Correlation of COX- 2 Expression in Colorectal Carcinoma with Clinicopathological Features
Sandhya VENKATACHALA1, Manjula RAJENDRAN2
1Department of Pathology, Apollo Hospitals, BANGALORE, INDIA
2Department of Government Sivagangai Medical College & Hospital, SIVAGANGAI, INDIA
Keywords: Adenocarcinoma, Colorectal neoplasms, Cyclooxygenase 2, Cyclooxygenase 2 inhibitors, Lymphatic metastasis
Colorectal carcinoma is the most common neoplasm of the gastrointestinal tract. COX-2 expression is upregulated in colorectal
carcinoma. Therefore its assessment would identify patients amenable to adjuvant anticyclooxygenase therapy. We studied COX-2
immunoexpression in colorectal adenocarcinoma and correlated it with clinicopathological features as age, gender, tumor location, tumor size,
degree of differentiation, and TNM stage.
Material and Method: Sixty-five consecutive cases of colorectal adenocarcinoma were retrieved from the records of the Pathology Department
of a tertiary care hospital. The tumors were categorized as low positive and high positive based on their total COX-2 scores, which is the sum of
proportion and intensity scores of immunostaining, and were correlated with clinicopathological features. Statistical analysis was done using the
Chi-square test and Kendall's Tau–b correlation method.
Results: COX-2 was expressed in 86.2% of cases including 90% of left colonic carcinomas and 77.3% of right colonic carcinomas. Lymph node
metastasis was present in 22.2%, 25% and 47.75% of COX-2 negative, low positive and high positive tumors respectively. High positive COX-2
cases constituted 33.3% of stage I, 58.8% of stage II, 80% of stage III and 100% of stage IV tumors. About 56.6% of well differentiated, 66.6%
of moderately differentiated and 100% of poorly differentiated carcinomas showed high COX-2 expression. The COX-2 overexpression was
associated with advancing depth of invasion (p=0.021), stage of tumor (p=0.05), more frequent lymph node metastasis and decreasing degree
Conclusion: The association of COX-2 overexpression with increasing stage and depth of invasion may justify the use of COX-2 inhibitors as an
adjuvant to chemo and radiotherapy.
Colorectal carcinoma is the fourth common cancer in the
world and second most common cause of cancer-related
. Epidemiological studies data have shown a lower
incidence of adenomas and carcinomas in patients taking
Non Steroidal Anti-Inflammatory Drugs (NSAIDs) for a
long time suggesting a pathogenic role for cyclooxygenase
(COX-2) in colonic tumorigenesis2-4
. COX-2, an
inducible isoform of cyclooxygenase is usually absent or
present in low levels in normal colonic epithelium and is
upregulated in colorectal carcinoma5
. Assessment of
this molecular factor would therefore help in identifying
the patients who are likely to benefit from COX-2 inhibitor
adjuvant therapy which attenuates the metastatic potential
of colorectal carcinoma, thereby improving the prognosis.
Therefore the present study aims to evaluate COX-2
expression in colorectal carcinoma and to correlate it with
clinopathological features as age, sex, tumor location, size,
depth of invasion, histological type, degree of differentiation,
lymph node metastasis and stage of the tumor.
Sixty-five consecutive resection specimens of colorectal
carcinoma diagnosed between January 2009 and December
2013 were retrieved from the records of the pathology
department at a tertiary care hospital. Hematoxylin and
eosin slides of the 65 cases were reevaluated in respect to
the location, histological type and grade of the neoplasm,
depth of invasion and lymph node metastasis. Clinical
details which included the age, gender, stage of tumor were
recorded. The representative slides were selected in each case
that included primary tumor and, lymph nodes or satellite
nodules. If the case had distant metastasis, the metastatic
deposits were also included. Immunohistochemical
staining for COX-2 was done by the following procedure6
. Four microns thick sections were taken, floated on to
Poly-L-Lysine coated slides. Deparaffinization followed by
dexylinization and rehydration was done. Antigen retrieval
was done by pressure cooking in citrate buffer (pH -6.0)
for 10 minutes. After peroxidase block, the sections were
covered with concerned primary antibody (Monoclonal Rabbit Anti-Human COX-2, THERMO Scientific) for
1 hour. Super enhancer was then added and left for 30
minutes. It was washed with TBS buffer 3 times. Super
sensitive Poly- HRP was added. The slides were treated
with colour development solution 3’3’ diaminobenzidine
(DAB) for 5-8 minutes. Counterstaining was done with
haematoxylin stain. The slides were air dried, cleared in
xylene and mounted with DPX.
A section of lung adenocarcinoma was used as positive
control and the negative control was the same tissue
incubated without secondary antibody. COX-2 expression
was evaluated using a method wherein both the percentage
of positive cells (proportion score) and the intensity of
staining (intensity score) were added to obtain the “total
COX-2 score”7,8. The proportion score was scaled
between 0 and 4 (0: No cells are positive, 1: 1% to 25%
cells are positive, 2: 26% to 50% cells are positive, 3: 51% to 75% cells are positive and 4: 76% to 100% cells are
positive). The intensity score was scaled between 0 and 3
(0: No staining, 1: Weak staining, 2: Intermediate staining,
3: Strong staining). The stratification of staining intensity
was made in comparison with the staining intensity of
inflammatory cells. When the staining intensity of tumor
cells was less than the inflammatory cells, it was considered
weak staining (intensity score-1). Similarly when tumor cell
staining was the same and stronger than inflammatory cells,
it was considered intermediate (score 2) and strong staining
(score 3), respectively (Figure 1A-D). “Total COX-2 score”
thus obtained was used to categorize COX-2 expression
of tumors as ”Negative (total score 0 to 2)”, “Low positive
(total score 3 to 4)” and “High positive (total score 5 to 7)”.
Click Here to Zoom
|Figure 1: A) Well-differentiated adenocarcinoma (H&E; x100). B) Strong COX-2 expression (COX-2; x100). C) Moderate COX-2
staining (COX-2; x400). D) Weak COX-2 expression (COX-2; x100) (Note the red arrow in C&D to compare with inflammatory cells).
COX-2 expression (total COX-2 score) was then correlated
with clinicopathological features using the statistical
methods of Chi-square test and Kendall (τ) Tau-b tests. The Chi-square test was used for determining correlation
between COX-2 expression and pathological features
such as depth of tumor, microscopic grade, lymph node
metastasis, and TNM stage. Relationship between age,
gender and COX-2 expression was determined by Kendall
Of the 65 cases of colorectal carcinoma included in the
study, 56 (86.2%) cases expressed COX-2 while 9 cases
(13.8%) were COX-2 negative. Forty-four (67.7 %) of the 56
positive cases, strongly expressed COX-2 while 12 (18.5 %)
weakly expressed COX-2. Mean age of presentation was 64
years-old ± Standard Deviation (Range: 38 years-93 years,)
with males (36 cases) slightly outnumbering the females
(29 cases). Forty cases (61%) were located in left colon,
while 22 (34%) and three cases (4%) were located in right
colon and transverse colon, respectively. Ninety percent
of left colonic carcinomas and 77.3% of right colonic
carcinomas expressed COX-2. Three stage I, 34 stage II, 25
stage III and three stage IV tumors were seen in the present
study with one pT1, three pT2, 48 pT3 and 13 pT4 tumors.
Lymph node metastasis was seen in 26 of the 65 cases (40%)
with 15 pN1 and 11 pN2 cases. Two of the three stage IV
cases showed metastatic deposits in both ovaries while one
showed deposits in uterus and fallopian tube.
COX-2 Negative Cases
Four of the nine COX-2 negative cases did not show any
expression of COX-2. However in the remaining five
cases, less than 25% of tumor cells weakly expressed COX-
2 accounting to a total score of 2 which was considered
negative. Five were located in the right colon while four
were in the left. No lymph node metastasis was seen in 7
of the nine (77.8%) COX-2 negative cases. Seven stage II and two stage III tumors were seen. Only 2 cases (22.2%)
had tumor in the lymph node and the tumor cells in one of
them were low positive for COX-2.
COX-2 Low Positive Cases
Of the twelve low positive tumors, three were located in the
right and nine in the left colon. Two stage I, seven stage
II, three Stage III tumors were seen. There was no stage IV
tumor. Nine cases (75%) had no evidence of tumor in the
lymph node. In two of three cases (25%) with lymph node
deposits, a similar COX-2 expression was seen in both the
primary tumor and the lymph node deposits while a higher
expression was seen in the lymph node deposits in the third
case in the N2 stage. The morphology of primary tumor
and the metastasis was however the same.
COX-2 High Positive Cases
The location of these 44 tumors was 27 (61.4%) in the
left colon, 14 (31.8%) in the right colon, and three in the
transverse colon. COX-2 high positive tumors included one
stage I, 20 stage II, 20 stage III and three stage IV tumors.
Lymph node deposits were seen in 21 cases (47.7%) of which
11 were in N1 stage and 10 were in N2 stage. Twenty-three
cases (52.3%) did not have lymph node metastasis. One
(2.2%) T2 tumor, 34 (77.3%) T3 tumors and 9 (20.5%) T4
tumors were present in this group. There was no T1 tumor.
Stage I, stage II, stage III and stage IV tumors constituted
one case (2.2%), 20 cases (45.5%), 20 cases (45.5%) and
three cases (6.8%) respectively. The organs involved by
distant metastasis in the three stage IV tumors were the
fallopian tube and ovary in two of them and an umbilical
nodule in the third. All of the mucinous and signet ring cell
carcinomas showed high COX-2 positivity (Figure 2A,B
Click Here to Zoom
|Figure 2: A) Mucinous carcinoma with extracellular mucin (H&E; x100). B) Strong COX-2 expression in the same tumor (COX-2; x400).
Click Here to Zoom
|Figure 3: A) Signet ring cell carcinoma infiltrating the muscle layer (H&E; x100). B) Strong COX-2 expression in signet ring cells (COX-
Lymph node metastasis was present in 22.2%, 25% and
47.8% of COX-2 negative, low positive and high positive
cases, respectively (Figure 4A,B). This difference however
was not statistically significant (p value=0.08)
Click Here to Zoom
|Figure 4: A) Strong COX-2 staining in 30% of metastatic tumor cells in lymph node. Red arrow shows capsule with few lymphoid cells
(COX-2; x100). B) Peritoneal nodule with COX-2 expressing signet ring cells (COX-2; x100).
The distribution of cases and correlation between COX-2
expression, stage, the depth of invasion, histopathological
grade and tumor type have been shown in Tables I-III.
Click Here to Zoom
|Table III: COX-2 expression in the different histological types and grade of colorectal carcinoma
COX-2 Expression and Location of Tumor
Among 65 cases 22 cases (33.9%) were located in right
colon, 40 cases (61.5%) were located in of left colon and three cases (4.6%) were located in the transverse colon.
Of the 22 right colonic carcinomas, 17 (77.3%) expressed
COX-2 and 5 (22.7%) were negative. The forty cases of
left colonic carcinoma included 4 (10%) COX-2 negative
tumors and 36 (90%) COX-2 positive tumors.
COX-2 Expression and Size of Tumor
The greatest diameter of the nine COX-2 negative tumors
varied between 2.5 cm and 6.0 cm (mean 4.6 cm ± SD)
while those of 56 COX-2 positive tumors ranged from 2.5
cm to 12.0 cm (mean 5.3 cm ±SD). The difference was not
statistically significant (p=0.27).
COX-2 Expression and Stage of Tumor
The correlation between depth of invasion and COX-2
expression was statistically significant (p=0.021). The stages
of tumors also bear a significant correlation with COX-2
expression, p value being equal to 0.05. The p values for
correlation with lymph node metastasis were 0.08.
COX-2 Expression and Other Clinicopathological
The p values for correlation with age, gender, and
microscopic grade were 0.82; 0.47; and 0.08 respectively.
Colorectal cancer is the fourth most common cancer in
the world and the second most common cause of cancer related death1
. Assessment of molecular prognostic
factors would be of great help for identifying patients who
would benefit from adjuvant therapies, thereby increasing
their survival. Cyclooxygenase-2 (COX-2) and its product
PGE2 play an important role at different levels in colorectal
carcinogenesis. It has been implicated from the initiation
stage to tumor progression9
. Some of its carcinogenetic
mechanisms include evasion of apoptosis, increasing bcl-
2 expression and inhibition of Fas-mediated apoptosis,
activation of the APC-β catenin pathway, increasing the
expression of vascular endothelial growth factor (VEGF),
decreasing E-cadherin expression favoring epithelialmesenchymal
transition (EMT) and increasing matrix
metalloproteinases-2 (MMP-2) thereby enhancing the
invasiveness and metastatic potential of the tumor cells10
. The present study was thus done to analyse the relation
between COX-2 expression and the biological characteristics
of colorectal carcinoma.
In the present study of the 65 colorectal carcinomas, 86.2%
of cases expressed COX-2 while 13.8% did not express.
Similar expression has been seen by Al-Maghrabi who
found 85% of colorectal carcinomas expressing COX-211. However other studies as published by Soumaoro et
al. have shown relatively fewer tumors (70%) expressing
The present study included fifty-nine (90.6%) stage II/
III tumors with only three (4.6%) of the stage I tumors
contributing to the high percentage of COX-2 expressing
tumors. The difference noted in studies by Al Maghrabi
and Soumaro could be attributed to the difference in
location of the tumors in their series. In the former study
by Al Maghrabi, similar to our study greater numbers of
left colon carcinoma were present, while in the latter a
greater number of right colon carcinoma were included.
Right colon carcinomas are more frequently associated
with microsatellite instability and lower or absent COX-
2 expression while left colon carcinomas are frequently
associated with loss of APC and hence increased COX-2
expression mediated by β catenin12,13. In the absence
of APC, β catenin translocates to the nucleus and increases
COX-2 levels. Nasir et al. and others found an inverse
correlation between COX-2 and APC expression and a
more frequent COX-2 expression in left colonic carcinoma13. In agreement with these findings, 90% of left colon
carcinomas were COX-2 positive while only 77.3% of right
colon carcinomas expressed COX-2 in the present study.
Lymph node metastasis was present in 22.2%, 25% and
47.8% of COX-2 negative, low positive and high positive
cases respectively. Though the values were not statistically
significant, these findings are in consistence with those of
Sheehan KM12 and Al-Maghrabi11 who also found
two to four times more frequent lymph node metastasis in
tumors with high COX-2 expression. Furthermore, in 76%
of stage III tumors with lymph node metastasis, a similar
staining intensity was seen in the primary tumor and lymph
node deposits, while in 16% of the cases, the primary
tumor was high positive, whilst the lymph node deposits
being low positive. However, no larger series are available
to compare these findings. With increasing stage of the
tumor, there was an increase in number of high COX-2
positive tumors from 33.3%, 58.8%, 80% to 100% in stage I,
II, III, IV tumors respectively. On the contrary the number
of low COX-2 expressing tumors gradually declined as
the stages increased and there was no COX-2 negative carcinoma in stage IV. These findings are in correlation
with other studies that used the Dukes classification12,14
or the AJCC staging systems11,15. Elzagheid et al. have
also found high COX-2 expression in association with
advanced stages of colorectal carcinoma while finding no
significant association with tumor grade and lymph node
status16. As the tumor depth of invasion progressed from
T1 to T4, invading the submucosa, muscularis propria,
serosa and beyond, an increase in the proportion of high
COX-2 positive carcinoma from 0% to 77% was observed
with a decline in the low COX-2 positive tumors from
T2 to T3. This association was statistically significant.
A similar relation between depth of invasion and COX-
2 overexpression has been documented by Soumaoro7 and Wu17 in their studies. The number of high
COX-2 positive tumors increased as the degree of tumor
differentiation decreased. The proportion of high COX-2
expressing tumors increased from 56.2% to 66.6% and 100%
in well differentiated, moderately differentiated and poorly
differentiated carcinomas. Our findings are consistent with
the studies of Masunaga14 and Al-Maghrabi11. We
also observed that all of the mucinous and signet ring cell
carcinomas showed high COX-2 positivity. These findings
are in agreement with those by Baba et al.18. A few others
have described a relatively low COX-2 expression in signet
ring cell carcinoma compared to the mucinous category19. Comparing the COX-2 scores with the tumor size
in the present study showed no statistically significant
correlation similar to other previous studies7,14. Further
comparing the total COX-2 scores of the primary tumor and
the distant metastasis in the three stage IV tumors in the
study, both the primary tumor and metastatic tumor were
found to be high COX-2 expressors, raising the possibility
for the role of COX-2 in tumor progression. However, more
stage IV tumors need to be evaluated to assess the statistical
significance of this correlation as there were only three
stage IV tumors in the present study.
The association between COX-2 expression and tumor
infiltrating lymphocytes (TIL) (CD8+) was not evaluated
in the present study. Nosho et al. have reported that the
number of TILs were increased in microsatellite instabile
tumors but decreased in COX-2 expressing tumors20.
In conclusion, COX-2 expression in colorectal carcinoma
was investigated in the present study. The tumors were
categorized as low positive and high positive based on their
total COX-2 scores, which is the sum of proportion and
intensity scores of staining. Left colonic carcinomas more
frequently expressed COX-2 than the right colonic tumors.
The association between COX-2 expression and depth of tumor invasion and the stage of tumor were statistically
significant. The deeper the depth of invasion, the greater
was the total COX-2 score. Additionally, the presence of
lymph node metastasis increased (22.2% to 25% to 47.7%)
with increasing levels of COX-2 expression (negative to
low positive to high positive) in the primary tumor. These
findings may justify the use of selective COX-2 inhibitors
such as Celecoxib, as an adjuvant to chemotherapy
and radiotherapy in or combined usage of celecoxib
with 5-fluorouracil which has been shown to improve
chemosensitivity in chemorefractory colorectal carcinoma
to improve recurrence free survival, disease free survival
and overall survival21,22.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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