Immunohistochemical analysis of p53, CD10 and Ki67 expression as prognostic biomarkers in urinary bladder carcinomas correlating with age, histomorphology, grade and stage: An institutional study of one and half year Mohapatra AS, Mohanty P, Pradhan S, Sabat DK,

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Immunohistochemical analysis of p53, CD10 and Ki67 expression as prognostic biomarkers in urinary bladder carcinomas correlating with age, histomorphology, grade and stage: An institutional study of one and half year

Ajit S Mohapatra¹, Pranita Mohanty¹, Sarita Pradhan¹, Debabrat Kumar Sabat²
¹ Department of Pathology, IMS and SUM Hospital, S‘O’A (Deemed to be University), Bhubaneswar, Odisha, India
² Department of Urology, IMS and SUM Hospital, S‘O’A (Deemed to be University), Bhubaneswar, Odisha, India

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Date of Submission	17-May-2021
Date of Decision	27-Oct-2021
Date of Acceptance	29-Nov-2021
Date of Web Publication	14-Nov-2022

Abstract

Background: Bladder carcinoma ranks tenth among all cancers worldwide predominantly affecting elderly males. Common risk factors being cigarette smoke and aniline dyes. Immunohistochemical markers play a pivotal role for its diagnosis and prognosis. Aim: To analyze the immunohistochemical expression of p53, CD10, Ki-67 in bladder cancers correlating with demographic features, pathological grade, and stage and to establish as prognostic biomarkers. Materials and Methods: Surgical samples of total of 70 cases of bladder tumor were collected, processed, stained in routine hematoxylin and eosin followed by immunohistochemistry of p53, CD10, and Ki67 markers performed on randomly selected 30 cases only. Results: Out of 70 cases 69 cases (98.6%) were carcinomas; urothelial carcinoma being 71.4% (n = 50) with male: female ratio = 7.7:1 and mean age = 61.81 ± 12.83 years. Out of 30 cases, p53 was positive in 50% of cases, 30% - negative and 20% - equivocal. p53 positive expression pattern was more in high grade (HG) than low grade (LG). Significant difference was observed in the mean p53 scoring (%) and different stages (P = 0.043). CD10 expression was negative in 56.6%, (1+) in 16.6%, and (2+) in 26.6% of cases and significant difference in CD 10 expression was observed between the high and LG (P = 0.001). Ki-67 labeling index was appreciably higher in HG than the LG tumor (32.49% ± 24.35%; 6.86% ± 8.1%). Majority of Ki-67 expression was observed in stage pT2, followed by the pT1 stage. Conclusion: Cocktail of p53, CD10, and Ki67 is useful as potential prognostic markers in bladder cancers.

Keywords: Markers, p53, transitional cell carcinoma, urinary bladder carcinoma

How to cite this URL:
Mohapatra AS, Mohanty P, Pradhan S, Sabat DK. Immunohistochemical analysis of p53, CD10 and Ki67 expression as prognostic biomarkers in urinary bladder carcinomas correlating with age, histomorphology, grade and stage: An institutional study of one and half year. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Apr 1]. Available from: https://www.jmau.org/preprintarticle.asp?id=361122

Introduction

Worldwide absolute incidence of urinary bladder cancer (UBC) ranks tenth among all cancers accounting for the sixth position in men and seventeenth in women.^[1] Approximately 3.0% of newly diagnosed cancer and 2.1% of cancer deaths are due to it. Most bladder cancers are epithelial in origin, and >90% are classified as urothelial (transitional) carcinoma (UC).^[2]

The pathogenesis of UC depends on a combination of genetic and environmental factors. It is common in industrial areas, (especially in those associated with petrochemicals) and their incidence is increased with exposure to cigarette smoke and arylamines. Other environmental factors include aniline dyes (particularly benzidine and beta naphthylamine, auramines, phenacetin, and cyclophosphamide) that predispose to bladder malignancy.^[3] Among parasitic infections Schistosoma hematobium is pathogenetically related to urothelial and squamous cell carcinoma (SCC) of the urinary bladder.^[4] In 2016, WHO classified pTa and pT1 tumors into LG and HG tumors with all detrusor muscle-invasive UCs (pT2, pT3, pT4) into high grade (HG) tumor. The treatment of UC is largely based on histological grade and stage of the tumor. Although histomorphology stands as a firm base for diagnosis yet immunohistochemistry (IHC) has an important role in confirming the urothelial origin at the metastatic site by differentiating it from mimickers of UCs.^[5]

IHC markers such as GATA3, Uroplakin III, p63, CK7, CK20, CK5/6 are employed in the diagnosis of UC. TP53 has a role in urothelial cell carcinoma progression as well as recurrence by its inactivation. Increased immunohistochemical detectability of TP 53 in the nucleus is considered as a surrogate marker for genetic deletion and/or mutation.^[6] Esrig et al. demonstrated that increased nuclear expression of TP 53 in formalin-fixed, paraffin-embedded sections of transitional cell carcinomas (TCCs) from patients undergoing cystectomy correlated with reduced survival and disease progression.^[7] CD10, a zinc-dependant metalloendoprotease is associated with bladder carcinoma pathogenesis and its progression. CD10 is thought also to affect the invasion and metastatic potential of tumor cells by altering the cellular microenvironment.^[8],[9] CD10 expression in intratumoral stromal cells may also contribute to tumor progression.^[10] Ki67 is a reliable proliferative marker used to access the aggressiveness of tumor and survival rate. Hence, the current study was aimed at analyzing the expression of p53, CD-10, and Ki-67 in UC and its correlation between different clinico-pathological features, histological grade, and stage of the tumor. The usefulness of CD10 and Ki67 labeling index as prognostic biomarkers in urothelial malignancy was also elucidated.^[11]

Materials and Methods

The present study was a hospital-based perspective study conducted in the department of pathology within a period of 1½ years (May 2019–November 2020), in the teaching hospital. Total 70 cases of those presented with painless/gross hematuria, chronic cystitis, or dysuria with radiologically suspected bladder malignancies were included in the study [Figure 1]a and [Figure 1]b. Biopsy samples (Transurethral Resection of Bladder Tumor [TURBT] biopsies and radical specimens) were received along with the relevant clinical data from the Urology department, grossed according to CAP guidelines (version 4.0.1.1), processed and then stained with Hematoxylin and eosin stain for routine microscopy.^{[12],[13],[14],[15]} Following it IHC for p53 (PathnSitu), CD10 (Dako), and Ki-67(Dako) markers were performed randomly on 30 cases only. Staining of p53 was scored semiquantitatively like negative: No staining or predominantly basally located urothelium positivity, partial or equivocal: Patchy labeling, with some cells above the basal layer showing strong positivity, positive: Strong and diffuse labeling of the area of concern (full urothelium thickness or otherwise, dependent on the distribution of the abnormal cells).^[16] CD10 was scored semiquantitatively based on the percentage of positive cells.^[5],[12] Score (0): <5% positive cells, (+1): Low expression in 5%–50% positive cells, (+2): Strong expression in >50% positive cells. Ki-67 was considered positive when 10% of cells showed nuclear positive expression.^[10],[17]

Figure 1: HG TCC case: (a) CT scan and (b) cystoscopy showing a solitary polypoidal growth. (c) Cystectomy specimen showing polypoidal growth along the lateral wall, (d and e) Microscopy showing papillary configuration of marked pleomorphic urothelium with and loss of polarity and increased mitosis (H and E, x10, x40)

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Statistical analysis of both histopathology and IHC features was done by using IBM SPSS version 22.0; IBM Corp., New York, USA. P < 0.05 was considered statistically significant and assessed using Chi-square test and ANOVA. Institutional ethical committee clearance was sought. (Ref. no./DMR/IMS.SH/SOA/180131. Date:4th Jan 2019.).

Results

Age and sex

The study group comprised a total of 70 cases, with the age limit of 37–88 years, mean age of 61.81 ± 12.83 years (median age-65 years). Overall age incidence was between 6^th and 8^th decades of life. There were 62 males (88.6%) and 8 females (11.4%) with male sex predominance showing male: female ratio of 7.7:1.

Chief complaints, operative procedure, gross appearance, tumor site

The commonest presention was painless hematuria (63 cases, 90%) followed by hematuria with urgency (n = 6, 8.6%) and hematuria with incontinence (n = 1, 1.4%). Correlating gender with chief complaints showed that 90% of patient had painless hematuria of which 90% were male. The preferred operative procedure was TURBT (n = 64, 91.4%) followed by radical cystectomy (n = 3, 4.3%), radical cystoprostatectomy (n = 2, 2.8%), and wide local excision (n = 1, 1.4%).The predominant gross appearance was papillary type (n = 30, 42.8%), followed by ill-defined mass (n = 8, 11.4%) and diffuse pattern accounting for 8 cases (11.4%) [Figure 1]c. Correlating gross appearance and operative procedure showed that out of total 91.42% (n = 64) patients operated for TURBT, 40.62% (n = 26) cases possess papillary appearances. The most common site detected was posterolateral wall (n = 18, 25.7%) followed by right lateral wall (n = 10, 14.2%) and the third most common was involvement of whole wall (n = 8, 11.4%) [Table 1].

Table 1: Demographic features of patients

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Histopathology

Histopathology analysis showed that out of the total seventy cases (n = 70) UB neoplasm, 69 (98.6%) were malignant cases (UBC) with n = 1 (1.4% case) reported as benign leiomyoma in a 37-year-old male. Among all cancers, UC/TCC was n = 50 (71.4%), followed by TCC with squamous differentiation (n = 5, 7.1%), PUNLMP (n = 4, 5.7%), sarcomatoid carcinoma (n = 2, 2.8%). Certain unusual histological forms were also encountered, i.e., SCC, large cell neuroendocrine carcinoma, small-cell neuroendocrine carcinoma, small-cell carcinoma, TCC with focal neuroendocrine differentiation, TCC with 70% sarcomatoid and 20% squamous differentiation and undifferentiated carcinoma; accounting for 1.4% each. Microscopically, papillary configuration was found to be the commonest ones (n = 48, 68.6%) followed by solid pattern (n = 13, 18.6%) [Figure 1]d, [Figure 1]e and [Figure 2]a, [Figure 2]b. Other configuration noted were papillary with solid pattern (n = 6, 8.6%), solid/nodular (n = 1, 1.4%), ulcerated (n = 1, 1.4%) [Table 1].

Figure 2: HG TCC case: (f) p53 positivity (80%), (g) CD10 positivity (score 2+), (h) Ki 67 (60%) (40x). LG TCC case: (a and b) (inset) - Photomicrograph showing well defined papillary architecture with mild nuclear pleomorphism, few mitotic figures in basal layer and intact umbrella cell layer (H and E, x4, x40). (c) Equivocal score of p53, (d) (1+) score for CD10 (x40)

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Grade and stage

Of the 70UBC cases, tumor grading/staging could be assessed in 61 cases of TCC according to the 2016 WHO classification and was graded as low grade (LG) and HG as grading/staging is applicable for TCC only (both pure and mixed forms). There were 19 (27%) cases of LG and 42 cases (60%) of HG tumors. In the current study, 50% (n = 35) of tumor were staged as pT2, 22.9% (n = 16) cases were in pTa and pT1 stage each and 1.4% (n = 1) case belong to each of pT3 and pT4 stage. In the HG, staging was available for 42 cases, among these 30 cases (71.4%) were associated with stage pT2, 10 cases (23.8%) were associated with pT1. In LG, staging was known for 19 cases and among these 16 cases (84.2%) were associated with pTa and 3 cases (15.8%) were in stage pT2 [Table 2].

Table 2: Association between grade and stage of urinary bladder cancer

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Association of grade and stage with patient age were also done in 61 cases out of total 70 cases. Of 42 HG cases; 61.9% (n = 26) were >64 years of age, while rest 38.1% (n = 16) were <64 years of age. In stage pT2 63.33% (n = 19) were >64 years while 36.67% (n = 11) were <64 years. Simillarly, out of 19 cases of LG tumors; 31.6% (n = 6) were >64 years of age and 68.4% (n = 13) were <64 years. In HG tumors; majority of patients found in stage pT2 (n = 30), of which 83.3% (n = 25) were male and 16.7% (n = 5) were female. The mean age of the patients of HG tumors with pT2 stage was 64.90 ± 13.56 years. Among LG tumors; number of cases were more in stage pTa (n = 16) rest belong to pT2 (n = 4).They were all males (n = 16) with mean age of 56.13 ± 12.08 years in pTa stage, while 75% were male and 25% were female in pT2 with mean age of 57 ± 5.83 years [Table 3].

Table 3: Overall association between tumor grade and stage with patients' age and gender

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Immunohistochemistry

IHC of p53, CD10, and ki67 IHC marker could be performed randomly on thirty cases (42.8%) only and was analyzed, correlated with age, gender, grade, stage [Figure 1]f, [Figure 1]g, [Figure 1]h and [Figure 2]c, [Figure 2]d. Fifteen cases (50%) - positive, nine cases (30%) - negative and six cases (20%) were stained equivocally for p53 with the mean age being 63.60 ± 14.11 years for positive cases. Cases with equivocal staining patterns had a mean age group of 66.5 ± 9.8 years with (P = 0.039) that was statistically significant. It was observed that the association between the p53 staining pattern and the grading of the tumor was statistically insignificant (P = 0.432). 92.3% of HG tumors showed positive staining for p53 while 7.7% were belonging to LG tumor [Table 4]. The majority of the p53 expression was seen in the pT2 stage showing positive 10 cases (66.7%), negative 4 cases (44.4%), and equivocal 3 cases (50%). However, the association between tumor stage and p53 expression was not significant (P = 0.154). Whereas on assessing the correlation between the p53 score (%) with that of the stage of the tumor, significant difference was observed in the mean p53 scoring (%) and different stages (P = 0.043) [Table 5].

Table 4: Association between grade and p53 expression pattern

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Table 5: Association between stage and p53 scoring

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CD10 score analysis showed: 54% (n = 16) - negative, 23% (n = 7) - score 2+ and 23% (n = 7) - score 1+. A statistical significance between staining pattern and intensity of CD10 with that of age was found in the study group with (P = 0.044) [Table 6]. Significant difference in CD10 expression was observed between the high and LG TCC (P = 0.001) [Graph 1]. The strong expression (2+) of CD10 was seen in 6 cases (85.7%) of HG, while only 1 (14.3%) case was observed in LG. The weak positive expression (1+) of CD10 was seen in 6 cases (85.7%) of HG, while only 1 (14.3%) case was observed in LG. Likewise, 9 (56.2%) cases showed negative CD10 expression in HG and 7 cases (43.8%) showed negative CD10 expression in LG (53.3%). The majority of weak positive CD10 expression was seen in pT2, i.e., n = 5 cases (71%). Most of the strong positive CD10 expression was also seen in pT2, i.e., n = 4 cases (57%). The negative CD10 expression was also high in pT2 i.e., 8 cases (50%) and was found to be statistically insignificant.

Table 6: Association of age with CD10 scoring

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In the present study, significant difference was observed between the grade and Ki-67 expression (%). The Ki67 expression was higher in HG (20 cases) with a mean expression index (%) of 32.49 ± 24.35 as compared to LG (7 cases) with an expression index (%) of 6.86 ± 8.1 that was statistically significant (P = 0.012). Majority of Ki-67 expression was present in stage pT2, showing 17 cases with a mean Ki-67 expression index of 35.49 ± 27.24, followed by pT1 having 5 cases with a mean Ki-67 expression index of 30.68 ± 34.97.

Follow-up

During follow-up; 78.57% of patients (n = 55) were lost to follow, 12.85% (n = 9) turned up for check cystoscopy, 5.71% (n = 4) were on chemotherapy. One patient (1.42%) who was diagnosed with LGNEC succumbed to death after 8 months postdiagnosis and lastly, one patient was yet to follow for check cystoscopy procedure. Out of seventy cases studied, tumor recurrence and progression were noted among nine cases (12.85%).

Discussion

TCC accounts for more than 90% of all bladder tumors.^[18] In the present study out of 70 cases, 88.6% were male and 11.4% were female with male: female ratio of 7.7:1. Similar studies by Atique et al. showed 90% males and 10% females, Ziaran et al. reported 80.4% male and Gupta et al. reported a male: female ratio of 8.6:1.^{[19],[20],[21]} However in Goyal et al. study, the male: female ratio was 5.25:1 that was lower in comparison to the current study.^[21] In the present study, the mean age of the patients was 61.81 ± 12.83 years and most of the cases (31.5%) were between the 6^th and 7^th decades of life. The mean age in Goyal et al. study (60.79 years), Matalka et al. study (60.6 years) and Vaidya et al. study (65 years) had been well correlated with the present study.^{[22],[23],[24]} Like the present study (90%), Goyal et al. reported painless hematuria in 91% of cases. So also cases who underwent TURBT (91.4%) were close to Goyal et al. study, (100%) and Atique et al.'s study (100%).^[19],[22] In our study, 42.8% had gross appearance of papillary UC, which was similar to Atique et al. study (44% of cases).

In the present study, histological papillary configuration was found to be the commonest ones (n = 48, 68.6%) followed by solid pattern (n = 13, 18.6%). A study by Schned et al. showed low-grade papillary lesions with a papillary configuration in 60% of tumors and 22.6% in high-grade papillary carcinomas while nonpapillary UC accounted for 10.1%.^[25] Koyuncuer et al. study included 125 patients, that had papillary configuration in 32.9%-pTa, in 45.9%-pT1 of all LG papillary UC and in 5%-pTa, in 20%-pT1, in 17.5%-pT2 of all HG papillary UC.^[26] The current study showed TCC (UC) as major (71.4%) histological type; 42 cases (60%) were HG tumors and 19(27%) LG tumor similar to Thakur et al. study that showed UC in 97.3% of patients, of which 55.5% HG and 44.5% LG tumors.^[27] Cheng and colleagues, Gupta et al. also had similar observations.^[21],[28] In Goyal et al. study 60.41% HG and 32.25% LG and in Sathya et al. study 62.85% HG and 25% LG were also observed.^[28],[29] Similar to the current study, Atique et al. reported 50 cases of UC of which (44%) had advanced-stage tumor invading lamina propria as well as muscles (pT2), followed by 18% noninvasive tumors (pTa) and 38% invasion restricted to lamina propria (pT1). The study by Koyuncuer et al. found >77% of patients with invasive carcinoma were pT1, in contrast to 23% of the patients with pT2, of the total of 125 patients.^[26]

The present study showed association between stage and grade; in 42 cases of HG tumors, 30 cases (71.4%) are associated with the pT2 stage and in 19 cases of LG tumors 16 (84.2%) are associated with pTa stage. It was in corroboration with Sasikumar et al. work, they evaluated 184 bladder cancer patients of which 89.2% were of UC cases. Further, stage pT2 was associated with 86.3% of HG tumors, whilst stage pTa and pT1 comprised 85.71% and 68.56% of LG tumors.^[30]

Out of 30 cases, p53 positive staining was seen in 50% of cases, 30% negative, and 20% equivocal. In Thakur et al. study, 76.4% showed positive p53, 19% showed equivocal and 4.6% showed negative p53 staining.^[28] Halimi et al. analyzed 100 cases of UC with positive p53 staining was seen in 57% of cases.^[31] Reddy reported 45.45% positive and 54.55% negative p53 staining which was in contrast to the present study observation.^[32] A recent study conducted in China showed 86% with high p53 expression and was associated with poor prognosis.^[33] In the current study; when correlated with age, p53 staining showed significant association (P = 0.03). Increased expression of p53 was also seen in HG tumors (92.3%) as compared to the LG tumors (7.7%). In Reddy et al.'s study, out of 33 patients, 91.91% were seen with TCC and 60% of high-grade tumors showed positive p53 expression, Ye et al.'s study 78% p53 positive staining in HG tumors.^[30],[34] In line with the present study report, Vetterlein et al. showed that 77.3% of males and 22.7% of females showed p53 positive, respectively (P = 0.9).^[35] Several studies have failed to find prognostic value of nuclear overexpression of TP53, independent of tumor grade and stage, in patients with superficial bladder tumors.^{[36],[37],[38],[39]} Hence, P53 is suggested as an independent predictor of outcome in invasive tumors.

CD10 expression was negative in 54%, (1+)-23% and (2+) - 23% of cases in the present study. In Bahadir et al.'s study CD 10 expression revealed negative in (57.7%) cases, (1+) - 41.4% and (2+) - 58.6% of cases. Also found 75.20% of CD 10 (2+) score associated with HG tumors.^[10] In the current study, CD 10 positive expression of 1+ score (85.7%) and 2+ score (85.7%) was associated with HG tumors and it was significant (P = 0.001). Mohammed et al. analyzed 49 paraffin blocks of TCC and among these 53.06% was HG and 46.93% was LG.^[40] In their study, 53.06% of positive CD10 expression was associated with HG tumor. Atique et al. also reported that 86.95% of CD 10 (2+) scoring was associated with HG tumors.^[19] A progressive increase in the immunostaining score of CD10 along with an increase in tumor grade suggests that upregulation in antigen expression may occur in higher-grade tumors. Although few literatures are available for CD10 expression in bladder cancers majority show an inverse correlation with tumor grade and a positive correlation with grade has been noted in others.^{[9],[10],[41]} It is thought to be associated with tumor progression in bladder cancer pathogenesis. However, the exact mechanism of action through which it regulates neoplastic processes is yet to know.

In the present study, the Ki-67 labeling index was significantly higher in HG as compared to the LG tumor (32.49 ± 24.35 vs. 6.86 ± 8.1; P = 0.012). Similar to the present study report, Vetterlein et al. reported that the Ki-67 labeling index is significantly higher in HG than that of the LG (45.8 ± 19.8 vs. 29.7 ± 16.1; P = 0.004).^[34] In our study, the Ki-67 labeling index is higher in the pT2 stage in 17 patients with the mean expression of 35.49 ± 27.24 as compared to the pT1 stage in 5 patients with a mean expression of 30.68 ± 34.97 but found to be fairly significant (P = 0.056). Similar to it Thakur et al. showed that positive expression of Ki-67 is higher in pT2 and it was found to be significant (P = 0.0001).^[27] Ki-67 overexpression was statistically significantly associated with advanced pathological stage, higher tumor grade, lymphovascular invasion, lymph node metastases, and increased probability of disease recurrence.^[11]

Conclusion

No single specific diagnostic/prognostic marker has been assigned for TCC; but a cocktail of IHC with P53, CD10, and Ki67 marker can be used as potential prognostic markers as all the biomarkers had shown strong association with higher grade and stage (pT2) of TCC Hence, further studies with the large number of cases should be done to validate current observation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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