PDF-957.pdf 381Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L Expression of Bcl-2 and Bax in Advanced or Metastatic Prostate Carcinoma Kazem Anvari,1 Mehdi Seilanian Toussi,1 Mahmoud Kalantari,2 Shahram Naseri,1Mahdi Karimi Shahri,1 Hassan Ahmadnia,3 Mehrdad Katebi,4 Abdolazim Sedighi Pashaki,1 Mahdieh Dayani,1 Maryam Broumand5 Purpose: To evaluate the correlation of Bcl-2 and Bax protein expressions with biochemical failure-free survival in patients with advanced or metastatic prostate Materials and Methods: This retrospective study was performed on patients with therapy. Samples were analyzed immunohistochemically for Bax and Bcl-2 ex- pression. The H-score was calculated for each sample based on intensity and per- Results: Thirty-seven patients (13 metastatic and 24 locally advanced) were eligi- - tween Bax expression and Gleason score, high Bcl-2 expression (H-score > 85) P months and 16 (range, 9 to 26) months, respectively. High Bcl-2 expression (P = P of lower biochemical progression-free survival. Conclusion: High Bcl-2 expression was associated with higher Gleason scores androgen deprivation therapy. Keywords: prostatic neoplasms, Bax protein, bcl-2, disease progression, survival Corresponding Author: Mehdi Seilanian Toussi, MD Department of Oncology, Omid Hospital, Kohsangi St., Alandasht Square, Mashhad, Iran Tell/Fax: +98 511 842 6936 E-mail: silanianm@mums. ac.ir Received April 2011 Accepted September 2011 1 Cancer Research Center, Department of Radiothera- py Oncology, Mashhad Uni- versity of Medical Sciences, Mashhad, Iran 2 Department of Pathology, Mashhad University of Medical Sciences, Mash- had, Iran 3 Department of Urology, Mashhad University of Medical Sciences, Mash- had, Iran 4 Department of Pathol- ogy, Bentolhoda Hospital, Mashhad, Iran 5Mashhad University of Medical Sciences, Mashhad, Iran Urological Oncology 382 | INTRODUCTION P -cal behavior from indolent tumors to ag- factors, which predict recurrence after treatment, include clinical stage, grade, and pretreatment se- (1) However, the above-mentioned prognostic factors are unable to predict the outcome in all subjects. The genetic factors play an essential role in tumor progression and in governing whether a prostate cancer is aggressive or indolent.(2-4) Almost all tissues have a system to remove dam- aged cell through programmed cell death or apo- ptosis.(5) Disturbance of programmed cell death can lead to accumulation of cells with impaired genome and eventually cancer. A number of genes regulate apoptosis, which include Bcl-2 family and tumor suppressor p53 gene.(6) Bcl-2 family proteins include both anti-apoptotic (eg, Bcl-2 and Bcl-xL) and pro-apoptotic (eg, Bax, BAK, - duce permeabilization of outer mitochondrial - chrome c into the cytosol, which in turn activates a group of cysteine protease called caspases leading to cell death. Meanwhile, anti-apoptotic proteins prevent cell death program through preserving mitochondrial membrane integrity and releasing cytochrome c.(7,8) A study by Tolonen and colleagues showed that normal epithelium of cancerous prostates contain multiple foci with high expression of Bax and Bcl-2.(9) 2 expression and Ki-67 index indicating higher proliferating rates in Bcl-2 positive tumors. - ly associated with high Gleason score tumors.(11,12) These results suggest a potential role for altered apoptosis in carcinogenesis. Androgen deprivation, which is the mainstay of mediated apoptosis in androgen dependant cells and regression of tumor. However, androgen in- dependent cancer eventually develops in all cases of metastatic disease, which is accompanied with Bcl-2, Bcl-xL, and McL-1 overexpression in most cases.(13,14) The results of some studies suggest an association between apoptotic pathway dysfunc- tion and worse outcome.(15-17) According to the result of a population–based can- cer registry, the age-standardized incidence rate of - son per year,(18) which is much lower than western countries. Meanwhile, in comparison with west- which can be explained partly by lack of national study was to investigate the association between Bax and Bcl-2 expression and time to progression undergoing androgen deprivation therapy. MATERIALS AND METHODS - Eligible subjects were patients with distant me- tastasis and/or lymph node metastasis (metastatic - who were referred to our department between were not followed up properly and those without required clinicopathological information. The usual imaging procedures were chest x-ray, abdominopelvic computed tomography scan, and bone scintigraphy. Thirty-seven eligible patients with retrievable pathological specimens and ad- equate clinical information were selected. The specimens were re-evaluated for determining their Gleason score by our pathologists. The Gleason Urological Oncology 383Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L Bcl-2 and Bax Protein Expression in Prostate Carcinoma | Anvari et al scores above 7 were considered as poorly differ- entiated (high Gleason score). Our samples con- sisted of 14 low and 23 high Gleason scores. Treatment and Follow-up The diagnosis was based on histologic evaluation of the prostate biopsy specimens. All non-meta- static cases underwent local and regional external beam radiotherapy. All the patients received an- drogen deprivation therapy, which included lute- inizing hormone-releasing hormone (LHRH) ago- Twenty-eight subjects received anti-androgen plus medical or surgical castration. All the patients were followed up every 3 months for the evidence of biochemical progression. Three consecutive rises in PSA was considered as biochemical pro- gression.(19) Immunostaining for Bax and Bcl-2 Multiple 4-μm-thick sections of representative cut for immunohistochemical studies. A Polymer- Based (EnVision™) immunohistochemical meth- od was used for the detection of Bax (Polyclonal Rabbit Anti-Human, Dako) and Bcl-2 (Monoclo- - tive lymph node with follicular hyperplasia was adding antibody was used as the negative control. All immunostained sections were examined by for evaluating Bax and Bcl-2 expression. Bax and Bcl-2 staining was cytoplasmic. Protein expres- sion was scored as negative, weak; faint cytoplas- mic staining, moderate; diffuse cytoplasmic stain, and strong; diffuse intense cytoplasmic stain. Furthermore, proportion of malignant cells which had positive staining was considered in reporting. Mild, moderate, and strong staining was consid- ered as positive. which considers both the intensity and percent- age of cells stained in each intensity. H-score was calculated as follows: (% of cells stained at intensity 1 × 1) + (% of cells stained at intensity 2 × 2) + (% of cells stained at intensity 3 × 3). A H- considered as positive for Bax or Bcl-2 expres- sion (Figures 1 and 2). The median H-score values were selected for distinction between the groups of high and low Bax or Bcl-2 expression. Figure 1. Positive Bax immunostaining (H-score = 240) in a pros- tate cancer with the Gleason score of 7. Figure 2. Positive Bcl-2 reaction (H-score = 200) in a prostate cancer with the Gleason score of 9. 384 | Statistical Analysis Progression-free survival was determined from the time of diagnosis to the time of biochemical failure or the last visit using Kaplan-Meier meth- od. We used Log-rank test for univariate compar- - sion model with backward stepwise selection of co-variates was used for multivariate analysis. - tion between the Bax and Bcl-2 expression and the Gleason score. RESULTS The participants included 13 patients with me- tastasis (9 distant and 4 lymph node metastasis) and 24 with locally advanced tumors. The median age of the patients was 73 years (range, 52 to 87 years). The median pretreatment PSA value was 17 ng/ mL (range, 7.5 to 96 ng/mL) for high-risk ad- - of metastatic cases. for all the subjects. We recorded Gleason scores > 7 in 23 (62.7%) patients, including 16 (66.6%) of high-risk advanced and 7 (53.8%) of metastatic cases. Treatment Results With a median follow-up period of 32 months biochemical progression, including 13 out of 13 (58.3%) in the locally advanced group. The me- dian time to the biochemical progression was 22 advanced group and 16 months (range, 9 to 26 months) in the metastatic group. The median biochemical recurrence-free survival rates for locally advanced and metastatic groups 21.87), respectively. Bax and Bcl-2 Expression Of 37 samples, 36 (97.3%) were positive for Bax - Figure 3. Association between Bcl-2 expression and biochemi- cal recurrence- free survival in patients with advanced or meta- static prostatic carcinoma. Figure 4. Association between pretreatment prostate-specific antigen and biochemical recurrent-free survival in patients with advanced or metastatic prostatic carcinoma. Urological Oncology 385Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L Bax expression and Gleason score (P = .28), high high Gleason score (P Table 2 shows the effect of different parameters on biochemical failure-free survival. Gleason Score and Bax expression did not affect biochemical progression-free survival. Meanwhile, as shown in Figures 3 and 4, patients with high Bcl-2 expres- sion (P P - ated with lower biochemical progression-free sur- was associated with relatively higher biochemical failure-free survival, the difference did not reach Bcl-2 expression (P (P - chemical progression-free survival. DISCUSSION - - ing androgen deprivation therapy. About 41.6% of high-risk locally advanced group had pretreat- subjects that could not be found via our imaging methods, such as bone scintigraphy and computed tomography scan. Hormonal treatment was the main treatment mo- dality for prolongation of survival in most of our patients. We used the median H-score for the dis- tinction between high and low expression of each - ical progression-free survival was shown for sub- ng/mL) and those with high Bcl-2 expression. The contribution of Bcl-2 to prostate carcino- Table 1. Correlation between Gleason score and Bax or Bcl-2 expression High Bax expression, n (%) High Bcl-2 expression, n (%) Gleason score: G 2 to 7, n = 14 G 8 to 10, n = 23 8 (57.1%) 9 (39.1%) 3 (21.4%) 16 (69.6%) P = .28 P = .004* *Statistically significant. Bcl-2 and Bax Protein Expression in Prostate Carcinoma | Anvari et al Table 2. The pathological features and progression-free survival rate Median biochemical failure-free survival (95% confidence interval), months P Bax .45High expression 24 (14.59 to 33.41) Low expression 20 (09.60 to 39.41) Bcl-2 .01*High expression 16 (09.60 to 22.30) Low expression 32 (22.35 to 41.03) Gleason score .54High (G 8 to 10) 18 (07.8 to 35.22) Low ( G 2 to 7 ) 24 (15.54 to 32.46) Pretreatment prostate-specific antigen, ng/mL .01*> 20 18 (14.11 to 21.88) ≤ 20 35 (23.02 to 36.98) *Statistically significant. 386 | genesis and hormone independence has been documented.(21) As the Bcl-2 family modulates apoptosis, altered expression of Bcl-2 might affect response to genotoxic stresses, including radio- therapy, hormone deprivation, or cytotoxic agents. Expression of Bcl-2 was shown to be associated with higher failure rate after radiotherapy in lo- calized prostate cancer.(22-25) Bcl-2 expression has also been associated with higher biochemical re- currence after radical prostatectomy.(26) predicting factors for time to progression included Gleason score, pretreatment PSA value, and na- dir PSA value after treatment initiation.(27-29) Tis- sue biomarkers might also be helpful in prediction of response duration in metastatic or advanced diseases. Following androgen ablation therapy, increased expression of Bcl-2 develops in tumor cells, which eventually leads to an androgen inde- pendent state. Zhou and colleagues used in-vivo model of an- molecular biology of progression from a hormone sensitive to hormone resistant state following cas- tration. Hormone resistant tumors had decreased apoptosis accompanied with augmented expres- sion of p53, p21/waf1, Bcl-2, Bax, and the Bcl-2/ Bax ratio compared to androgen-sensitive tumors. (31) Bcl-2 expression in newly diagnosed metastatic patients might be less responsive to hormone ma- nipulation.(13) As Bax is a pro-apoptotic agent, its expression is expected to be associated with increased tumor sensitivity to radiotherapy or systemic therapy. However, the results of studies have been incon- reported worse prognosis for patients with Bax ex- pression.(17) Meanwhile, in a group of patients un- dergoing external beam radiotherapy, lower Bax expression was associated with worse prognosis; however; the difference was not statistically signif- icant. Patients with increased Bcl-2/Bax ratio had higher risk of failure following radiotherapy.(22) a study by Amirghofran and colleagues, Bax ex- pression was not correlated with apoptosis index suggesting presence of nonfunctional Bax pro- tein. Mutated or dysfunctional Bax may appear overexpressed, while negatively affect apoptotic Bax expression in a tumor specimen was consid- ered as under- or overexpression relative to the Pollack and associates showed that higher Bcl-2 expression and altered Bax expression were asso- ciated with higher biochemical failure rates.(16) a group of patients with locally advanced prostate cancer who underwent radiotherapy, more favora- ble outcome were associated with negative Bcl-2 and normal Bax, especially in those who received short-term androgen ablation therapy plus radio- therapy.(15) Altered Bax expression seems to be a more reliable predictor of outcome compared to Bax overexpression. As the role of apoptotic modulator, especially Bcl- 2, has been documented in development of hor- mone independent prostate cancer, these genes were considered as a target for development of novel treatments. Bcl-2 antisense oligonucleotides might counteract anti-apoptotic survival mecha- nisms and enhance hormone and chemotherapy sensitivity.(32-36) CONCLUSION We concluded that high Bcl-2 expression is as- sociated with worse biochemical progression-free - tients undergoing hormone manipulation therapy. ACKNOWLEDGEMENTS Urological Oncology 387Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L This study was supported by a grant from the Vice- chancellorship for Research, Mashhad University of Medical Sciences. CONFLICT OF INTEREST None declared. REFERENCES 1. Kattan MW, Eastham JA, Stapleton AM, Wheeler TM, Scar- dino PT. A preoperative nomogram for disease recurrence following radical prostatectomy for prostate cancer. J Natl Cancer Inst. 1998;90:766-71. 2. Qian J, Jenkins RB, Bostwick DG. Determination of gene and chromosome dosage in prostatic intraepithelial neo- plasia and carcinoma. Anal Quant Cytol Histol. 1998;20:373- 80. 3. Xu J, Zheng SL, Isaacs SD, et al. 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