Expression of Ki-67, C-erbB2 and EGFR in TCC of the urinary bladder and their correlation with tumor grading

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Article Information:

Group: 2008
Subgroup: Volume 10, Issue 2
Date: April 2008
Type: Original Article
Start Page: 95
End Page: 99


  • SA Shamsdin
  • Department of Immunology, Fasa University of Medical Sciences, Fasa, Fars, Iran
  • D Mehrabani
  • Gastroenterohepathology Research Center, Shiraz University of Medical Science, Nemazee Hospital, Shiraz, Fars, Iran
  • M Hosseinzadeh
  • Department of Pathology, Fasa University of Medical Sciences, Fasa, Fars, Iran


      Affiliation: Department of Immunology, Fasa University of Medical Sciences
      City, Province: Fasa, Fars
      Country: Iran
      Tel: 98-731-2220994-6
      Fax: 98-731-2227091


Background: Transitional cell carcinoma (TCC) of the bladder is the most common malignancy of the urinary tract. Staging and grading of TCC of the bladder and its biomarkers can be indicators of the prognosis. This study was performed to evaluate the expression of Ki-6, EGFR and c-erbB2 in bladder TCC and their correlation with the tumor grading using immunohistochemistry method.


Methods: Immunohistochemical staining of paraffin blocks of 60 cases of primary TCC was performed and the results were correlated with grading of the tumor on H & E staining.  


Results: The expression of EGFR was significantly higher than c-erbB2 and Ki-67 in TCC of the bladder (p<0.05). This increase was higher in 3 and 4 grades for EGFR and c-erbB2 when compared with EGFR and Ki-67 (p<0.05).


Conclusion: Expression of EGFR and c-erbB2 is useful in the prognosis of grading and tumor aggressiveness.

Keywords: Transitional cell carcinoma; Ki-67; c-erbB2; EGFR; Grading; Bladder

Manuscript Body:



Transitional cell carcinoma comprises 90% of all primary tumors of the urinary bladder. Among the environmental causal factors, chemical agents are the most important ones.1 Therefore, this tumor is more common in industrial areas especially the petrochemicals and aryl amines.2,3 Cigarette smoking, opium and other environmental factors such as aniline dyes play a role in TCC of the bladder. Most cases of TCC of the bladder are present in patients older than 50 years but they can also occur in young adults and even children.4,5 Men are affected more often than women and whites more than blacks. Painless hematuria is the most common form of clinical presentation.6 Identification of biomarkers may improve the screening and diagnosis of TCC, characterizing the malignancy and determining the prognosis. Biomarkers should be non-invasive, rapid, easy, and accurate with high sensitivity and specificity. The validity of biomarkers is measured by their ability to differentiate the stage and grade of the tumor.7 Proliferating antigens (ki-67 antibody, proliferation cell nuclear antigen) and the murine monoclonal antibody ki-67 react with nuclear antigen that is related to cell proliferation. Increased ki-67 immunostaining in TCC has been reported with higher tumor grade, stage, and recurrence.5,8

The EGFR is part of a subfamily of four closely related receptors including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). The receptors exist as inactive monomers, which are dimerized after ligand activation. This causes homodimerization or heterodimerization between EGFR and another member of the erb receptor family. After ligand binding, the tyrosin kinase interacellular domain of the receptor is activated, with autophosphorilation of the intracellular domain, which initiates a cascade of intracellular events.9 Oncogenes (C-erbB2, C-MYC, and C-ras) of malignant transformation can be due to genetic changes. C-erbB2 is a transmembrane glycoprotein over-expressed in high stage and grade of bladder tumors.10,11 Among growth factors (EGF, TGF-ß, FGF and VEGF), EGFR is a transmembrane glycoprotein activated by binding of either EGF or TGF-α.. In malignancies, the pattern of distribution of EGFR involves all layers. Several reports have shown that up-regulated expression of EGFR in bladder cancer tissue is correlated with an increase in tumor stage and grade.7 Increased expression of C-erbB2 oncogen product has been found immunohistochemically in 10% of the grade II tumors, in 60% of the grade III, and in 100% of the adenocarcinomas.12



Materials and Methods


From Jan. 2004 to Dec. 2006, the paraffin blocks of 60 patients with primary superficial TCC of the urinary bladder referring to Nemazee or Faghihi hospitals affiliated to Shiraz University of Medical Sciences and Shariati Hospital affiliated to Fasa University of Medical Sciences in southern Iran were enrolled. The patients mean age was 62 years, and the male:female ratio was 3.6:1. The patients' follows up were determined as the number of months from the date of the diagnostic surgical procedure to the date of the most recent cystoscopy (48 to 120 months). Tumor recurrence was defined as reappearance of the tumor after the initial treatment with at least one tumor free cystoscopy. Survival was the period between diagnosis and death. Cancer related death was that caused by bladder carcinoma. The end point of our study was disease free, progression free, and overall cancer specific survival. Tumor size was defined as the largest tumor measure and the specimens were stratified as < 3 cm, 3-5 cm, and > 5 cm. Hematoxylin and eosin stain compromised primary tumors and their recurrences were re-evaluated by pathologists. The resulting grade and stage were in accordance with the 2004 WHO.

After selection of proper satisfactory paraffin blocks, the sections were deparaffinized, rehydrated and washed in PBS (pH=7.2). Dew was axed in xylene, rehydrated in graded ethanol, and washed for five minutes with phosphate buffer saline. For antigen retrieval, the sections were boiled in 10 mM citrate buffer (pH=6). Endogenous peroxides were blocked by incubation of the slides for 30 minutes with 3% hydrogen peroxide in methanol. After washing with TBS (Tris Buffer Saline), the sections were incubated with the primary mouse monoclonal antibodies against C-erbB2 (Dako Cytomation, Glostrup, Denmark; final dilution, 1:300), EGFR (DakoLtd, Ontario, Canada; final dilution, 1:200) and Ki-67 (Dako Cytomation, Glostrup, Denmark; without dilution). While, incorporating positive and negative controls, after 30 minutes the sections were washed twice at room temperature and were incubated with secondary antibodies, biotinylated goat antibody to mouse immunoglobulins, for 30 min. After washing, the slides were incubated with the streptavidin HRP complex (horseradish peroxidase) for 30 min. Diaminobenzidin solution was used as the final chromogen, and the sections were counterstained with Mayer’s hematoxylin before mounting. C-erbB2 and EGFR cell membrane specific immunoreactivity were scored by estimating the percentage of positive tumor cell as follows: score 0, no immunoreactive cell; score +1, positivity in < 5% cancer cells; score +2, positivity in 5-50% cancer cells; and score +3, positivity in > 50% of cancer cells. The specimens were considered positive for EGFR and C-erbB2 expression when the score was +2 or +3. The nuclei in morphologically malignant cells were considered positive for the ki-67 antigen when they showed dark brown granular staining.

The association between the different pathological characteristics and the expression of EGFR, C-erbB2 and ki-67 were correlated with expression of markers and WHO grading was undertaken using SPSS software, version 13 (Chicago, IL, USA). Statistical significance was evaluated by Chi-Square test. AP value < 0.05 was considered significant.





The mean age at diagnosis (13 women and 47 men) was 62 years (range, 31-91). Most of patients were in age range of 61-71 years (P=0.001). The male-female ratio was 3.6:1 (P=0.04). The difference between two sexes and the age groups was statistically significant. According to the last recommendations of WHO, 13 (21.7%) tumors were classified as papilloma (G1), 17 (28.3%) as papillary neoplasm of low malignant potential (grade G2), 10 (16.7%) as low grade carcinoma (grades G3), and 20 (33.3%) tumors as high grade carcinoma (grade G4). Tumor size ranged from 1 to 8 cm (mean: 4.02). In relation to stage progression and recurrence, there was an association between grade and sex, age and over-expression of EGFR (P<0.05). EGFR expression was observed in 32 out of 60 (53.3%) cases (Figure 1). A strong correlation was observed between increased EGFR expression and high grade tumors (P<0.05, Table 1). 35 out of 60 (58.3%) patients showed expression of C-erbB2. This expression was not statistically significant (P=0.08, Figure 2) A strong correlation was found between increased C-erbB2 and increased expression of EGFR. The association of C-erbB2 and EGFR expression was observed in high grade tumors (grade III and IV) (P<0.05) (Table 1). Ki-67 was positive in 25 out of 60 (41.7%) patients. Expression of Ki-67 and c-erbB2 were all associated with an increased tumor grade, but this difference was not statistically significant (Table 1).


Fig 1: EGFR: IHC, Avidin Biotin Method (x 400).



Table 1: Relationship between expression of Ki-67, EGFR, C-erbB2 and Grade of tumor in transitional cell

P value

Grade 4

Grade 3

Grade 2

Grade 1



(20) % 

(10) %

(17) %

(13) %

P=0. 5

(10) 50

(4) 40

(6) 35.3

(5) 38.5



(17) 85

(8) 80

(4) 23.53

(3) 23.08



(14) 70

(6) 60

(8) 47.05

(7) 53.85



(20) 100

(10) 100

(12) 70.6

(9) 69.3



A P value <0.05 was considered significant, EGFR: Epithelial Growth Factor Receptor


Fig 2: C-erbB2: IHC, Avidin Biotin Method, (x 400).



During the two year follow up, and after all patients had been treated with BCG, 20 (34%) patients remained free of disease but 10 (17%) had recurrence of their tumor. The stage progression was seen in 30 (50%) patients and in their follow up re-biopsies. EGFR expression was found in 24 out of 32 (75%) metastatic patients (p<0.005), whereas C-erbB2 expression was found in 18 out of 35 (51%) metastatic patients. A significant association was found between EGFR expression and a lower survival. In contrast, C-erbB2 expression was not significantly correlated with the disease relapse (p=0.06). Over-expression of the cell cycle marker, ki-67 (15%) was associated with a slightly increased risk of recurrence, but this difference never reached a statistical significance.





Different studies indicated that alteration in cell cycle regulation is a key event in determining of biological behavior of bladder cancer.13,14 There are many known environmental factors that play significant role in the genesis of this tumor. For future planning and increasing the survival of patients with bladder cancer, determination of prognosis of the disease seems necessary which expression of EGFR and C-erbB2 will be crucial. Although traditional prognostic factors such as grading, staging, age, sex, invasion, etc are very important for this purpose.1

In the present study, ki-67 was overexpressed in 41.7% of patients and no significant correlation was found between the ki-67 index and tumor grade, however was noticed in patients with tumors and a high ki-67 index value. Only a few studies have investigated the issue of immunohistochemical markers in predicting the outcome of TaT1 bladder urothelial cell carcinoma since the introduction of the WHO grading scheme for bladder urothelial tumors.15,16 Studies have focused mainly on P53 and ki-67 and suggested that they might be related to progression and recurrence, respectively. However, studies including survival analysis are limited by Quintero et al.5 Recently; molecular phenotyping has provided another dimension to the characterization of the biological potential of tumors that may help a better prediction of their clinical outcome. Our results are also consistent with previous reports by Tsuji et al. and okamura et al.17,18 Pich et al. found that the ki-67 was the best predictor of recurrence in noninvasive bladder tumors.19 Yan et al. suggested that the ki-67 could be used to identify higher risk of a first recurrence.20 Van Rhijn et al. found that a high proliferation was related to a poor survival.21

In this study, we evaluated the expression of both EGFR and C-erbB2 in four different grades of TCC by immunohistochemistry. Moreover, EGFR and C-erbB2 were evaluated as potential prognostic indicators of disease progression. A significant increase in EGFR expression was observed in patients with increased tumor grade (grade III and IV). These results are in agreement with reports of Qiuseppe et al. and Scher et al. in prostate cancer patients.22,23

Our study showed a significant association between EGFR expression and lower survival, and also we found C-erbB2 expression not to be significantly correlated with diseases relapse. Popov et al. showed that EGFR expression was significantly correlated with the disease relapse in patients.24 These results are in agreement with research of Qiuseppe et al. in prostate cancer.25 Lipponen also found that C-erbB2 expression predicted recurrence-free survival in superficial tumors, and in survival analysis moderate or intense expression of C-erbB2 oncoprotein was related to decreased survival probability.25 Expression of some of the molecular factors such as P53, Ki-67, c-erbB2, EGFR, MDM2 and so on have significant role for determination of prognosis of superficial bladder TCC.26

In this study we found that after patient's treatment with BCG, 56.7% patients remained free of disease and 16.7% of patients had recurrence of their tumor. These results are in agreement with a research of Scher et al.23

We showed that C-erbB2 and EGFR immunostaining in superficial urothelial carcinoma of the urinary bladder were correlated well with the grade of tumor and can be used as an important prognostic factor. Over expression C-erbB2 is a target for treatment with Herceptin.





This project was supported by a grant from Fasa University of Medical Sciences (grant no. 17) and in part by Shiraz Institute for Cancer Research. We would like to thank Fars Hospital-based Cancer Registry Personnel at Nemazee Hospital too.

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