Survivin and Bcl-2 Expression in Prostatic Adenocarcinomas

Aberration in the control of cell proliferation and/or cell death is required for tumor establishment.1 Inhibition of apoptosis, or programmed cell death, may be involved in the pathogenesis of cancer by prolonging cell life and facilitating retention of deleterious mutations.2,3 Several inhibitors of apoptosis related to the baculovirus IAP (inhibitors of apoptosis) gene have been identified in humans, mice, and Drosophila.4,5 Among the regulators of cell death, IAP proteins, which contain cysteine/histidine baculovirus IAP repeats and a carboxyl terminal ring finger, have been described6 and have emerged as modulators of an evolutionarily conserved step in apoptosis, which may potentially involve the direct inhibition of terminal effected caspases 3 and 77 downstream of bcl-2.8 Recently, a novel and structurally unique member of the IAP proteins, survivin, a bifunctional protein that suppresses apoptosis and regulates cell division, has been described.9 Survivin, a 16.5-kd cytoplasm protein located on chromosome 17q25 and encoding 142 amino acids, has a single baculovirus IAP repeat and no carboxy ring finger that inhibits apoptosis induced by growth factor (IL-3) withdrawal when transfected in B-cell precursors.10 Survivin messenger RNA (mRNA) and protein is found ubiquitously in fetal tissues and, other than in the thymus and placenta, is completely down-regulated in normal adult tissues. Survivin is expressed in transformed cell lines and in many common human malignancies, including cancers of the lung, prostate, colon, pancreas, and breast.10 

Bcl-2, an antiapoptotic protein,11 has been implicated in the pathogenesis of hematologic and epithelial malignancies, including carcinomas of the lung, colon, breast, and prostate.12–23 Bcl-2 and survivin contribute independently to embryonic and fetal development.24,25 As inhibitors of apoptosis, both proteins have been implicated in promoting cancer, although the distribution of Bcl-2 and survivin expression varies widely in both normal and transformed cell types.19 Studies of survivin in prostate cancer have been limited, and we know of no published reports relating survivin expression to established prognostic factors or disease outcome. Bcl-2 expression, however, has been studied extensively in benign prostate tissues,26 preneoplastic conditions,18 and cancer cell lines.27 Studies of the association of Bcl-2 expression with prognostic variables and clinical outcome in prostate cancer have yielded conflicting results.18,20–23 

We randomly selected 138 patients who had undergone radical prostatectomy for biopsy-proven prostate adenocarcinoma (PAC). All patients were seen at the Albany Medical Center Hospital (Albany, NY) between 1987 and 1997. Hematoxylin-eosin–stained slides from each radical prostatectomy specimen were reviewed, and a Gleason grade28 and pathologic stage29 were assigned. Tumors were classified as high grade when the combined Gleason score was 7 or above and as low grade when the combined score was 6 or below. Serum prostate-specific antigen (PSA) levels were obtained from the patients' medical records. Serum PSA was measured by the Hybritech tandem method (Beckman Coulter, Inc, Brea, Calif). A postsurgical elevation of the PSA level from a baseline level of 0 ng/mL (0 μg/L) to greater than 0.4 ng/mL (0.4 μg/L) on 2 consecutive occasions was considered as biochemical evidence of disease recurrence. Follow-up information was obtained from review of the patient's medical records.

Immunohistochemical staining for Bcl-2 and survivin proteins was performed by an automated method on the Ventana ES immunohistochemistry instrument (Ventana Medical Systems Inc, Tucson, Ariz) using an indirect biotin-avidin diaminobenzidine (DAB) detection system on contiguous formalin-fixed, paraffin-embedded 4-μm sections from a representative block in each case. Following deparaffinization to water, the antigenic determinant sites were unmasked in EDTA with steam for 60 minutes for Bcl-2 detection. No antigen retrieval was used for the detection of the survivin protein. The primary antibody used for Bcl-2 was predilute monoclonal mouse anti-human Bcl-2 (clone Bcl-2/100/D5; Ventana) for 32 minutes at 37°C. The secondary antibody was biotinylated goat anti-mouse immunoglobulins (Dako Corporation, Carpinteria, Calif) at a dilution of 1:250. The primary antibody for survivin was polyclonal goat anti-human survivin (Santa Cruz Biotechnology, Santa Cruz, Calif) at a dilution of 1:40 for 32 minutes at 37°C. The secondary antibody was biotinylated rabbit anti-goat immunoglobulins (Dako) at a dilution of 1:400. After the development of the color with DAB, the slides were counterstained with hematoxylin. Similarly processed sections from human tonsil and colon adenocarcinoma were used as positive controls for Bcl-2 and survivin immunostaining, respectively. To confirm the specificity of both of the primary antibodies, negative control slides were run with every batch, using a predilute negative control reagent (Ventana) at the same concentration as that of the primary antibody.

Immunoreactivity for both survivin and Bcl-2 was interpreted without prior knowledge of any of the clinicopathologic parameters. The intensity of staining and the distribution of cytoplasmic positivity were considered in the semiquantitative assessment of the immunohistochemical results for both antibodies. The distribution of staining in the tumor cells was graded as focal (≤10%), regional (11%–50%), or diffuse (>50%). The intensity of cytoplasmic staining was subjectively graded as weak, moderate, or intense. Cases in which the staining patterns were categorized as intense diffuse, intense regional, moderate diffuse, and moderate regional were considered positive for expression of the 2 proteins. Cases that were categorized as intense focal, moderate focal, and all 3 distributions of weak staining were considered to be negative.

Five-micrometer, formalin-fixed, paraffin-embedded sections were stained by the Feulgen method and analyzed for DNA content with the CAS 200 Image Analyzer (Cell Analysis Systems, Lombard, Ill). After the instrument was calibrated against similarly stained tetraploid rat hepatocytes, the DNA content of the PACs was measured in a minimum of 100 tumor cells, and the tumor DNA index was determined by comparison with the control diploid cells of the benign prostatic epithelium. All the tumor cell histograms were reviewed without knowledge of the tumor grade, stage, recurrence status, or immunohistochemical results. A DNA index of 0.77 to 1.22 was considered to be diploid. Peaks in the tetraploid region containing less than 15% of the total cell population were considered to be the G₂M components of diploid cell populations. Tumors with tetraploid peaks greater than 15% and hyperdiploid, nontetraploid peaks were considered to be nondiploid (aneuploid).30 

Statistical comparisons were carried out with the STATA software (Stata Corporation, College Station, Tex). The χ2 test was used to determine the significance of the associations between protein expression and pathologic variables. Disease recurrence analysis was performed with univariate models and by the Kaplan-Meier method. Multivariate analysis, including clinicopathologic parameters and expression of each protein, was performed using the Cox proportional hazards model, with outcome defined as postsurgical biochemical evidence of disease recurrence. A postsurgical elevation of the PSA level from a baseline level of 0 ng/mL (0 μg/L) to greater than 0.4 ng/mL (0.4 μg/L) on 2 consecutive occasions was considered as biochemical evidence of disease recurrence. The level of significance was set at P ≤ .05.

The mean age of the patients was 66 years (range, 49–94 years) and the mean preoperative PSA level was 12.4 ng/mL (12.4 μg/L, range 1.6–87.8 μg/L). Of the 138 PACs, there were 77 (56%) low-grade (Gleason score ≤ 6) and 61 (44%) high-grade (Gleason score ≥ 7) tumors. At prostatectomy, there were 84 (61%) organ-confined tumors (stages I and II) and 54 (39%) advanced-stage (stages III and IV) tumors. Of the 96 cases previously analyzed for total DNA content, 68 (71%) were diploid and 28 (29%) were nondiploid. Follow-up information was available for all patients, of whom 52 (38%) had biochemical postsurgical disease recurrence. The mean follow-up was 55 months (range, 2–132 months).

The immunostaining pattern for both proteins was cytoplasmic with tumor cells showing moderate to intense positivity as opposed to relatively weaker expression in benign elements (Table 1). One hundred thirteen (82%) of 138 PACs expressed survivin. We found no correlation between survivin expression and prognostic variables, including grade (Figure 1), stage, ploidy status, and recurrence.

Positive staining for Bcl-2 was found in 95 (69%) of the 138 PACs. In a subgroup of intensely positive cases (41/95), Bcl-2 protein expression correlated with nondiploid DNA content and recurrence, while showing a trend for a correlation with tumor stage. Fourteen (50%) of 28 nondiploid PACs expressed Bcl-2, as compared to 17 (25%) of the 68 diploid tumors (P = .02) (Figure 2). A trend toward association of Bcl-2 expression with tumor stage was noted in 21 (39%) of 54 advanced-stage PACs staining positively for Bcl-2, in comparison with 20 (24%) of 84 early-stage tumors (P = .07). On univariate analysis, 25 (48%) of the 52 tumors that recurred expressed Bcl-2, as compared with 16 (19%) of the 86 PACs that did not relapse (P < .001) (Figure 3). No significant coexpression was observed between Bcl-2 and survivin. On multivariate analysis, with outcome defined as postsurgical biochemical evidence of disease recurrence, advanced stage was the only predictor of disease recurrence (Table 2). Preoperative serum PSA levels were available in 75 (54%) of the 138 cases, and when added as a covariate to the Cox model, PSA level (P = .001) was significantly associated with disease recurrence, along with advanced stage (P = .02) and aneuploidy (P = .02).

Survivin, a newer member of the IAP family, has been implicated in tumor progression. Survivin overexpression detected by immunohistochemistry and reverse transcriptase–polymerase chain reaction has been correlated with adverse prognosis in a variety of human malignancies, including carcinomas of the stomach,31 pancreas,32 lung,33 esophagus,34 colon,35–37 and breast.38 Survivin expression has consistently correlated with the apoptotic index and progression through the cell cycle.31–39 Survivin is associated with the microtubules of the mitotic spindle and is expressed in the G₂M phase of the cell cycle.39 Disruption of the survivin-microtubule interaction leads to loss of survivin function and increased proapoptotic caspase-3 and -7 activity.39 

In the present study, the majority of PACs overexpressed survivin. Survivin expression has been reported previously in PAC clinical samples, both by immunohistochemistry and by in situ hybridization.10 Survivin expression has been associated with neuroendocrine differentiation of both normal and malignant cells of the human prostate.40 Our study of survivin expression in PACs did not link overexpression of the protein with adverse prognostic variables or disease recurrence. These findings are similar to previous studies, in which survivin did not predict disease outcome in gastric,31 lung,33 esophageal,34 and breast38 cancers. Interestingly, survivin mRNA levels in tumors of the lung33 and esophagus34 have been linked with decreased survival on univariate analyses. Curiously, nuclear, but not cytoplasmic, survivin expression, which was not seen in the PACs in this study, has been correlated with a favorable prognosis in gastric cancer.31 

Extensive studies of Bcl-2 expression have confirmed its role as a major antiapoptotic protein.41 Originally linked with the t(14;18) translocation in follicular non-Hodgkin lymphoma, altered Bcl-2 expression has now been linked to adverse outcome in a variety of solid tumors.13–16,41–44 In normal prostate tissues, Bcl-2 expression is restricted to the basal cells.18–23 Bcl-2 expression levels and their respective correlation with disease outcome have varied in different primary solid tumors. Interestingly, increased Bcl-2 expression has been linked with favorable disease outcome in breast,45 colorectal,13,16 and bronchogenic carcinomas,46 whereas other immunohistochemistry-based studies of colorectal47 and lung14,15 cancers revealed no prognostic significance. In prostate cancer, proapoptotic molecules, such as PTEN, p53, Bin1, TGF-β, and Par-4, and antiapoptotic proteins, such as Bcl-2/Bcl-xL, NF-κB, IGF, caveolin, and Akt, have been linked to progression of prostate cancer.48 Bcl-2 overexpression in prostate cancer is not related to the t(14;18) chromosomal translocation.21 In the present study, the majority of PACs (69%) expressed Bcl-2, which was significantly associated with disease recurrence. In other studies, Bcl-2 expression in PACs has ranged from 13.4% to 29%.12,22–23 In the present study, Bcl-2 expression was associated with nondiploid PACs, a finding also reported by others.23 

In the present study, we identified no correlation between Bcl-2 and survivin expression. This finding is at variance with studies by others, who found a coassociation of Bcl-2 and survivin expression in breast38 and gastric49 adenocarcinomas. Bcl-2 and survivin may contribute to cancer evolution and multidrug resistance by working through different apoptotic pathways. Survivin inhibits paclitaxel-induced apoptosis, but is ineffective on microtubule-depolymerizing drugs, such as nocodazole or vincristine.50 Bcl-2 inhibits apoptosis induced by all drugs, independent of microtubule assembly and disassembly functions50 As the field of targeting cancer therapy by overcoming resistance to apoptosis progresses, further studies of both survivin and Bcl-2 expression and regulation appear warranted.