoptosis-related proteins. As an example, Bax expression of the above-described populations is shown (right column, Figure 3).

In addition, the apoptosis-related proteins investigated in this study can be extended to other known members of the Bcl-2 family (A1, Bcl-w, Bak, Bad, Bcl-xS) or members of the recently emerging important family of inhibitors of apoptosis proteins, IAPs: XIAP, ILP2, c-IAP1, c-IAP2, ML-IAP, NIAP, Survivin, Apollon.28,29,30,31,32 Moreover, the availability of many directly labeled monoclonal antibodies and polyclonal antibodies makes this method very flexible so that it can be used in several studies using cells in suspension, especially in studies aimed at small populations that cannot be measured with conventional methods such as Western blot analysis.

In conclusion, the multiparametric flow cytometric protocol presented in this paper permits a very accurate analysis of apoptosis-related proteins in a complex mixture of cell populations such as that found in follow-up AML bone marrow containing MRD cells and can give important data on the role of apoptosis-resistance in AML. An ongoing study using this approach will offer great help to unravel the mechanisms contributing to the emergence of MRD and chemotherapy failure in AML, and will eventually help to direct future therapies.

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Correspondence to G J Schuurhuis.

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The Dutch Cancer Foundation financially supported this work.

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van Stijn, A., Kok, A., van der Pol, M. et al. A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia. Leukemia 17, 780–786 (2003). https://doi.org/10.1038/sj.leu.2402885

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Keywords

  • minimal residual disease
  • apoptosis resistance
  • acute myeloid leukemia
  • Bcl-2
  • Bcl-xL
  • Bax
  • Mcl-1

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