Colorectal cancer is the third most common cancer in the world and the second most common cause of cancer-related death (Ricchi et al., 2003). During 2000, in the United States, 130 200 new cases of colon cancer and rectal cancer were reported (Nelson et al., 2001). Despite numerous attempts to detect cancer at an early stage, the overall long-term outcome of patients curatively resected has not significantly changed in the last decade, the 5-year survival rate being 60% (Ratto et al., 1998). In Egypt, colorectal carcinoma (CRC) shows unique characteristics that are different from those reported in western countries. Unlike the western societies, where most of the cases of CRC are elderly, 35% of the Egyptian patients with CRC are under 40 years of age. Moreover, they are of high grade, high stage, and showed more mutations (Bahnassy et al., 2002).
Earlier diagnosis of colorectal cancer, better knowledge of its clinicohistological prognostic factors, and its surgical treatment combined or not with chemotherapy or radiation therapy have contributed to the improved outcome of affected patients. The most significant and independent prognostic factors accepted to date in CRC remain the tumor node metastasis (TNM) stage and residual disease after initial surgery (Shepherd et al., 1989). Assessment of molecular prognostic factors associated with a distinct prognostic outcome would, therefore, be of great help for identification of patients who are likely to benefit from adjuvant therapies, leading to an improvement in prognosis (Soumaoro et al., 2004).
It is now commonly believed that most cancers result from accumulations of genetic mutations involving genes, mostly cell cycle regulators, which are responsible for either stimulation or inhibition of cell cycle growth and progression. These mutant genes have been recently described as cancer-causing genes (Bahnassy et al., 2002).
In colon cancer patients, histological stage has been considered as the most important predictor of recurrence. However, for better management of patients, especially those within the same stage, additional factors should be examined (Kouraklis et al., 2006). Recently, it has been demonstrated that cyclin D1 acts as an oncogene in vitro and in vivo (Resnitzky and Reed, 1995). Once cells have entered the cell cycle, they are normally committed to go on and divide. Hence, the so-called G1 cyclins have a predominant role in pushing cells toward progression. The cyclin D1/cdk4, cyclin D1/cdk6, and cyclin E/cdk2 complexes are the main regulators of the G1→S transition, each of them controlling a different and discrete rate-limited step (Hartwell and Kastan, 1994). Among these, cyclin D1 presents the strongest evidence of a potential contribution to the multistep process of oncogenesis (Bartkova et al., 1994). However, a limited number of studies have examined the association of cyclin D1 expression and prognosis of patients with different malignant tumors (Stendahl et al., 2004), whereas others have evaluated it in colon adenocarcinoma with equivocal results (Palmqvist et al., 1998). According to the available published studies, the role of cyclin D1 in colorectal carcinoma remains controversial with some studies showed positive prognostic values and other contradictory studies showed negative prognostic values. These results suggest that the role of cyclin D1 in colorectal carcinoma is complex and multiple.
Two other proteins that are known to play an important role in the growth and development of many tumors are cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR). These proteins are abundantly expressed in many tumor types, and have been correlated with a bad prognosis in colorectal cancer as well. By looking at the expression of these proteins, it might be possible to predict which patients will respond well to chemotherapy and/or radiotherapy, and the promising proteins might be useful targets for therapy in patients with rectal cancer (Debucquoy et al., 2009).
EGFR is a 170 kD receptor tyrosine kinase encoded by the c-erb-B (HER-1) protooncogene (Cohen et al., 1982). It is expressed in various solid tumors, including colorectal, prostate, head and neck, and lung cancers, and also in certain normal tissues (Salomon et al., 1995). When it is bound by ligands, such as EGF and transforming growth factor, EGFR undergoes conformational changes that activate its intracellular tyrosine kinase activity, initiating autophosphorylation and downstream signal transduction pathways (McCune and Earp, 1989). Activation can mediate a variety of cellular responses, including gene expression, cell proliferation, and cell survival. Dysregulation of the EGFR signaling pathway because of EGFR overexpression, genetic aberrations, or other causes leads to malignant transformation. Recent studies have shown that EGFR expression is present in approximately 60–80% of CRCs, and the receptor has emerged as a rational target for anticancer therapy in these tumors (Bhargava et al., 2006).
COX-2 is overexpressed in the majority of human colon cancers. Interestingly, COX-2 has been reported to be significantly increased in up to 85% of human sporadic CRC (Buchanan and DuBois, 2006). COX-2 converts arachidonic acid to prostaglandins and related eicosanoids and promotes inflammation and cell proliferation. Supporting the importance of COX-2 in colorectal carcinogenesis, randomized trials have shown that aspirin and COX-2-selective inhibitors reduce risk of recurrent adenoma among high-risk patients (Ogino et al., 2008). COX-2 may also induce resistance to apoptosis, alter extracellular matrix adhesion, modulate tumor angiogenesis, and increase metastatic potential (Soumaoro et al., 2004).
Moreover, a study was conducted that showed that celecoxib, a specific COX-2 inhibitor, suppressed both incidence and multiplicity of azoxymethane-induced colon cancer in rats by 93 and 97%, respectively (Kawamori et al., 1998). In another previous clinical trial, there was evidence that rofecoxib, another specific COX-2 inhibitor, significantly decreased the number and size of rectal polyps in familial adenomatous polyposis patients (Higuchi et al., 2003). However, it is controversial whether COX-2 expression in itself is a prognostic factor for local recurrence and/or survival of patients with colorectal cancer (Soumaoro et al., 2004). Despite the well-accepted role of COX-2 in tumor development, studies are conflicting regarding prognostic significance of COX-2 in colorectal cancer, with some supporting and others refusing an independent adverse effect of COX-2 overexpression. COX-2 overexpression has been positively associated with p53 alteration and inversely associated with microsatellite instability, which generally predicts longer survival of patients with colon cancer. Moreover, COX-2 and p53 seem to regulate each other in a complex manner. Thus, effect of COX-2 on patient survival can possibly be confounded by p53 alteration, microsatellite instability, and other related molecular events (Ogino et al., 2008).
The aim of this study was to immunohistochemically evaluate the expression of cyclin D1, EGFR, and COX-2 in CRC, and to correlate this expression with different patients' clinicopathological characteristics to determine whether these markers could be used as prognostic factors for CRC.
Materials and methods
Tissue microarray preparation
This is a preliminary study that consisted of 50 cases of formalin-fixed and paraffin-embedded CRCs, obtained from Pathology Department, Faculty of Medicine, Cairo University and National Cancer Institute during the period from October 2008 to November 2009. The specimens were subjected to sectioning at 4 μm thickness, hematoxylin and eosin staining, and routine microscopic evaluation. One tumor spot was chosen under microscopy for each case and was marked on the corresponding spot on the tissue block. Then, cyclinderic tissue columns were punctured with a tissue arrayer in the marked area and were transferred to corresponding receiver pore of the prepared block. The tissue array block was then completed according to the predetermined scheme. The block was heated at 40°C for 15 min, and the surface was flattened for subsequent section of 4 μm thickness. The tissue microarray preparation (TMA) technique was used for cost reduction and easier interpretation (Kononen et al., 1998). The TMA block was serially cut on three charged slides for immunohistochemical staining.
Tissue sections were dewaxed in xylene for 30 min, rehydrated through graded alcohol then put in phosphate-buffered saline (PBS), then immersed in 3% hydrogen peroxide at room temperature for 10 min to inhibit endogenous peroxidase activity. After washing with PBS, the sections were subjected to antigen retrieval in boiling sodium citrate buffer (0.01mol/l, pH 6.0) for 10 min (microwave 450 W). After cooling at room temperature, and washing with PBS and distilled water sequentially. The following diluted antibodies were applied for a period of 45 min each at room temperature; polyclonal rabbit anticyclin D1 (M-20), (Santa Cruz Biotechnology Inc., California, USA, diluted 1 : 200), polyclonal goat anti-EGFR (1005), (Santa Cruz Biotechnology Inc., diluted 1 : 200), and polyclonal goat anti-COX-2 (N-20; Santa Cruz Biotechnology Inc., diluted 1 : 200; Wu et al., 2003).
The slides were then washed three times in PBS. The Envision system, peroxidase, mouse (Dako, Glustrup, Denmark) was applied for 10 min, followed by washing in PBS and using 3,3-diamino-bezidine-tetrahydrochloride as chromogen for 10 min. The slides were rinsed well in tap water for 5 min. The counterstain was performed using Mayer's hematoxylin for 1 min, and slides were dehydrated in ascending grades of alcohol. Then, the slides were cleared in xylene for three changes, and then Canada balsam and cover slips were applied. The positive control used was a case of mantle cell lymphoma for cyclin D1, human placenta for EGFR, and normal kidney for COX-2. Negative controls were obtained by replacing the primary antibody by nonimmunized rabbit or mouse serum.
The pattern of cyclin D1 immunostaining in positive cases was characterized either as nuclear only, or cytoplasmic and nuclear or cytoplasmic only. Percentage of positive population was scored as follows: score 0 if less than 1%; score +1 if more than 1% and less than 20%; score +2 if more than 20% and less than 50%; score +3 if more than 50% (Kouraklis et al., 2006).
Epidermal growth factor receptor
The positive staining was defined as the presence of membranous or cytoplasmic staining. The percentage of stained cells was assessed as follows: score 0 if less than 1%; score +1 if 1–10%, +2 if 10–50%; and +3 if more than 50% (Bibeau et al., 2006).
Positive immunostaining was considered when there was mainly cytoplasmic and occasionally nuclear staining. Extent of staining was scored as 0 (0%), +1 (1–20%), +2 (21–50%), and +3 >51% (Bibeau et al., 2006). Statistical analysis was conducted with SPSS for Windows (SPSS, UK Ltd., Woking, Surrey, UK). Results were considered statistically significant at a P value of 0.05 or less.
The age of the 50 cases included in this study ranged from 25 years to 76 years, with a mean age of 55.04±13.406 years. Most of the cases were men (56%). Ninety percent of the cases were grade 2 and 56% were tumor Dukes stage C. The lymph node metastatic cases were 58%. Margin-free cases were 92%. Tumor necrosis was seen in only 42% of cases, vascular emboli in 62% of cases, perineural invasion in only 36% of cases, and budding in 66% of cases. Invasive tumor border was seen in 64% of cases, whereas the rest showed pushing border. There was a slight predominance of right colon incidence representing 46% of cases. Tumors reaching the fat represent 86% of cases, whereas those reaching the muscle were only 14% of cases. All clinical and histological characteristics of the cases are summarized in Table 1.
Tumor markers expression
Positive cyclin D1 expression was detected in 96% of cases (Fig. 1), positive EGFR expression was seen in 74% of cases (Fig. 2), and positive COX-2 expression (Figs. 3 and 4) was noticed in 92% of cases. Further clarifications are in Table 1.
Correlation between tumors markers expression and patients' characteristics
No significant correlation was found between cyclin D1 expression and different prognostic factors of CRC. However, cyclin D1 expression was seen more in lower-grade than in higher-grade tumors (97.8% in grade 2 vs. 80% in grade 3), but this was an insignificant finding (P>0.05). Data are shown in Table 2.
Epidermal growth factor receptor
EGFR expression correlates with some poor prognostic factors. In grade 2 cases, 71.1% showed positive staining, whereas all grade 3 cases were positively stained. In addition, cases with lymphovascular emboli showed positive staining in 87.1% of cases, whereas only 52.6% of cases without vascular emboli showed positive staining. These results show statistical significance (P<0.05).
Cases with metastatic lymph nodes showed positive staining in 82.8% of cases, whereas only 61.9% of node-free cases were positively stained. Eighty-one percent of cases showing necrosis were positively stained, whereas 69% of cases without necrosis showed staining. Positive staining was also seen in 81.8% of cases showing budding, as opposed to 58.8% in cases with no budding. Tumors reaching the muscle showed positive staining in 71.4% of cases, whereas tumors reaching till fat showed positive staining in 74.5% of cases. However, no significant statistical correlation was found between these characteristics and EGFR expression (P>0.05). Data are shown in Table 3.
All cases with necrosis show positive staining for COX-2, whereas only 86.2% of cases without necrosis show positive staining. Positive staining was also seen in 97% of cases with budding, but in only 82.4% of cases with no budding. Tumors reaching the muscle showed positive staining in 71.4% of cases, whereas tumors reaching till fat showed positive staining in 95.4% of cases. However, all these results show no significant correlation (P>0.05).
Surprisingly, COX-2 expression seems to correlate with more differentiated tumors, as 95.6% of grade 2 tumors show positive immunostaining, opposing to 60% only of grade 3. This shows statistical significance (P<0.05). Another significant correlation was noticed between Cox-2 expression and tumor location as the highest percentage of colorectal carcinomas showing positive immunostaining were located in the left colon (90.9%) (P <0.05). Data shown in Table 4.
In this study of human CRC, the expression of cyclin D1 protein was positive in 96% of cases, but showed lack of correlation with the clinicopathological tumor characteristics. These findings goes with the study by Kouraklis et al. (2006) in which 111 cases of CRC were conducted showing cyclin D1 expression in 63.9% of cases. None of the patient characteristics mentioned in the study (age, tumor location, tumor grade, tumor stage, lymph node or hepatic metastasis, and vascular invasion) correlated with cyclin D1 immunostaining.
In contrast to our results, the study conducted by Bahnassy et al. (2002) showed that 68% of their studied 60 CRC cases were positively stained for cyclin D1. Moreover, they stated that cyclin D1 immunostaining correlated with poor prognostic factors such as tumor grade, lymph node metastasis, and tumor invasion. Another study conducted by Chen et al. (2007) showed positive cyclin D1 immunostaining in 58 of 85 CRC cases, which correlated with tumor stage, speculating that cyclin D1 aberrations may involve in the colon cancer development and may give the tumor an invasive potential.
This study showed that EGFR expression correlated with only two of the patient characteristics, which are tumor grade and lymphovascular invasion. In grade 2 cases, 71.1% showed positive staining, whereas all grade 3 cases were positively stained. In addition, cases with lymphovascular emboli showed positive staining in 87.1% of cases, whereas only 52.6% of cases without vascular emboli showed positive staining. All other patient characteristics given attention to in this study showed no significant relationship with EGFR expression. A study conducted by Zlobec et al. (2007) with 867 CRC cases showed EGFR expression in 64.4% of cases. EGFR overexpression was not found to be associated with tumor stage or vascular invasion, but only showed marginally significant associations with tumor grade.
A relatively contradictory study conducted by Spano et al. (2005) with 148 CRC cases showed EGFR expression in 97% of cases and found that the only prognostic factor that seems to correlate with EGFR expression was the tumor stage, using TNM staging system not Dukes' that was used in our study. In contrast, a study was conducted by Spindler et al. (2006), which included 193 CRC cases, and showed EGFR staining in 51% of cases. There was no correlation between EGFR immunostaining and the different clinicopathological parameters in terms of age, sex, tumor location, and TNM tumor staging system.
In this study, COX-2 expression showed correlation with only two patient clinicopathological characteristics, tumor grade and tumor location. COX-2 expression in our study significantly correlated more with the differentiated tumors (G2>G3). Another significant correlation was noticed between COX-2 expression and tumor site, as the highest percentage of CRCs showing positive immunostaining were located in the left colon (90.9%). No other tumor properties correlated with COX-2 expression. A similar result was conducted by Ogino et al. (2008) who studied 662 cases of CRC with positive COX-2 staining in 83% of cases. In their study, they found a highly significant correlation between COX-2 expression and tumor site (proximal>distal) and tumor grade (low>high).
The study conducted by Ceccarelli et al. (2005) showed one similar and another contradictory result compared with our study. They found a similar significant correlation between COX-2 expression and tumor grade (low>high), but showed a significant correlation between COX-2 expression and budding (EGFR increases as tumor budding increases).
Completely opposite to our results, Soumaoro et al. (2004) conducted a study on 288 CRC cases showing COX-2 staining in 70.8% of cases. They concluded that COX-2 overexpression significantly correlated with prognostically worse clinicopathologic variables, including tumor size (the larger the size the higher the expression), depth of invasion (highest expression in pT3), lymph node metastasis (metastatic>nonmetastatic), lymphovascular invasion (cases with lymphvascular invasion>cases with no lymphovascular invasion), and tumor recurrence. There was no significant correlation between COX-2 expression and age, sex, tumor site, or tumor grade. As for the study conducted by Wu et al. (2003), it was clear that COX-2 expression had no association with clinicopathological features, such as differentiation, invasion depth, vessel emboli, and TNM staging. Their results were similar to those found by Fux et al. (2005) who stated that they did not find any relationship between COX-2 expression and patient prognosis or survival.
In our opinion, the discrepancies in results between our study and other studies may be attributed to differences in population type (Egyptian vs. non Egyptian), size, characteristics, lack of standardization of methods, and differences in the choice of cutoff levels.
From this study, it is concluded that cyclin D1 expression does not correlate with any of the studied patient characteristics. EGFR overexpression correlated with higher tumor grade and presence of lymphovascular invasion. Thus, it could be considered as a poor prognostic factor. Finally, COX-2 overexpression correlates with lower tumor grade and tumors located in the left colon. Therefore, COX-2 could be considered as a good prognostic factor for CRC.
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