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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1046-1056
Simultaneous Activity of MRP1 and Pgp Is Correlated With In Vitro
Resistance to Daunorubicin and With In Vivo Resistance in Adult Acute
Myeloid Leukemia
By
Ollivier Legrand,
Ghislaine Simonin,
Anne Beauchamp-Nicoud,
Robert Zittoun, and
Jean-Pierre Marie
From Université Paris 6, Formation de Recherche Claude Bernard,
E 9912 INSERM, and Service d'Hématologie, Hôpital
Hôtel-Dieu, Paris, France.
 |
ABSTRACT |
In adult acute myeloid leukemia (AML), the weight of the
contribution of the combined activity of Pgp and MRP1 to drug
resistance is not known. To address this question, we compared the
activity of these proteins to the in vitro resistance to daunorubicin
(DNR), etoposide, and cytosine arabinoside (Ara-C), using the
calcein-AM uptake and the 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl]
tetrazolium bromide (MTT) assay in 80 adult AML patients. We found no
correlation or only a weak correlation between the in vitro drug
resistance to DNR and etoposide and MRP1 or Pgp expression or function
when tested separately. However, a strong correlation was observed between the simultaneous activity of MRP1 and Pgp (quantified as the
modulation of calcein-AM uptake by cyclosporin A and probenecid) and
the LC50 of DNR (r = .77, P < .0001). This
emphasized the role of these two proteins, not separately, but together
in the resistance to DNR. In contrast, Mvp/LRP expression did not
correlate with the LC50 of DNR. A high level of simultaneous activity
of Pgp and MRP1 was predictive of a poor treatment outcome (for
achievement of CR [P = .008], duration of relapse-free
survival [RFS; P = .01], and duration of
overall survival [OS; P = .02]). In addition, high LC50 of DNR and high LC50 of etoposide together were also predictive of a poor treatment outcome (for duration of RFS [P = .02] and duration of OS [P = .02]). The
unfavorable cytogenetic category was more closely associated with the
combined activity of both MRP1 and Pgp (P = .002) than with
the activity of Pgp or MRP1 separately. This could explain the poor
prognosis and the in vitro resistance to daunorubicin in this group of
patients. These data suggest that treatment outcome may be improved
when cellular DNR and etoposide resistance can be circumvented or
modulated. Modulation of not only Pgp but also MRP1 could be essential
to attain this aim in adult AML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MULTIDRUG RESISTANCE (MDR) of some
cancers, particularly acute myeloid leukemia (AML), remains a major
obstacle to successful chemotherapy. The best-characterized resistance
mechanism in AML which has been shown to be associated with poor
outcome is mediated by the MDR1 gene and expression of membrane P
glycoprotein.1 But alternative proteins, such as the more
recently recognized multidrug-associated protein (MRP1)2 or
the lung-resistance protein (Mvp/LRP),3 may also contribute
to the resistance to anthracyclines and etoposide in AML. However, the
role of these two proteins are still under discussion.4-10
In several publications, the expression of Pgp did not correlate with
its function of drug efflux.1,11 For this reason,
determining the functional role appears to be more informative than
quantification of MDR proteins. In previous studies, we have shown in
cell lines and in AML that the quantification of
calcein-acetoxymethylester (calcein-AM) uptake (with or without
specific modulator[s] of MRP1 and/or Pgp) can be used to assess the
activity of both MRP1 and Pgp.10,12 Calcein-AM, which is a
substrate of both Pgp and MRP1, becomes fluorescent after the cleavage
of calcein-AM by cellular esterases, producing a measurable fluorescent
derivate calcein in flow cytometry.10,12-16
Despite its association with clinical resistance, MDR1
expression was not correlated with in vitro resistance to daunorubicin (DNR) and etoposide, using the quick and semi-automatized 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl] tetrazolium bromide (MTT) assay, in
several studies.17-19 However, both we and others have
shown that MDR1 and MRP1 gene overexpression emerged in
a sequential manner during selection of different leukemic cell lines
by drugs.20-22 The overexpression of the MRP1 gene
preceded that of the MDR1 gene; afterward, MRP1 and
MDR1 were co-overexpressed. In light of these results,
MRP1 gene overexpression is probably an early event in the
development of drug resistance, and clinical trials that modulated only
Pgp might have limited or no success. In addition, both we and van der
Kolk et al have shown that MRP1 was functional in fresh leukemic blast
cells.10,23 Therefore, it is important to study the
combined activity of Pgp and MRP1. To date, no study has analyzed the
correlations between the simultaneous activity of Pgp and MRP1 and in
vitro and in vivo drug resistance to DNR or etoposide, even though
coexpression of these two proteins is common in adult AML.4
Understanding of these relationships can help unravel the mechanisms of
resistance to anthracyclines and etoposide which are clinically
relevant in adult AML. In addition, such studies can emphasize the
contribution of the combined activity of Pgp and MRP1 in comparison to
other mechanisms.
Therefore, we have studied the contribution of Pgp, MRP1, and Mvp/LRP
expression and Pgp and MRP1 function (using calcein-AM) to the in vitro
resistance to DNR and etoposide and to the in vivo treatment result in
80 adult AML patients.
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MATERIALS AND METHODS |
Patients.
Between July 1995 and December 1997, 80 samples from adult AML patients
(60 de novo and 20 relapsed AML) were successfully tested. The
diagnosis was based on French-American-British (FAB) criteria.24,25 Immunophenotyping was performed by using
flow cytometry. Promyelocytic leukemia (AML3) patients were excluded from the study (because of retinoic acid treatment). For each patient,
several clinical and biological characteristics were analyzed (age,
white blood cell [WBC] count at diagnosis, CD34 expression, and
karyotype). Unfavorable karyotypes were defined as t(9;22) or
abnormalities of chromosomes 5 or 7, abnormalities of 11q2.3 band, or
complex abnormalities. Inversion in chromosome 16 (inv 16) or t(8;21)
indicated good prognosis, and the other karyotypes, including normal,
indicated intermediate prognosis.26 Only untreated de novo
AML patients (60 patients) were analyzed for treatment outcome. De novo
AML patients, in our department, were included in the European
Organization for the Research and Treatment of Cancer
(EORTC) leukemia cooperative group protocols (AML-13 for
patients  60 years old, and AML10 for patients <60 years old). In
induction phase, all the patients received a standard dose of cytosine
arabinoside (Ara-C) (100 mg/m2/d × 10 days),
etoposide, and one anthracycline (DNR, idarubicin, or mitoxantrone × 3 days) at random.
Level of MDR1, MRP1, and Mvp/LRP mRNA expression.
The level of MDR1, MRP1, and Mvp/LRP mRNA expression measured by
reverse transcriptase-polymerase chain reaction (RT-PCR) was described elsewhere.4,10,27 The variations between
samples in the cDNA synthesis were normalized by their relative
quantities of  2 microglobulin (2m)
amplified by 23 cycles of PCR. The normalized yield of MDR products
relative to 2m were then compared with those of A549 cells for MRP1
and Mvp/LRP (a cell line that expressed a high level of MRP1 and
Mvp/LRP) and to those of HL60/DNR for MDR1 (a cell line that expressed
a high level of MDR1), which were defined as 1 arbitrary unit. All
samples contained more than 80% of leukemic cells. Percentage of blast
cells was determined by the May-Grünwald-Giemsa staining and by
immunophenotyping performed by flow cytometry. We performed this test
in 75 of the 80 patients. Correlations with clinical outcome were
largely performed using results of RT-PCR as a continuous variable, in
accordance with consensual recommendations.28-30
Levels of Pgp, MRP1, and Mvp/LRP protein expression.
Pgp, MRP1, and Mvp/LRP protein expression was measured by labeling
fresh viable cells with the UIC2, MRPm6, and LRP56 monoclonal antibodies (MoAbs), respectively, and phycoerythrin (PE)-labeled second
antibody as described before.10 The expression of MDR proteins was established with blast cells selected by CD34 antibody (HPCA2 clone; Becton Dickinson, Le Pont de Claix, France)
(two-color assays) or other markers (for example CD33/CD7, CD33/CD2,
CD33/CD19, or CD33/CD22 by three-color assays) whenever possible, or
with physical characteristics only if blast cells did not express
characteristic markers. Fluorescence was analyzed on a FACSORT flow
cytometer (Becton Dickinson). Values were expressed as adjusted for
control, ie, the ratio of MoAb fluorescence/control antibody
fluorescence. We performed this test in 75 of the 80 patients.
Correlations with clinical outcome were largely performed using the
fluorescence ratio as a continuous variable, in accordance with
consensual recommendations.28-30
Functional analysis of Pgp and MRP1 using calcein-AM.
Cells exposed to the nonfluorescent calcein-AM become fluorescent after
the intracytoplasmic cleavage of calcein-AM by cellular esterases which
produced the fluorescent derivate calcein. Both Pgp and MRP1 actively
extruded calcein-AM.12,13 When we measured calcein-AM
uptake by flow cytometry, we assessed the amount of fluorescent calcein
that had been converted from nonfluorescent calcein-AM. When the Pgp
and/or MRP1 proteins were active, less calcein-AM was retained and less
was converted to fluorescent calcein. Therefore, calcein-AM uptake
(with specific modulators of Pgp and/or MRP1) could be used to assess
whether Pgp and/or MRP1 were functional.10,12-15,31,32 In
our previous studies, calcein-AM uptake ± cyclosporin A (CsA)
provided in AML cells a functional test as specific and sensitive as
Rh123 ± CsA,10 the most specific and sensitive Pgp
functional test.15,31 Calcein-AM uptake ± probenecid also provided a functional test for MRP1 in leukemic cells.
Probenecid was used as specific modulator of MRP1 activity.10,12,33
We performed this functional test in 40 adult AML patients among the 60 de novo AML patients. Cells were incubated with 0.1 µmol/L of
calcein-AM for 15 minutes at 37°C in RPMI medium without or with
modulators (only CsA [2 µmol/L] for Pgp function, only probenecid
[2 mmol/L] for MRP1 function, or both CsA and probenecid together to
assess the simultaneous activity of Pgp and MRP1). Cells were washed
twice in cold phosphate-buffered saline (PBS) and samples were analyzed
with a FACSORT flow cytometer. One example is shown in
Fig 1. All samples were analyzed without
fixation. All the data were calculated as the ratio of drug
fluorescence with modulator(s) divided by drug fluorescence without
modulator after subtraction of the fluorescence of the control. Dead
cells were gated out following scatter characteristics.34
The function of MDR proteins was established with blast cells selected
as above. Correlations with clinical outcome were largely performed
using fluorescence ratio as a continuous variable, in accordance with several consensual recommendations.28-30
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| Fig 1.
One example of Pgp and MRP1 activity quantified by the
effect of probenecid ± CsA (modulators of MRP1 and Pgp, respectively)
on the level of calcein-AM uptake. Cell fluorescence (A) without
modulator, (B) with probenecid, (C) with CsA, and (D) with both
probenecid and CsA together. The results were calculated as the ratio
of drug fluorescence with modulator divided by drug fluorescence
without modulator after subtraction of the fluorescence of the control.
For this example the ratios were 1.51 with probenecid (which quantified
MRP1 activity); 1.7 with CsA (which quantified Pgp activity); and 2.66 with both probenecid and CsA (which quantified the combined activity of
MRP1 and Pgp).
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MTT cytotoxicity test.
In vitro sensitivity of cells to DNR, Ara-C, and etoposide was
determined by planting 2 × 105 cells in a 200-µL
growth medium, without any specific growth factor, containing several
dilutions of the drug in 96-well microtiter plates. Each concentration
of drugs was repeated in six wells. After incubation for 3 days at
37°C with 5% CO2, cell viability was determined using
the MTT assay as described by Plumb et al.35 Briefly, 20 µL of MTT (5 mg/mL in PBS) was added to each well and incubated for 6 hours. The medium and MTT were then removed from the wells by
centrifugation, and formazan crystals were dissolved in 200 µL of
dimethyl sulfoxide (DMSO). The absorbance was recorded in a microplate
reader (Model MR5000; Dynatech Laboratories, France) at
the wavelength of 550 nm. The effect of drug on growth inhibition could
be assessed as: % of Growth Inhibition = 1  [(Absorbance of
Drug-Treated Cells/Absorbance of Untreated Cells) × 100]. The lethal concentration 50% (LC50) was determined as the drug
concentration that resulted in a 50% growth inhibition. Samples were
considered evaluable if the drug-free control wells contained more than
80% of leukemic cells before and more than 70% of leukemic cells
after 3 days of culture. The MTT assay gave reliable results under
these conditions.36 Percentage of blast cells was
determined by the May-Grünwald-Giemsa stain and by
immunophenotyping that was performed by flow cytometry. In vitro drug
resistance was defined as the LC50 more than the plasma peak
concentration achieved in pharmacologic studies (etoposide 60 µmol/L,
DNR 0.85 µmol/L, Ara-C 4 µmol/L).37-39
Statistical analysis.
Clinical and biological factors were investigated for their influence
on remission rate by the  2 or Fisher's exact tests for
binary variables and by the Mann Whitney U test for continuous
variables. Correlations among levels of expression of continuous
variables were estimated using the Spearman rank coefficient. The rate
of (1) relapse-free survival (RFS) was measured from
establishment of complete remission (CR) until relapse or death from
any cause, with observation censored for patients last known alive
without report of relapse; and (2) overall survival (OS) was measured
from diagnosis until death from any cause, with observation censored
for patients last known alive. RFS and OS were estimated by the method
of Kaplan and Meier40 and compared by the log-rank test and
the Breslow-Gehan-Wilcoxon test. However, data were largely
reported and analyzed as continuous variables, as in the consensus
recommendations.28-30 Analyses of prognostic factors for
treatment outcomes were based on proportional hazards regression models
for RFS and OS.41 Significance was defined as a two-tailed
P value of  .05.
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RESULTS |
Expression and function of MDR variables.
We have performed both mRNA detection by RT-PCR and protein detection
by flow cytometry in 70 of 80 patients. The correlation between RT-PCR
and flow cytometry was good for MRP1 gene expression (r = .87, P < .0001) (Fig 2A). All
the negative samples in RT-PCR (a sensitive technique) had a
fluorescence ratio of MRP1 protein expression 1.4 and all samples
with a fluorescence ratio >1.4 expressed MRP1 mRNA (Fig 2A).
Therefore, we have used this threshold of positivity (1.4) for MRP1
protein expression. With this cut-off, 34% of patients expressed MRP1
protein. There was also a strong correlation between MRP1 expression by
RT-PCR or flow cytometry and MRP1 activity (Fig 2B, r = .81, P < .0001; Fig 2C, r = .81, P < .0001, respectively). In some cases, the ratios of fluorescence in the MRP1
protein activity assay ranged from 0.72 to 1. This was probably caused
by variations in cellular uptake of calcein-AM in the experiments.
Conversely, fluorescence ratios of up to 1.28 can also represent some
experimental variability. Therefore, we have used this threshold of
positivity (1.28) for MRP1 protein activity (Fig 2B and C). With this
cut-point, 27% of patients expressed a functional MRP1 protein.
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| Fig 2.
(A) Correlation between MRP1 mRNA expression by RT-PCR
and MRP1 protein expression by flow cytometry. All patients negative by
RT-PCR (a very sensitive technique) expressed a fluorescence ratio from
0.81 to 1.4 in the MRP1 protein detection assay. Therefore, the
threshold of positivity of 1.4 (horizontal dotted line) was used (with
this cut-off of fluorescence ratio, 34% of patients expressed MRP1
protein). (B) Correlation between the effect of probenecid on
calcein-AM uptake and MRP1 mRNA expression by RT-PCR in 40 patients.
Three patients positive by RT-PCR assay were negative in the MRP1
activity assay. (C) Correlation between the effect of probenecid on
calcein-AM uptake and MRP1 protein expression by flow cytometry in 40 patients. One patient positive by flow cytometry assay was negative in
the MRP1 activity assay. In some cases, the ratios of fluorescence in
MRP1 protein activity assay ranged from 0.72 to 1 (B and C). This was
probably caused by variations in cellular uptake of calcein-AM in the
experiments. Conversely, the fluorescence ratios of up to 1.28 can also
represent some experimental variability (area between the two vertical
dotted lines) (B and C). With these thresholds of positivity, there was
7.5% discordance between RT-PCR and functional assays (3 of 40 samples
were MRP1+/activity) and 2.5% between protein detection and
functional assays (1 of 40 samples was MRP1+/activity).
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The correlation between RT-PCR and flow cytometry was also strong for
MDR1 gene expression (r = .95, P < .0001)
(Fig 3A). All the negative samples in
RT-PCR had a fluorescence ratio of Pgp expression 1.3 and all samples
with a fluorescence ratio >1.3 expressed MDR1 mRNA (Fig 3A).
Therefore, we have used this threshold of positivity (1.3) for Pgp
expression. With this cut-point, 51% of patients expressed MDR1. There
was also a correlation between MDR1 mRNA expression and Pgp function
(Fig 3B, r = .63, P < .0001) and between Pgp
expression and Pgp function (Fig 3C, r = .66, P < .0001). In some cases, the ratios of fluorescence in Pgp activity assay
ranged from 0.61 to 1. This was probably because of variations in
cellular uptake of calcein-AM in the experiments. Conversely, the
fluorescence ratios of up to 1.39 can also represent some experimental
variability. Therefore, we have used this threshold of positivity
(1.39) for Pgp activity (Fig 3B and C). With this cut-point,
35% of patients expressed a functional Pgp.
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| Fig 3.
(A) Correlation between MDR1 mRNA expression by RT-PCR
and Pgp expression by flow cytometry (r = .95, P < .0001). All patients negative by RT-PCR expressed a fluorescence ratio
from 0.7 to 1.3 in the Pgp detection assay. Therefore, the threshold of
positivity of 1.3 (horizontal dotted line) was used (with this cut-off
of fluorescence ratio, 51% of patients expressed Pgp). (B) Correlation
between the effect of CsA on calcein-AM uptake and MDR1 mRNA expression
by RT-PCR in 40 patients (r = .63, P < .0001). (C)
Correlation between the effect of CsA on calcein-AM uptake and Pgp
expression by flow cytometry in 40 patients (r = .66, P < .0001). In some cases, the ratios of fluorescence in Pgp
activity assay ranged from 0.61 to 1. This was probably caused by
variations in cellular uptake of calcein-AM in the experiments.
Conversely, the fluorescence ratios of up to 1.39 can also represent
some experimental variability (area between the two vertical bold
lines) (B and C). With these thresholds of positivity, 14 patients
(35%) were Pgp+/activity+, 12 patients (30%) were
Pgp/activity. However, as previously described,1,6,11
discrepant cases were identified, including 13 samples (32.5%)
Pgp+/activity and 1 sample (2.5%) Pgp/activity+.
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As previously reported,10 there was a weak correlation
between Mvp/LRP and MRP1 protein expression (r = .29, P = .04), but no correlation between Mvp/LRP and Pgp (r = .03, P = .84) and between MRP1 and Pgp expression
(r = .25, P = .10) (data not shown). In contrast, there
was a weak correlation between MRP1 and Pgp activity (r = .39, P = .008) (Fig 4).
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| Fig 4.
Graphic representation of samples (A) with simultaneous
activity of both Pgp and MRP1 (6 patients; 15%); (B) with MRP1
activity, without activity of Pgp (5 patients; 12.5%); (C) with Pgp
activity, without activity of MRP1 (7 patients; 17.5%); and (D)
without activity of both Pgp and MRP1 (22 patients; 55%). The area
included between the two horizontal bold lines represents the samples
with a negative activity of Pgp (fluorescence ratios of samples which
ranged between 0.61 and 1.39 represent experimental variability). The
area included between the two vertical dotted lines represented the
samples without activity of MRP1 (fluorescence ratios of samples which
ranged between 0.72 and 1.28 represent experimental variability). There
was a weak correlation (r = .39, P = .008) between
MRP1 and Pgp activity. *Two samples.
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We also analyzed 40 patients for combined Pgp and MRP1 activity (Fig
4). In these patients, we found 6 (15%) samples with simultaneous
activity of Pgp and MRP1, 22 (55%) samples without Pgp and MRP1
activity, 13 samples (32%) with Pgp activity, and 11 samples (27%)
with MRP1 activity. Therefore, 18 (45%) samples had functional
activity of one or both proteins (Fig 4).
The percentage of patients who expressed Pgp or MRP1 (protein or
function) is shown in Table 1.
MDR parameters and other in vitro resistance variables.
No statistically significant correlation was found between the level of
Pgp expression and the LC50 of DNR (r = .29, P = .10), etoposide (r = .09, P = .62), and Ara-C (r = .12, P = .37) and between the level of MRP1 expression and the
LC50 of DNR (r = .33, P = .07), etoposide (r = .23, P = .12) and Ara-C (r = .14, P = .50)
(data not shown). There was also no correlation between the level of
Mvp/LRP expression and the LC50 of DNR, etoposide, and Ara-C (data not shown).
Similarly, we found no correlation or only a weak correlation between
the LC50 and both the effect of CsA on the level of calcein-AM uptake,
which quantified only Pgp activity (r = .36, P = .02 for DNR; r = .03, P = .85 for etoposide; and r = .25, P = .16 for Ara-C)
(Fig 5A and B) and the effect of probenecid on the level of calcein-AM uptake, which quantified only MRP1 activity
(r = .47, P = .002 for DNR; r = .24, P = .14 for etoposide and r = .08, P = .61 for
Ara-C) (Fig 5C and D). In contrast, there was a strong correlation
between the LC50 of DNR and the combined effect of probenecid and CsA
(which quantified the simultaneous activity of Pgp and MRP1) on the
level of calcein-AM uptake (r = .77, P < .0001) (Fig 5E), a weak correlation between the LC50 of etoposide and
the combined effect of probenecid and CsA on the level of calcein-AM
uptake (r = .54, P = .007) (Fig 5F) and no correlation
between the LC50 of Ara-C and the combined effect of probenecid and CsA
on the level of calcein-AM uptake (r = .27, P = .20)
(data not shown).
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| Fig 5.
Correlations between Pgp activity (measured by the effect
of CsA on calcein-AM uptake) (y-axis, A and B) and the LC50 of DNR
(x-axis, A) and the LC50 of etoposide (x-axis, B); between MRP1
activity (which was measured by the effect of probenecid on calcein-AM
uptake) (y-axis, C and D) and the LC50 of DNR (x-axis, C) and the LC50
of etoposide (x-axis, D); and between the simultaneous activity of MRP1
and Pgp (which was quantified by the combined effect of probenecid and
CsA on calcein-AM uptake) (y-axis, E and F) and the LC50 of DNR
(x-axis, E) and the LC50 of etoposide (x-axis, F). When the combined
effect of CsA/probenecid on calcein-AM uptake is analyzed, some cases
had a fluorescence ratio which ranged from 0.59 to 1 (E and F). As for
protein detection, this was probably caused by variations in cellular
uptake of calcein-AM in the experiments. A similar variation in the
opposite direction (1.41) can also represent some experimental
variability. Therefore, we have used this threshold of positivity
(1.41) for the simultaneous activity of Pgp and MRP1 (horizontal dotted
line). With this cut-off, 52% of patients expressed combined activity
of both MRP1 and Pgp.
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MDR parameters and in vivo resistance.
Sixty untreated AML patients were evaluable for clinical
response. Sixty-five percent of patients achieved CR. Variables
influencing CR are shown Table 2. CR rate
significantly decreased with increasing MDR1 gene
expression (0.197 ± 0.022 v 0.092 ± 0.087, P = .04 by RT-PCR; 3.93 ± 2.27 v 2.10 ± 0.59, P = .04 by flow cytometry) and with increasing MRP1 gene
expression (0.756 ± 0.312 v 0.212 ± 0.341, P = .05 by RT-PCR; 1.90 ± 0.43 v 1.38 ± 0.53, P = .05 by flow cytometry). However, CR rate
was not associated with the level of Mvp/LRP expression by both assays
(RT-PCR and flow cytometry) (Table 2). Patients who achieved CR also
had a lower activity of Pgp (1.17 ± 0.27 v 1.55 ± 0.37, P = .05), a lower activity of MRP1 (1.12 ± 0.36 v
1.39 ± 0.22, P = .05) and a lower simultaneous activity of
MRP1 and Pgp (1.35 ± 0.47 v 2 ± 0.54, P = .008)
than patients who did not (Table 2). While the CR rate significantly decreased with increasing LC50 of DNR (0.56 ± 1.32 v 0.29 ± 0.28, P = .05), it was not associated with the level of
LC50 of etoposide and Ara-C. When the threshold of positivity was used
for in vitro MDR variables, we obtained the same results
(Table 3).
RFS and OS decreased significantly with increasing LC50 of Ara-C
(P = .02 and P = .001, respectively), and with
increasing simultaneous activity of Pgp and MRP1 (P = .01 and
P = .02, respectively) (Table 4).
However, the expression (by both RT-PCR and flow cytometry) and the
activity of Pgp and MRP1 separately did not influence RFS or OS (data
not shown).
Interestingly, while the LC50 of DNR and LC50 of etoposide were not
separately associated with RFS and OS, patients with both high LC50 DNR
(0.85 µmol/L) and high LC50 etoposide (60 µmol/L) had higher
risks of relapse or death than other patients
(Fig 6a and c). Similarly, patients with
high combined activities of Pgp and MRP1 also had a higher risk of
relapse or death than other patients (Fig 6b and d).
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| Fig 6.
Relation between the in vitro drug resistance to DNR and
etoposide together (a and b) or the simultaneous effect of both CsA and
probenecid on calcein-AM uptake (b and d) and the probability (which
analyzed the combined activity of Pgp and MRP1) of RFS and OS in
untreated de novo AML patients.  Compared by the log-rank test;
compared by the BreslowGehan-Wilcoxon test.
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Other prognostic factors and correlation with in vitro resistance
variables.
The effect of other well-known variables such as age, cytogenetics, WBC
count at diagnosis, and CD34 expression on clinical response were also
analyzed. RFS and OS were significantly poorer for patients with
unfavorable cytogenetics (P = .04 and P = .01, respectively) and decreased significantly with increasing age (P = .03 and P = .01, respectively) and increasing WBC
(P = .03 and P = .04, respectively) (Table 4). CD34
expression was not a prognostic factor for RFS and OS.
Table 5 shows significant
associations between older age and high mRNA MDR1 expression (P = .01), high Pgp expression (P = .01), high effect of CsA on
calcein-AM uptake (P = .009), high level of LC50 of DNR
(P = .05), and high level of LC50 of Ara-C (P = .04).
Unfavorable cytogenetic effect was correlated with a high
effect of both probenecid and CsA on calcein-AM uptake (P = .002), a high level of LC50 of DNR (P = .03), and a high level
of LC50 of Ara-C (P = .02). High WBC count at
diagnosis was associated with a high level of Mvp/LRP
(P = .02).
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Table 5.
Significant Associations Between Well-Known Clinical
and Biological Variables on Clinical Response in AML and In Vitro
Resistance Variables
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| |
DISCUSSION |
While both MRP1 and Pgp may confer resistance to different families of
drugs in AML, including anthracyclines and etoposide, the relative
importance of these two genes is not known. Several studies reported
the sequential expression of MRP1 and Pgp in drug-selected cell
lines.20-22 At clinically relevant concentrations of
doxorubicin or homoharringtonine, resistance to these drugs was related
to MRP1 overexpression, but not to MDR1 expression in human myeloid
leukemia cell lines.20,22 Only when cell lines were exposed
to drugs for a prolonged time period or selected for relatively
high-level drug resistance did Pgp/MDR1 overexpression become apparent.
Similar findings occur in other cell lines (murine leukemia, small cell
lung cancer cells).21,42,43 In light of these results,
MRP1 gene overexpression is probably an early event in the
development of drug resistance, and MRP1 and Pgp could be coexpressed
in AML cells exposed to pharmacological doses of cytotoxic drugs. In
clinical samples of AML, MRP1 overexpression ranged from 7% to
30%.4,7,8,44-49 MRP1 overexpression was more frequent in
drug-refractory or relapsed patients than in drug-sensitive patients in
one study,4 but not in others.46,47 Similarly,
the coexpression and correlation between MRP1 and MDR1 are under
debate. These contradictory results might be partially caused by
differences in the composition of samples and experimental methods, as
well as differences in the definition of overexpression. To date, few
data on the coexpression of these two genes in AML cells have been
reported, and the combined functionality of these two proteins in
clinical samples has not been studied. We have shown, in previous
studies, that calcein-AM uptake can be used to assess whether MRP1
and/or Pgp are functional and to assess the simultaneous activity of
MRP1 and Pgp in fresh leukemic cells.10,12 In our present
study, 45% of the AML samples studied exhibited a functional activity
of one or both proteins. Taken together, these previous and present
reports suggest that MRP1 and Pgp need to be considered together and
that clinical trials that selectively modulate Pgp are likely to
achieve limited success. Therefore, we analyzed the contribution of the
combined activity of Pgp and MRP1 to in vitro and in vivo resistance to
chemotherapy in AML patients.
In our study, the absence of or only weak correlations between MRP1 or
Pgp expression or function (tested separately) and in vitro drug
resistance to DNR and etoposide were in agreement with other
data17-19 and could be partly explained by the separate analysis for the two resistance genes. In agreement with this, we have
found a good correlation between the simultaneous activity of MRP1 and
Pgp and in vitro resistance to DNR, which emphasizing the role of these
two proteins together in the resistance to DNR in adult AML. In
addition, probenecid, the modulator of MRP1 used in this study, has
been associated with an increased accumulation of DNR and with the
correction of the altered distribution of DNR in leukemic cell
lines.33
Similarly, we have shown that the combined activity of MRP1 and Pgp was
a prognostic factor for treatment outcome (achievement of CR, and
duration of RFS and OS), but not MRP1 or Pgp separately (for RFS and
OS). In several other studies, the prognostic value of MRP1 expression
is discussed.4,7,8,44-49 However, in these studies only one
technique was used and functionality was not assessed. As for
MDR1,15,28-30 an elaboration of consensus recommendations would be required.
In contrast, preliminary studies using the MTT assay for the prediction
of chemoresistance in adult AML suggest that it may be helpful for
risk-group stratification in adult AML.50,51 In addition,
this test has a strong value in the prediction of clinical response in
childhood leukemias.52,53 It was also shown that in vitro
drug resistance determined with the differential staining cytotoxicity
(DiSC) assay, based on the same concept as the MTT assay, was related
to survival in adult AML.54 Therefore, we used the MTT
assay to assess the in vitro resistance to drugs. In our study,
patients who exhibited both high LC50 of DNR and high LC50 of
etoposide, but not etoposide alone, had a poorer prognosis than other
patients, underlying the importance of these two drugs. In clinical
trials, it was unclear whether the addition of etoposide improved
treatment outcome in adult AML.55-57 But in Bishop's
randomized study, which included 264 patients, there was an additional
benefit from the use of etoposide specifically confined to patients
ages less than 55 years.58 There has not been another large
randomized trial comparing anthracycline + Ara-C ± etoposide as
induction chemotherapy. Nevertheless, our finding is in accordance with
the fact that etoposide could be effective in the treament of AML. We
also showed that the combined activity of MRP1 and Pgp in resistance to
etoposide appears less important than their role in resistance to DNR.
We have shown that the well-known prognostic factors in
AML,59 age and cytogenetics, were associated with both Pgp
and MRP1 expression and function. Age has already been
correlated with Pgp expression and function.6,7 In our
study, the unfavorable cytogenetic category was better associated with
the combined activity of both MRP1 and Pgp than with the activity of
Pgp or MRP1 separately. This could explain the poor prognosis and the
in vitro resistance to daunorubicin in this group of patients.
Modulation of not only Pgp but also MRP1 could be essential, in this
category of patients, to improve the results of treatment.
As in our previous report,10 we found a good correlation
between mRNA expression detected by RT-PCR and protein expression detected by flow cytometry for both MDR1 and MRP1
genes. As previously described, we have identified discrepant cases
between Pgp expression and function.1,6,11 In contrast, for
MRP1, discrepancy between protein expression and function were
uncommon. 28-30 We recommend the detection of MRP1
expression by flow cytometry. In addition, a functional
test using calcein-AM uptake assay could be used to assess the activity
of MRP1 (we found 7% of cases with a discrepancy between MRP1
expression and function) and the simultaneous activity of both MRP1 and
Pgp. Other functional tests can assess the activity of
MRP1.60 A critical evaluation of these different assays
would be useful.
Unlike the results obtained with MRP1 and Pgp, we found, as recently
reported by both Leith et al7 and us,10 that
the level of Mvp/LRP expression is not correlated with treatment
outcome in adult AML, in contrast to other studies.5,9 In
our study, the level of Mvp/LRP expression was not correlated with in
vitro resistance to DNR or etoposide. Therefore, a causal mechanistic relationship of Mvp/LRP with drug resistance is still lacking. But, as
we recently reported, the discrepancy in the clinical significance of
Mvp/LRP expression may be related to the methodology used.61
In conclusion, all the data presented here support the hypothesis that
the modulation of both Pgp and MRP1, by agents such as probenecid and
PSC833, may simultaneously increase the percentage of CR, the
percentage of RFS, and survival duration in adult AML patients by
increasing blast cell DNR ± etoposide cytotoxicity. The concentration of probenecid that reverses MRP1 function is clinically achievable in vivo.33 However, because of the
relatively small patient numbers in our study, a multicenter study will
be required to evaluate all of these resistance parameters.
| |
FOOTNOTES |
Submitted October 9, 1998; accepted March 29, 1999.
Supported in part by a grant from ARC (Grant No. 9637).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ollivier Legrand, MD, PhD,
Hôpital Hôtel Dieu, 1 place du parvis Notre Dame, Service
d'hématologie, 181 Paris Cedex 04, France; e-mail:
olivier.legrand{at}htd.ap-hop-paris.fr.
| |
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Z. Benderra, A. M. Faussat, L. Sayada, J.-Y. Perrot, R. Tang, D. Chaoui, H. Morjani, C. Marzac, J.-P. Marie, and O. Legrand
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M. Qadir, K. L. O'Loughlin, S. M. Fricke, N. A. Williamson, W. R. Greco, H. Minderman, and M. R. Baer
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Z. Benderra, A.-M. Faussat, L. Sayada, J.-Y. Perrot, D. Chaoui, J.-P. Marie, and O. Legrand
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J. Thomas, L. Wang, R. E. Clark, and M. Pirmohamed
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J. E. Kolitz, S. L. George, R. K. Dodge, D. D. Hurd, B. L. Powell, S. L. Allen, E. Velez-Garcia, J. O. Moore, T. C. Shea, E. Hoke, et al.
Dose Escalation Studies of Cytarabine, Daunorubicin, and Etoposide With and Without Multidrug Resistance Modulation With PSC-833 in Untreated Adults With Acute Myeloid Leukemia Younger Than 60 Years: Final Induction Results of Cancer and Leukemia Group B Study 9621
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D. Mahadevan and A. F. List
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R. B. Walter, B. W. Raden, M. R. Cronk, I. D. Bernstein, F. R. Appelbaum, and D. E. Banker
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R. W. Robey, K. Steadman, O. Polgar, K. Morisaki, M. Blayney, P. Mistry, and S. E. Bates
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T. Brooks, H. Minderman, K. L. O'Loughlin, P. Pera, I. Ojima, M. R. Baer, and R. J. Bernacki
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G. D. Leonard, T. Fojo, and S. E. Bates
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R. B. Walter, B. W. Raden, T. C. Hong, D. A. Flowers, I. D. Bernstein, and M. L. Linenberger
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O. Legrand, J.-Y. Perrot, G. Simonin, M. Baudard, and J.-P. Marie
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O. Legrand, J.-Y. Perrot, M. Baudard, A. Cordier, R. Lautier, G. Simonin, R. Zittoun, N. Casadevall, and J.-P. Marie
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TOP
ABSTRACT
INTRODUCTION