Expression of p53 and mdm2 and Their Significance in Recurrence of Superficial Bladder Cancer1

PATHOLOGY

Original Paper

RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp

Expression of p53 and mdm2 and Their Significance in Recurrence of Superficial Bladder Cancer* Burçin Tuna, Kutsal Yörükog˘lu, Emre Tüzel*, Merih Güray, Ug˘ur Mungan*, Ziya Kırkalı* Departments of Pathology and *Urology, Dokuz Eylül University School of Medicine, Izmir, Turkey

Summary

Introduction

The purpose of this study is to evaluate the expression of p53 and mdm2 and to determine whether they may be used as additional predictors of recurrence in superficial transitional cell carcinoma of the bladder. Paraffin sections of 80 patients with superficial transitional cell carcinoma of the bladder, who were treated with transurethral resection, were stained with p53 and mdm2 antibodies using the standard avidin biotin immunoperoxidase method. Nuclear staining for both p53 and mdm2 was calculated as the percentage of labeled nuclei out of a total number of tumor cells counted. The percentage of p53- and mdm2-positive cells showed a significant relationship with tumor grade and recurrence (p = 0.002 and p = 0.016; p = 0.01 and p = 0.003, respectively). In addition, a weak inverse relationship was found between p53 and mdm2 values (r = –0.184). p53 and mdm2 reactivities are valuable parameters in predicting recurrence in superficial bladder cancer. Thus, mdm2 expression appears to play a role in predicting biologic behavior in superficial transitional carcinoma of the bladder.

Bladder cancers are heterogeneous with respect to their histopathologic characteristics, morphologic growth pattern, and clinical behavior. Alterations in proto-oncogenes and tumor suppressor genes are thought to play a role in bladder cancer pathogenesis [23]. The p53 gene, a tumor suppressor gene, is mapped to chromosome 17q13. In various tumors such as breast, colon, and gastric carcinoma, p53 alteration has proven to be an independent prognostic factor [22, 30, 32]. The prognostic significance of p53 alteration with respect to disease recurrence, progression, and death owing to bladder cancer has been evaluated by numerous investigators, and p53 overexpression was shown to be related to poor prognosis in both superficial and muscle-invasive bladder cancer [15, 20, 21, 25, 26]. The mdm2 gene, a proto-oncogene located on chromosome 12, encodes for a 90-kd nuclear protein. Mdm2 proto-oncogene was originally isolated as an amplified sequence in spontaneously transformed murine cell line [19]. Mdm2 overexpression may be considered an alternate pathway for p53 inactivation [16, 21]. The results regarding the functions of p53, mdm2, and prognosis, described in various studies, are controversial.

Key words: Bladder cancer – Recurrence – p53 – mdm2

*

Supported by a grant from Dokuz Eylül University Research Fund (Grant #: 0909.20.03.04). Pathol. Res. Pract. 199: 323–328 (2003)

Address for correspondence: Burçin Tuna, Dokuz Eylul University, School of Medicine, Department of Pathology, Inciralti, 35340 Izmir, Turkey. Phone and Fax: +90 232 259 59 59-3447. E-mail: [email protected] 0344-0338/03/199/05-323 $15.00/0

324 · B. Tuna et al.

In this study, we examined their expression in pTa and pT1 primary bladder cancers, as solid data is lacking. The results were correlated with stage, grade, tumor size, numbers, and the presence of carcinoma in situ (CIS). Furthermore, we evaluated the prognostic impact of p53 and mdm2 expression on the prediction of recurrence.

Materials and Methods Eighty patients with pTa-pT1 primary bladder TCC were included in the study. Of the 80 patients, 77 were men (96%), and the mean age was 64 years (range 27–87). Histopathologic evaluation revealed pTa tumors in 40% and pT1 in 60%. Histologic differentiation was grade I in 30 (38%) tumors, grade II in 36 (45%), and grade III in 14 (18%) tumors (Table 1). After initial transurethral resection (TUR), patients were followed 3 times per month in the first year, 4 times per month in the second year, 6 times per month for 2 years, and yearly thereafter. This schedule was repeated for patients with recurrence. Tumors were staged according to the TNM staging system [29] and graded according to the grading schema proposed by World Health Organization (WHO) [18]. The presence of carcinoma in situ in random biopsies was noted. The number of recurrences, tumor progression, and the time until the first recurrence were determined. The time until the first recurrence was defined as the period between the first TUR and the first recurrence. Recurrent tumors with invasion of the detrusor muscle or beyond (pT2, pT3, and pT4) were accepted as tumor progression. Primary tumor size and number were assessed cystoscopically. The histological sections of all cases were reviewed, and the tumor tissues obtained from the paraffin blocks were mounted onto polyL-lysin-coated slides. The standard streptavidin biotin immunperoxidase method was used for immunostaining with p53 (NeoMarker, 738P007, California, USA; dilution 1/200), and mdm2 (NeoMarker, 291P906, California, USA; dilution 1/50) antibodies. The sections were deparaffinized and rehydrated; endogenous peroxidase activity was blocked using a 0.3% solution of hydrogen peroxide at room temperature for ten minutes. The sections were then heated in citrate buffer (0.01 mol/l, pH 6) for epitope retrieval in a microwave oven (3 times for 10 minutes at 700 W), then allowed to cool to room tem-

perature for 20 minutes. Primary antibodies were applied for 30 minutes at room temperature and washed in TRIS buffer. Linking antibody and streptavidin peroxidase complex (DAKO LSAB Kit, K0675, Carpintera, California, USA) were added consecutively for 10 minutes at room temperature and washed in TRIS buffer. Peroxidase activity was visualised with 3,3′-diaminobenzidine tetrachloride (DAB; Sigma Chemical Co, St. Louis, Mo., USA). The sections were counterstained with Mayer’s hematoxylene. Known positive controls (breast carcinoma for both p53 and mdm2 ) were also stained simultaneously. Negative controls were stained by ommitting the primary antibody incubation. Any dark, brown nuclear staining was considered positive, indicating abnormal stabilization of p53 or overexpression of mdm2 (Figs. 1 and 2, respectively). Nuclear p53 and mdm2 immunos-

Fig. 1. Nuclear p53 expression in transitional cell carcinoma. Original magnification ×200.

Table 1. Histopathologic distribution of tumors Patologic stage

Grade –––––––––––––––––––––––––––––––––––––––––– 1 2 3

Total

Ta T1 Total

22 8 30

32 48 80

7 29 36

13 11 14

Fig. 2. Nuclear staining of mdm2 in tumor cells. Original magnification × 200.

p53 and mdm2 Expression in Superficial Bladder Cancers · 325

taining were calculated as the percentage of labeled nuclei out of the total number of tumor cells counted [11]. The correlation of p53 and mdm2 expression with conventional prognostic factors (pathologic stage, histologic grade, presence of carcinoma in situ, recurrence and progression) were evaluated by χ2 test. The relationship of various parameters, including age, sex, tumor size, and number, was investigated by one-way ANOVA test. The correlation between clinicopathologic parameters was assessed using Spearman correlation test. Recurrence-free periods were calculated by Kaplan-Meier curves. p values of < 0.05 were considered significant. The variables were grouped so that they could be applied to statistical analysis according to the best cut-off values obtained from corresponding single-factor receiver operating characterictics (ROC) curves with respect to the end-points; cut-off levels were 5% and 20% for p53 and mdm2 indices, respectively. The significant parameters were further evaluated by Cox proportional hazards model, and independent prognostic factors for recurrence were defined. For statistical analysis, the commercially available software Scientific Package for Social Sciences (SPSS 11.0) was used.

tients (8%) had three, and 5 patients (6%) had four recurrences. As only 2 patients had invasive progression, we did not perform statistical analysis for this parameter. The median follow-up was 27 months (range 1 to 115; Mean ± SD = 32.43 ± 20.08). Seventy-seven percent and 71% of the tumors showed positive staining with p53 and mdm2 antibodies, respectively (Table 2). The percentage of p53-positive cells showed a significant relationship with tumor grade and recurrence. Thus, p53 immunoreactivity was higher in high grade and in recurrent tumors than in low grade and in non-recurrent tumors (p = 0.002 and p = 0.016, respectively). High percentage p53 staining predicted recurrence with a sensitivity of 92% and specificity of 39% (positive predictive value = 62%, and likelihood ratio = 1.51). (Fig. 3). The percentage of p53 immunoreactivity did

Results The size of the primary tumor ranged between 5 and 70 mm (Mean ± SD = 29.2 ± 17.2). The number of the primary tumor ranged between 1 and 10 (Mean ± SD = 3.6 ± 2.9). Thirteen (14.5%) had carcinoma in situ in the epithelium adjacent to the tumor or in random biopsies. Forty-two of the tumors (47.7%) developed 1– 4 recurrent tumors within 1–29 (7.5 ± 7.6) months. Sixteen patients (20%) with recurrent tumors and 21 patients (26%) without recurrence had intracavitary epirubicin or BCG installations (p = 0.17). Patients undergoing intracavitary therapy regimen were selected randomly in the scope of a clinical study protocol. Twenty-two patients (28%) had one, 9 patients (11%) had two, 6 paTable 2. p53 and mdm2 expressions according to histologic grade and recurrence status

pTaG1 pTaG2 pTaG3 pT1G1 pT1G2 pT1G3

n

Recurrence ––––––––––––––––––––––––––– p53* mdm2*

Recurrence + ––––––––––––––––––––––– p53* mdm2*

22 7 3 8 29 11

5/7 1/3 1/1 2/2 9/4 3/0

5/5 2/1 0/1 2/2 5/11 5/3

5/7 2/2 0/2 0/4 5/8 2/1

6/4 3/0 1/0 1/3 11/5 7/1

*: the numbers representing: p53 < 5%/p53 > 5%, mdm2 < 20%/mdm2 > 20%

Fig. 3. Prognostic relevance of a) p53, and b) mdm2 immunohistochemistry in 80 patients with superficial transitional cell carcinoma (a) p = 0.016, (b) p = 0.03

326 · B. Tuna et al.

not correlate with stage and CIS. On the other hand, an inverse relationship was found between mdm2 immunoreactivity and tumor grade and recurrence (p = 0.01 and r = – 0.321; p = 0.003 and r = – 0,408, respectively). There was no correlation between mdm2 expression and tumor size, number, tumor stage, and the presence of carcinoma in situ. In addition, a weak inverse relationship was found between p53 and mdm2 expressions (r = – 0.184). When the patients with CIS were excluded, similar results were achieved. Both p53 and mdm2 expression correlated with recurrence (p = 0.05, p = 0.046, respectively). The time until the first recurrence for p53 < 5% cases ranged between 1 and 21 months (Mean ± SD = 5.58 ± 5.54). In p53 > 5% cases, recurrences developed between 1 and 29 months (Mean ± SD = 9.33 ± 8.95). For mdm2 < 20% and mdm2 > 20% cases, the time until the first recurrence ranged from 1 to 27 and from 3 to 29 months, respectively (Mean ± SD = 7.04 ± 7.42; 8.75 ± 8.43, respectively) Cox proportional hazards model was applied with p53 and mdm2, which were prognostically significant in univariate analysis for evaluating the interaction of these factors with respect to recurrence. Multivariate analysis showed that both parameters were not independent prognostic factors.

Discussion The biologic behavior of superficial bladder cancer is dependent on several prognostic factors, such as tumor number, size, grade, the presence of carcinoma in situ, and prior recurrence rate [13]. In a recent study of the same patient population, we found the recurrence rate to be associated with increased tumor number [24]. The time until the first recurrence was related to tumor size, proliferative activity, and tumor grade. Higher tumor grade was correlated with increased tumor number, proliferative activity, and tumor stage. Tumor size and proliferative activity was found to be useful in the estimation of the time until the first recurrence, and tumor grade was helpful in predicting prognosis [24]. Although these indicators of prognosis were found to be the most important factors in predicting the recurrence and the progression of superficial bladder tumors, the biologic basis for the subsets of disease is not completely understood [8, 12, 33]. Cellular proliferation is regulated by cell cycle associated protein complexes. p53 is a tumor suppressor gene and has a vital role in the regulation of the cell cycle. Alterations in p53 gene result in the production of an abnormal and usually dysfunctional protein, consequently causing cell cycle arrest [4]. Loss of cell cycle control causes cells to proliferate [28]. As a conse-

quence, an additional event such as p53 inactivation would transform a small percentage of these cells into a more invasive stage, leading to clinical tumor progression. This model for bladder tumor progression has important implications, because some tumors share the same initiation event. Loss of cell cycle control appears to occur at the early stages of tumorigenesis and subsequently leads to tumor progression. The prognostic value of p53 in muscle–invasive TCC of the bladder is well described [5, 21]. In addition, mutations in p53 have been detectable in over 50% of muscle-invasive tumors and carcinoma in situ. However, the role of p53 overexpression as a prognostic indicator in superficial TCC is not clear [8, 12]. The prognostic impact of p53 in superficial bladder cancer is controversial [2, 9, 12, 27]. Furthermore, several studies have demonstrated that p53 reactivity is increased in higher grade and stage bladder cancer and is associated with disease progression [3, 6, 17, 21]. Current data suggests that immunohistochemical assessment of p53 may be useful in the management of locally advanced bladder tumors and in the selection of patients for possible bladder preservation or adjuvant chemotherapy [1]. Although the precise functions of the mdm2 gene product are not completely understood, it has been shown that mdm2 protein has a tumorigenic potential [7]. It has been demonstrated that mdm2 protein binds directly to the p53 protein and inhibits its transactivating ability [19]. Mdm2 protein was found to interact with RNA and to bind many cellular proteins, such as p53. mdm2 aminoterminal transactivating region can interact with the aminoterminal transactivating region of p53 and neutralizes the tumor-suppressor activity of the latter. In DNA-damaged cells, the overexpressed p53 transactivates mdm2 gene, and the induced mdm2 protein downregulates wild-type p53 functions [31]. Overexpression or amplification of mdm2 gene is common in tumors such as soft tissue sarcomas and lymphoblastic leukemia; it is less common in bronchogenic carcinoma and bladder cancer [14] and has been related to more aggressive behavior and poorer survival in these diseases. The prognostic value of mdm2 was has also been investigated in superficial bladder cancer in a small number of studies, and mdm2 overexpression was found to be more common in lowgrade lesions and in papillary Ta tumors than in muscleinvasive tumors [10, 22]. Mdm2 gene expression may be an early event in urothelial tumorigenesis. This is in contrast to p53 overexpression, which is a late event in bladder cancer progression. This finding suggests that mdm2 expression may have a potential role in tumorigenesis. In this study, mdm2 values were lower in high-grade and in recurrent tumors, whereas p53 immunoreactivity was found to be higher in these lesions. As previously

p53 and mdm2 Expression in Superficial Bladder Cancers · 327

mentioned, our data supports the idea that mdm2 expression may be an early event in bladder cancer progression, whereas p53 expression is a late event in bladder cancer progression. Our results suggest that p53 and mdm2 reactivities may be valuable parameters in predicting recurrence in superficial bladder cancer. Our data supports the hypothesis that p53 inactivation and mdm2 overexpression are alternative mechanisms that inactivate the same regulatory pathway for cell growth suppression. Thus, mdm2 expression seems to play a role in predicting biologic behavior in superficial transitional carcinoma of the bladder. Continuous overexpression of mdm2 appears to result in the inactivation of p53 without gene aberration. P53 gene inactivation may require mdm2 overexpression, and persistent overexpression of mdm2 causing prolonged inactivation of p53 may be considered an alternate pathway for p53 inactivate or dysfunction. Further research is required to determine the true prognostic value of mdm2 oncoprotein expression in superficial and invasive bladder cancer.

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