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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1086-1099
By
From the Departments of Pathology and the Cancer Center,
University of New Mexico School of Medicine, Albuquerque, NM; the Fred
Hutchinson Cancer Research Center and the Department of Medicine,
University of Washington, and the Southwest Oncology Group (SWOG)
Statistical Center, Seattle, WA; the Cleveland Clinic, Florida
Division; the Department of Pathology, City of Hope Cancer
Center Program, Duarte, CA; the Department of Pathology, St Jude
Children's Hospital, Memphis, TN; and the Southwest Oncology Group
Leukemia and Cytogenetics Programs, San Antonio, TX.
Therapeutic resistance is a major obstacle in the treatment of acute
myeloid leukemia (AML). Such resistance has been associated with rapid
drug efflux mediated by the multidrug resistance gene 1 (MDR1; encoding
P-glycoprotein) and more recently with expression of other novel
proteins conferring multidrug resistance such as MRP1 (multidrug
resistance-associated protein 1) and LRP (lung resistance protein). To
determine the frequency and clinical significance of MDR1, MRP1, and
LRP in younger AML patients, we developed multiparameter flow
cytometric assays to quantify expression of these proteins in
pretreatment leukemic blasts from 352 newly diagnosed AML patients (median age, 44 years) registered to a single clinical trial (SWOG 8600). Protein expression was further correlated with functional efflux
by leukemic blasts [assessed using two substrates: Di(OC)2 and Rhodamine 123] and with the ability of MDR-reversing agents to
inhibit efflux in vitro. MDR1/P-glycoprotein expression, which was
highly correlated with cyclosporine-inhibited efflux, was noted in only
35% of these younger AML patients, distinctly lower than the frequency
of 71% we previously reported in AML in the elderly (Blood
89:3323, 1997). Interestingly, MDR1 expression and functional drug
efflux increased with patient age, from a frequency of only 17% in
patients less than 35 years old to 39% in patients aged 50 years
(P = .010). In contrast, MRP1 was expressed in only 10% of
cases and decreased with patient age (P = .024). LRP was
detected in 43% of cases and increased significantly with increasing
white blood cell counts (P = .0015). LRP was
also marginally associated with favorable cytogenetics (P = .012) and French-American-British (FAB) AML FAB subtypes (P = .013), being particularly frequent in M4/M5 cases. Only
MDR1/P-glycoprotein expression and cyclosporine-inhibited efflux were
significantly associated with complete remission (CR) rate
(PMDR1 = .012; Pefflux = .039) and resistant disease (RD; PMDR1
= .0007; Pefflux = .0092). No such correlations
were observed for MRP1 (PCR = .93;
PRD = .55) or LRP (PCR = .50; PRD = .53). None of these parameters were
associated with overall or relapse-free survival. Unexpectedly, a
distinct and nonoverlapping phenotype was detected in 18% of these
cases: cyclosporine-resistant efflux not associated with MDR1, MRP1, or
LRP expression, implying the existence of other as yet undefined efflux
mechanisms in AML. In summary, MDR1 is less frequent in younger AML
patients, which may in part explain their better response to therapy.
Neither MRP1 nor LRP are significant predictors of outcome in this
patient group. Thus, inclusion of MDR1-modulators alone may benefit
younger AML patients with MDR1(+) disease.
RESISTANCE TO CHEMOTHERAPY is one of the
major obstacles to the effective treatment of patients with acute
myeloid leukemia (AML).1,2 While nearly 80% of younger AML
patients may initially achieve a complete remission with current
therapeutic regimens, the majority of these patients relapse with
resistant disease; even with aggressive therapy, the overall 5-year
survival rate is only 35%.1-3 One of the
best-characterized drug-resistance mechanisms in AML is active drug
extrusion mediated by the multidrug resistance protein 1 (known as MDR1
or P-glycoprotein). MDR1-mediated drug resistance is of particular
interest as MDR1 confers cross-resistance to a wide variety of
structurally unrelated antineoplastic drugs used in the therapy of AML.
In addition, MDR1 modulators such as verapamil, cyclosporine A (CsA),
and its nonimmunosuppressive analogue PSC833 can reverse MDR1-mediated
resistance in vitro and in vivo. Large clinical trials have been
developed to assess the benefits of incorporation of first- or
second-generation MDR1 modulators (such as PSC833) into AML
therapy.4-7 While several trials are ongoing, encouraging
results have already emerged from one recently completed randomized
trial that incorporated CsA in the treatment of patients with high-risk
AML.7 This trial found that both overall survival (OS) and
relapse-free survival (RFS) were significantly improved among patients
whose treatment included CsA. Three-year OS was 22% among CsA-treated
patients compared with 6% among the control group, while 3-year RFS
was also significantly improved at 43% compared with
10%.7
MDR1 is frequently expressed in AML and is associated with lower
complete remission (CR) rates and, in some studies, shorter overall
survivals for patients treated with standard therapeutic regimens.8-14 Our recent studies focusing on elderly AML
patients (with a median age of 68 years) found that MDR1 was expressed in greater than 70% of cases; in these patients, the CR rate was significantly and independently associated with MDR1 expression, secondary AML, and unfavorable cytogenetics. CR rate ranged from 81%
in patients with MDR1( Although multidrug resistance mediated by MDR1 appears to be of
biologic and clinical importance in AML, it is now apparent that other
novel proteins may be associated with a multidrug resistant (MDR)
phenotype. One such protein, the multidrug resistance-associated protein MRP1, is distantly related to MDR1, and like MDR1, lowers intracellular drug accumulation by promoting drug efflux.15 Although the exact mechanism of MRP1-mediated drug transport is not yet
fully understood, recent studies indicate that MRP functions as a
transporter of glutathione S-conjugates.16-18 Very recent studies have also reported the existence of multiple MRP1 homologs, and
their relative roles in conferring resistance are as yet
undefined.19 Another protein associated with an MDR
phenotype is the lung resistance protein LRP. LRP was recently shown to
be a component of the major vault protein complex located in the
cytoplasm and at the nuclear pore.20 As nuclear pore
complexes mediate the bidirectional transport of substances between the
nucleus and cytoplasm, it has been hypothesized that LRP may confer
resistance by altering drug transport between the cytoplasm and the
nucleus. While the biologic and clinical significance of these
alternative MDR-associated proteins has not been extensively studied in
AML, preliminary studies on limited numbers of patients have reported
conflicting results.21-28
To determine the frequency of the expression and the biologic and
clinical significance of MDR1 in younger AML patients and to contrast
these results with our prior studies in elderly patients,8 we undertook a retrospective study of a large cohort of previously untreated AML patients registered to a single Southwest Oncology Group
therapeutic trial (SWOG 8600).29 In addition, the
availability of this cohort gave us the opportunity to assess the
biologic and clinical significance of MRP1 and LRP in a large number of uniformly treated AML patients. The results of these studies were correlated with pretreatment clinical parameters, cytogenetics, and
therapeutic outcome.
Patients.
All biologic samples were obtained at initial diagnosis before therapy
from patients registered to a previously reported single Southwest
Oncology Group study, SWOG study 8600: a randomized investigation of
high-dose versus standard-dose cytosine arabinoside (Ara-C) with
daunorubicin in patients with previously untreated AML.29
In this study, patients with previously untreated AML were randomized
on a 2-to-1 basis to daunomycin 45 mg/m2 for 3 days and
standard-dose Ara-C versus daunomycin and high-dose Ara-C. Those
patients on the standard-dose arm who achieved CR after 1 or 2 cycles
of therapy were again randomized to consolidation therapy with either
daunomycin and standard-dose Ara-C versus daunomycin and high-dose
Ara-C. Patients who achieved CR on the high-dose induction were
assigned to consolidation with daunomycin and high-dose Ara-C. Thus,
this study asked, in a randomized fashion, whether standard-dose or
high-dose Ara-C was superior for induction, and among those induced
with standard-dose Ara-C, whether standard-dose or high-dose Ara-C was
superior for consolidation. This study included patients with an
antecedent hematologic disorder and treatment-related AML as well as de
novo AML. However, information about such patient histories was not
collected. After a brief preliminary trial for patients 65 years or
older, the study was limited to those less than 65 years old at
diagnosis. The high dose of Ara-C was initially defined as 2 g/m2/d for patients Biologic studies.
Blasts from pretreatment bone marrow or peripheral blood samples were
enriched upon sample receipt by density gradient separation, and an
initial immunophenotyping panel performed to confirm the myeloid
lineage of the leukemia. In cases where extra cells remained, these
were aliquotted and cryopreserved in 90% fetal calf serum and 10%
dimethyl sulfoxide (DMSO) at Expression of the drug resistance proteins MDR1, MRP1, and LRP.
MDR1 expression by leukemic blasts was measured using 2 MDR1-specific
antibodies, MRK16 (Kamiya Biotech, Thousand Oaks, CA) and MM4.17
(courtesy of D. Cohen, Novartis, East Hanover, NJ) in
three-color flow cytometric assays where blasts were costained with
MRK16/MM4.17, the hematopoietic stem/progenitor cell antigen CD34, and
the pan-myeloid antigen CD33, as previously described.31 MRK16/MM4.17 staining was detected in a 2-step approach using first
biotin-labeled anti-mouse antibody (1 µg/106 cells;
Becton Dickinson, San Jose, CA) to detect MRK16/MM4.17, and then RED613 (Becton Dickinson) to detect the biotin label. This
approach allows accurate analysis of MRK16/MM4.17 staining in a
phenotypically gated myeloid blast population and correlation of MDR1
protein, CD34, and CD33 expression. The biotin-avidin detection system
augments the signal obtained with dim staining with MRK16/MM4.17. The
method is identical to that we have previously described with the
exception of the use of a lower concentration of second-step biotin
anti-mouse antibody in this protocol to decrease nonspecific staining
of leukemic blasts.31 Appropriately matched isotype
controls (same IgG subclass at the same protein concentration as the
antibody tested) were used in all assays. The MDR1(+) DOX cell lines
and MDR 1( Functional efflux.
To assess functional drug efflux and correlate efflux with MDR1
expression, the abilities of leukemic blasts to efflux the fluorescent
dyes Di(OC)2 and Rhodamine 123 (Rh123) were measured in
single-color flow cytometric assays, as described.31 The fluorescent dye Di(OC)2 is an MDR1 substrate; however,
unlike other MDR1 substrates it does not appear to be transported by MRP1 and may thus be a more specific substrate than other drugs/dyes for assessment of MDR1-mediated transport.36 In contrast,
Rh123 appears to be transported both by MDR1 as well as by MRP1,
although, as observed by us and described by others, MRP1-mediated
transport takes place more slowly.37,38
Measurement of protein expression and efflux.
Analyses were performed on a FACScan flow cytometer using Lysis II
software (Becton Dickinson). Expression of CD34 was reported as
positive, weakly positive, or negative by comparing staining of
leukemic blasts with CD34 to an isotype control.39;40 In
addition, for the positive cases, it was noted whether the entire blast
population or only a subpopulation stained with CD34. Expression of the
drug resistance markers MDR1, MRP1, and LRP on gated leukemic blasts
compared with control cells was measured using a modification of the
Kolmogorov-Smirnov (KS) statistic, denoted D, which measures the
difference between two distribution functions and generates a value
ranging from 0 to 1.0.41 The method was modified by
ascribing a negative value to cases where the median fluorescence
intensity of the control cells was brighter than that of
antibody-stained cells. Because the KS statistic sensitively identifies
even small differences in fluorescence, it is useful in detection of
low-level antigen expression as occurs frequently with MDR1 expression
in primary patient samples.31,42 MRK16/MM4.17 staining
intensity was categorized for descriptive purposes as follows: bright
(D Cytogenetic analysis.
Cytogenetic studies on pretreatment bone marrow or unstimulated blood
samples were performed using standard G-banding with trypsin-Giemsa or
trypsin-Wright's staining in SWOG-approved cytogenetics laboratories.
ICSN 1995 guidelines were followed for clonal definition and
description of individual and numerical karyotypic
anomalies.43 Karyotypes were considered normal diploid if
no clonal abnormalities were detected in a minimum of 20 metaphases
examined and if 2 cell-processing methods were used. Each karyotype was
independently reviewed by at least 3 members of the SWOG Cytogenetics Committee.
Statistical methods.
Demographic and clinical data for patients on this study were collected
with quality-control review according to standard procedures of the
SWOG. Expression of MDR1, MRP1, LRP, and efflux were represented as
quantitative variables using the KS statistic D, and categorized for
descriptive purposes as described above. Correlation among multidrug
resistance and efflux variables was estimated using Spearman's rank
order correlation coefficient (rS). Unweighted
least squares and logistic regression (LR) analyses were performed to
identify variables predictive of MDR1, MRP1, or LRP expression or
efflux.44,45 Standard criteria were used to define CR and
relapse.29 Patient response was coded as CR, partial
response (PR), stable disease, early death (death before any assessment
of response), and not adequately assessed. OS was measured from
randomization until death from any cause, with observation censored for
patients last known alive. According to the protocol, patients were not
to be transplanted in first CR. Thus, data regarding bone marrow
transplantation (BMT) were not collected for study 8600, and
consequently survival was not censored at BMT. RFS was measured from
establishment of CR until relapse or death from any cause, with
observation censored for patients last known alive without report of
relapse. Distributions of OS and RFS were estimated by the method of
Kaplan and Meier.46 Analyses of prognostic factors for
treatment outcomes were based on LR models for CR and proportional
hazards (PH) regression models for OS and RFS.45,47 These
included both simple regression models to examine the separate effects
of individual prognostic factors, and multiple regression models to
examine the joint effects of multiple factors. The potential prognostic
factors available for this study are shown in
Tables 1 and
2. Statistical significance is
represented by two-tailed P values. Analyses were based on
clinical and biologic data available March 11, 1997.
Patient characteristics.
SWOG study 8600 accrued 863 patients from November 1986 through
December 1991 (Table 1). The SWOG Leukemia Repository had sufficient cryopreserved pretreatment material to retrospectively study
one or more of the following: CD34, MDR1, MRP1, LRP expression, and
functional efflux in 387 of these 863 cases. Because specimen submission was not initially a mandatory eligibility requirement on
this clinical trial, 275 (71%) of the 387 patients with specimens entered the study after 1988, compared with 497 (58%) of the entire 863 patients. Submission of specimens to SWOG-approved laboratories for
cytogenetic analysis during this time period was also not required and
was limited to 109 patients, of whom 86 (79%) entered the study after
1988. Of the 387 cases with cells available, 35 were subsequently
excluded because the diagnosis of AML was not confirmed by the SWOG
Leukemia Pathology Committee either because of inadequate materials (25 cases), or because morphologic and immunophenotypic data were
diagnostic of a different entity (acute lymphoblastic leukemia,
myelodysplasia, myelofibrosis). The remaining 352 cases are the subject
of this study. These 352 cases came from patients registered at 81 SWOG-affiliated centers.
Drug resistance and efflux phenotypes of AML cases.
Using the MDR1-specific antibody MRK16 and sensitive multicolor flow
cytometric assays, MDR1 proteins were detected on the leukemic blasts
of 123 of 351 cases (35%; CI, 30% to 40%; Table 2;
Fig 1). These MDR1+ cases
included 64 (18%; CI, 14% to 22%) with very bright staining (D
Correlation of drug-resistant phenotype with clinical, morphologic,
and biologic disease factors.
Expression of MDR1 and CsA-inhibited efflux varied with age, FAB
subtype, and CD34 expression. There was a progressive increase of MDR1
expression with age in this patient cohort (P = .010 for MRK16). Moderate or bright MDR1 expression was seen in 19 of 109 (17%)
patients under age 35, in 33 of 121 (27%) ages 35 to 49, and in 47 of
121 (39%) over 50 years old. A similar pattern was seen for
CsA-inhibited Di(OC)2 efflux (P = .0056).
Significant heterogeneity of MDR1 expression was observed among the FAB
categories (with M0, M6, M7, M0/M5, and other AML pooled into a single
category) (P = .0001). As shown in
Table 4, moderate/bright MDR1 expression was relatively more frequent in M1 and M2 cases (81 of 217, 37%) and
less frequent in M3, M4, and M5 (11 of 115; 10%). A similar association was seen for CsA-inhibited Di(OC)2 efflux
(P = .0001; Table 4). As we have previously reported in elderly
patients,8 MDR1 expression was highly associated with
expression of the stem and progenitor cell antigen CD34 (P < .0001). Only 12 of 147 (8%) patients with no or weak CD34 had
moderate/bright MDR1 expression, compared to 17 of 70 (24%) cases with
strong CD34 expression in a subpopulation of blasts, and 70 of 134 (52%) with uniform strong CD34 expression by the blasts. A similar
association was seen between CD34 expression and CsA-inhibited
Di(OC)2 efflux (P < .0001). There were no
significant associations of MDR1 or efflux with any of the other
variables in Table 1. Essentially the same results were obtained using
CsA-inhibited Rh123 efflux or MM4.17 staining.
Cytogenetic analysis.
Because of the lack of submission requirements in the early stages of
this clinical trial, cytogenetic studies performed and reviewed in
approved SWOG cytogenetic laboratories were available on only 96 of the
352 cases in this study. Fifteen cytogenetic studies were deemed
inadequate after review by the SWOG Cytogenetics Committee, largely due
to the examination of insufficient metaphases. Of the 81 acceptable
cases, 34 (42%) had normal karyotypes. Abnormalities associated with
de novo AML were found in 22 cases (27%), including 11 cases (14%)
with t(8;21), 4 cases (5%) with inv(16)/t(16;16), 5 cases (6%) with
t(15;17), and 2 (2%) with t(6;9). In addition, abnormalities of 11q
were quite frequent, being present in 10 cases (12%). In contrast,
abnormalities associated with myelodysplasia and secondary AML were
less frequently detected in this patient group, with only 9 cases
(11%) showing Correlation of clinical, morphologic, and biologic disease factors
with CR rate.
Of the 352 patients, 185 (53%; CI, 47% to 58%) achieved a CR,
including 139 (39%) after 1 induction course.29 Of the 213 patients who did not achieve CR after 1 induction, 98 received a second
induction course on protocol and 46 (47%) of these 98 achieved CR.
Thirty-one patients (9%) were classified as early deaths (all after a
single induction attempt), and another 41 (12%) were not adequately
assessed for response (including 1 who refused protocol therapy and 2 who received 2 induction attempts). The remaining 95, who had resistant
disease, are discussed further below. Induction therapy with
standard-dose Ara-C was given to 245 (70%) of the patients, while 107 (30%) received high-dose Ara-C (see Materials and Methods; Patients).
There was no significant difference in CR rate between these 2 groups,
with 127 (52%) of patients treated with standard-dose Ara-C, and 58 (54%) of those treated with high-dose Ara-C achieving CR. Although the
CR rate was slightly lower for patients over age 50 (57 of 121, 47%)
compared with younger patients (128 of 231, 55%), the CR rate did not
vary significantly with age in simple logistic regression analysis (P = .18).29 CR rate also decreased slightly but
not significantly with increasing WBC counts in these patients
(P = .12 and .18 without and with adjustment for the effect of
FAB, respectively).
Correlation with resistant disease.
Ninety-five (27%) of the 352 patients had disease that was resistant
to induction therapy. This includes 27 who achieved only partial
response (including 10 after 2 induction attempts) and 68 with stable
disease (40 after 2 induction attempts). Simple logistic regression
analyses of resistant disease were generally complementary to the
corresponding analyses of CR. The resistant disease rate increased with
increasing MDR1 (detected by MRK16) expression (P = .0007) and
CsA-inhibited Di(OC)2 efflux (P = .0092), but not
with expression of LRP (P = .53) or MRP1 (P = .55), or CD34 (P = .16; Table 5). Very similar results were obtained
when patients with partial response were excluded from the resistant disease group (data not shown). Resistant disease was also correlated with FAB subtype (P = .0097), being more frequent in M1 (30 of 72 or 42%) compared with all others (65 of 280 or 23%). As noted above, M1 was also associated with greater MDR1 expression and CsA-inhibited Di(OC)2 efflux. However, even after adjusting
for the effects of FAB in multiple logistic regression analysis,
resistant disease was still correlated to MDR1/MRK16 expression
(P = .0069), although interestingly not to CsA-inhibited
Di(OC)2 efflux (P = .104). When the effect of high
Ara-C dose on resistant disease was examined, there was no significant
interaction between Ara-C dose and MDR1 expression and resistant
disease (P = .97 without adjustment for FAB, .95 after
adjustment for FAB). The RD rate increased slightly but not
significantly with increasing WBC count (P = .70 and .93 without and with adjustment for the effect of FAB, respectively).
Correlation with OS and RFS.
Three hundred of the 352 patients have died and the remaining 52 have
been in follow-up a median 6.0 years (range, 8 months to 9.4 years).
The estimated median survival was 11.7 months (95% CI, 9.4 to 13.5 months). OS was significantly associated with 3 factors in multiple
proportional hazards regression analysis; OS decreased significantly
with increasing patient age (P < .0001), increasing
peripheral blood blast count (P = .0077), and varied significantly among FAB categories (P < .0001). After
adjusting for these factors there was no association between OS and
MDR1 (P = .59), MRP1 (P = .81), LRP (P = .53),
CsA-inhibited efflux (P = .23), or CD34 expression (P = .93; Table 5). The estimated distributions of OS are shown by MDR1
expression in Fig 1. There was no significant interaction between the
effects of Ara-C dose and MDR1 expression in the analysis of OS, either
without (P = .90) or with (P = .63) adjustment for the
effects of age, blast count, and FAB category. OS decreased slightly
with increasing WBC count; this was marginally significant in the
simple proportional hazards regression analysis (P = .028), but
not after adjusting for the effects of age, peripheral blast count, and
FAB (P = .38).
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ABSTRACT
INTRODUCTION
) de novo AML with favorable or
intermediate cytogenetics, to 13% in patients with MDR1(+) secondary
AML and unfavorable cytogenetics. Resistant disease was likewise
associated independently with MDR1 and unfavorable
cytogenetics.
50 and 3 g/m2/d for age
<50. However, after 2 years the Data Monitoring Committee for the
study reduced the dose to 2 g/m2/d for age <50. The
standard dose of Ara-C was 200 mg/m2 for all ages
throughout the study. The diagnosis of AML was confirmed by central
histopathologic review by the SWOG Leukemia Pathology Committee using
standard French-American-British (FAB) criteria as modified by
SWOG.
D < 0.30), dim (0.15 
