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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 4  |  Page : 695-699
A study on expression pattern of cyclooxygenase-2 in carcinoma of cervix


1 Department of Pathology, Burdwan Medical College, West Bengal, India
2 Department of Pathology, Institute of Postgraduate Medical Education and Research, Kolkata, India
3 Department of Pathology, Medical College, Kolkata, India

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Date of Web Publication6-Jan-2012
 

   Abstract 

Objective: The purpose of the present study was to determine the differential expression pattern of cyclooxygenase-2 (COX-2) in patients of carcinoma of uterine cervix and its correlation with tumor differentiation and lymphovascular invasion. Materials and Methods: Seventy (70) cases of cervical carcinoma were included (20 in-situ, 42 invasive squamous cell, and 8 cases of adenocarcinoma). Formalin-fixed paraffin-embedded tissue sections were stained by Hematoxylin and Eosin. Immunohistochemistry for COX-2 were performed on these blocks. Results: A higher expression of COX-2 was seen in invasive squamous cell carcinoma than in in-situ carcinoma (P = 0.002). Five out of eight cases of adenocarcinoma showed strong positivity for COX-2 antibody. Among the histopathological correlates, tumor differentiation did not show a positive correlation (P = 0.717), while lymphovascular invasion was associated with positive staining in majority of the cases (P = 0.001). Conclusion: Expression of COX-2 is more in cases of invasive than in in-situ carcinoma. Adenocarcinomas showed a strong expression of COX-2. A positive association of COX-2 expression and the presence of lymphovascular emboli were found in the present study. COX-2 inhibitors need to be studied as a therapeutic adjunct for the treatment of carcinoma cervix.

Keywords: Cyclooxygenase-2, carcinoma of cervix, lymphovascular invasion

How to cite this article:
Bandyopadhyay R, Chatterjee U, Mondal SK, Nag D, Sinha SK. A study on expression pattern of cyclooxygenase-2 in carcinoma of cervix. Indian J Pathol Microbiol 2011;54:695-9

How to cite this URL:
Bandyopadhyay R, Chatterjee U, Mondal SK, Nag D, Sinha SK. A study on expression pattern of cyclooxygenase-2 in carcinoma of cervix. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Apr 24];54:695-9. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/695/91495



   Introduction Top


Metabolites of arachidonic acid are critical for numerous biological processes. Their role in the development of cancer is studied extensively. Cyclooxygenase-1 (COX-1) is constitutively expressed in many tissues including kidneys, lungs, gastrointestinal tract (GIT), and is responsible for producing cytoprotective prostaglandins. The second isoform of the cyclooxygenase enzyme, cyclooxygenase-2 (COX-2), shares significant sequence homology and catalytic activity with COX-1. It is constitutively expressed only in placenta, macula densa of kidney, and brain. [1] A variety of extracellular and intracellular stimuli (LPS, IL1, TNF, EGF, TGF α, IFN g, PAF) can rapidly induce COX-2 at sites where it is not expressed constitutively. [2]

The role of COX-2 has been extensively studied in colorectal cancers. It has been found that approximately 50% of adenomas and 80 - 85% of adenocarcinomas show increased expression of COX-2. [3] Cancer of the cervix has been the most important cancer in women in India over the past two decades. [4] Until now, only a few studies have been undertaken to evaluate the role of COX-2 in carcinomas of cervix. We tried to evaluate the role of COX-2 in carcinomas of cervix by studying the expression pattern in different types of carcinomas.

Aim

This study was undertaken to show the differential expression pattern of COX-2 in carcinoma cervix. Both in-situ and invasive squamous cell carcinomas and invasive adenocarcinomas were included to note any difference in expression in these groups. An attempt was also made to correlate the expression pattern with tumor differentiation and lymphovascular invasion.


   Materials and Methods Top


Seventy cases of carcinoma cervix (20 in-situ, 42 invasive squamous cell carcinoma [SCC], and 8 adenocarcinomas [Stage II A, FIGO]) were studied over a period of two years. Twenty cervical biopsies and 50 hysterectomy specimens were included in the study. Formalin-fixed paraffin-embedded tissue sections were stained with Hematoxylin and Eosin. The histologic grade, depth of invasion, and presence of lymphovascular invasion were noted. Lymph node status was noted in hysterectomy specimens. Immunohistochemistry for COX-2 was performed using rabbit monoclonal antibody to COX-2 and supersensitive polymer based detection system (Biogenex). Formalin fixed, paraffin embedded tissue sections were used for staining. After deparaffinization, tissue sections were rehydrated using descending grades of alcohol and water. Antigen retrieval was done by heat treatment using microwave oven. Tris buffer saline (TBS) was used for washing. Peroxide blocking was done for 10 minutes. This was followed by power block for 10 minutes, cleaning and incubation with primary antibody overnight at 4°C. Slides were washed again in TBS and incubated with secondary antibody for 30 minutes. After washing and cleaning, link label was added and incubated for 30 minutes. After washing in buffer, DAB was added and incubated for 10 minutes. After washing, slides were counterstained with Hematoxylin. Positive staining showed cytoplasmic and membrane positivity. Cases of colon carcinoma were taken as positive controls. Positive staining pattern was graded as: undetected, low (expressed in 10% tumor cells), moderate (10-50% positive tumor cells), and high (>50% positive tumor cells). Statistical analysis was performed with Fisher's two-tailed exact test (with Freeman Halton extension). For the statistical purpose, the expression pattern was classified as expressed (including low, moderate and high positivity) and not expressed (including the undetected cases).


   Results Top


In the carcinoma in-situ (CIN) III SCC group, the age at presentation ranged from 25 to 47 years. Invasive SCC cases presented between 35-47 years of age. Out of 42 cases of invasive SCC, we noted nine keratinizing, 21 large cell non keratinizing (LCNK) and 12 small cell non keratinizing tumors. Studying the COX-2 expression pattern in in-situ SCC, a low rate (20% of cases) of positivity was noted [Figure 1] and [Figure 2]. However, in the invasive SCC group, percentage of positive cases was higher [Table 1] and [Figure 3]. Classifying the COX-2 expression in two groups as expressed and non-expressed, there was a significant difference between the CIN III and invasive SCC with expression being more in the invasive cases (P = 0.002) [Table 1]. In the invasive SCC, positive expression was noted in all the three types. No relation was noted between COX-2 expression pattern and histological differentiation of the tumor (P = 0.717) [Table 2]. Cases of invasive SCC were studied for lymphovascular invasion. Out of 26 cases showing lymphovascular invasion, positive COX-2 expression was seen in 20 (77%) cases [Table 3]. Analyzing the results, the frequency of COX-2 expression was found to be significantly more in lymphovascular invasion-positive tumors (P = 0.001; Relative Risk 3.08). We encountered 30 cases of metastatic involvement of different lymph nodes. Thus, the COX-2 expression did not vary between the cases showing metastasis and those without lymph node involvement. COX- 2 positivity was found in cases of adenocarcinoma of cervix [Figure 4]. All the positive cases showed moderate to high positivity and the intensity of staining was also higher than SCC. However, comparing this group with the SCC group, there was no statistically significant difference in COX-2 expression between them (P = 0.112) [Table 4].
Table 1: COX-2 expression pattern in carcinoma cervix (n = 70)

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Table 2: COX-2 expression in different grades of squamous cell carcinoma of cervix (n = 42)

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Table 3: Distribution of cases regarding COX-2 expression and lymphovascular invasion (total 42)

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Table 4: Comparison of COX-2 expression pattern in squamous cell carcinoma and adenocarcinoma

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Figure 1: Photomicrograph showing negative COX-2 staining in a case of in-situ squamous cell carcinoma (IHC, ×200)

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Figure 2: Photomicrograph showing moderate COX-2 positivity in a case of in-situ squamous cell carcinoma (IHC, ×200)

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Figure 3: Photomicrograph showing strong COX-2 expression in a case of invasive grade 2 squamous cell carcinoma (IHC, ×400)

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Figure 4: Photomicrograph showing strong COX-2 expression in a case of cervical adenocarcinoma (IHC, ×100)

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   Discussion Top


It is well known that the concentration of prostaglandin in tumor tissues is much greater than the corresponding normal tissues. [2] Cyclooxygenase, the key enzyme in the prostaglandin metabolism, has been studied extensively to find out its potential role in the development and progression of tumors. It has been noticed that COX-2 expression is involved in both early and late stages of carcinogenesis. [5] Alteration in the COX-2 expression has been observed more frequently in several types of malignant tumors because COX-2 is present independently in cells during the early stages of cell differentiation or replication. [6],[7] Increased COX-2 expression has been demonstrated in high grade SCC of esophagus, [8] adenomatous and metaplastic lesions of stomach, [9] pre-neoplastic lesions of lung, pre-invasive neoplasias of breast, [10] bladder, [11] and pancreas. [12]

Studies on the expression of COX-2 in cervical cancer are relatively few. It has been shown to be expressed in dysplastic epithelium and in invasive carcinomas. The number and intensity of positive COX-2 staining has been shown to increase with the severity of dysplasia. [13] But the expression of COX-2 is lower in in-situ carcinomas compared to invasive carcinomas. [14] Dai et al. reported that COX-2 expression was positive in 45% of cases of CIN. [15] Similar results have been showed by Farley et al.[13] Dersun et al. have shown 25% positivity in CIN-3 and 55% in invasive SCC. This group has suggested that COX-2 may participate in the progression of cervical SCC. Similarly, in the present study, we encountered a lower rate of positivity in CIN; only 20% of CIN shows positivity for COX-2 compared to 66.6% of invasive SCC. The difference in expression between these two groups was found to be statistically significant.

Relationship of COX-2 expression with tumor grade has been studied extensively, but the results do not support a relationship between higher grade and COX-2 expression. Significantly increased COX-2 expression has been reported in well differentiated tumors. [16] In the present study, the difference in COX-2 expression between the keratinizing, LCNK, and small cell non keratinizing tumors was not significant. Several investigators have found similar results while comparing different histological grades of the tumor. [17],[18],[19]

COX-2 expression has been shown to be an independent poor prognostic factor in patients receiving radiation therapy. [20] There was a significant difference in five-year disease-free survival in tumors expressing COX-2. Other studies have demonstrated association of elevated COX-2 expression with parametrial invasion and lymph node metastasis. [21] These studies point toward a role of COX-2 as a prognostic marker. Lymphovascular invasion has been found to be associated with positive COX-2 expression in other tumors like carcinoma of the breast. [22] Presence of lymphovascular invasion is an established parameter of prognosis in cervical carcinomas. It has been reported that the presence of lymphovascular invasion increases the risk of lymph node metastasis and tumor-related death and reduces the overall and disease-free survival. [19] A positive association of COX-2 expression and the presence of lymphovascular emboli was found in the present study. This observation again points to the probable role of COX-2 expression as a prognostic marker and it may be used in conjunction with other major criteria in modifying the postoperative adjuvant therapy in SCC. [23],[24],[25] However, some investigators have found no significant correlation between the COX-2 expression and tumor response in patients who have undergone radiotherapy. [26] Only a few studies have evaluated association between COX-2 expression and lymph node metastasis. One of them found an association of COX-2 expression and lymph node metastasis, [27] where as another reported an association between COX-2 expression and lymphovascular invasion and larger tumor size, but not with parametrial involvement, lymph node metastasis recurrences, and survival. [19] One large series with 196 cases has reported an association of COX- 2 expression with lymph node metastasis. [28] In our series, COX-2 expression was seen in a higher number of cases with metastatic disease. However, the difference was not found to be statistically significant.

COX-2 expression has also been demonstrated in adenocarcinomas of cervix. In the present study, adenocarcinomas showed a stronger positivity than SCC and in higher percentage of cases. But this difference did not prove to be of statistical significance. The number of cases might have been very small to draw any conclusion from this. Other studies have shown greater proportion (73.9%) of positivity for adenocarcinomas. [20] Some studies have suggested unfavorable treatment response in cases of adenocarcinomas showing high COX-2 expression. [29]

In conclusion, our study shows that COX-2 expression is more in cases of invasive SCC than its in-situ counterpart. Positive COX-2 expression has a strong association with presence of lymphovascular invasion. Cervical adenocarcinomas show strong positivity with intense staining pattern. Studies with larger number of cases are required to evaluate the role of COX-2 in cervical adenocarcinomas and the potential therapeutic role of COX-2 inhibitors in such cases.


   Acknowledgment Top


Dr. Avijit Hazra, Associate Professor, Department of Pharmacology, Medical College, Kolkata for the statistical help provided.

 
   References Top

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2.Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 1999;18:7908-16.  Back to cited text no. 2
    
3.Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, Dubois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107:1183-8.  Back to cited text no. 3
    
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[PUBMED]  Medknow Journal  
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6.Smith WL, Langenbach R. Why there are two cyclooxygenase isozymes. J Clin Invest 2001;107:1491-5.  Back to cited text no. 6
    
7.Dannenberg AJ, Altorki NK, Boyle JO, Dang C, Howe LR, Weksler BB, et al. Cyclooxygenase-2: A pharmacological target for the prevention of cancer. Lancet Oncol 2001:2;544-51.  Back to cited text no. 7
    
8.Shamma A, Yamamoto H, Doki Y, Okami J, Kondo M, Fujiwara Y, et al. Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus. Clin Cancer Res. 2000;6:1229-38.  Back to cited text no. 8
    
9.Lim HY, Joo HJ, Choi JH, Yi JW, Yang MS, Cho DY, et al. Increased expression of cyclooxygenase-2 protein in human gastric carcinoma. Clin Cancer Res 2000;6:519-25.   Back to cited text no. 9
    
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11.Shirahama T. Cyclooxygenase-2 expression is up-regulated in transitional cell carcinoma and its preneoplastic lesions in the human urinary bladder. Clin Cancer Res 2000;6:2424-30.  Back to cited text no. 11
    
12.Maitra A, Ashfaq R, Gunn CR, Rahman A, Yeo CJ, Sohn TA, et al. Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia: An immunohistochemical analysis with automated cellular imaging. Am J Clin Pathol 2002;118:194-201.  Back to cited text no. 12
    
13.Farley J, Uyehara C, Hashiro G, Belnap C, Birrer M, Salminen E. Cyclooxygenase-2 expression predicts recurrence of cervical dysplasia following loop electrosurgical excision procedure. Gynecol Oncol 2004;92:596-602.  Back to cited text no. 13
    
14.Kim JY, Lim SJ, Park K, Lee CM, Kim J. Cyclooxygenase-2 and c-erbB-2 expression in uterine cervical neoplasm assessed using tissue microarray. Gynecol Oncol 2005;97:337-41.  Back to cited text no. 14
    
15.Dai Y, Zhang X, Peng Y, Wang Z. The expression of cyclooxygenase-2, VEGF and PGs in CIN and cervical carcinoma. Gynecol Oncol 2005;97:96-103.  Back to cited text no. 15
    
16.Chen YJ, Wang LS, Wang PH, Lai CR, Yen MS, Ng HT, et al. High cyclooxygenase expression in cervical adenocarcinomas. Gynecol Oncol 2003;88:379-85.  Back to cited text no. 16
    
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18.Lee JS, Choi YD, Lee JH, Nam JH, Choi C, Lee MC, et al. Expression of cyclooxygenase-2 in adenocarcinoma of the uterine cervix and its relation with angiogenesis and tumor growth. Gynecol Oncol 2004;95:523-9.  Back to cited text no. 18
    
19.Dursun P, Yuce K, Usubutun A, Ayhan A. Cyclooxygenase-2 expression in cervical intraepithelial neoplasia III and squamous cell cervical carcinoma, and its relation with clinicopathologic variables. Int J Gynecol Cancer 2007;17:164-73.  Back to cited text no. 19
    
20.Kim YB, Kim GE, Cho NH, Pyo HR, Shim SJ, Chang SK, et al. Overexpression of cyclooxygenase-2 is associated with a poor prognosis in patients with squamous cell carcinoma of the uterine cervix treated with radiation and concurrent chemotherapy. Cancer 2002;95:531-9.  Back to cited text no. 20
    
21.Ryu HS, Chang KH, Yang HW, Kim MS, Kwon HC, Oh KS. High cyclooxygenase-2 expression in Stage IB cervical cancer with lymph node metastasis or parametrial invasion. Gynecol Oncol 2000;76:320-5.  Back to cited text no. 21
    
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23.Delgado G, Bundy B, Zaino R, Sevin BU, Creasman WT, Major F. Prospective surgical-pathological study of disease-free interval in patients with stage IB squamous cell carcinoma of the cervix: A Gynecologic Oncology Group study. Gynecol Oncol 1990;38:352-7.  Back to cited text no. 23
    
24.Crissman JD, Makuch R, Budhraja M. Histopathologic grading of SCC of the uterine cervix. An evaluation of 70 stage Ib patients. Cancer 1985;55:1590-6.  Back to cited text no. 24
    
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26.Kang MK, Park W, Choi Y-L, Cho EY, Ahn G, Nam H, et al. The effect of cyclooxygenase-2 expression on tumor volume response in patients treated with radiotherapy for uterine cervical cancer. J Korean Med Sci 2009;24:1170-6.  Back to cited text no. 26
    
27.Kim MH, Seo SS, Song YS, Kang DH, Park IA, Kang SB, et al. Expression of cyclooxygenase-1 and -2 associated with expression of VEGF in primary cervical cancer and at metastatic lymph nodes. Gynecol Oncol 2003;90:83-90.  Back to cited text no. 27
    
28.Khunamornpong S, Settakorn J, Sukpan K, Srisomboon J, Ruangvejvorachai P, Paul S, et al. Cyclooxygenase-2 expression in squamous cell carcinoma of the uterine cervix is associated with lymph node metastasis. Gynecol Oncol 2009;112:241-7.   Back to cited text no. 28
    
29.Jung YW, Kim SW, Kim S, Kim JH, Cho NH, Kim JW, et al. Prevalence and clinical relevance of cyclooxygenase-1 and -2 expression in stage IIB cervical adenocarcinoma. Eur J Obstet Gynaecol Reprod Biol 2010;148:62-6.  Back to cited text no. 29
    

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Ranjana Bandyopadhyay
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DOI: 10.4103/0377-4929.91495

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