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1 MRIMS Journal of Health Sciences 2016;4(1) pissn: , eissn: Original Article A study of and p-53 immunostaining expressions in colonic carcinomas Shashi Kiran K 1, V. Hari Shanker 2, Triveni Bhopal 3, Kandukuri Mahesh Kumar 4 1 Consultant Pathologist, Omega Cancer Hospital, Banjara Hills, Hyderabad 2 Professor, Department of Pathology, Malla Reddy Institute of Medical Sciences, Hyderabad 3 Professor, Department of Pathology, MNJ Cancer Hospital, Osmania Medical College, Hyderabad 4 Assistant Professor, Department of Pathology, Malla Reddy Institute of Medical Sciences, Hyderabad Corresponding Author: Dr. Shashi Kiran K shashikayetha@gmail.com Abstract: Background: Colorectal cancer burden has been steadily rising in women. It was the fourth most common cancer in 1975 and has reached the second position by 1990, with about 49% increase in the number of cases globally over a period of 15 yrs. From , the rate of people dying from colorectal cancer has varied, depending on their race and ethnicity. Objec: To study and p-53 immunostaining expressions in colonic carcinomas. Methods: The present study is both a retrospec and a prospec study. The study is based on histomorphological evaluation of 30 resected specimen of Colorectal Carcinomas received at the department of Pathology, Osmania General Hospital in a 2 year period from June 2009 to May The most representa block for all 30 cases was then selected for immune histochemical analysis of p-53 protein and Oncoprotein for p-53 immunostaining, we used the DO-7 monoclonal antibody (dilution 1:30; DAKO), and, for, the monoclonal antibody 124 was used (dilution 1:80; DAKO). Results: Posi staining for p-53 protein was detected in 56.67% ( 17/30 ) of colorectal adenocarcinomas. There is statistically no significant association observed between p53 expression and Dukes stage, TNM stage or Histological grade (p value > 0.05). Similarly no association was found between expression of p-53 and other patient variables, such as age, sex, location of tumor, & tumor differentiation. In the present study statistically there is no significant association between & p-53 either individually or combined together with any of the parameters (like age, sex, tumor location, Dukes stage, Histological grade & TNM stage). Conclusion: Though not statistically significant, to comment as the optimism expressed by certain investigators due to the low number of cases taken up for the present study, a large study group is needed to comment on the prognostic significance of the & p-53 immuno profile individually and in a combined variation to predict the clinical outcome. Key words:, p-53, immunostaining INTRODUCTION: Colorectal cancer burden has been steadily rising in women. It was the fourth most common cancer in 1975 and has reached the second position by 1990, with about 49% increase in the number of cases globally over a period of 15 yrs. From , the rate of people dying from colorectal cancer has varied, depending on their race and ethnicity. Black people were more likely to die of colorectal cancer than any other group. White people had the second highest rate of deaths from colorectal cancer, followed by people who are Hispanic, American Indian/Alaska Na, and Asian/Pacific Islander. 1 Epidemiological studies indicate that the etiology of colorectal carcinoma is closely related to the environmental factors, with genetic factors playing an important but less obvious role. The former are largely dietary, particularly in terms of fats and animal proteins and lack of fibre and fresh vegetables. 2 One hypothesis indicates that excess dietary fat may result in increased production of bile acids, which may MRIMS Journal of Health Sciences, Vol. 4, No. 1, January-March 2016 Page 28

2 Shashi Kiran K et al. A study of and p-53 immunostaining expressions in colonic carcinomas themselves become carcinogenic by the action of bacteria in the bowel lumen. Colonic cancer is mostly age related, & the incidence increases with increase in age. The mean age of incidence is 62 years. However in high-risk areas 8% of patients are under the age of 50 years (Western countries, Japan etc.) Male sex, increasing age, presence of long standing IBD (Inflammatory bowel disease), and familial predisposition are strong risk factors. Prognosis of young patients differs from that of more elderly patients. 3 Though the incidence is little higher in males than females. Cancer of right colon is more frequent in females of all ages, whereas cancer of left colon is more frequent in females under 50 years and in males above 70 years. 4 Present study was undertaken to study and p-53 immunostaining expressions in colonic carcinomas. MATERIAL AND METHODS The present study is both a retrospec and a prospec study. The study is based on histomorphological evaluation of 30 resected specimen of Colorectal Carcinomas received at the department of Pathology, Osmania General Hospital in a 2 year period from June 2009 to May Due importance was given to record a brief clinical history with age, Inpatient registration number, biopsy number, presenting symptoms and signs. The specimens were received in 10% formalin. Thorough gross examination was carried out and salient features were noted down. Length of the bowel both above and below the lesion was recorded. After initial gross examination, the specimen was opened length wise along the antimesteric border. The most representa block for all 30 cases was then selected for immune histochemical analysis of p53 protein and Oncoprotein for p-53 immunostaining, we used the DO-7 monoclonal antibody (dilution 1:30; DAKO), and, for, the monoclonal antibody 124 was used (dilution 1:80;DAKO). Table 1: Primary Antibodies Test name Test code and clone CODE: ONCOPROTEIN IR614/IS614 CLONE:124 P53 PROTEIN CODE: IR616/IS616 CLONE: DO-7 Method Peroxidase antiperoxidase Peroxidase antiperoxidase Appropriate posi and nega controls were used for each antibody. For posi controls for expression, we used tissue sections from normal lymph nodes. For posi controls for p53 expression, we used a known case of laryngeal carcinoma with diffuse p53 nuclear accumulation. The primary antibody was omitted in the nega controls. Immunohistochemistry Reagents: Phosphate Buffered Saline (PBS) ph 7.4; Antigen Retrieval Solution: Tris-EDTA buffer ph 9.0, Sodium chloride, 3% Hydrogen Peroxide; Monoclonal mouse anti human p53 antibody (dilution 1:30; DAKO) and monoclonal antibody 124 (dilution 1:80; DAKO). Diaminobenzidine (DAB) reagent, and Harris haematoxylin. Procedure: 1. Sections of 3 micron thickness were taken on poly-lysine coated slides. 2. Sections were deparaffinised using xylene. 3. Sections were hydrated in graded alcohols and brought to water. 4. The slides were kept in jar containing antigen retrieval solution. Keep for 6-10 cycles (each cycle 3 minutes) in microwave after setting the temperature. 5. After cooling the slides were washed with distilled water for 2minutes. 6. Two washes with PBS. 7. To block the endogenous Peroxidase, the slides were immersed in a coplin jar containing 3% Hydrogen peroxide in methanol for 5 minutes. 8. The slides were laid flat in a humidified chamber and sections were covered with the blocking reagent for 10 minutes. This was done to block the nonspecific background staining. 9. The blocking reagent was then drained off and excess reagent was wiped away carefully. 10. Without washing the slides were incubated for 30 minutes with primary antibody. 11. The slides were rinsed with buffer. 12. Sections were incubated with super enhancer reagent for 20 minutes. 13. The slides were rinsed with buffer. 14. Sections were then incubated with Poly-HRP reagent for 30 minutes. 15. The slides were rinsed with buffer. 16. Sections were counter stained with haematoxylin for 1 minute. 17. Sections were washed under tap water for 2 minutes. 18. Dehydrated and mounted with DPX. Results: Posi brown stain in nuclei and/ cytoplasm Controls: Formalin-fixed, paraffin-embedded sections of normal human lymph node served as a posi control for in all slide runs. A known case of laryngeal carcinoma with a high level of nuclear p-53 immunoreactivity was used as a posi control for this oncoprotein. Nega control slides were processed with each slide run and excluded the primary antibody but included all other steps of the procedure. immunoreactions were evaluated semi-quantitaly according to the criteria established by van Slooten and 5 colleagues taking the following parameters into consideration. Intensity of cell staining (I) and the fraction of stained cells (F). The intensity of cell (I) staining was classified as: 0 (nega), MRIMS Journal of Health Sciences, Vol. 4, No. 1, January-March 2016 Page 29

3 Shashi Kiran K et al. A study of and p-53 immunostaining expressions in colonic carcinomas 1 (weakly stained), 2 (moderately stained) and 3 (strongly stained). The fraction of stained cell (F) was classified as: I (05%), II (25-75%) & III (75-100%). The final score was the result of the combination of the two parameters (I and F) and ranged from 0 to 6. Cases with a final score 3 were classified as posi for. For p53 we considered only tumor cells with distinct nuclear immunostaining for p-53 as posi and considered the tumor posi only if nuclear accumulation was identified in 10% of all malignant cells in a tissue section. We chose this cut-off value of 10% positivity because this value showed the highest concordance between immuno histochemical detection of p53 and point mutations of the p53 gene as detected by single-strand conformational polymorphism analysis (Grizzle et al. 1998). 6 RESULTS The present study colorectal carcinomas & p53 expression is both a retrospec & a prospec study. The material for study comprised of resected specimens of colon and rectum received in the upgraded department of pathology at Osmania General Hospital, Hyderabad for a period of 2 years (from June 2009 to May 2011). In the present study we have selected only resected colorectal carcinoma specimens and not the colonoscopic biopsies. Total 30 consecu cases of colorectal carcinomas were collected & all are primary adenocarcinomas. The p53 protein is known to regulate apoptosis via the - 2/BAX pathway. Na p53 inhibits gene expression by transcriptional activation of the pro-apoptotic gene BAX. Despite the defined role of protein in suppression of apoptosis, in colorectal cancer the evidence suggests that expression is correlated with favorable parameters and a better prognosis, and p53 expression with worst prognosis. Of 30 colorectal adenocarcinomas, 33.33% (10/30) of tumors were classified as expressing (immunostaining score 2). In addition, diffuse cytoplasmic staining of expression was observed in basal cells of the normal mucosa of colonic crypts adjacent to the tumor but usually not in normal colonic epithelium distant from the tumor. (Table 13) (See figure 13) There is a statistically no significant association observed between expression and Dukes stage, TNM stage or Histological grade (p value > 0.05). Similarly no association was found between expression of and other patient variables, such as sex, age, location of tumor, & tumor differentiation. Posi staining for p53 protein was detected in 56.67% (17/30) of colorectal adenocarcinomas. There is statistically no significant association observed between p53 expression and Dukes stage, TNM stage or Histological grade (p value > 0.05). Similarly no association was found between expression of p53 and other patient variables, such as age, sex, location of tumor, & tumor differentiation. There is a statistically no significant association observed between combined & p53 expression and Dukes stage, Histological grade, or TNM stage (p-value > 0.05). Similarly no association was found between combined immuno histochemistry ( & p-53) and other patient variables, such as age, sex, location of tumor, & tumor differentiation. In the present study statistically there is no significant association between & p53 either individually or combined together with any of the parameters (like age, sex, tumor location, Dukes stage, Histological grade & TNM stage). DISCUSSION When the immuno profile was assessed, it is found that majority of the cases had p53 expression. In the present study there were 5 cases with positivity to both p53 and 2, 3 cases were in Duke s B stage & 2 cases were in Duke s C stage. There were 5 cases of 2 posi and p53 nega, of which 2 & 3 cases were in the Duke s B & C stage respecly. 12 cases were of 2 nega but p53 posi, of which 6 cases belonged to stage Duke s B & C stage. 8 cases were nega to both p53 and 2, of which 6 were in Dukes stage B & 2 were in Dukes. There is no inverse correlation observed in this study between & p53 expression, & no statistical significance observed between & p53 expression alone or in combination with Dukes stage, TNM stage & Tumor grade. However the present study is similar to that observed by Sinicrope et al 7 and Tollenaar et al 8 with no prognostic significance of & p53 expression either alone or in combination. There was no correlation between the present study & the other studies - AJM Watson et al 9, Manne et al 10, Nicholas FS Watson et al 11, Giatromanolaki et al 12, Zhao Dan ping et al 13, Chandrakumarshanmugam et al 14 which showed invere correlation between & p53 expression, having better prognosis with posi & p53 nega group. CONCLUSION: Though not statistically significant, to comment as the optimism expressed by certain investigators due to the low number of cases taken up for the present study, a large study group is needed to comment on the prognostic significance of the & p53 immunoprofile individually and in a combined variation to predict the clinical outcome. REFERENCES: 1. Office for national statistics. Cancer Registrations in England ons.gov.uk/ons /search/ index.html. Cancer registrations. 2. Kim YI Vegetables, fruits and colorectal cancer risk; what should we believe? Nutr Rev. 2001: 59; Parkin DM Global cancer statistics in the year 2000; Lancet Oncol.2000:2; MRIMS Journal of Health Sciences, Vol. 4, No. 1, January-March 2016 Page 30

4 Shashi Kiran K et al. A study of and p-53 immunostaining expressions in colonic carcinomas 4. Peter Boyle, J S Langman. ABC of colorectal cancer Epidemiology, Clinical review : BMJ volume 321, 30 September 2000 bmj.com 5. van Slooten HJ, Clahsen PC, van Dierendonck JH, Duval C, Pallud C, Mandard AM, A Delobelle- Deroide, CJH van de Velde, and MJ van de Vijveret. Expression of in node-nega breast cancer is associated with various prognostic factors, but does not predict response to one course of periopera chemotherapy. Br J Cancer 1996; 74: Grizzle WE, Myers RB, Manne U, Srivastava S Immunohistochemical evaluation of biomarkers in prostatic and colorectal neoplasia. In Hanausek M, Walaszek Z, eds. John Walker s Methods in Molecular Medicine Tumor Marker Protocols. Totowa, NJ, (1998) Humana Press, Frank A. Sinicrope, San Bao Ruan, Karen R. Cleary, L. Clifton Stephens, J. Jack Lee, and Bernard Levin. and p53 Oncoprotein Expression during Colorectal Tumorigenesis. CANCER RESEARCH, (January15, 1995):55, RAEM Tollenaar, JHJM van Krieken, H-J van Slooten, DJ Bruinvels, KMJ Nelemans, LJ van den Broek, J Hermans and JH van Dierendonck. Immunohistochemical detection of p53 and BcI in colorectal carcinoma: no evidence for prognostic significance. British Journal of Cancer (1998) 77(11), AJM Watson, AJ Merritt, LS Jones, JN Askew, E Anderson, A Becciolini, M Balzi,CS Potten and JA Hickman Evidence for reciprocity of and p53 expression in human colorectal adenomas and carcinomas. British Journal of Cancer (1996) 73, Upender Manne, Russell B. Myers, Cecilia Moron, Robert B. Poczatek, Stephenie Dillard, Heidi Weiss, David Brown,Sudhir Srivastava Andwilliam E. Grizzle. Prognostic Significance of Expression and P53 Nuclear accumulation in Colorectal Adenocarcinoma. Int. J. Cancer (Pred. Oncol.) (1997): 74, Nicholas FS Watson, Zahra Madjd, Duncan Scrimegour, Ian Spendlove, Ian O Ellis, John H Scholefield and Lindy G Durrant. Evidence that the p53 nega / posi phenotype is an independent indicator of good prognosis in colorectal cancer: A tissue microarray study of 460 patients. World Journal of Surgical Oncology 2005, 3: Alexandra Giatromanolaki, George P. Stathopoulos, Eleni Tsiobanou, Costandina Papadimitriou, Vassilios Georgoulias, Kevin C. Gatter, Adrian L. Harris, Michael I. Koukourakis. Combined Role of Tumor Angiogenesis,, and p53 Expression in the Prognosis of Patients with Colorectal Carcinoma. American Cancer Society / Volume 86 / Number ZHAO Dan-ping, DING Xiao-wen, PENG Jia-ping,ZHENG Yi-xiong, ZHANG Su-zhan Prognostic significance of and p53 expression in colorectal carcinoma. Zhejiang Univ SCIENCE B (12): Chandrakumar Shanmugam, Venkat R. Katkoori, Nirag C. Jhala, William E. Grizzle, Gene P. Siegal and Upender Manne p53 Nuclear Accumulation and Expression in Contiguous Adenomatous Components of Colorectal Adenocarcinomas Predict Aggressive Tumor Behavior. J Histochem Cytochem : 305. Table 2: & P53 IHC EXPRESSION Feature MRIMS Journal of Health Sciences, Vol. 4, No. 1, January-March 2016 Page 31-2 posi - 2 nega P val ue P53 posi P53 nega P val ue Sex Male (19) Fem ale (11) Age < 45 years (8) > years (22) Immuno reactivity Site of Proxi tumor mal colo n (12) Dista l colo n (12) Rect um (6) Dukes staging A (0) B (17) C (13) Histopath ological grading G1 (16) TNM stage G (5) G3 (9) I (10) 73 II (7) III (13)

5 Shashi Kiran K et al. A study of and p-53 immunostaining expressions in colonic carcinomas Table 3: Combined effect of and p53 expression Feature P - valu e Sex Male (19) Female (11) Age (years) < 45 (8) > (22) Immuno reactivity Site of tumor Proxim al colon (16) Distal colon & rectum (14) Dukes stage A (0) B (17) C (13) Histopathologi cal grade G1 (16) G2 (5) G3 (9) TNM Stage I (10) II (7) III (13) Cite this article as: Shashi Kiran K, Hari Shanker V, Bhopal T, Mahesh Kumar K. A study of and p-53 immunostaining expressions in colonic carcinomas. MRIMS J Health Sciences 2016;4(1): Source of Support: Nil. Conflict of Interest: None. MRIMS Journal of Health Sciences, Vol. 4, No. 1, January-March 2016 Page 32