Download Elevated cyclooxygenase-2 expression is associated with

J Med Dent Sci 2005; 52: 189–193
Original Article
Elevated cyclooxygenase-2 expression is associated with histological grade in
invasive ductal breast carcinoma
Emiko Takeshita, Takayuki Osanai, Tetsuro Higuchi, Labile Togba Soumaoro and Kenichi Sugihara
Department of Surgical Oncology, Graduate School of Medicine, Tokyo Medical and Dental University
Purpose: The aim of this study was to evaluate
the expression of cyclooxygenase-2 in human
breast cancer using immunohistochemistry and to
determine whether the expression of cyclooxygenase-2 is associated with clinicopathological factors in invasive ductal breast carcinoma.
Methods: Cyclooxygenase-2 expression was
investigated by immunohistochemistry in 30 invasive ductal breast carcinoma specimens and relationships between cyclooxygenase-2 expression
and age, histological grade, histological type,
nodal status, and hormone receptor status were
evaluated.
Results: Cyclooxygenase-2 expression was
found in 56.7% of the tumor samples and was related to histological grade (P＜0.01) and histological
type (P＜0.001).
Conclusions: Our results suggest that
cyclooxygenase-2 expression has an important
role in tumor differentiation in invasive ductal
breast carcinoma.
Key words:
cyclooxygenase-2, breast carcinoma,
histological grade
Corresponding Author: Kenichi Sugihara
Department of Surgical Oncology, Graduate School of Medicine,
Tokyo Medical and Dental University
1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Tel: +81-3-5803-5261 Fax: +81-3-5803-0139
E-mail: [email protected]
Received May 6; Accepted June 10, 2005
Introduction
Epidemiological studies have suggested the
chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs) in colorectal cancer1,2,3.
Cyclooxygenase (COX) is the major cellular target of
NSAIDs
and
regulates
the
synthesis
of
prostaglandins. Two isoforms of COX are recognized;
COX-1, the constitutively expressed isoform, and
COX-2, the inducible type, which is induced by proinflammatory cytokines, growth factors, and mitogens4.
Numerous studies have emphasized that COX-2
plays an important role in the prevention of colon cancer5,6,7,8, and may be involved in tumorigenesis of the
esophagus9,10, stomach11,12, and other solid
tumors13,14,15. More recent studies indicate that COX-2
contributes to cell proliferation, mediates inhibitory
effects on apoptosis, modulates the production of several proangiogenic factors, and increases cell invasiveness16,17,18.
In early studies, the effects of NSAIDs on breast cancer risk were underestimated19. In contrast, analysis of
prospective data has recently indicated the protective
effects of the regular aspirin, ibuprofen and other
NSAIDs against the development of breast cancer20,21. Animal studies have demonstrated that selective COX-2 inhibitors can reduce tumor incidence and
multiplicity22,23.
In human breast cancer, several studies have
shown that expression of COX-2 mRNA and protein is
elevated, and COX-2 has been implicated the pathogenesis of the disease13,24,25,26. Ristimäki et al. have
studied large numbers of breast tumor samples and
showed a significant correlation between elevated
COX-2 protein expression and a number of clinico-
190
E. TAKESHITA et al.
pathological variables, including tumor stage, hormone receptor status and HER-227. However, other
groups have reported that COX-2 expression is not
associated with some of these factors28,29,30. Thus, the
relationships between COX-2 expression and tumor
characteristics remain uncertain. The aim of this
study was to assess whether expression of COX-2 protein is associated with clinicopathological factors in
invasive ductal breast carcinoma by immunohistochemistry.
Materials and Methods
Patients
Surgical specimens from 30 patients diagnosed as
having primary invasive ductal breast carcinoma and
operated on between 1999 and 2001 at the
Department of Surgical Oncology of Tokyo Medical and
Dental University were studied. The Human Subjects
Committee at Tokyo Medical and Dental University
approved the research protocol. All patients gave written consent to participate in the study.
Immunohistochemistry
For immunohistochemical examination of COX-2,
we used a universal immunoenzyme polymer method.
Formalin-fixed, paraffin-embedded specimens were
serially sectioned at a thickness of 3 Òm, placed onto
MAS-coating slides. Specimens were deparaffinized,
rehydrated, and antigen was retrieved using an autoclave at 121°C for 20 minutes in 10 mM sodium citrate
buffer (pH 6.0), then treated with 3% hydrogen peroxide
in methanol solution at room temperature for 15 minutes to quench endogenous peroxidase activity.
Nonspecific reactivity was blocked by treating slides
with 10% normal goat serum for 10 minutes. The primary antibody used for immunohistochemical staining
was anti-human COX-2 monoclonal antibody
(Cayman Chemical Co., Ann Arbor, MI, USA) at a dilution of 1:500 and applied overnight at 4°C. Next,
slides were incubated with labeled polymer (NHistofine Simple Stain MAX PO MULTI, Nichirei Co.,
Tokyo, JAPAN) for 30 minutes at room temperature.
Sections were incubated in 0.02% 3, 3’-diaminobenzidine tetrahydrochloride (Dojin Chemical Laboratory,
Co., Kumamoto, JAPAN) and 0.06% hydrogen peroxide
in 50mM Tris-Hcl (pH7.6) for 5 minutes. Finally, slides
were counterstained with 1% Meyer’s hematoxylin. A
known COX-2-positive colonic cancer sample was
used as a positive control. As a negative control, tissue
J Med Dent Sci
sections were treated with normal mouse IgG serum
instead of primary antibody 31.
Evaluation of COX-2 immunohistochemistry
COX-2 immunohistochemical staining was scored
independently by two investigators (E.T. and T.O.)
who were blinded as to the sample origins. Evaluation
of COX-2 expression was semiquantitatively estimated
by intensity and area of positive cells, modifying the
methods of Ristimäki et al. 27: Negative staining; no
staining or weak diffuse cytoplasmic staining (may
contain stronger intensity in less than 10% of cancer
cells); Elevated staining, moderate to strong granular
cytoplasmic staining in 10-100% of cancer cells.
Association between COX-2 staining and clinicopathological factors was then investigated.
Pathological factors
Histological grade was assigned according to the
Nottingham modification of the Bloom and Richardson
grading system. The grade was obtained by summing
the scores for tubule formation, nuclear pleomorphism, and mitotic count, each of which was given 1, 2,
or 3 points. Grade I invasive carcinomas had 3 or 4
points, grade II neoplasms had 5 to 7 points, and grade
32
III tumors had 8 or 9 points . The histological type of
human breast cancer investigated was invasive ductal
carcinoma, which was classified as papillotubular carcinoma, solid-tubular carcinoma or scirrhous carcinoma
according to the Japanese Breast Cancer Society criteria33. When combined histological type was
observed, predominant type was selected for evaluation.
Statistical Analysis
The correlations between COX-2 expression and
several clinicopathological parameters were analyzed
by Á2 test. P values of less than 0.05 were considered
significant. For statistical evaluation, we used the
StatView software package (version 5.0; SAS Institute
Inc., Cary, NC).
Results
Patient characteristics
Mean patient age was 55 (range, 31-75) years.
Patient characteristics, including histological type, histological grade, hormone receptor status, and nodal
status are shown in Table 1.
CYCLOOXYGENASE -2 EXPRESSION IN BREAST CARCINOMA
191
Table 1. Relationships between elevated COX-2 expression and clinicopathological factors
COX-2 Expression
Elevated COX-2 expression was found in 57% (17
out of 30) of breast cancer samples. Representative
examples of COX-2 expression are shown in Fig. 1 (1ab). COX-2 staining was granular and localized predominantly in the cytoplasm of the tumor cells.
Negative staining was shown in Fig. 1b. In some
tumor samples, enhanced COX-2 staining was
observed in the perinuclear area (Fig. 1c). Staining in
normal-appearing epithelia was of no intensity
(Fig. 1d). Within the same tissue sections, stromal
staining for COX-2 in endothelial cells, lymphocytes,
and fibroblasts was weakly detected adjacent to COX2 expressing tumor epithelia (Fig. 1e).
COX-2 Expression and Tumor Characteristics
Elevated expression of COX-2 was more frequent in
high grade tumors (grade III) than in lower grade
tumors (grade I and grade II) (P＜0.01, Table 1).
Histological type (papillotubular and solid-tubular carcinoma vs. scirrhous carcinoma) was also associated
with elevated COX-2 expression (P＜0.001, Table 1). All
samples of scirrhous carcinoma showed elevated
COX-2 expression, while 73.3% and 100% of papillotubular and solid-tubular samples were negative. No
significant correlations were observed between COX-2
expression and age, estrogen receptor (ER), progesterone receptor (PgR), or lymph node status (Table 1).
Fig. 1. Immunohistochemical expression of cyclooxygenase-2
(COX-2) in samples of invasive ductal breast carcinoma.
COX-2 staining is detected in the cytoplasm of the tumor cells.
Scirrhous carcinoma, grade III shows elevated expression of COX-2
(1a, ×40). Example of negative expression: solid tubular carcinoma,
grade II. (1b, ×40). COX-2 protein is strongly expressed in perinuclear area of the tumor cells (1c, ×400). Normal breast epithelia are
shown with negative staining (d, ×200). Immunoreactive COX-2 is
detected in stromal cells surrounding the tumor cells (e, ×200).
Discussion
In the present study, we found that elevated expression of COX-2 was associated with higher histological
grade. This suggests that COX-2 has important role in
tumor differentiation in invasive ductal breast carcinoma. Elevated COX-2 expression was previously found
to be associated with tumor size and depth in colorectal and gastric cancer34 35 and with grade of dysplasia in
colorectal adenoma36. In those studies, however, no
evaluation was conducted on COX-2 expression and
tumor differentiation.
In breast cancer studies, our analyses of correlation
between COX-2 expression and histological factors are
consistent with the results of others27,37,38, but they also
contradict some reports28,30,39,40,41. In a recent study,
192
E. TAKESHITA et al.
Shim et al. showed that COX-2 expression was
detected in almost all ductal carcinoma in situ specimens, with increased COX-2 staining correlating with
higher nuclear grade42. In the present findings, association between lower histological grade (I and II) versus
higher grade (III) was compared to COX-2 expression.
Based on O’Reilly’s description, histological grades I
and II were combined to one category of “well-differentiated” tumors, and only grade III tumors were analyzed as a separate, single group43. The presence of
significant correlations in our study group may have
resulted from the close relationship between histological grade and histological type of invasive ductal carcinoma; almost all scirrhous carcinoma samples exhibited elevated COX-2 expression and most scirrhous carcinoma belonged to grade III.
According to the classification by the Japanese
Breast Cancer Society, there are three types of invasive
ductal carcinoma and prognosis of patients with papillotubular type is known to be the best, while scirrhous is
33
the worst . Elevated COX-2 expression would therefore
be a marker of poor differentiation, and may reflect the
prognosis of invasive ductal carcinoma. Some investigators have reported that elevated COX-2 expression is
associated with a significantly worse disease-free survival and overall survival, and have evaluated COX-2 as
one of prognostic factors27,44,45.
COX-2 in malignant tumors is thought to be
involved in tumorigenesis. Shattuck-Brandt et al. have
reported that COX-2 expression was observed in both
epithelial and tumor stromal cells46. In our samples,
COX-2 immunoreactivity was detected with weak
intensity in stromal cells surrounding the tumor cells.
Moreover mastopathy and adenosis adjacent to COX-2
positive carcinomas also expressed COX-2 otherwise
mastopathy alone didn’t show COX-2 positivity (data
not shown). This observation supports the notion that
COX-2 in stromal cells acts to promote tumors and
exerts paracrine effects on nearby carcinoma cells47.
One study has emphasized the significant roles of
fibroblasts and endothelial cells in the intestinal polyp
development through COX-2 expression, while little
COX-2 expression was seen in macrophages48. In
contrast, other groups found that COX-2 was
expressed predominantly by macrophages in the stroma47,49. COX-2 expression in breast cancer stromal
cells has not been clarified. One reason may be that it
is difficult to distinguish stromal cells from tumor cells
because of the unique morphology of breast cancer.
The potential value of COX inhibitors for breast cancer is currently under examination50. However, it is not
J Med Dent Sci
known which group of patients could gain the most benefit from COX inhibitors. Our results showing the positive correlation between elevated COX-2 and higher histological grade suggest that COX-2 inhibitors have a
potential therapeutic effect on grade III scirrhous invasive ductal breast carcinoma.
Acknowledgments
We thank Ms. Yoko Takagi for valuable technical
assistance.
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