Investigation of p53, c-erbB-2, PCNA immunoreactivity, DNA content, AgNOR and apoptosis in bladder carcinoma as prognostic parameters

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Cancer Letters 126 (1998) 143–148

Investigation of p53, c-erbB-2, PCNA immunoreactivity, DNA content, AgNOR and apoptosis in bladder carcinoma as prognostic parameters Meral Koyuncuog˘lu a ,*, Aydanur Kargı a, Sultan Cingo¨z a, Ziya Kırkalı b a

Department of Pathology, Dokuz Eylu¨l University, Medical School, 35340 ˙Inciraltı, ˙Izmir, Turkey b Department of Urology, Dokuz Eylu¨l University, Medical School, ˙Izmir, Turkey

Received 21 August 1997; received in revised form 28 November 1997; accepted 1 December 1997

Abstract The association between known prognostic variables such as TNM stage, histological grade and mutant p53 tumor suppressor gene product, c-erbB-2 oncoprotein, DNA ploidy and cell kinetic data, including mitoses, PCNA expression, AgNOR scores and apoptosis, was investigated in 29 transitional cell carcinoma (TCC) cases. A positive correlation between the histologic grade and all the studied parameters, except for c-erbB-2 expression, and a positive correlation between the stage and histological grade, DNA ploidy, mitoses, apoptosis and p53 expression were found. The results of this study are in accordance with some of the previous studies, except for apoptosis which had been studied for the first time in TCCs. Although we found a statistically significant correlation between the apoptosis and both tumor stage and histological grade, the predictive value of apoptosis as an independent prognostic factor remains to be established in a larger series.  1998 Elsevier Science Ireland Ltd. Keywords: Bladder carcinoma; p53; c-erbB-2; PCNA; DNA; Apoptosis; AgNOR; Mitoses

1. Introduction Transitional cell carcinoma of the bladder is one of the most highly prevalent malignancies in the middleaged population and accounts for more than 45 000 new cases diagnosed per year in the USA. Approximately 70–80% of bladder cancers are superficial without an invasion deeper than the lamina propria. Superficial bladder cancers were divided into pTa and

* Corresponding author. Tel.: +90 232 2595959; fax: +90 232 2590541.

pT1 by UICC in 1978. Sixty to 70% of superficial cancers are of pTa tumors without any invasion of the lamina propria. Large series have confirmed depth of invasion to be a generally useful prognostic index. pT1 tumors have a significantly worse prognosis than pTa tumors and any degree of muscle infiltration significantly worsens survival [1,2]. Large studies have shown a positive correlation between the histologic grade by WHO and the clinical stage by UICC [1–3]. However, TCCs of the bladder vary greatly in their biological behavior. Fifty to 70% of superficial TCCs (pTa/T1) will eventually recur and 10–20% will progress to muscle invasion (pT2 or

0304-3835/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0532-6


M.l. Koyuncuog˘lu et al. / Cancer Letters 126 (1998) 143–148

greater). About 50% of patients with muscle invasive tumors subsequently develop metastases [1,2,4]. Tumor recurrence or progression cannot be predicted by histological criteria alone. Therefore, there has been an extensive search for prognostic parameters in order to more accurately predict the tumor’s biological behavior. The aim of this study was to examine the proliferative indices, including PCNA expression, AgNOR counts and mitotic index, c-erbB-2 oncoprotein and mutant p53 gene protein expression, DNA content and apoptotic index of TCCs and to investigate how they correlate with the tumor grade and stage.

2. Materials and methods The study included 32 bladder cancer biopsy specimens from 29 patients consisting of 29 initial and three follow-up biopsies in three patients with recurrent disease. The biopsies were obtained by transurethral resection in 25 cases, during open surgery in four cases and by cold-cup forceps during control cystoscopy from cystoscopically evident tumors in three cases. The median age was 60 years (range 27–82 years). Twenty-four of the cases were men and five were women. Formalin-fixed paraffin-embedded tissue specimens from all cases were analyzed for patho-

Table 1 The results of all the tested parameters for each case Case no.

1 2 3 4 5 6a 7a 8b 9 10 11 12b 13 14c 15b 16b,c 17b,c 18 19 20a 21 22 23 24 25a 26b 27 28 29b a

Grade (WHO)

G2 G2 G2 G3 G3 G2 G1 G3 G3 G2 G3 G3 G3 G3 G3 G2 G2 G3 G2 G2 G2 G1 G1 G2 G1 G2 G2 G2 G2

Free of disease. Died of disease. c Recurrence. b

Stage (TNM)

T1 T3a T1 T1 T1 Ta Ta T2 T3a T1 T3a T3a T2 T2 T1 T1 Ta T3b T3a T1 T1 Ta Ta Ta Ta T3a Ta Ta T1

Mitoses count (in 1000 cells) 12 5 1 8 11 1 0 2 1 3 2 1 1 26 31 2 0 2 0 0 0 1 0 0 0 0 0 0 0

Apoptosis count (in 1000 cells) 3 1 3 3 4 0 0 3 8 1 20 10 4 12 7 4 4 6 0 0 0 0 0 1 0 1 0 6 0

AgNOR (in PCNA 100 cells) (10 HPF)

80 90 46 90 90 40 95 70 66 34 56 90 100 85 70 150 90 70 90 50 53 60 50 90 65 80 146 140 86

45 – – – 44 – – 159 184 – 891 – 920 749 753 – – 695 – – – – – – – – 6 460 89




– – – – ++ – – ++ – – +++ – ++ +++ +++ – – +++ – – – – – – – – – – –

+ + +++ ++ + + ++ + + + – +++ +++ +++ ++ +++ +++ +++ +++ ++ + ++ ++ + – ++ +++ ++ +++

DNA content

0.3741 0.3512 0.3179 0.3214 0.3309 0.2801 0.2935 0.5370 0.3767 0.3717 0.7591 0.3865 0.4010 0.3519 0.5077 0.3886 0.3150 0.3518 0.4134 0.3099 0.2961 0.3650 0.2873 0.2206 0.3573 0.3374 0.3379 0.2619 0.4441


M.l. Koyuncuog˘lu et al. / Cancer Letters 126 (1998) 143–148

logical grade (WHO) [5] and stage (modified UICC TNM system) [6]. Immunohistochemical studies were performed by the streptavidin–biotin–peroxidase method. PC-10 was used at a dilution of 1:50 for detection of PCNA in paraffin sections. c-erbB-2 and p53 were used at dilutions of 1:100 and 1:50, respectively. All three monoclonal antibodies were obtained from Dakopatts. The colloidal silver nitrate technique was employed for the demonstration of NORs. For the detection of DNA content, gallocyanin chromalum stain was performed. Since both RNA and DNA were stained in this technique, 10% perchloric acid was used in order to eliminate the RNA staining. 2.1. Assessment of staining For the evaluation of p53 and c-erbB-2 antibodies, tumors were classified as negative if there was no staining. The immunoreactive cases were graded as (+) if there were less than 10% stained tumor cells, as (++) if there were 10–50% stained tumor cells and as (+++) if there were more than 50% stained tumor cells. The immunoreactive tumor cells for PC-10 were counted in 10 high-power fields and the cases were graded as low, moderate or high PC-10 reactive if there were less than 500 stained cells, between 500 and 1000 positive cells and more than 1000 positive cells, respectively. For the NOR enumeration, the number of nuclear dots (AgNORs) from 100 randomly selected neoplastic cells were counted using a ×100 oil immersion objective. A microspectrophotometer was used at 550 nm in order to examine the stained DNA content. The number of apoptotic cells per 1000 tumor cells were counted by using a ×100 oil immersion lens objective and mitotic figures per 1000 tumor cells were counted Table 2 Distribution of cases according to stage and histological grade Case (n) G1 (n)

G2 (n)

G3 (n)

Case (n) %

pTa pT1 pT2 pT3a pT3b Total

6 5 – 3 – 14

– 2 5 3 1 11

10 7 5 6 1 29

4 – – – – 4


34.5 ,0.01 24.1 17.3 20.7 3.4 100.0

Table 3 Relationship between mitotic index, apoptotic index and histological grade

Mitotic index (10 HPF) (in 1000 cells) 0 0–5 6–10 .10 Apoptotic index (10 HPF) (in 1000 cells) 0–5% 6–10% .10%

G1 (n)

G2 (n)

G3 (n)


3 1 – –

6 7 – 1

– 7 1 3


1 – –

8 2 –

4 2 2


using a ×40 objective on hematoxylin–eosin stained slides. 2.2. Statistical analysis Univariate analysis, using the Wilcoxon test, the Kruskal–Wallis test and rank correlation, and stepwise multiple regression analysis were performed.

3. Results The results of all the tested parameters for each case are shown in Table 1. The division of our cases according to histological grade and clinical stage is shown in Table 2. A statistically significant relationship between the histological grade and clinical stage (P , 0.01) was found. The relationship between mitotic index, apoptotic index, PCNA positivity, p53 positivity, c-erbB-2 positivity, DNA content and histological grade is shown in Tables 3 and 4. All the investigated parameters, except for c-erbB-2, were found to be positively correlated with histological grade (P , 0.05) when analyzed with the Kruskal– Wallis and Wilcoxon tests. The statistical analysis showed a positive correlation between stage and grade, DNA content, mitoses, apoptosis and mutant p53 expression. No such relationship was found between stage and PCNA, AgNOR and c-erbB-2 expression. The rank correlation between stage and grade and all the other parameters, including DNA content,


M.l. Koyuncuog˘lu et al. / Cancer Letters 126 (1998) 143–148

Table 4 Relationship between histologic grade, p53, c-erbB-2 and PCNA expression Immunoreactivity

Histologic grade G1 (n)

p53 c-erbB-2 PCNA

G2 (n)

G3 (n)













4 – 4

– – –

– 3 –

– – –

15 – 8

– 6 5

– 3 1

– 6 –

3 1 2

– 3 3

3 3 5

4 4 1

mitotic index, apoptotic index, PCNA and p53 positivity, are shown in Table 5. The results of all the tested parameters in the initial and follow-up biopsies of three recurrent cases are shown in Table 6. By stepwise multiple regression analysis a significant correlation between grade and mitotic activity, DNA content and apoptotic index was found. Apoptotic index also showed a significant correlation with stage and also p53 immunoreactivity. Mitotic activity was related to AgNOR counts.

4. Discussion Epidemiological, pathological and clinical evidence supports the pathogenesis of bladder cancer as a multistage process with an often unpredictable course characterized by a significant number of recurrences and frequent evolution toward infiltrating disease. Many prognostic factors have been described in an attempt to predict the recurrence or progression of bladder cancers. Among all the investigated clinical, biochemical, histopathological and cytogenetic factors, the histological grade and stage are the best accepted and most widely used parameters as discriminants of prognostic value [1–9]. It has been proved that the pathological stage is the most potent predictor of survival and the grade is the most sensitive predictor of tumor stage. As it was shown in previous studies, we found a significant correlation between the tumor’s pathological stage and grade [8,10]. Therefore, we have compared the results of all the other investigated parameters with the histological grade and stage in order to assess their prognostic value. Several studies have shown a significant correlation between cell kinetic data, including mitotic indices, PCNA and AgNOR scores and flow cytometric S-

phase fraction measurements, with histologic grade and stage in bladder cancer [10,11]. We have studied the mitotic index, PCNA expression and AgNOR counts and the results of our study are in good agreement with previous studies. It is thought that disease progression is associated with genomic instability. Therefore, measurement of DNA content may be useful in determining progression. In fact, DNA ploidy seems to be an important prognostic marker in bladder cancers and a correlation between DNA ploidy and histopathology has been reported [2,8,12]. Although our results support the relationship between DNA ploidy, histologic grade and stage, it remains to be proven whether this information is additional to conventional histopathology in larger series with follow-up data. The pathogenesis of bladder cancer is mainly unknown. The carcinogens probably cause mutations in the genes involved in growth regulation and abnormal oncoproteins can be detected in bladder cancer. Expression of c-erbB-2 is related to epidermal growth factor and to the rapid proliferation of cancer cells. Although previous studies showed that c-erbB-2 was Table 5 Relationship between tumor stage, grade and prognostic parameters (rank correlation test)

Grade DNA PCNA MI Apoptosis p53 AgNOR c-erbB-2


Histologic grade

,0.01 ,0.01 .0.05 ,0.05 ,0.05 ,0.01 .0.05 .0.05

,0.05 ,0.01 ,0.01 ,0.01 ,0.01 ,0.05 .0.05

Values given are P-values.


M.l. Koyuncuog˘lu et al. / Cancer Letters 126 (1998) 143–148 Table 6 Diagnostic parameters in the initial and follow-up biopsies of cases with recurrence pTG








Initial pT2G3 Recurrence pT3aG3 Initial pTaG2 Recurrence pT1G2 Initial pTaG2 Recurrence pTaG2

0.3519 0.4766 0.5658 0.3886 0.3150 0.3307

749 1320 506 – – –

26 2 2 2 0 0

12 12 0 4 4 0

85 80 140 150 90 90

+++ ++ – – – –

+++ +++ +++ +++ +++ ++

expressed in bladder cancers, its role has not been established [13]. Some studies have shown frequent expression of c-erbB-2 in higher stage bladder cancer, whereas others have shown a less frequent association [9,13]. We did not find a significant positive correlation between the c-erbB-2 expression and histological grade and stage. However, the value of c-erbB-2 expression as an independent prognostic factor was not investigated and its clinical significance remains controversial. Mutations on chromosome 17p 13 in the p53 gene result in increased cellular concentration of abnormal p53 oncoprotein which becomes detectable by immunohistochemical methods. Abnormal p53 protein results in uncontrollable cell growth. p53 gene mutations have been observed particularly in invasive bladder cancers [13,14]. The expression of p53, like that of c-erbB-2, is probably related to rapid proliferation of tumor cells. Our study demonstrated a significant correlation between the mutant p53 expression, histological grade and stage. Apoptosis is the genetically-mediated mechanism controlling deletion of individual cells in normal and malignant cells. Continuous or spontaneous cell death is also an inherent property of malignant neoplasms in which reduction of cell mass may be achieved through necrosis or apoptosis. Experimental studies have shown that apoptosis in tumors, like cell proliferation, may be influenced by numerous oncogenes and tumor suppressor genes, including bcl-2, cmyc, Ha-ras, ABL and p53. It may also be deregulated by a variety of intra- and extracellular factors, but the trigger mechanisms that initiate the process are largely unknown [15,16]. Although the extent of apoptosis has been examined in several experimental tumor types, little data exist concerning the extent of apoptosis in spontaneous human tumors [17]. In a

few studies, a positive correlation between the apoptotic indices and histological malignancy grades were reported for prostatic adenocarcinomas and nonHodgkin’s lymphomas [18,19]. This study constitutes the first study that correlates apoptosis with the histological grade and stage in bladder cancers. Positive correlations between apoptosis and histological grade and stage were found. In conclusion, we have demonstrated a positive correlation between histological grade and stage. In addition, histological grade was correlated with proliferation indices, including mitosis, PCNA expression and AgNOR counts, apoptosis and mutant p53 protein expression and DNA content in bladder cancer, by univariate analysis. By stepwise multiple regression analysis the significant correlations between grade and mitotic activity, grade and DNA content, grade and apoptotic index, apoptotic index and stage and mitotic activity and AgNOR counts were found. However, the clinical significance and the contribution of each of these parameters as a prognostic factor independent from the histological grade and stage are unknown and remain to be proven by further studies. References [1] P.D. Abel, Prognostic indices in transitional cell carcinoma of the bladder, Br. J. Urol. 62 (1988) 103–109. [2] J.A. Witjes, L.A.L.M. Kiemeney, G.O.N. Oosterhof, F.M.J. Debruyne, Prognostic factors in superficial bladder cancer, Eur. Urol. 21 (1992) 89–97. [3] J. Makinen, Y. Collan, A. Heikkinen, Transitional cell tumours of the urinary bladder, Eur. Urol. 4 (1978) 176–181. [4] I. Moch, G. Sauter, D. Moore, M.J. Mihatsch, F. Gudat, F. Waldman, p53 and erbB-2 protein overexpression are associated with early invasion and metastasis in bladder cancer, Virchows Arch. A. Pathol. Anat. 423 (1993) 329–334.


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