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ORIGINAL ARTICLE
Year : 2010  |  Volume : 26  |  Issue : 2  |  Page : 206-212
 

Can p53, Ki-67 and bcl-2 predict biochemical failure after radical prostatectomy?


1 Hacettepe University Medical Faculty Urology Department, Ankara, Turkey
2 Hacettepe University Medical Faculty Pathology Department, Ankara, Turkey

Date of Web Publication12-Jul-2010

Correspondence Address:
B Baseskioglu
ESOGU Med.Fac. Urology Department, Ankara
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-1591.65390

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   Abstract 

Background and Objective : To analyze p53, Ki-67 and bcl-2 expressions immunohistochemically and their predictive role in biochemical recurrence after radical prostatectomy.
Materials and Methods : Seventy one patients who had undergone radical prostatectomy between 1992 and 2001 were randomly selected. Tissue microarrays were constructed from their radical prostatectomy specimens. They contained four cores from neoplastic and additional four cores from corresponding non-neoplastic regions. Gleason score ranged from 6-9, and pathological stage ranged from T2N0Mx to T3BN1. Staining for bcl-2 was scored visually taking percent negative, weak, moderate and strong positivity into consideration. Strong immunoreactivity was considered positive for p53. Ki-67 index was measured as the percentage of positive nuclei among tumor cells. Statistical analysis was performed to explore correlations between staining patterns and clinicopathological prognostic parameters.
Results : The follow-up period extended from 13 to 112 months with a mean 60 (48 ± 23, 2) months. Of all, 38.02% had no evidence of disease, 52.1% were alive with disease and 9.8% were died during follow-up. The expression of p53, Ki-67 and bcl-2 in tumors were 39%, 76% and 5% respectively. While the secretory layer showed negative or weak bcl-2 staining in most cases, expression in basal cells was often stronger. Statistical analysis revealed differences in staining between normal and carcinoma for all three markers. There was no correlation between staining patterns and time to biochemical relapse. On the other hand, cases with higher Gleason sum showed the tendency for over expression of p53, Ki-67 and bcl-2 although the differences were not statistically different. Multivariate analysis revealed CMS group and seminal vesicle invasion as the independent predictors of PSA failure (log rank P = 0.0039 and P = 0.001, respectively).
Conclusion : The proteins bcl-2, p53 and Ki-67 were expressed at a different rate in normal and neoplastic prostate tissue. Bcl-2 was mainly expressed by basal cells in normal glands. p53 and Ki-67 expression were increased in most prostate carcinomas. However, overall expression levels did not correlate with biochemical recurrence in this study.


Keywords: Bcl-2, ki-67, microarray, p53, prostate cancer


How to cite this article:
Baseskioglu B, Akdogan B, Baydar D E, Ozen H. Can p53, Ki-67 and bcl-2 predict biochemical failure after radical prostatectomy?. Indian J Urol 2010;26:206-12

How to cite this URL:
Baseskioglu B, Akdogan B, Baydar D E, Ozen H. Can p53, Ki-67 and bcl-2 predict biochemical failure after radical prostatectomy?. Indian J Urol [serial online] 2010 [cited 2019 Jun 26];26:206-12. Available from: http://www.indianjurol.com/text.asp?2010/26/2/206/65390



   Introduction Top


Prostate cancer is the most common malignancy throughout the world among the males over middle age and the second leading cause of cancer-related deaths in western countries. Radical prostatectomy (RP) is the most preferred treatment option for patients with localized prostate cancer. Widespread usages of nerve-preserving technique resulted in a decrease in post-operative complications and increase in quality of life concerning predominantly continence and erectile functions, which made the technique more popular. However, 30-56% of the patients will recur in the first decade following surgery and even some will develop metastatic disease. [1] Many studies focused on to find accurate predictors of recurrence after RP over the past decade. The role of PSA and histopathological parameters in prognostic stratification of patients is still limited. New molecular-genetic researches are expected to bring new prognosticators to better define patients who will have more benefit from the surgery or who are in high risk group for the recurrence and could be better if treated with other modalities.

Certain studies focused on P53, Ki-67 and bcl-2 expressions to clarify the unknowns in biology and molecular genetics of prostate cancer. Conflicting evidence exists regarding prognostic influence of those markers. Some showed that they were significant prognostic parameters determining the recurrences, [2] whereas others have reported insignificant correlations. [3] Today, there is no unfailing indicator that can be used to foretell the disease prognosis in daily uro-oncology clinical practice.

In this study, the value of these three cell cycle proteins to predict recurrence was assessed together with other clinical and the histopathological parameters in intermediate and high risk prostatic carcinoma patients treated by radical prostatectomy at a single tertiary care center.


   Materials and Methods Top


A total of 71 randomly selected prostate carcinoma patients who had been defined in intermediate to high risk group according to D'amico et al . criteria [4] and undergone radical prostatectomy (RP) between 1992 and 2001 were included in this study. Surgery was performed with retropubic approach, under the supervision of the same surgeon (HO). None of the patients received pre-operative hormonal treatment or radiotherapy. Sixty nine cases had pelvic lymphadenectomy. Pre-operative data including demographic, clinical and laboratory records were collected on all patients from the hospital charts.

Histopathological features of all cases were re-evaluated by the single pathologist (DEB). Tumor volume (estimated by simple eye-balling), Gleason score, localization, status of uni- versus bilaterality, focus number, surgical margins, presence of extra-prostatic extension, lymphovascular invasion, seminal vesicle invasion, lymph node metastasis and pathological stage (according to 2002 TNM classification) were noted.

Tissue microarray (TMA) construction

Tissue microarrays were constructed by 0.6 mm tissue cores from 71 radical prostatectomies using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD). They contained four cores from the index tumor (defined as most significant cancer nodule which was the largest tumor focus with the highest Gleason score and stage) and additional four cores from corresponding non-neoplastic regions for each case. Control tissues were placed in an orderly pattern throughout each array. Four-΅m sections from the array blocks were cut for H-E and immunostaining. H-E sections were scanned to record the tissue type (non-neoplastic normal glands, prostatic intraepithelial neoplasia and adenocarcinoma) represented in each core.

Immunohistochemistry

A total of 4-μm thick tissue sections taken from TMAs on charged slides were deparaffinized and rehydrated. Slides for bcl-2 were heated in citrate buffer steam (0.01 M, pH 8.0) for 20 min; the ones for p53 and Ki-67 were exposed to high temperature target retrieval in Dako target retrieval solution (cat#S1700) for 40 minutes to accomplish antigen unmasking. Endogenous peroxidase activity in sections was inactivated by Dako Endogenous Enzyme Block (S2001) for 5 min. Then the sections underwent incubation with primary antibodies. Anti-p53 antibody (Dako cat# M7001) was used at a dilution of 1:800 in antibody diluents buffer (ChemMate ADB250) for 45 minutes. Anti-Ki-67 antibody (Zymed cat# 18-0192) was applied at a dilution of 1:400 with overnight incubation at 4΀C. Anti-bcl-2 antibody (Dako cat# M0887) was used at a dilution of 1:200 for 45 minute incubation. They were then washed in PBS (phosphate buffered saline). Specific immunoperoxidase stainings were developed using the DAKO Envision+ system (K 4007 horseradish peroxidase, Mouse Envision 3, 3'-diaminobenzidine Plus kit, DAKO) with 3, 3'-diaminobenzidine (DAB) as chromogen. DAB-stained slides were counterstained with hematoxylin, dehydrated, and mounted with a cover-slip.

Quantization of immunohistochemistry

Immunostainings were evaluated microscopically for the represented tissue types (normal glands, prostatic intraepithelial neoplasia or carcinoma) in each core in the tissue microarrays. Intense nuclear staining involving more than 1% of the cells was considered positive for p53. Ki-67 labeling index (labeling frequency %) for benign and neoplastic areas was calculated by the following formula: [number of positive nuclei/total number of represented cells] Χ100. <10% was scored as 1+ while ΃ 10% as 2+. Intensity and extent of cytoplasmic staining were recorded for bcl-2, visually taking per cent negative, weak, moderate and strong positivity into consideration. Moderate or intense level of staining involving more than 5% of the cells were considered positive, anything less than that was regarded as negative.

For the statistical analysis, Statistical Package for Social Sciences (SPSS) version 15.0 was used.


   Results Top


Mean age and PSA values (±sd) at diagnosis were; 62.1 ± 6.5 years and 9.8 ± 6.5 ng/ml, respectively. Gleason score ranged from 6-9, and pathological stage ranged from T2N0 to T3BN1. Grade and stage characteristics of the cases were displayed in [Table 1]. Five patients had pelvic lymph node metastasis. Extraprostatic extension, seminal vesicle invasion, lymphovascular involvement and positive surgical margins were identified in 46 (64, 7%), 18 (25, 3%), 11 (15, 4%) and 31 (43, 7%) of the radical prostatectomies respectively.

Mean clinical follow-up for the patients was 60.48 (± 23.2). Biochemical relapse was observed in 36 (50.7%) patients. The mean time from surgery to recurrence was 36.4 months. Overall 5 year recurrence-free survival rate was 72.2%.

TMAs and immunohistochemistry

Twenty seven cores contained only stromal tissue, but no prostate glands. Tissues from additional 19 cores were lost during the procedures for immunohistochemistry. Eventually no tumor tissue was available for immunohistochemical studies in two cases. One or more tissue cores containing non-neoplastic prostatic glands that could be evaluated were available for 66 different prostatectomies.

Nuclear p53 expression was found in more than 1% of cells in 29/69 tumors (42%). These tumors were considered p53 positive. Most of the time, positive p53 staining involved more than 50% of tumor cells [Figure 1]. Only four cases showed P53 over expression in non-neoplastic glands. In two of these, the glands were associated with adjacent chronic inflammation. The third had positive cells only at basal layer, not in secretory cells.

A high Ki-67 proliferation index (>10%) was found in 10% of 69 prostate cancer [Figure 2]. One case had Ki-67 positivity above 20%. None of the normal glands expressed Ki-67 over 10% although 1-10% positivity was detected in 40 cases among 69 where non-neoplastic glandular prostate tissue was available for evaluation.

Bcl-2 showed diffuse cytoplasmic staining and typically restricted to the basal cell layer of normal glands [Figure 3]. 27 cases expressed bcl-2 in normal non-neoplastic acini (40.9%). In 10 of them basal cells were accompanied by positive secretory cells albeit in a lesser extent and intensity. Only four tumors (5.8%) revealed bcl-2 expression which was focally and involved less than 20% of neoplastic area [Figure 4].

There was no statistical correlation between p53, Ki-67 labeling and bcl-2 stainings.

None of the markers provided important prognostic information. There was no significant association of p53, Ki-67 or bcl-2 with pathologic stage, histological grade or the other prognostic variables as well as disease outcome in terms of biochemical failure or 5 year-disease free survival [Table 2]. In the univariate analyses, high Gleason score, bilateralism, presence of extraprostatic extension, seminal vesicle invasion, lymphovascular invasion, surgical margin positivity, pathologic stage, tumor volume over 5% and D'Amico high risk group were found significant for higher chance of biochemical failure [Table 3]. Gleason score, tumor focality (unifocal versus multifocal), status of seminal vesicle invasion and surgical margins, and CMS risk grouping were the determinants of five-year disease free survival [Table 4]. Multivariate analysis including these five parameters showed that only state of the seminal vesicle involvement and the CMS grouping proved to be of independent prognostic value, the relative risk of progression being 3.3 (P = 0.001) and 2.1 (P = 0.039), respectively [Table 5].


   Discussion Top


There has been ongoing interest to molecular pathways leading to malignant transformation and progression in prostate cancer. P53, Ki-67 and bcl-2 are the most intensely investigated gene products in various tumors which are involved in the regulation of cell cycle and/or apoptosis. In this study, we have examined P53, Ki-67 and bcl-2 expression immunohistochemically in intermediate and high risk prostate cancer and aimed to investigate their relationship with prognosis.

The p53 is a tumor suppressor gene, takes an important a role in cell cycle control, DNA repair, and apoptosis; [5],[ 6] p53 gene mutations commonly occur in a wide variety of human malignancies. The most frequent form of p53 mutations are of the missense type and can result in an increased half-life of the p53 protein, making it detectable by immunohistochemistry. [7] Several previous studies have shown that alterations of the p53 are less frequent in prostate cancer than in other common tumors. [8],[9],[10] The prevalence of p53-positivity in prostate tumors has been found in the range of 4% -60%. [11],[12] Our study showing 42% p53 immune staining falls in between. We have also observed that normal prostate glands displayed positive reaction for p53 in four cases. However, in two of these, the glands were intimately associated with prominent inflammatory infiltrate, which can induce up-regulation of the protein understandably. In another case, p53 positive cells were noted interestingly in the basal layer. We could not determine a significant association between p53 overexpression and prognosis although RFS was found to be lower in those with (+) p53 expression, compared to those (-). Risk factors for recurrence such as high Gleason score, EPU, surgical margin (+) and involvement of the seminal vesicle were also more frequently detected in p53 (+) patients, but these didn't reach statistical significance.

Bcl-2 is known to have strong inhibitory effect on apoptosis. [13] So it is likely that bcl-2 overexpression can facilitate prostate cancer progression through prolonged tumor cell survival and an increased net tumor.

Additionally, cells that are unable to undergo apoptosis can accumulate secondary genomic aberrations. Thus, the accumulation of additional genomic aberrations may be ultimately responsible for tumor progression and poor prognosis of Bcl-2-positive prostate carcinomas. There are studies, claiming that increase in bcl-2 expression can be an important marker for PSA recurrence following radical prostatectomy. [9] Previous clinical studies have suggested a poor prognosis of hormonally treated Bcl-2-positive prostate carcinomas. [14],[15] Increased bcl-2 expression during the early stage of prostate cancer is reported in 32-41%. [16] Interestingly, in our series, the rate of tumor bcl-2 staining is very low, being only 5.8%. Rather benign prostate glands are frequently stained with the strongest expression by the basal cells. We could not observe any correlation between bcl-2 expression in the tumor and prognostic variables or PSA recurrence. Our results are similar to those of Borre et al, who have found that positivity of bcl-2 is 10% in prostate cancer and it is not an independent prognostic factor. [17]

Several groups have reported that an increased tumor cell proliferation is of prognostic relevance in prostate cancer. [18],[19] Ki-67 is a proliferative nuclear antigen. There are studies where Ki-67 labeling index (LI) appears as an independent prognostic factor prostate cancer. [20] Moul et al, have stated that, patients with a high MIB-1 score -a monoclonal antibody to recombinant parts of Ki-67- experienced recurrence 3.1 times more than others. [21] We have observed a high (over 10%) Ki-67 LI in 10% of our tumors. However, we could not see a prognostic impact of Ki-67 proliferative activity in our patient set.

The analysis of biochemical failure in our patient series has showed a strong association between Gleason score, bilateralism, extraprostatic extension, seminal vesicle invasion, lymphovascular invasion, surgical margin positivity, pathologic stage, tumor volume and CMS. This is consistent with previous reports. [22],[23],[24] Multiparameter analysis by the Cox proportional hazard model has showed that only Gleason score and a CMS are independent predictors of 5-year disease free survival.

In summary, overexpression of p53, bcl-2 and high Ki-67 proliferative index do occur in prostate cancer. They may be important in the carcinogenesis; however, we could not find them influential in the disease progression. Attempts should go on to discover better biomarkers to better predict failure after RP. We have to admit that, genetic biomarker studies on prostate cancer are still in their infancy stages.

 
   References Top

1.Catalona WJ. Surgical management of prostate cancer: Contemporary results with anatomic radical prostatectomy. Cancer 1995;75:1903.  Back to cited text no. 1      
2.Bauer JJ, Sesterhehn I, Mostofi F, McLeod DG, Moul JW. Elevated levels of apopitosis regulator proteins p53 and bcl-2 are independent prognostic biomarkers in surgically treated clinically localized prostate cancer. J Urol 1996;156:1511.  Back to cited text no. 2      
3.Brooks JD, Bova GS, Ewing CM. An uncertain role for p53 gene alterations in human prostate cancer. Cancer Res 1996;56:3814-22.  Back to cited text no. 3      
4.D'Amico AV. Combined modality staging for localized adenocarcinoma of prostate. Oncology (Williston Park) 2001;15:1049-59; discussion 1060-2, 1064-5, 1069-70, 1073-5.   Back to cited text no. 4      
5.Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell 1992;70:523-6.  Back to cited text no. 5      
6.Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor gene. Nature 1991;351:453-6.  Back to cited text no. 6      
7.Finlay C, Hinds P, Tan T, Eliyahu D, Oren M, Levine A. Activating mutations for transformation by p53 produce a gene product that forms a hsc7O-p53 complex with an altered half-life. Moll Cell Biol 1988;8:531-9.  Back to cited text no. 7      
8.Mirchandini D, Zeng J, Miller GJ, Cote DK, Burman P. Heterogeneity in intratumour distribution of p53 mutations in human prostate cancer. Am J Pathol 1995;147:92.  Back to cited text no. 8      
9.Schlomm T, Iwers L, Kirstein P, Jessen B, Kφllermann J, Minner S, et al. Clinical significance of p53 alterations in surgically treated prostate cancers. Mod Pathol 2008;21:1371-8.  Back to cited text no. 9      
10.Byrne R, Horne W, Robinson M, Bishop R, Hamdy F. The expression of waf-1, p53 and bcl-2 in prostatic adenocarcinoma. BJU Int 1997;79:190-5.  Back to cited text no. 10      
11.Nariculam J, Freeman A, Bott S, Munson P, Cable N, Brookman-Amissah N, et al. Utility of tissue microarrays for profiling prognostic biomarkers in clinically localized prostate cancer: The expression of BCL-2, E-cadherin, Ki-67 and p53 as predictors of biochemical failure after radical prostatectomy with nested control for clinical and pathological risk factors. Asian J Androl 2009;11:109-18.   Back to cited text no. 11      
12.Stackhouse G, Sesterhehn I, Bauer J, Mostofi FK, Moul JW. p53 and bcl-2 immunohistochemistry in pretreatment prostate needle biopsies to predict recurrence of prostate cancer after radical prostatectomy. J Urol 1999;162:2040.  Back to cited text no. 12      
13.Korsmeyer SJ. Bcl- 2 initiates a new category of oncogenes: Regulators of cell death. Blood 1992;80:879-86.  Back to cited text no. 13      
14.McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung LW, Hsieh JT, et al. Expression of the protooncogene Bcl- 2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 1992;52:6940-4.  Back to cited text no. 14      
15.Colombel M, Symmans F, Gil SO, Chopin D, Benson M, Olsson CA, et al. Detection of the apoptosis-suppressing oncoprotein Bcl- 2 in hormone-refractory human prostate cancers. Am J Pathol 1993;143:390-400.  Back to cited text no. 15      
16.Takahiro I, Takehiko S, Taizou S, Toru Y, Tomomi Y, Naoko K, et al. Androgen receptor, Ki-67, and p53 expression in radical prostatectomy specimens predict treatment failure in Japanese population. Urology 2005;66:332-7.  Back to cited text no. 16      
17.Levine AJ, Momand J, Finlay A. The p53 tumour supressor gene. Nature 1991;351:453.  Back to cited text no. 17      
18.Visakorpi T. Proliferative activity determined by DNA flow cytometry and proliferating cell nuclear antigen (PCNA) immunohistochemistry as a prognostic factor in prostatic carcinoma. J Pathol 1992;168:7-13.  Back to cited text no. 18      
19.Harper ME, Glynne JE, Goddard L, Wilson DW, Matenhelia SS, Conn IG, et al. Relationship of proliferating cell nuclear antigen (PCNA) in prostatic carcinomas to various clinical parameters. Prostate 1992;20:243-53.  Back to cited text no. 19      
20.Bubendorf L, Sauter G, Moch H, Schmid HP, Gasser TC, Jordan P, et  al. Ki-67 labeling index: An independent predictor of progression in prostate cancer treated by radical prostatectomy. J Pathol 1996;178:437-41.  Back to cited text no. 20      
21.Gali S, Gaboardi F, Rozzola A, Ferrari A. Immunohistochemical expression of bcl-2, p53, ki-67 and choromograms in prostatic carcinoma: Correlation with stage and grade. J Urol 1995;153:483.  Back to cited text no. 21      
22.Catalona WJ, Smith DS. Five year tumour recurrence rates after anatomical radical retropubic prostatectomy for prostate cancer. J  Urol 1994;152:1837.  Back to cited text no. 22      
23.Huakaas S, Halversen O, Hostmark J, Akslen L. Is preoperative serum prostate-specific antigen level significantly related to clinical recurrence after radical retropubic prostatectomy for localized prostate cancer? BJU 2006;97:51.  Back to cited text no. 23      
24.Kupelian P, Katcher J, Levin H, Zippe C, Klein E. Correlation of clinical and pathologic factors with rising prostate-specific antigen profiles after radical prostatectomy alone for clinically localized prostate cancer. Urology 1996;48:249-60.  Back to cited text no. 24      


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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