| Abstract|| |
Background: Urothelial carcinoma is common urinary malignancy responsible for a significant proportion of cancer morbidity and mortality. We carried out the present study to demonstrate the clinicohistopathological features and to correlate the p53 and Ki-67 immunoexpression with grade and stage of bladder carcinomas. Materials and Methods: We investigated 110 cases of bladder tumor. Grading and staging were done according to the WHO-2004 and American Joint Committee on Cancer-TNM staging recommendations. Immunohistochemical staining for p53 and Ki-67 was performed in all the cases, categorized as high and low expression taking 20% positivity as cutoff value. Statistical analysis was done using McNemar's Chi-square test and Fisher's exact test. Results: There were 61 cases of high grade and 49 cases of low grade exhibiting urothelial carcinoma as the most common variant (97.3%). Muscle invasive carcinomas (pT2) noted in 29 cases whereas 23 and 58 cases revealed stage pTa and pT1, respectively. Evaluation of p53 and Ki-67 immunoexpression showed a significant association with histological grade and stage individually and also in combination (P < 0.05). Conclusion: Our results corroborate with the opinion that combined use of p53 and Ki-67 immunomarkers may provide additional prognostic information along with histological grading and staging in bladder carcinomas.
Keywords: Bladder, grade, Ki-67, p53, stage, urothelium
|How to cite this article:|
Thakur B, Kishore S, Dutta K, Kaushik S, Bhardwaj A. Role of p53 and Ki-67 immunomarkers in carcinoma of urinary bladder. Indian J Pathol Microbiol 2017;60:505-9
|How to cite this URL:|
Thakur B, Kishore S, Dutta K, Kaushik S, Bhardwaj A. Role of p53 and Ki-67 immunomarkers in carcinoma of urinary bladder. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Jun 25];60:505-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/505/222964
| Introduction|| |
Urothelial carcinoma comprises approximately 90% of all primary bladder tumors with gross or microscopic painless hematuria as common presentation. The gold standard for diagnosing bladder carcinoma is cystoscopy and biopsy of suspicious area., The management and posttreatment monitoring of superficial bladder cancers are largely dependent on stage and grade because approximately 70% of superficial urothelial carcinomas may recur and 10%–20% progress to a higher stage, grade, or metastatic disease.
Some previous studies have shown p53 mutations and cell proliferation markers to play an important role in urothelial carcinoma pathogenesis and also as prognostic factors.,, Therefore, immunohistochemical expression of p53 and proliferation marker Ki-67 may be helpful in proper staging and grading of bladder carcinomas. This study has been carried out to present the clinicohistopathological features of bladder carcinomas and also to demonstrate the correlation of p53 and Ki-67 immunoexpression with grade and stage of bladder carcinomas at first presentation.
| Materials and Methods|| |
In the present study, we included a total of 110 cases of urinary bladder carcinomas diagnosed during the past 3 years. The Institutional Ethical Committee has vetted and approved the study. Relevant history and clinicoradiological findings were recorded on the designated proforma. Tissue samples in 10% buffered formalin were received as TURBT chips or bladder biopsy in histopathology section and processed. 3–5 μ thick H and E stained sections were analyzed for histopathological diagnosis.
For immunohistochemistry, extra 2-4 μ thick sections were taken on Poly-L lysine coated slides and subjected to antigen retrieval by microwave heat method. Immunohistochemical staining of p53 and Ki-67 was performed using primary antibodies DO7 (By cell marque: Key code-CMC45329022; at 1:300 dilution in Tris-buffered saline) and SP6 (By cell marque: Key code-CMC27531021; at 1:300 dilution in Tris-buffered saline), respectively. DO7 mouse monoclonal antibody reacts with an epitope in the N-terminal extreme of the p53 protein and recognizes both mutants as well as wild-type p53 proteins. Sections were incubated with the secondary biotinylated antibody and avidin-biotin peroxidase complexes for 30 min. Reaction products were revealed with diaminobenzidine as the chromogen and sections were counterstained with Harris's hematoxylin to enhance nuclear detection. Sections of colonic carcinoma tissue (for p53) and chronic tonsillitis tissue (for Ki-67) were used as positive controls. For negative controls, primary antibody was substituted with phosphate buffer saline in duplicate sections.
Nuclear positivity was seen as dark brown color on bluish background. Percentage of immunopositive cells was calculated by counting at least 1000 tumor cells in areas of maximum positivity. The results were interpreted taking the cutoff value as 20% and divided into three categories as immunonegative, <20% as low, and >20% as a high expression for both immunomarkers.
Subsequently, the data were studied, tabulated, and correlated with histopathological diagnosis. Statistical analysis was done using McNemar's Chi-square test (with DAG Stat software; by Mackinnon A. 2000) and Fisher's exact test (by SPSS windows 15.0 program; SPSS Inc., Chicago), and results were highlighted in [Table 1] and [Table 2] respectively. We accepted P < 0.05 as statistically significant.
|Table 1: Distribution pattern of p53 and ki-67 immunohistochemical staining according to histology, grade, and stage|
Click here to view
|Table 2: Correlation of p53 and Ki-67 immunoexpression according to stage and grade of bladder tumors|
Click here to view
| Results|| |
Majority of the patients were males (97 cases, 88.2%) and only 13 cases (11.8%) were reported in females accounting for male:female ratio of 7.46:1 in this study. The median age of presentation for males was 55 years whereas, in females, it was 62 years. A maximum number of cases were seen in the age group of 41–60 years (50 cases, 45.5%) followed by age group of 61–80 years (46 cases, 41.8%). No case was reported up to 20 years of age. Majority of male patients (48 cases) were in between 41 and 60 years of age. However, in females, highest number (8 cases) was observed in 61–80 years of age group [Table 3].
|Table 3: Distribution pattern of bladder carcinoma patients according to age, gender, histologic type, grade, and stage of bladder tumors|
Click here to view
The most common clinical presentation in our study was gross or microscopic hematuria seen in 100 cases (90.9%). Hematuria alone was seen in 44 cases (40%) whereas its association with urinary tract symptoms or lower abdominal pain was seen in 35 cases (31.82%) and 21 cases (19.09%), respectively. Seven cases presented with only UTI and two with lower abdominal pain alone. Only one patient was asymptomatic and diagnosed as an incidental finding.
The most common histological variant was urothelial carcinoma seen in 107 cases (97.3%), followed by squamous cell carcinoma (2 cases) and adenocarcinoma (1 case). According to recommendations of WHO-2004, all cases were graded as low (LG) and high grade (HG). Forty-nine cases (44.5%) were reported as LG whereas rest of the 61 cases (55.5%) as HG tumors including squamous cell carcinoma and adenocarcinoma [Figure 1]a, [Figure 1]d and [Figure 2]a, [Figure 2]d. Staging of all the cases was also done according to American Joint Committee on Cancer-TNM classification. In this study, 81 patients (73.6%) revealed nonmuscle invasive bladder carcinoma, of which 23 cases (20.9%) had stage pTa (without lamina propria invasion) and 58 cases (52.7%) were of stage pT1 (with lamina invasion) whereas the remaining 29 cases (26.4%) showed stage with pT2 (detrusor muscle invasive disease) [Table 3]. Patients with stage T1 were further classified as pT1HG (32 cases) and pT1 LG (26 cases) depending on their grade.
|Figure 1: (a) Section showing low-grade urothelial carcinoma (H and E, ×100). (b) Section showing high expression of p53 in low-grade urothelial carcinoma (IHC, ×100). (c) Section showing high expression of Ki-67 in low-grade urothelial carcinoma (IHC, ×100). (d) Section showing high-grade urothelial carcinoma (H and E, ×100). (e) Section showing high expression of p53 in high-grade urothelial carcinoma (IHC, ×100). (f) Section showing high expression of Ki-67 in high-grade urothelial carcinoma (IHC, ×100)|
Click here to view
|Figure 2: (a) Section showing glandular arrangement of tumor cells; suggestive of adenocarcinoma of urinary bladder (H and E, ×100). (b) Section showing high expression of p53 in adenocarcinoma (IHC, ×100). (c) Section showing high expression of Ki-67 in adenocarcinoma (IHC, ×100). (d) Section showing small sheets and nests of round to polygonal neoplastic squamous cells with evidence of keratinization; suggestive of squamous cell carcinoma of urinary bladder (H and E, ×100). (e) Section showing low expression of p53 squamous cell in carcinoma (IHC, ×100). (f) Section showing high expression of Ki-67 in squamous cell carcinoma (IHC, ×100)|
Click here to view
It was observed that, out of 110 cases of urinary bladder carcinoma, 84 cases (76.4%) showed high p53 expression, 21 cases (19.0%) showed low p53 expression, and 5 cases (4.6%) were immunonegative for p53. High expression of p53 was seen mainly in HG tumors (52 cases) as compared to 32 cases of LG tumors [Figure 1]b and [Figure 1]e. Low expression was noticed in 13 patients of LG tumors and in only 8 cases of HG tumors. This difference of p53 expression was statistically significant (P = 0.0001; df = 1) while taking grading into account. A maximum number of patients with high p53 expression (42 cases) was observed in stage pT1 as compared to stage pTa (17 cases) and pT2 (25 cases). Only three cases each of pTa and pT2 stage showed low immunoexpression whereas stage pT1 showed 15 cases. In terms of staging, these results were also statistically significant (P = 0.000001; df = 1). Both cases of squamous cell carcinoma showed low p53 expression whereas single case of adenocarcinoma revealed high expression [Figure 2]e and [Figure 2]b. Out of five immunonegative patients, three were of pTa stage and one each of pT1 and pT2 stage including single case of HG urothelial carcinoma [Table 1].
In this study, low Ki-67 immunoexpression was noted in 56 cases and high expression in 54 patients. LG tumors showed mainly low expression (48 cases) and HG tumors exhibited high expression (53 cases) [Figure 1]c and [Figure 1]f. All 29 cases of muscle invasive tumors (pT2) revealed high Ki-67 immunoexpression including squamous cell and adenocarcinoma [Figure 2]f and [Figure 2]c whereas all patients with pTa stage showed low expression. In pT1 staged carcinomas, low and high expression was noticed in 33 and 25 cases respectively. Difference was found to be statistically significant in terms of grading (P = 0.01; df = 1) and staging pT1 (pTa + pT1/pT2, P = 0.000001; pTa/pT1, P = 0.000001; pT1/pT2, P = 0.000002; df = 1) highlighted in [Table 1] and [Figure 3].
|Figure 3: (a) Section showing infiltrative tumor cell nests with p53 overexpression in muscle invasive urothelial carcinomas (IHC, ×100). (b) Section showing scattered tumor cells in between smooth muscle bundles exhibiting high Ki-67 labeling index in muscle invasive urothelial carcinomas (IHC, ×400)|
Click here to view
| Discussion|| |
Urinary bladder carcinoma is a common multistage progressive malignancy ranking 9th in worldwide cancer incidence and responsible for significant mortality and morbidity. Although the incidence of urothelial carcinomas increases with age, smoking, petrochemicals, and arylamines exposure, its burden is also growing due to shifting of smoking habits, tobacco use, industrialization, and urbanization from developed to developing countries in the past few decades.,,
In our study, majority of the patients were male and belonged to middle to older age group. Similar findings were described in other studies as well.,, Furthermore, hematuria was a most common presenting symptom which is in concordance with the findings of other authors.,,,, About 10% or 20% patients presented with microscopic or gross hematuria, respectively, are diagnosed subsequently as bladder carcinoma after evaluation. In the present study, predominant histologic variant was urothelial carcinoma. Similar data were also published by other authors also.,,, Adenocarcinomas are uncommon bladder tumors accounting for approximately 2% of all bladder carcinomas which coincides with our study. Squamous cell carcinoma was noted in about 1.8% of cases. Beltran et al. also described its incidence as 1%–3% all vesical tumors.
A reliable and identical staging and grading system for bladder carcinomas by histopathological analysis are important for categorizing the patients for better treatment options among institutions and also to stratify them into distinct groups prognostically. About 80% of the patients are diagnosed as superficial nonmuscle invasive carcinoma at first presentation. In this study, nonmuscle invasive tumors were noted in approximately 74% cases (Ta + T1 stage); similar findings were reported in other studies as well.,,, Therefore, it is also important to mention the presence of detrusor muscle in the biopsy specimen as it may lead to understanding in many patients. We divided urothelial carcinomas as low and HG depending on the cellular atypia, polarity, epithelial thickness, and mitosis (WHO-2004 grading system). HG tumors (39 cases) were more common in older patients more than 60 years of age group coinciding with similar finding noted by Joshi et al. We found 44.5% cases with LG carcinomas similar to few other studies., We also observed about 58 cases of T1 stage included both noninvasive LG and HG urothelial tumors whereas Ta stage revealed all patients with LG only.
As there is more risk of tumor recurrence, stage progression, and tumor-related mortality with increasing grade and infiltrative tumor pattern, histopathological grading and staging of urothelial carcinomas can be an important tool in determining the prognosis of the disease. However, these pathologic variables exhibit limited ability to predict the response toward treatment more so where are different treatment approaches and follow-up schedules. Hence, there is a clear need for molecular studies which may be helpful for accurate prediction of the behavior of these cases.
Cell proliferation and mutations in cell cycle regulatory genes are hallmarks in various tumors including urothelial carcinomas., In carcinogenesis, p53 is the most common gene mutation observed in carcinomas of lung, breast, colon, and bladder. The p53 gene is a tumor suppressor gene located on chromosome 17p; important for genome stability, response to genotoxic stimuli, and activation of apoptosis. However, its molecular basis affecting cellular functions and tumorigenesis remains unclear. In normal tissues, p53 gene product is 393-amino acid nuclear protein with shorter half-life while mutated p53 gene product having longer half-life is accumulated which allows its detection by immunohistochemistry easily.
Proliferation abnormalities resulting from disruption of cell cycle regulators and also abnormal cellular proliferation can be easily detected by immunohistochemistry using Ki-67 labeling index which is a measure of cell growth fraction and therefore also of biological aggressiveness of malignancy. Nuclear antigen Ki-67 coded by gene on chromosome 10 is absent in resting cells (G0 phase) and hence exclusively positive in the nuclei of proliferating cells., In the present study, we have taken 20% nuclear positivity as the cutoff value for low and high immunoexpression of p53 and Ki-67 as this discriminating cutoff value has been more predictive in terms of tumor recurrence, progression and disease free survival in some of the previous analytical studies.,,,
Significant association of grade and stage (superficial versus muscle invasive) with p53 positively stained tumor cells was observed while taking 20% as cutoff value (P = 0.0001 and P = 0.000001) [Figure 3]a. Similar behavior of this marker was noted by some other authors in tumor grade and/or stage or clinical outcome.,,,,, Serth et al. found disease progression in p53 overexpression (12/14 cases) whereas only one case out of 55 patients in <20% p53 positivity. It can be inferred from the results that higher p53 expression may be an indicator for tumor progression and early failure of local therapy; also for early surgical intervention especially for superficial carcinomas. Although, a few investigators described opposite results as well.,
We also observed p53 immunonegative cases which may signify the presence of whether inactivation of p53 or other alternative genetic pathways in the molecular pathogenesis of urothelial tumors. A few previous analysis suggest alternative immunomarkers as well.,,,, Positive significant correlation of Ki-67 labeling index with histological grading and staging of urothelial carcinomas (P< 0.05); similarly described by other studies also.,,,,, All muscle invasive carcinomas revealed high expression of Ki;67; also no immunonegative case was observed [Figure 3]b.
In our study, percentage of p53 positively stained tumor cells was ranging from 0% to 70% and overall mean value of p53 expression was 31.8% (pTa = 24.45%, pT1 = 31.77%, pT2 = 35.93%) whereas median value was observed as 37.5%. Mean Ki-67 labeling index was 18.4% (pTa = 6.69%, pT1 = 15.13%, and pT2 = 33.48%) and median value was 22% [Table 2]. We observed that mean expression of Ki-67 was consistently more increasingly changed with staging as compared to p53 expression. Furthermore, there was more difference between expression of these two markers in pTa and pT1 stage (P = 0.0001) as compared to pT2 [Table 2].
| Conclusion|| |
Our results corroborate with the opinion that combined use of p53 and Ki-67 immunomarkers in urinary bladder carcinomas may provide additional prognostic information along with histological grading and staging. Few studies also described similar results., Unfortunately, there was great difficulty to follow up the patients because of their referral to higher centers. However, p53 overexpression may be important for stratifying the high-risk patients, especially in superficial urothelial carcinomas. However, we need further larger multi-institutional studies with reliable, reproducible, and equivalent standardized immunohistochemical techniques and use of other biomarkers as well as for adding more information regarding the unpredictable course of bladder carcinomas.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rosai J. Urinary tract: Bladder. In: Rosai and Ackerman's Surgical Pathology. 10th
ed., Vol 1. New Delhi: Elsevier; 2011. p. 1257-64.
Eble JN, Sauter G, Epstein JI, Sesterhenn IA, editors. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. Lyon: IARC Press; 2004. p. 90-123.
Burkhard FC, Markwalder R, Thalmann GN, Studer UE. Immunohistochemical determination of p53 overexpression. An easy and readily available method to identify progression in superficial bladder cancer? Urol Res 1997;25 Suppl 1:S31-5.
Serth J, Kuczyk MA, Bokemeyer C, Hervatin C, Nafe R, Tan HK, et al.
p53 immunohistochemistry as an independent prognostic factor for superficial transitional cell carcinoma of the bladder. Br J Cancer 1995;71:201-5.
Popov Z, Hoznek A, Colombel M, Bastuji-Garin S, Lefrere-Belda MA, Bellot J, et al.
The prognostic value of p53 nuclear overexpression and MIB-1 as a proliferative marker in transitional cell carcinoma of the bladder. Cancer 1997;80:1472-81.
Ploeg M, Aben KK, Kiemeney LA. The present and future burden of urinary bladder cancer in the world. World J Urol 2009;27:289-93.
Gupta P, Jain M, Kapoor R, Muruganandham K, Srivastava A, Mandhani A. Impact of age and gender on the clinicopathological characteristics of bladder cancer. Indian J Urol 2009;25:207-10.
] [Full text]
Biswas RR, Mangal S, Guha D, Basu K, Karmakar D. An epidemiological study of cases of urothelial carcinoma of urinary bladder in a tertiary care centre. J Krishna Inst Med Sci Univ 2013;2:82-8.
Laishram RS, Kipgen P, Laishram S, Khuraijam S, Sharma DC. Urothelial tumors of the urinary bladder in Manipur: A histopathological perspective. Asian Pac J Cancer Prev 2012;13:2477-9.
Joshi HN, Makaju R, Karmacharya A, Karmacharya RM, Shrestha B, Shrestha R, et al.
Urinary bladder carcinoma: Impact of smoking, age and its clinico-pathological spectrum. Kathmandu Univ Med J (KUMJ) 2013;11:292-5.
Al-Bazzaz PH. Stage of urinary bladder cancer at first presentation. Saudi J Kidney Dis Transpl 2009;20:628-31.
] [Full text]
Sathya M, Chinnaswamy P. Urinary bladder cancer: A clinicopathological and histological study. J Med Sci 2014;14:206-9.
Pudasaini S, Subedi N, Prasad KB, Rauniyar SK, Joshi BR, Bhomi KK. Cystoscopic bladder biopsies: A histopathological study. Nepal Med Coll J 2014;16:9-12.
Beltran H, Robinson BD, Tagawa ST. Primary squamous cell carcinoma of the urinary bladder presenting as peritoneal carcinomatosis. Adv Urol 2010; Vol. 2010 Article ID179250.
Pagano F, Garbeglio A, Milani P, Bassi P, Pegoraro V. Prognosis of bladder cancer; risk factors in superficial transitional cell carcinoma. Eur Urol 1987;13:145-9.
Margulis V, Shariat SF, Ashfaq R, Sagalowsky AI, Lotan Y. Ki-67 is an independent predictor of bladder cancer outcome in patients treated with radical cystectomy for organ-confined disease. Clin Cancer Res 2006;12:7369-73.
Shariat SF, Tokunaga H, Zhou J, Kim J, Ayala GE, Benedict WF, et al.
p53, p21, pRB, and p16 expression predict clinical outcome in cystectomy with bladder cancer. J Clin Oncol 2004;22:1014-24.
Santos L, Amaro T, Costa C, Pereira S, Bento MJ, Lopes P, et al.
Ki-67 index enhances the prognostic accuracy of the urothelial superficial bladder carcinoma risk group classification. Int J Cancer 2003;105:267-72.
Dalbagni G, Parekh DJ, Ben-Porat L, Potenzoni M, Herr HW, Reuter VE. Prospective evaluation of p53 as a prognostic marker in T1 transitional cell carcinoma of the bladder. BJU Int 2007;99:281-5.
Luo Y, Zhang X, Mo M, Tan Z, Huang L, Zhou H, et al.
High Ki-67 immunohistochemical reactivity correlates with poor prognosis in bladder carcinoma: A comprehensive meta-analysis with 13,053 patients involved. Medicine (Baltimore) 2016;95:e3337.
Mumtaz S, Hashmi AA, Hasan SH, Edhi MM, Khan M. Diagnostic utility of p53 and CK20 immunohistochemical expression grading urothelial malignancies. Int Arch Med 2014;7:36.
Koyuncuer A. Immunohistochemical expression of p63, p53 in urinary bladder carcinoma. Indian J Pathol Microbiol 2013;56:10-5.
] [Full text]
van Rhijn BW, van der Kwast TH, Vis AN, Kirkels WJ, Boevé ER, Jöbsis AC, et al.
FGFR3 and P53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma. Cancer Res 2004;64:1911-4.
Wang L, Feng C, Ding G, Zhou Z, Jiang H, Wu Z. Relationship of TP53 and Ki67 expression in bladder cancer under WHO 2004 classification. J BUON 2013;18:420-4.
Turk NS, Aybek Z, Duzcan E. Ki-67, p53, Bcl-2 and bax expression in urothelial carcinomas of urinary bladder. Turk J Pathol 2010;26:25-30.
Dr. Brijesh Thakur
Department of Pathology, SGRRIM and HS, Patel Nagar, Dehradun - 248 001, Uttarakhand
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]