Assessment of Correlation between Cyclo oxygen ase-2 Expression and Clinicopathological Features of Colorectal Carcinoma

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1 Med. J. Cairo Univ., Vol. 84, No. 1, September: , Assessment of Correlation between Cyclo oxygen ase-2 Expression and Clinicopathological Features of Colorectal Carcinoma EMAN E. MOHAMED, M.Sc.*; EHAB M. HASSANIN, M.D.*; MAHA L. ZAMZAM, M.D.* and NOHA R. NOUFAL, M.D.** The Departments of Clinical Oncology* and Pathology**, Faculty of Medicine, Suez Canal University, Ismailia, Egypt 1029 is the sixth cancer both in males and females representing 4.5% and 3.6% of the total cancers with Age-Standardized Rates (ASRs) per 100,000 populations [2]. Experimental, epidemiologic, and clinical data provide strong evidence for a causative link between chronic inflammation and CRC risk [3]. One specific mechanism through which inflammation leads to both colitis-associated and sporadic carcinogenesis is the pro inflammatory Cyclooxygenase (COX) pathway. COX2 has been shown to play an important role in carcinogenesis in various organ systems, such as colorectal cancer, breast cancer, lung cancer, esophageal cancer and pancreatic cancer [4]. Recently multiple studies have shown that COX-2 is expressed at high levels in 80-90% of colorectal adenocarcinomas [5,6] and selective inhibition of COX-2 reduces colorectal carcinogenesis [7,8]. Several recent studies have reported a 40-50% lower risk of CRC in people who are continuously taking aspirin or other Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) [9]. The mechanism by which NSAIDs prevent colorectal carcinogenesisis not clear, but one possibility involves inhibition of Cyclooxygenase (COX) [4]. A meta-analysis of 23 studies (4567 patients) that evaluated the correlation between COX-2 overexpression and survival in patients with CRC reached a conclusion that COX-2 expression was associated with poor Overall Survival (OS) in CRC [10]. Although Cyclooxygenase-2 is believed to be an important enzyme in the pathogenesis of CRC, correlations between the expression of COX-2 with

2 1030 Assessment of Correlation between COX-2 Expression & Clinicopathological Features tumor growth and distant metastasis is still under investigations. In the current study we examined correlation between COX-2 expression and other clinicopathological features of CRC as COX-2 inhibitors may represent a new and very promising group of chemotherapeutic agents with great potential for both CRC in prevention. Material and Methods The study was a retrospective descriptive study applied on thirty patients who attended the Clinical Oncology and Nuclear Medicine Department of Suez Canal University at Ismailia during period from January 2012 to January All patients underwent colectomy as a surgical procedure for tumour excision and pathologicaly proved to have CRC were included. The correlation between expression of cyclooxygenase 2 gene COX-2 and other clinicopathological features of CRC which affects prognosis was examined. Demographic criteria and clinical data of patients were obtained from archived files in the oncology clinic. Hematoxylin Eosin (H & E) stained paraffin sections from all tumours were reviewed. Prognostic factors were stratified according to the new categorization published by the College of American Pathologists in collaboration with a multidisciplinary group of clinical (medical oncology, surgical oncology and radiation oncology), pathologic and statistical experts as follow [11]. Category I factors: Pathological staging was done based on the (TNM) staging system applied by the American Joint Committee on Cancer (AJCC) [12] as follow: - Local extent of the tumour (pt). - Regional Lymph Node Metastasis (pn). - Distant Metastasis (M): Evidence of distant metastasis proved by either pathological examination or reported in patients' medical records. - Lymphovascular invasion: Based on identification of tumor within an endothelial-lined channel or surrounded by an elastic lamina [13]. Category IIA factors: - Histological grade: Samples were graded based on a 2-tiered grading system (high grade and low grade) [14]. - Circumferential Resection Margin (CRM) [11]. Category IIB factors: - Histologic type: All other subtypes of colorectal cancer other than gland-forming adenocarcinoma were excluded. Specimens were classified into Non Mucinous Adenocarcinoma (NMA) and Mucinous Adenocarcinoma (MA). Immunohistochemistry: Representative paraffin sections of normal mucosa and resected tumour tissue were tested with the antibody against COX2. Cytoplasmic expression of COX-2 antibody was evaluated according to Allred Scoring Guidelines [15]. Statistical analysis: Values are presented in mean and standard deviation. The significance of differences among proportions was evaluated by Pearson's Chi square test. All analyses were performed using the statistical package for social science, SPSS, version 18, Results were considered significance at p- value <0.05. Results Thirty patients with CRC were included in the present work. They consisted of 12 males and 18 females with ages ranging from 20 to 60 years (mean ± 48.3 years). The most common site in the study was left side of colon and rectum (46.7%) followed by right side (40%) and least were transverse and double sites each (6.7%). Histopathologic examination of colectomy specimens revealed that 22 cases were non mucinous adenocarcinomas and eight cases were mucinous adenocarcinoma. Histological grading of all specimens revealed 13 (43.3%) were low grade and 17 (56.7%) were high grade (Figs. 1,2). Fifty percent of cases (15 patients) were T3 followed by 8 cases were T4 (27.7%) and 7 cases were T2 (23.3%). Lymph node metastasis was reported in 63.3% of cases. Most of the studied cases were negative for metastasis (56.7%). Lymphovascular invasion was present among 60% of specimens and was negative in 40% of specimens. Examinations of circumferential margins were negative in all specimens. Immunohictochmistry showed cytoplasmic positive expression of 87% of cases while 13% were negative for COX2. Intensity of COX2 expression was assessed to be moderate in 60% of cases, 26.7% showed faint intensity and 13.3% were negative. Forty percent of specimens showed positive expression for COX2 in more than 66% of the whole specimen (PS=5) (Fig. 3) and 27% were

3 Eman E. Mohamed, et al expressing COX2 in 33 to less than 66% (PS=4) (Fig. 4), only 13% were negative. There was statistically significance correlation between COX2 expression and histological type and metastasis. Significant correlation was achieved between smoking and COX2 expression. There was no significant correlation between COX2 expression and other studied clinicopathological parameters (Table 1). Table (1): Correlation between COX2 expression and different clinicopathologic features of the studied cases. Item No. of cases (n=30) Percent (%) COX2 +ve (%) COX2 ve (%) p- value Age: 20-< < < < Gender: Male Female GIT diseases: Adenomatous polyps Smoking: Smokers * No smokers BMI: Underweight Normal weight overweight Obesity Histopathological site: Rt. side Lt. side Transverse colon Double sites Pathological type: Mucinous * Non mucinous Grading: Low grade High grade Depth of invasion "T": T T T Lymph node "N": N N N Metastasis "M": M * M Dukes stage: B C D Lymphovascular invasion: Yes No (*p<0.05).

4 1032 Assessment of Correlation between COX-2 Expression & Clinicopathological Features Fig. (1): Low grade adenocarcinoma, (X200). Fig. (2): High grade adenocarcinoma invading fat, (X100). Fig. (3): Moderate cytoplasmic expression of COX2 in low grade adenocarcinoma, (PS=5), (X100). Fig. (4): Moderate cytoplasmic expression of COX2 in high grade adenocarcinoma, (PS=4), (X100). Discussion The present study revealed that the mean age of the cases was 48.3 years old with more than half of the patients older than 50 years old. Our data matched the global age incidence of CRC which reveals that 90% of new cases and 93% of deaths arise in people aged 50 and older [16]. Although in the current study there was predominance of the female gender representing 60% of the sample and 40% were males, Murphy et al., have demonstrated that CRC incidence is higher in males than females [17]. This can be explained by the small sample size in our study that cannot be an indicator for sex distribution in CRC. Histopathological examination of colectomy specimens revealed that 22 cases (73.3%) were non mucinous adenocarcinomas; eight cases (26.7%) were mucinous adenocarcinoma. This generally corresponds to the incidence rate of MA in most Western countries that ranges from 11 to 20% [18,19]. In our study 13% of patients were obese and more than half of the patients were overweight, this keeps with cancer statistics in United Kingdom which estimated 13% of bowel cancers in the UK are linked to overweight or obesity [20]. Percentage of smokers in the study was 27% of the study cases. In UK, it is estimated that 8% of bowel cancer cases are linked to tobacco smoking [21]. This difference can be explained by the small sample size in our study which cannot represent the whole population. In the present study, 87% of CRC patients expressed COX-2, in keeping with the results of previous studies which showed that COX-2 was expressed in 85%-95% of CRCs [5,22]. In agreement with other studies, examined slides showed that COX2 expression was observed mainly in tumour area; normal colonic mucosa adjacent to the COX2 positive tumours was not stained or occasionally stained weakly for COX- 2 [23,24].

5 Eman E. Mohamed, et al as they classified into well, moderate and poorly differentiated. When correlating COX2 with distant metastasis, we observed a significant correlation between COX2 expression and distant metastasis (p=.038), tumours with more COX2 expression revealed more distant metastasis than did those with no expression. Similar results were in [29] who worked on 94 CRC patients; in which COX2 expression has been reported in all 54% of CRC cases. This data also supports results of Tomozawa et al., [26] who have reported that COX2 overexpression was significantly associated with tumour recurrence, and especially with haematogenous metastasis. In contrast to these results, however, the study by Kazem et al., [30] which failed to establish any correlation between COX2 overexpression and metastasis. A trend with borderline significance was observed between COX2 and Dukes stage where more than two thirds of COX2 positive patients were advanced Dukes stages C and D ( p=.06). This is in agreement with a study showed that COX2 expression was correlated with advanced tumour stage [29]. Contrary to our results, recent study of revealed that COX-2 levels didn't correlated with Dukes stage [30,31]. These discrepancies may be related to the use different scoring systems or antibodies employed in immunohistochemistry. We concluded that COX-2 protein overexpression suggests its role in cell proliferation and carcinogenesis. Significant correlation was reached between COX2 and distant metastasis and with Dukes' classification. COX2 might be an early marker of neoplastic transformation and progression of CRC. References 1- SIEGEL R.L., K.D. MILLER and A. JEMAL: Cancer statistics, CA: A cancer journal for clinicians, 65 (1): 5-29, IBRAHIM A., et al.: Cancer in Egypt, Gharbiah: Triennial Report of , Gharbiah Population-based Cancer Registry. Cairo: Middle East Cancer Consortium, SCHOTTENFELD D. and J. BEEBE-DIMMER: Chronic inflammation: A common and important factor in the pathogenesis of neoplasia. CA: A Cancer Journal for Clinicians, 56 (2): 69-83, TOMOZAWA S., et al.: Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX- 2 inhibitor, JTE-522. British journal of cancer, 81 (8): , 1999.

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