1042 Correspondence | JNCI Vol. 100, Issue 14 | July 16, 2008
Reduced Expression of
Primary Breast Cancer
Recently reported results of a phase 2 study
combining the aromatase inactivator
exemestane with the cyclooxygenase-2
(COX-2) inhibitor celecoxib for the treat-
ment of breast cancer showed that patients
receiving the combined treatment had
response rates and clinical benefit rates that
were similar to those of the group receiving
exemestane only (1). The rationale for
using a COX-2 inhibitor for the treatment
of breast cancer is based on several reports
that COX-2 is frequently overexpressed in
breast cancer ( 2 ) and that elevated levels of
COX-2 in breast cancer specimens were
associated with tumor progression and poor
prognosis ( 3 ). The disappointing effect of
celecoxib in this clinical study indicates that
COX-2 may not be as crucial for the pro-
gression of human breast cancer as previ-
ously hypothesized. A possible explanation
for the unexpected findings could be that
the reported overexpression of COX-2 in
human breast cancer was not correct. In
one previous study, only the tumor samples
were analyzed, and the expression of COX-
2 was correlated with clinical parameters
such as disease-free survival or lymph node
metastasis without comparison to healthy
tissue ( 3 ). In studies that compared COX-2
expression in tumor and healthy tissues,
COX-2 expression was mainly analyzed
by immunohistochemistry ( 4 – 6 ). These
immunhistochemical methods, however,
were not quantitative and would strongly
depend on the quality of the antibody and
the staining protocol and also on the selec-
tion of the analyzed region. Only a few
studies analyzed the expression of COX-2
by reverse transcription – polymerase chain
reaction (RT – PCR) ( 4 – 6 ), but measure-
ments with conventional RT – PCR are also
not quantitative. Recent real-time RT –
PCR analyses ( 7 ) used just one housekeep-
ing gene for normalization, which could
lead to incorrect interpretations because
the expression levels of housekeeping genes
can vary considerably between tumor tis-
sues and healthy tissues.
To quantitatively measure the expres-
sion of COX-2 in breast cancer tissues,
tumor-adjacent tissues, and healthy breast
tissues, we performed real-time RT – PCR
analysis and normalized the expression of
COX-2 to the mean expression of four dif-
ferent housekeeping genes. The expression
of COX-2 mRNA was decreased in the
breast cancer samples (mean COX-2 expres-
sion: 0.3, 95% confi dence interval [CI] = 0.2
to 0.4) in comparison with tumor-adjacent
tissues (mean expression: 2.0, 95% CI = 1.3
to 2.7) and healthy tissues (mean expression:
1.0, 95% CI = 0.5 to 1.4) ( Figure 1 ). The
median expression of COX-2 in tumor tis-
sues was only 20% of the median expression
in healthy tissues ( P < .001) and only 10%
of that of tumor-adjacent tissues ( P < .001).
Furthermore, none of the tumor samples
with relative high COX-2 expression
exceeded the range of COX-2 expression
found in healthy tissue samples or tumor-
adjacent samples. Some tumor samples
showed only about 1% of the mRNA levels
measured in tumor-adjacent tissues.
Our results indicate that COX-2 is
expressed at reduced levels in human breast
cancer, not overexpressed as previously
reported. Furthermore, we did not fi nd any
correlation of COX-2 mRNA expression in
the tumor tissues with the mRNA expres-
sion of HER2/neu, estrogen receptor alpha,
or the progesterone receptor (data not
shown). The loss of COX-2 expression in
established breast cancers that we observed
could explain the failure of celecoxib to
inhibit tumor growth in the cited clinical
trial ( 1 ).
DANIEL F . LEGLER
1. Dirix LY , Ignacio J , Nag S , et al . Treatment
of advanced hormone-sensitive breast cancer
in postmenopausal women with exemestane
alone or in combination with celecoxib . J Clin
Oncol. 2008 ; 26 ( 8 ): 1253 – 1259 .
2. Subbaramaiah K , Norton L , Gerald W , et al .
Cyclooxygenase-2 is overexpressed in HER-
2/neu-positive breast cancer: evidence for
Figure 1 . Expression of cyclooxygenase-2 (COX-2) in breast cancer tissues, tumor-adjacent tis-
sues, and healthy breast tissues. Real-time reverse transcription–polymerase chain reaction was
performed with custom-designed TaqMan Low Density Arrays (Applied Biosystems, Rotkreuz,
Switzerland). Expression profi les of COX-2 and several other cancer-related genes of 48 breast
cancer tissues, 41 tumor-adjacent tissues, and 12 breast tissue samples from healthy women
obtained during plastic – aesthetic surgery were compared (for detailed description of patients
and methods see Supplementary Material, available online). To correct for possible variations in
the expression of housekeeping genes, the expression of COX-2 was normalized to the mean
expression of four housekeeping genes ( GAPDH , UBC , TBP , and B2M ). Relative expression was
calculated with the ? ? Ct method. The horizontal line indicates the median relative expression of
each group. *** P < .001 vs tumor adjacent tissues, +++ P < .001 vs healthy breast tissues. P values
(two-sided) were calculated using the Mann – Whitney test.
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This work was supported by the Stiftung für
Forschung in Tumordiagnostik und Prävention,
9006 St Gallen, Switzerland.
Affiliations of authors: Biotechnology Institute
Thurgau at the University of Konstanz, Kreuzlin-
gen, Switzerland (EMB, DFL, HJS, GF); Tumor
Center ZeTuP, St Gallen, Switzerland (EMB, DFL,
HJS, GF); Tumor Center ZeTuP, Chur, Switzerland
(EMB, DFL, HJS, GF) .
Correspondence to: Gregor Fürstenberger, MD,
Tumor Center ZeTuP, Rorschacherstrasse 150,
9006 St Gallen, Switzerland (e-mail: gfuersten-
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