Role of Immunohistochemistry Markers (p53 and CEA), in Study of Colorectal Tumours

Colorectal cancer is the third most common cancer in men and the second in women globally. There is a marked variation in the incidence of colorectal carcinoma worldwide, where western countries having high rate compared to others. p53 tumour suppressor gene is one of the most intensively studied tumour markers in the colorectal tumours. Two markers were used, p53 (oncoprotein p53) and CEA (carcinoembryonic antigen) in the study. The 102 cases of paraffinembedded samples were processed for the immunohistochemistry examination. After the analysis of the selected patients regarding the antibodies distribution, statistical analysis was performed. The current study showed that there was a statistically significant correlation existing between p53 and CEA in each tumour type irrespective of its histological grades. The immunohistochemistry (IHC) was performed on 4-μm thick sections from 10% formalinfixed paraffin-embedded tissue blocks.


INTRODUCTION
Colorectal cancer is the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases, 9.2% of the total) worldwide [1][2][3]. Almost 55% of the cases occur in more developed regions. There is wide geographical variation in incidence across the world and the geographical patterns are very similar in men and women: incidence rates vary ten-fold in both sexes worldwide [4,5], the highest estimated rates being in Australia/New Zealand (ASR 44.8 and 32.2 per 100,000 in men and women respectively), and the lowest in Western Africa (4.5 and 3.8 per 100,000). Mortality is comparatively higher (52%) in low developed countries than the developed countries (694,000 deaths, 8.5% of the total); this reflects a poorer survival in those regions. There is less variability in mortality rates worldwide (sixfold in men, four-fold in women), with the highest estimated mortality rates in both sexes in Central and Eastern Europe (20.3 per 100,000 for men, 11.7 per 100,000 for women), and the lowest in Western Africa (3.5 and 3.0, respectively) [6].
Risk factors for development of colorectal carcinoma are old age, History of colorectal adenoma Inflammatory intestinal conditions, Inherited syndromes (familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, which is also known as lynch syndrome, gardner syndrome, old field syndrome, Turcott syndrome, Zanca syndrome, Peutz-Jeghers syndrome, juvenile polyposis syndrome}, family history of colorectal carcinoma, radiation therapy for cancer [7][8][9]. People taking high calorie diet along with sedentary life style habits are more prone to develop colorectal carcinoma [10,11]. Diabetes and obesity increases the risk of developing colorectal carcinoma. Statistical studies show that smoking, alcohol and animal fat consumption are the major predisposing factors [12][13][14][15]. Majority of Colorectal carcinomas remain asymptomatic for years. They most often present with fatigue and weakness as these bulky lesions bleed readily and cause anaemia [16].
In this prospective study of intestinal tumours, incidence with respect to age, sex, site, and histomorphological features of various tumours in large intestine were studied. The present study also evaluates the role of tumour suppressor gene protein p53 and cell surface glycoprotein CEA in intestinal neoplasms and its prognostic value in colorectal adenocarcinomas.

MATERIALS AND METHODS
The prospective study conducted in Department of Pathology, Sree Balaji Medical College and Hospital, Chennai, during the period of October 2015 to September 2017. Based on inclusion and exclusion criteria, the samples were screened for the study. A total of 104 samples were included in the studies which were received from General Surgery and Medical Gastroenterology as resected specimens and mucosal biopsies. Proper consents were taken from patients whose sample was included in the study. Patients' particulars, brief clinical history and clinical examination findings were recorded.

Inclusion Criteria
All cases proved to be adenoma or adenocarcinoma of colorectal region by histopathology irrespective of age and both of the sexes were included for the study.

Exclusion Criteria
All non-neoplastic lesions such as inflammatory polyps and cases with incomplete clinical data were excluded from the study.

Method of Data Collection
All the cases enrolled in the study as specified by the above criteria were assessed. Of the 104 cases, 100 cases were found to be suitable for the study. Clinical history and colonoscopy findings were tabulated. Adequate samples were taken from the growths in these specimens.
The tissues so obtained were processed and sections were cut at 4 microns. Hematoxylin and eosin staining of the sections were done and histopathological [17] findings were studied. Histopathological images were taken using Canon DSLR 700D with AmScope microscope adapter mounted on Olympus CH20i. Fiji (Image-J), an open source software, was used to analyse the images captured by the camera.

Relation of Regions Involved and Types of Colorectal Tumours
From the 100 cases studied, the most affected region of large intestine is rectum (50%) followed by sigmoid colon (23%) then caecum (12%). In contrast, the mucinous carcinoma showed caecum with higher number of frequency (71.43% of mucinous carcinoma). The Fig. 1 show distribution of different types of tumours in particular regions of large intestine, in the case studied. The statistical analysis using Pearson's Chi squared test showed p-Value of 0.000 which less than 0.01, indicating the statistics for site distribution of particular tumour type is highly significant.

p53 Scoring
For scoring p53 expression, all the p53 stain slides were given a score from Immunoreactive scoring (IRS) system devised, in which the intensity of the staining and the percentage of stained were multiplied. the score is interpreted In the study, benign and pre-malignant cases show negative and weak expression, and malignant cases show stronger expression of p53. The proportion of expression of p53 in benign and malignant cases are shown in Fig. 3.
The study also shows that there is strong relation between the expression of p53 and the lesion type (benign and malignant) with p-Value of 0.000, measured using Pearson's Chi squared test.
In overall tumour types, the p53 is found to be expressed to a greater extent in case of adenocarcinoma as shown (P= 0.000). Mucinous carcinoma also shows strong expression of p53.
The staging of the resected tumor cases is done using 'AJCC Colon and Rectum Staging System' (7 th edition). Staging is done only in resected specimens only, to eliminate the possibility of error due to lack of full clinical details particularly metastasis, the stage III and stage IV have been put in one single category.
The Fig. 5 show the expression of CEA in different grades of adenocarcinoma, and Fig. 6 show the expression of CEA in adenoma cases with different degree of dysplasia. It is evident that CEA is 100% expressed in grade-I (well differentiated), and reduced in grade-II (moderately differentiated) when compared to grade-I. CEA expression is more in higher grades of dysplasia compared to low grade. There is poor evidence of relation between CEA expression and histological grades of adenocarcinoma, and with the grades of dysplasia in adenoma. The p-Value are found to be statistically not significant in histological grading of adenocarcinoma (P = 0.440) and adenoma (P = 0.619).
In overall tumor types, the CEA is found to be expressed to a greater extent in case of adenocarcinoma as shown (P= 0.000). Mucinous carcinoma also shows strong expression of CEA.

Comparing p53 and CEA Expression
From the 100 cases studied, a two-way comparison table of p53 and CEA expressions shows correlation which is statistically significant with p-Value of 0.002. The Fig. 8 shows means of p53 expression and CEA expression in stacked (100 percent) bar graph. The figure shows proportion of p53 and CEA expressions in each tumor type irrespective of its histological grades.

DISCUSSION
Colorectal cancer is the third most common cancer in men and the second in women globally. There is a marked variation in the incidence of colorectal carcinoma worldwide, where western countries having high rate compared to others. Almost 55% of the cases occur in more developed regions. In India, c cancer ranks 8 th and rectal cancer ranks 9 among men. For women, colon cancer ranks 9 The incidence of both small and where western countries having high rate compared to others. Almost 55% of the cases occur in more developed regions. In India, colon and rectal cancer ranks 9 th among men. For women, colon cancer ranks 9 th . e incidence of both small and large bowel cancers are low in India [18]. The incidence of colorectal cancer in India is 3.6% when compared worldwide (10%) [6].
Carcinomas of the large bowel present in a range of macroscopic appearances. These vary The incidence of India is 3.6% when Carcinomas of the large bowel present in a range of macroscopic appearances. These vary somewhat with the anatomical site of origin. Conventionally a number of distinct macroscopic forms of large bowel cancer have been recognized as polyploidy, exophytic or fungating, ulcerating, stenosing, and diffusely infiltrating. 5 year relative survival ranges from greater than Conventionally a number of distinct macroscopic ancer have been recognized as polyploidy, exophytic or fungating, ulcerating, stenosing, and diffusely infiltrating. 5year relative survival ranges from greater than 90% in patients with stage I disease to slightly greater than 10% in patients with stage disease. Screening has been shown to reduce colorectal cancer incidence and mortality, but organized screening programs are still to be implemented in most countries [   p53 tumor suppressor gene is one of the most intensively studied tumor markers in the colorectal tumors [25,26]. In this study 100 cases of colorectal tumors are selected for the study of p53 expression by immunohistochemistry in various grades and types which included 7 cases of mucinous carcinomas. When compared to the findings of other studies, there are some variation. These differences may be due to the use of different scoring system and inter observer variability. In this study, the p53 shows strong expression in 11 (21%) cases from 52 cases of adenocarcinoma. The criteria devised by Agnieszka Halon et al. [27] for categorising the cases as overexpression of a marker, is based on a modified IRS system [ criterion, it is found that in the present study 65.4% of cases of adenocarcinoma show p53 overexpression.
; Article no.JPRI.66772 proportion of CEA expression in different tumor 11.5% (n=7) and melanoma 3.3% (n=2). Similar are observed in a study conducted by Fatimah Biade Abdulkareem et al. [24] where 87.1% were adenocarcinoma (comprising of well differentiated adenocarcinoma 55.5%, moderately differentiated carcinoma 21% and poorly differentiated adenocarcinoma 10.7%) p53 tumor suppressor gene is one of the most intensively studied tumor markers in the . In this study 100 cases of colorectal tumors are selected for the study of immunohistochemistry in various grades and types which included 7 cases of mucinous carcinomas. When compared to the findings of other studies, there are some variation. These differences may be due to the use of different scoring system and interver variability. In this study, the p53 shows strong expression in 11 (21%) cases from 52 cases of adenocarcinoma. The criteria devised ] for categorising the cases as overexpression of a marker, is stem [22]. Using this criterion, it is found that in the present study 65.4% of cases of adenocarcinoma show p53 The relation of p53 expression in colorectal carcinoma with histological, clinical, prognostic features using follow-up data and concluded that p53 expression occurred as a late event and was associated significantly associated with advanced stage of disease, early relapse and death. J Walker et al. [28] in their study found that the stage is the most accurate prognostic factor for survival. They concluded that p53 overexpression in colorectal carcinoma correlated with poor prognosis.
In the present study, p53 protein overexpression is seen in relatively high percentage of patients, which indicates that p53 mutation plays an important role in development of CRC.
CEA is a cell surface glycoprotein normally expressed in foetal tissue and transcriptionally silent in adults. CEA functions as an intercellular adhesion molecule and plays an important role during development. However, CEA overexpression is associated with a variety of cancers of epithelial origin.
Monoclonal or polyclonal antibodies may identify several antigens belonging to glycoproteins of CEA family. Inflamed or necrotic tissues react with CEA antibodies, making up a specific pattern. The new variants of monoclonal CEA antibodies react only with epitopes of "true" CEA 80-100% of colorectal cancers make a strong and diffuse positive staining [29,6]. In this study 100 cases of colorectal tumors are selected for the study of CEA expression by immunohistochemistry in various grades and types.
When compared to the findings of other studies, there are some variation. These differences may be due to the use of different scoring system and inter-observer variability. In the present study, 96.2% of cases of adenocarcinoma show CEA positivity. Since its sensitivity is high, it may be useful in differentiating tumor tissue from normal tissue. it was found that 98.8% of tumor tissues stained positive for CEA and differential CEA expression within normal/tumor pairs was appreciable, providing evidence that CEA is a reliable marker for differentiating between normal and tumor tissue [29,30]. It was found that the tissue expression for CEA in CRC was 94%. Monika Cerna et al (2006) did a quantitative estimation of CEA in the tumour tissue, it was observed that almost all tissue sample stained positive for CEA. They also found that there were differences in staining of CEA between tumor types and there was no relationship to staging or clinical development [6,16,30].
The present study shows that CEA have poor association with great and stage of the colorectal carcinoma (P=0.1000). And the study also shows that CEA is negative for melanoma which is in accordance with the study done by Selby WL et al (1992) where they found malignant melanoma showed no reactivity to monoclonal CEA. Though there was a clear evidence of association between the EA and colorectal cancer, the present study suffered by the limited number of cases. Thus this study also warranted for further level analysis to optimizing the role of CEA in cancers.

CONCLUSION
This is a prospective study undertaken in the Department of Pathology over a period of 2 years, from October 2015 to September 2017. Resected specimens and mucosal biopsies of large intestinal tumors were included for the study. The number of samples included for the study is 100, of which biopsy specimens were 54 (54%) and resected specimens were 46 (46%). In this study age, sex and site of the lesion were recorded. After H&E staining of the sections obtained from the specimen, histomorphological grading of colorectal tumors was done. Immunohistochemistry using p53 and CEA antibodies were done to assess the role of their expressions in various types of colorectal tumors. Results were tabulated and analyzed.
In colorectal tumors p53 expression increases with the histomorphological grades. P53 protein overexpression has been associated with a worst overall survival after cancer diagnosis. In the present study, 65.4% of cases of adenocarcinoma show p53 overexpression. The p53 protein overexpression is seen in relatively high percentage of patients, which indicates that p53 mutation plays an important role in development of CRC. In colorectal tumors p53 expression increases as the stage progresses and also there is significant correlation between high p53 levels and stage of colorectal carcinomas. It is evident that p53 can be used as a prognostic marker to assess aggressiveness of the colorectal tumors.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The study was approved by the Institutional Ethics Committee.