Expression of the Breast Cancer Resistance Protein in Breast

Cancer
Ian F. Faneyte, Petra M. P. Kristel, Marc Maliepaard, et al.

Clin Cancer Res 2002;8:1068-1074.

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1068 Vol. 8, 1068 –1074, April 2002
Expression of the Breast Cancer Resistance Protein in
Breast Cancer
Ian F. Faneyte, Petra M. P. Kristel,
Marc Maliepaard, George L. Scheffer,
Rik J. Scheper, Jan H. M. Schellens, and
Marc J. van de Vijver1
Departments of Experimental Therapy [I. F. F., P. M. P. K., M. M.,
J. H. M. S.], Pathology [I. F. F., M. J. v. d. V.], and Medical Oncology
[J. H. M. S.], The Netherlands Cancer Institute/Antoni van
Leeuwenhoek Hospital, and Department of Pathology, Free
University Hospital [G. L. S., R. J. S.], 1066 CX Amsterdam, the
Netherlands
ABSTRACT
Purpose: The breast cancer resistance protein (BCRP)
is involved in in vitro multidrug resistance and was first
identified in the breast cancer cell line MCF7/AdrVp. The
aim of this study was to investigate the role of BCRP in
resistance of breast cancer to anthracycline treatment.
Experimental Design: BCRP mRNA was determined
with real-time reverse transcription-PCR and immuno-
staining in nine breast cancer cell lines and in samples of 25
primary breast carcinomas and 27 patients who received
preoperative anthracycline-based therapy. Tumor response
to treatment and patient survival were recorded.
Results: In cell lines, only MCF7 and BT20 had BCRP
mRNA levels coinciding with membrane-bound immuno-
staining. In clinical samples, BCRP expression varied widely
(range, 0.01– 0.86). With immunohistochemistry, BCRP was
detected in vessels and normal breast epithelium but not in
tumor cells. There was no difference in BCRP expression
between anthracycline-naı̈ve and treated tumor samples.
BCRP expression was not associated with decreased re-
sponse or survival.
Conclusions: There is no indication that elevated BCRP
expression in breast carcinomas confers resistance to an-
thracyclines. Expression was not detectable with immuno-
histochemistry.
INTRODUCTION
MDR2 frequently poses a problem in breast cancer patients
with metastatic disease. Response rates of first-line chemother-
Received 6/27/00; revised 11/26/01; accepted 11/30/01.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
To whom requests for reprints should be addressed, at Department of
Pathology, Netherlands Cancer Institute/Antoni van Leeuwenhoek Hos-
pital, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Phone:
31-20-5122751; Fax: 31-20-5122759; E-mail: mvijver@nki.nl.
2
The abbreviations used are: MDR, multidrug resistance; BCRP, breast
cancer resistance protein; RT-PCR, reverse transcription-PCR; PBGD,
Downloaded from clincancerres.aacrjournals.org on April 29, 2014. © 2002 American Association for Cancer
Research.
Clinical Cancer Research
apy in metastatic breast cancer range from 50 to 70%; the
response rate to subsequent chemotherapy regimens is signifi-
cantly less as a result of MDR (1). As yet no explanation for this
MDR phenomenon in breast cancer has been found.
The BCRP (ABCG2) is a recently characterized xenobiotic
half-transporter protein. It is a member of the ATP-binding
cassette protein family and functions as an energy-dependent
efflux pump (2, 3).
BCRP was first identified in the breast cancer cell line
MCF7/AdrVp (2), which has a multidrug-resistant phenotype,
notwithstanding the addition of a MDR1/P-glycoprotein block-
ing agent (verapamil). The parental cell line MCF7 also ex-
presses BCRP but at much lower levels (4, 5). In vitro, elevated
expression of BCRP causes resistance against at least two
classes of drugs (anthracyclines and topoisomerase I inhibitors),
which are used in the treatment of cancer patients (6 –9).
Whether BCRP is also involved in clinical drug resistance
is unknown. Studies published to date describing expression of
BCRP in clinical material did not investigate such a role. Ex-
pression levels in blast cells of leukemia patients were found to
be highly variable, but no correlation was described between
BCRP expression and exposure to chemotherapy or sensitivity
to cytotoxic treatment (10). Antibody staining of a panel of 41
human solid tumor samples of mixed origin and 16 cytospins of
acute myeloblastic leukemia (11) showed no reactivity, except
for weak staining in one of two small intestinal adenocarcinoma
samples.
Recent studies have shown that BCRP activity can be
effectively inhibited by several modulating agents (12–14). It is
therefore of clinical interest to gain insight into the possible
contribution of BCRP to drug resistance in breast cancer.
We have studied the expression of BCRP mRNA and of the
BCRP protein in breast cancer cell lines and clinical breast
carcinoma specimens. By analyzing both chemotherapy-naı̈ve
and anthracycline-treated patients, we have aimed to detect a
possible up-regulation of expression caused by chemotherapy.
We have also studied the correlation of BCRP expression in
tumor samples with response of the tumor to anthracyclines.
MATERIALS AND METHODS
Cell Lines. Cell line controls with a known expression of
BCRP were the small cell lung carcinoma cell line GLC4/ADR
(very low expression), the ovarian carcinoma cell line Igrov
(low expression), its drug-selected variant Igrov/T8 (high ex-
pression), and the partially reverted subline Igrov/T8(p75)-40
(intermediate expression; Ref. 15). Breast cancer cell lines an-
alyzed were 1.6.2.6, BT20, CAMA1, HBL100, MCF7,
MPL13E, SKBR3, T47D, and ZR75-1. All cell lines were
porphobilinogen deaminase; FEC, 5-fluorouracil, epi- or doxorubicin,
and cyclophosphamide.

cultured in a 10% FCS-enriched medium with L-glutamine,
penicillin, and streptomycin. The Igrov variants were grown in
RPMI 1640 (Life Technologies, Inc., Paisley, Scotland); the
other cell lines were grown in DMEM (Life Technologies, Inc.).
To maintain the high expression of BCRP in Igrov/T8, these
cells were exposed to topotecan at a 950 nM concentration for
1 h weekly. Igrov/T8(p75)-40 cells were cultured for 75 pas-
sages as Igrov/T8 and then for 40 passages in standard RPMI
1640 (no topotecan added). GLC4/ADR cells were cultured in
1120 nM doxorubicin permanently, until 4 days before harvest-
ing for analysis.
Tissue Samples. All materials were obtained from the
Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital
Tissue Bank (fresh-frozen in liquid nitrogen upon surgery and
preserved at ⫺70°C), where they were originally stored between
1984 and 1999. Normal human liver was used in both RNA
analyses and immunohistochemistry as a positive control for
BCRP expression.
RNA Isolation and Analysis. RNA from 52 breast can-
cer specimens was isolated and analyzed. Twenty-five patients
were chemotherapy-naı̈ve: 18 patients had primary operable
tumors (n ⫽ 13) or residual tumor (n ⫽ 5) after radiotherapy for
locally advanced T3 (⬎5 cm) breast cancer; 7 specimens were
biopsies from a primary tumor (n ⫽ 2) or subclavicular lymph
node with metastasis (n ⫽ 5) of patients entered in a clinical
trial, described below.
Twenty-seven patients had been treated previously with
anthracycline-based chemotherapy (2 also with radiotherapy):
23 patients who took part in a clinical trial, described below, and
who received neoadjuvant FEC treatment; and 4 patients with
locally advanced breast cancer that had initially responded to
anthracycline-based chemotherapy [up to seven cycles of FEC
(60 mg/m2 of epi- or doxirubicin)] and had become progressive.
Immunohistochemistry was performed with breast cancer
specimens from: 20 patients who had received no prior treat-
ment (the RNA isolated from two corresponding frozen tissue
samples was also analyzed); 4 patients who took part in the trial
described below and who received neoadjuvant FEC treatment
(the RNA isolated from all of the corresponding frozen-tissue
samples was also analyzed); and 3 patients with progressive
locally advanced breast cancer that had initially responded to
anthracycline-based chemotherapy (the RNA isolated from all 3
corresponding frozen tissue samples was also analyzed).
In all patients treated with preoperative chemotherapy,
surgery was performed within 8 weeks of the last administration
of chemotherapy.
Histology. For all patients, paraffin slides were used for
typing and grading. Histological grading was performed accord-
ing to criteria described by Elston and Ellis (16).
Patients from High-Dose versus Normal-Dose Chemo-
therapy Trials. We assessed 30 tumor samples with RT-PCR
from a group of patients, treated previously in a Phase II
prospective clinical trial comparing high-dose versus normal-
dose chemotherapy (17). In this trial, 97 patients with operable
breast cancer and extensive axillary lymph node involvement
were entered. Patients received three cycles of epirubicin (120
mg/m2), 5-fluorouracil (500 mg/m2), and cyclophosphamide
(500 mg/m2) every 3 weeks before surgery. After surgery (mod-
ified radical mastectomy or wide local excision and axillary
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Clinical Cancer Research 1069
lymph node dissection) and radiotherapy, the patients were
randomized to receive either one more course of FEC or FEC
followed by a high-dose regimen with peripheral blood progen-
itor cell support. All patients received additional treatment with
tamoxifen.
Tumor samples were taken before treatment (diagnostic
biopsies) and after three cycles of FEC (surgical specimens)
given as a neoadjuvant. We quantified the response to neoad-
juvant treatment clinically, according to criteria of the Union
International Contre Cancer (18). In addition, we studied the
surgical specimens for histopathological evidence of response.
Survival. We determined both overall survival and disease-
free survival from the start of neoadjuvant treatment. End points
were death for overall survival and death or any recurrence of
breast cancer for disease-free survival. Survival comparisons
between the two randomly assigned treatment groups have been
published (17). The outcome after 6 years of follow-up was
identical for both treatment groups. For the analysis, we have
therefore used trial-patient data, regardless of the treatment arm
they were assigned to. For the other patients, the survival data
were available in The Netherlands Cancer Institute’s patient
database.
RNA Isolation. For total RNA isolation from either cells
or tissue specimens, the RNAzol B kit (Tel-Test, Inc., Friend-
swood, TX) was used according to the manufacturer’s protocol.
RNA was isolated from approximately 7.5 ⫻ 106 cells taken
from culture after trypsinization or from 10 to 20 30-␮m slices
of the cryopreserved tissue samples. Staining of both ribosomal
bands on a 1% agarose gel with ethidium bromide was used to
judge the quality of the isolated RNA.
Northern Blot Analysis. Ten ␮g of total RNA per tested
sample were denatured at 65°C in the presence of formamide
and were run on a 1% formamide-agarose gel and blotted onto
a nylon filter (Hybond-N⫹; Amersham Pharmacia Biotech,
Little Chalfont, Buckinghamshire, United Kingdom).
The 2.4-kb BCRP message (National Center for Biotech-
nology Institute GenBank no. AF098951; Ref. 2) was detected
with a 224-base probe, synthesized by PCR amplification with
Igrov/T8 cDNA as template. The forward primer was 5⬘-CAA
CCA TTG CAT CTT GGC TG-3⬘, and the reverse primer was
5⬘-CAA GGC CAC GTG ATT CTT CC-3⬘. The annealing
temperature was 60°C. Initially, 40 amplification cycles were
run, after which the PCR product was purified by electrophore-
sis on a 1% agarose gel and the Qiaex II gel extraction kit
(Qiagen, Hilden, Germany). In a second PCR amplification with
[␣-32P]dCTPs added to the reaction mixture, 12 ng of the
purified product were radiolabeled in 40 amplification cycles,
with the same primers and PCR conditions. The filter was
hybridized overnight at 65°C with the radioactive probe (1.7 ⫻
106 cpm), after prehybridization (60 min at 65°C) with salmon
sperm DNA. After hybridization, the filter was washed in 2⫻
and 1⫻ SSC ⫹ 1% SDS (15 min at 55°C), and films (X-OMAT
AR; Eastman Kodak Co., Rochester, NY) were exposed for 3
days and for 1 week at ⫺70°C.
TaqMan Quantitative RT-PCR. In a cDNA reaction
(100 ␮l), 2.5 ␮g of total RNA were used as template. Quanti-
fication of the message was performed with a TaqMan real-time
RT-PCR assay (P.E. Applied Biosystems, Foster City, CA),
according to the manufacturers’ protocol.
1070 BCRP Expression in Breast Cancer
The housekeeping gene PBGD served as the internal ref-
erence and was amplified parallel to BCRP for every sample, in
separate vessels. The oligonucleotide primers used for PBGD
(National Center for Biotechnology Institute GenBank no.
NM000190; Ref. 19) amplification were 5⬘-ACG ATC CCG
AGA CTC TGC TTC-3⬘ (forward) and 5⬘-GCA CGG CTA
CTG GCA CAC T-3⬘ (reverse), with an expected product length
of 87 bp. The VIC-labeled probe (P.E. Applied Biosystems)
used for PBGD detection was 5⬘-CAG CCT CCT TCC AGG
TGC CTC AGG-3⬘. Primers for BCRP amplification were 5⬘-
CAC AAC CAT TGC ATC TTG GC-3⬘ (forward) and 5⬘-GCT
GCA AAG CCG TAA ATC CA-3⬘ (reverse), with an expected
product length of 74 bp. The 6-carboxyfluorescein-labeled
probe (P.E. Applied Biosystems) used for BCRP detection was
5⬘-TCA TGG CTT CAG TAC TTC AGC ATT CCA CG-3⬘.
The PBGD amplicon spans an exon in the genomic DNA
sequence.
The constituents of each PCR (25 ␮l) were 2.5 ␮l of cDNA
(or dH2O), 2⫻ (forward and reverse) 0.5 ␮l of primer 15
pmol/liter each (Isogen Bioscience BV, Maarssen, the Nether-
lands), 2.5 ␮l of fluorescent-labeled probe 1 ␮mol/liter (P.E.
Applied Biosystems), 6.5 ␮l of dH2O, and 12.5 ␮l of TaqMan
Universal PCR Mastermix (P.E. Applied Biosystems).
Product amplification was performed up to 50 PCR cycles,
after uracil removal (2 min at 50°C) and polymerase activation
(10 min at 95°C). Each two-step PCR cycle comprised denatur-
ing (15 s at 95°C), annealing, and extending (1 min at 60°C).
A positive control (liver) and negative controls with no
template and genomic DNA as template were added in every
experiment. All assays were run in triplicate.
Immunohistochemistry. Immunohistochemistry was per-
formed on cytospins and on 5-␮m sections of frozen tissue
according to a protocol published previously (20). We used two
mouse monoclonal antibodies against BCRP, BXP34 (11) and
BXP21 (21).
Cytospins and tissue slides were air-dried overnight and
subsequently fixed in acetone (10 min), blocked with normal
goat serum (5%; 30 min), incubated with the primary antibody
(1:50; 60 min), and quenched in 0.15% H2O2 containing PBS
(10 min).
The monoclonal antibody was detected with horseradish
peroxidase-labeled goat antimouse IgG (1:100; 60 min; DAKO
A/S, Glostrup, Denmark), FITC-conjugated tyramine (1:500; 10
min; NEN Life Science Products, Inc., Boston, MA), and horse-
radish peroxidase-labeled rabbit IgG anti-FITC (1:100; 60 min;
DAKO A/S). Bound peroxidase was developed with 2.5% 3-
amino-9-ethyl-carbazole and 0.02% H2O2 in sodium acetate
(pH 5.0).
All reagents were diluted in a 1% bovine albumin solution
in PBS except for FITC-tyramine, which is diluted in the sup-
plier’s amplification fluid. Antimouse IgG solutions were ad-
mixed with 10% normal human serum to prevent nonspecific
binding. The primary antibody was replaced with 1% bovine
albumin solution in PBS as negative control for every cell line
and tissue sample tested.
Quantification of Results. For quantification of the RT-
PCR results, fluorescent signal intensities were plotted against
the number of PCR cycles on a semilogarithmic scale. The
amplification cycle, at which the first significant increase of
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Research.
fluorescence occurred, was designated the threshold cycle (CT).
This was done for all samples to be tested parallel with a
standardization series, with known concentrations of Igrov/
T8(p75)-40 cDNA as template. The CT of each sample was then
compared with those in the standardization series, and the cor-
responding concentration of Igrov/T8(p75)-40 cDNA was read
out as the expression level of the tested gene. This process is
fully automated and carried out by the TaqMan with the man-
ufacturers’ software.
We divided the expression values of BCRP mRNA by
those of PBGD mRNA. This returned the expression ratio in
each tested sample of the BCRP versus PBGD mRNA, as a
measure of the relative expression level. The expression level of
BCRP mRNA in a tested sample was thus defined as the ratio of
BCRP mRNA and PBGD mRNA expression in that sample
relative to the ratio of BCRP mRNA and PBGD mRNA in
Igrov/T8(p75)-40.
Two independent observers (M. V. and I. F.) judged the
immunohistochemical staining as: ⫹⫹, 100% membrane-bound
staining; ⫹, membrane-bound staining in up to 75% of cells;
⫹/⫺, cytoplasmic staining in up to 25% of cells; and ⫺, no
staining. The two observers scored all completely negative and
strongly positive staining results identically. Discordance only
occurred assessing the cell lines HBL100, 1.6.2.6, and CAMA1.
Because both observers did score positive staining to some
extent in these cells but not as clearly specific as in the positive
samples, we agreed to define a third category for immunohis-
tochemical staining, ⫹/⫺.
Statistical Analyses. All data were analyzed with SPSS
10.0.7 for Windows. For correlation between BCRP expression
levels and tumor parameters, we used the two-sided Spearman’s
test for nonparametric correlation. Differences of expression
levels between groups of samples were assessed with the two-
sided Mann-Whitney U test (nonnormal distribution). BCRP
expression was tested for association with outcome in a Cox
regression model.
RESULTS
BCRP mRNA and protein expression were assessed in a
panel of cell lines. The results of these analyses are summarized
in Table 1.
With TaqMan analysis, an increased BCRP expression
compared with Igrov/T8(p75)-40 (level 1.0) was found in
Igrov/T8 cells (level 4.04) and liver (level 1.26). Low expres-
sion was detected in the parental Igrov cell line (level 0.02) and
in GLC4/ADR (level 0.0008). Northern blot hybridization and
immunostaining detected BCRP in Igrov/T8 and in liver. Posi-
tive BCRP Northern blot hybridization of Igrov/T8(p75)-40 was
found previously by Maliepaard et al. (21). Staining of Igrov/T8
was bound to the membrane and occurred in all cells. In Igrov/
T8(p75)-40 cytospins, membrane-bound staining occurred in
50 –75% of cells. A bile canalicular staining pattern was seen in
50 –75% of cells in liver sections.
Expression of BCRP mRNA in breast cancer cell lines
varied widely (median, 0.06; range, levels 0.0001– 0.48). In two
breast cancer cell lines with the highest mRNA levels (MCF7,
level 0.48; BT20, level 0.18), BCRP could be detected by
Northern blot hybridization. In these cells, BCRP expression
 Clinical Cancer Research 1071

Table 1 BCRP expression measured in controls and breast cancer cell lines, with TaqMan real-time PCR, Northern blotting, and
immunohistochemistry with two monoclonal antibodies
TaqMan real-time Northern
PCRa blotb Immuno BXP21c Immuno BXP34c
Controls
Igrov/T8 4.0 ⫹ ⫹⫹ ⫹⫹
Igrov/T8(p75)-40 1.0 ⫹d ⫹ ⫹
Igrov 0.02 ⫺ ⫺ ⫺
Liver 1.3 ⫹ ⫹ ⫹
GLC4/ADR 0.0008 ⫺ NDe ⫺d
Breast cancer cell lines
MCF7 0.48 ⫹ ⫹ ⫹
BT20 0.18 ⫹ ⫹ ⫹
HBL100 0.14 ⫺ ⫹/⫺ ⫹/⫺
1.6.2.6 0.11 ⫺ ⫹/⫺ ⫹/⫺
CAMA1 0.06 ⫺ ⫹/⫺ ⫹/⫺
SKBR3 0.03 ⫺ ⫺ ⫺
T47D 0.008 ⫺ ⫺ ⫺
ZR75-1 0.008 ⫺ ⫺ ⫺
MPL13E 0.0001 ⫺ ⫺ ⫺
a
Ratio of BCRP and PBGD in samples versus in Igrov/T8(p75)-40.
b
Autoradiogram after hybridization with BCRP-specific probe; ⫹, signal at 2.4 kb; ⫺, no signal.
c
Results of immunostaining with BXP21 and BXP34 mouse monoclonal antibodies against BCRP; ⫹⫹, 100% membrane-bound staining; ⫹,
membrane-bound staining in up to 75% of cells; ⫹/⫺, cytoplasmic staining in up to 25% of cells; ⫺, no staining.
d
See Maliepaard et al. (21).
e
ND, not done.

was also detected immunohistochemically in the cell membrane.
None of the cell lines with expression levels below BT20 had
detectable BCRP on Northern blot or membrane-bound immu-
nostaining. Cytoplasmic staining did occur in three breast cancer
cell lines with lower BCRP levels (HBL100, level 0.14; 1.6.2.6,
level 0.11; CAMA1, level 0.06).
A threshold for the detection of membrane-bound staining
with both antibodies appears to lie at a BCRP mRNA expression
of ⬃0.15-fold the level in Igrov/T8(p75)-40. All staining pat-
terns observed in controls and breast cancer cell lines are shown
in Fig. 1.
Having thus established the levels of BCRP expression in
breast cancer cell lines and the BCRP levels needed for positive
immunohistochemistry, we analyzed clinical breast carcinoma
samples of 25 chemotherapy-naı̈ve and 27 anthracycline-treated
patients.
Fig. 2 shows the distribution of BCRP mRNA expression
levels measured in breast cancer cell lines (n ⫽ 9) and 52 tumor
samples (median, 0.11; range, 0.01– 0.86), subdivided by treat-
ment history. The expression of BCRP was higher in carcinoma
samples (mean, 0.18) than in cell lines (mean, 0.11), but this
difference was not statistically significant (P ⫽ 0.13). Disre-
garding MCF7 as an outlying observation, the difference be-
tween cell lines and samples was statistically significant (P ⫽
0.04).
We further summarized findings in subgroups of patient
samples in Table 2. There was not a statistically significant
difference between samples of untreated (mean, 0.19) and
treated (mean, 0.18) patients (P ⫽ 0.09).
Seven patients received radiotherapy before surgery. The
mean BCRP expression in these tumors was 0.14. This is below
the mean expression in all samples (0.18) and does not indicate
a trend of BCRP induction after radiotherapy.
Of 27 patients analyzed after anthracycline-based chemo-
therapy, clinical response was recorded in 22, and survival data
were available for all. One patient had a complete remission, 13
had a partial remission, and 8 had stable disease. No correlation
was found between BCRP mRNA expression levels and tumor
response to therapy. Patients with stable disease did not have
increased BCRP levels (0.23) compared with responsive patients
(0.18; P ⫽ 0.54). Overall survival and disease-free survival
were not significantly associated with BCRP mRNA expression
in a Cox regression model.
High tumor malignancy grade was associated with a de-
creased BCRP mRNA expression (␳ ⫽ ⫺0.33; P ⫽ 0.02).
Grades 1 and 2 tumors (n ⫽ 28) had a mean BCRP mRNA
expression of 0.23 versus grade 3 tumors with 0.14 (P ⫽ 0.03).
With two monoclonal antibodies against BCRP, we stained
frozen sections of 9 breast cancer samples in which we had
detected relatively high BCRP expression levels. In none of the
samples could specific tumor cell staining be observed. Addi-
tionally we stained tumor sections of 18 breast carcinoma spec-
imens with unknown expression of BCRP. Again, no specific
staining of tumor cells was seen.
Positive staining of vascular endothelium and of a part of
the normal epithelium in large mammary ducts did occur with
both antibodies. This pattern has been described previously by
Maliepaard et al. (21) and served as an internal positive control.
No staining of lymphoid cells was seen, and the expression of
BCRP mRNA was not correlated with the percentage of cancer
cells or with the amount of lymphoid infiltrate present in the
sample.
DISCUSSION
In this study, we investigated whether the expression of
BCRP in breast cancer may play a role in resistance to epiru-
bicin-based chemotherapy. The substrate specificity of BCRP

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1072 BCRP Expression in Breast Cancer
has become an issue of debate recently because various mutated
forms of the protein were shown to behave differently (22).
However, the ability of BCRP, in wild type or mutated form, to
transport anthracyclines still stands. More specifically, Litman
et al. (8) reported that epirubicin is a substrate of BCRP.
In a panel of controls with a known expression of BCRP,
we first showed that there is a good correlation of all three
methods of detecting BCRP (real-time RT-PCR, Northern blot-
ting, and immunohistochemistry). From this we conclude that
immunostaining of the cell membrane with both monoclonal
antibodies against BCRP (BXP21 and BXP34) is associated
with marked expression of BCRP mRNA. We defined a thresh-
old mRNA level for the immunohistochemical detection of 0.15
relative to the expression in Igrov/T8(p75)-40 cells. Basal ex-
pression of BCRP in most breast carcinoma cell lines was below
the threshold of immunohistochemical detection.
With immunohistochemistry, we did not detect BCRP in
tumor cells, whereas staining was positive in surrounding ves-
sels and normal ducts. This indicates that there is a marked
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Research.
Fig. 1 Results of BCRP stain-
ing with BXP34 in A, Igrov/
T8(p75)-40; B, liver; C, vascu-
lar endothelium in a breast
carcinoma sample; D, CAMA1;
E, MCF7. Bar, 56 ␮m.
contribution of non-tumor cells to the BCRP mRNA measured
in tumor specimens.
Because tumor cells in clinical samples did not stain with
the BCRP antibodies, our results do not support the hypothesis
of singular carcinoma cells in a breast tumor with an increased
expression of BCRP, which can survive chemotherapy and
become drug-resistant subclones by selection.
Clinical MDR development in cancer cells could also be
caused by increased expression of intrinsically present BCRP
levels after treatment with cytotoxic agents. We found no evi-
dence to support that putative mechanism. We did not detect
increased expression levels of BCRP mRNA in treated versus
untreated carcinoma samples. There was even a trend toward
more BCRP expression in untreated samples. As we have
shown, BCRP detected in tumor samples largely originates in
the vascular endothelium. It may well be that one effect of
chemotherapy exposure is a decreased vascular density in the
tumor. BCRP levels also did not exceed the immunohistochem-
istry detection threshold after cytotoxic treatment, as should be
 Clinical Cancer Research 1073

We conclude that basal levels of BCRP in breast cancer

cells are low and cannot be detected with immunohistochemis-
try. There is no apparent effect of in vivo anthracycline treat-
ment on BCRP expression in breast cancer. Furthermore, BCRP
expression in breast carcinoma does not seem to negatively
influence the response of the tumor to anthracycline-based treat-
ment, nor does it impair overall or disease-free survival of
patients. BCRP is therefore unlikely to be responsible for clin-
ical drug resistance as seen in breast cancer patients. Decreased
BCRP expression may be a marker of loss of differentiation in
epithelial breast tissue.

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