Cysteine

Context.-Controlled cell death is mediated by apoptosis-specific genes, tumor suppressor genes, and oncogenes. The caspase family is a group of at least 15 known cysteine proteases that serve as initiator and effector molecules of the apoptosis pathway. On activation, caspases cause cell shrinkage, condensation of chromatin, fragmentation of DNA, and the formation of blebs in the cytoplasmic membrane.

Objectives.-The patterns of cysteine protease protein (CCP) 32 (caspase-3) expression have been determined in normal human tissues and a variety of tumors, and have been shown to correlate with the outcome in breast cancer and linked to resistance to chemotherapy in other tumors. This study was performed to determine whether CPP32 is expressed in prostatic adenocarcinoma and to define its relationship with outcome variables.

Design.-Formalin-fixed, paraffin-embedded radical prostatectomy specimens from 211 patients with prostatic adenocarcinoma were evaluated for CPP32 expression by immunohistochemistry. Hematoxylin-eosin-stained slides were reviewed, and tumors were graded based on the Gleason grading system. Tumors were scored for CPP32 expression semiquantitatively, based on the staining intensity and distribution patterns. These results were compared with Gleason grade and clinical and pathologic stages.

Results.-One hundred thirty-three (63%) of 211 cases showed high expression of CPP32, whereas expression was low in 78 (37%) cases. One hundred three (49%) of 211 cases had a high Gleason score (7 and above). Of 103 cases with a high Gleason score, 74 (72%) showed high CPP32 expression. Strong cytoplasmic staining for CPP32 in high-grade tumors was statistically significant (P = .01). Also, by linear regression analysis a significant correlation was seen between the Gleason score and the cytoplasmic CPP32 expression (P = .001). Expression of CPP32 did not correlate with either clinical stage (P = .28) or pathologic stage (P = .60); however, this study included very few patients with stage IV disease.

Conclusion.-The correlation between CPP32 and high tumor grade suggests a CPP32-related high turnover rate in high-grade prostatic adenocarcinoma. Moreover, strong correlation with Gleason grade, a powerful predictor of disease progression and overall survival, suggests potential usefulness of CPP32 as a prognostic factor, especially in limited biopsy samples.

(Arch Pathol Lab Med. 2004;128:649-652)

Apoptosis, or programmed cell death, is a continuous process that is vital for the development, propagation, and timely turnover of normal tissues. Certain apoptosis-specific genes, oncogenes, and tumor suppressor genes are involved in this process of controlled cell death. The process of apoptosis is carried out in an orderly fashion, such that reduction in cell turnover rate or increase in the cell survival time results in neoplastic processes.1 Cysteine protease protein (CCP) 32 (caspase-3) represents a group of cysteine proteases involved in the induction of programmed cell death.2 This programmed cell death is an integral part of normal tissue hemostasis and is also seen in pathologic conditions.34 CPP32 is a novel human apoptotic gene that was cloned from human jurkat T lymphocytes. Its expression has been shown to be significantly high in cell lines of lymphocytic origin, and it was suggested to be an important mediator of apoptosis in the immune system.5

CPP32 is a member of the interleukin-1 [beta]-converting enzyme family of the mammalian proteases, which specifically cleaves substrates at the C-terminal side of the aspartic acid residues. Members of this family have been implicated in apoptosis, and CPP32 is thought to act as a control mediator of programmed cell death in mammalian cells. More than 10 caspases have been isolated and identified, of which CPP32 has been studied the most. The pattern of CPP32 protein expression has been observed in normal human tissues and a variety of neoplastic processes, both benign and malignant. The expression of CPP32 is variable in different normal tissues. Prostate secretory epithelial cells show high expression as compared to basal cells. On the other hand, CPP32 expression is low in cells of thymic cortex, which are more vulnerable to apoptosis as compared with those in medulla.1 Its expression has also been linked to prognosis, especially in breast cancer, where its expression is associated with poor outcome.6 Studies in leukemias have shown that defects in the mechanisms of programmed cell death by caspase-activated pathways can lead to chemoresistant disease and decreased patient survival.7 CPP32 has also been studied in Hodgkin lymphoma. The Reed-Sternberg cells of classic Hodgkin lymphoma show CPP32 expression, whereas it is lacking in popcorn cells of nodular lymphocyte-predominant Hodgkin lymphoma. Expression of CPP32 is also lacking in low-grade B-cell non-Hodgkin lymphomas.8 Few studies have looked at CPP32 expression in prostatic adenocarcinoma and the results have been conflicting.2,9 This study was undertaken to study the expression of CPP32 in prostatic adenocarcinoma (PAC) and its relationship with outcome variables.

MATERIALS AND METHODS

Specimen Collection, Tumor Grading, and Pathologic Staging

Two hundred eleven patients who underwent radical prostatectomy for biopsy-proven PAC between 1995 and 1999 were selected. Hematoxylin-eosin-stained, whole-mount slides from each totally embedded radical prostatectomy case were reviewed, and a Gleason score and pathologic stage were assigned. During review, 1 block from each case was identified based on the presence of adequate tumor and the representative nature of the overall Gleason score. Tumors were classified as high-grade when the combined Gleason score was 7 or higher and as low-grade when the combined score was 6 or lower. Serum prostate-specific antigen was measured by the Hybritech tandem method (Beckman Coulter, lnc, Brea, Calif). A postsurgical elevation of the prostatespecific antigen level from a baseline level of O ng/mL to greater than 0.4 ng/mL 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.

lmmunohistochemistry

Immunohistochemical staining for CPP32 protein was performed by an automated method on the Ventana ES (Ventana Medical Systems, lnc, Tucson, Ariz), using an indirect biotin-avidin diaminobenzidine (DAB) detection system on contiguous 4-µm, formalin-fixed, paraffin-embedded sections from a representative block in each case. Following deparaffinization to water, the antigenic determinant sites for CPP32 were unmasked in citrate buffer with steam for 60 minutes. The primary antibody used for CPP32, was an immunoglobulin Gl class mouse antihuman caspase-3 clone JHM62 (Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom), at a dilution of 1:25 for 32 minutes at 37°C. The secondary antibody was biotinylated goat anti-mouse immunoglobulin (Dako Corporation, Carpinteria, Calif) at a dilution of 1:250. After development of the color with DAB, the slides were counterstained with hematoxylin. Similarly processed sections from human adenoid tissue were used as positive controls for CPP32 immunostaining. To confirm the specificity of the primary antibody, negative control slides were run with every batch, using an isotype-matched immunoglobulin at the same concentration as that of the primary antibody. Expression of CPP32 protein was essentially cytoplasmic.

Staining Interpretation

Immunoreactivity for CPP32 was interpreted without prior knowledge of any of the clinicopathologic parameters. The intensity and distribution of cytoplasmic staining in the tumor cells were considered in the semiquantitative assessment of the immunohistocbemical results for the antibody. The intensity of cytoplasmic staining was subjectively graded as weak, moderate, or intense. The distribution of staining in the tumor cells was graded as focal (50% tumor staining). Slides were scored for CPP32 expression semiquantitatively based on staining intensity and distribution, according to the following 10-point scale: O, none; 1, weak focal; 2, weak regional; 3, weak diffuse; 4, moderate focal; 5, moderate regional; 6, moderate diffuse; 7, intense focal; 8, intense regional; and 9, intense diffuse.

Cases in which the staining patterns were categorized as intense diffuse, intense regional, and moderate diffuse were considered overexpression of the protein.

Statistical Analysis

Statistical comparisons were carried out with STATA software (Stata Corporation, College Station, Tex). The [chi]^sup 2^ test was used to determine the significance of the associations between protein expression and prognostic variables. Potential correlations between clinicopathologic parameters and expression of protein were examined by linear regression analysis using the Stata command "regres."10 The level of significance was set at P

RESULTS

Clinicopathologic Data

The mean age of the patients was 65 years (range, 45-98 years), and the mean preoperative prostate-specific antigen level was 12.6 ng/mL. Of the 211 PACs, there were 108 (51%) low-grade (Gleason score or = 7) tumors. The distribution of clinical stages of these cases was 52 (37%) stage I, 85 (61%) stage II, 1 (0.7%) stage III, and 1 (0.7%) stage IV.

The distribution of pathologic stages was 5 (2%) stage I, 139 (68%) stage II, 54 (26%) stage III, and 6 (3%) stage IV.

CPP32 Expression by lmmunohistochemistry

The immunostaining pattern of CPP32 protein was essentially cytoplasmic, but was occasionally nuclear with tumor cells showing weak (Figure, A), moderate (Figure, B), and intense (Figure, C), positivity, as opposed to weaker and inconsistent expression in benign elements, such as basal cells of glands, lymphocytes, and stromal cells. One hundred thirty-three (63%) of the 211 cases showed high expression of CPP32, while 78 (37%) showed low expression (Table). One hundred three cases (49%) had a high Gleason score (7 and higher). Of the 103 cases with a high Gleason score, 74 (72%) showed high CPP32 expression. Strong cytoplasmic staining for CPP32 in high-grade tumors was statistically significant (P = .01; see Table).

High Gleason score correlated with cytoplasmic CPP32 expression by linear regression analysis (P = .001). There was an insufficient number of high-stage cases to examine the relationship of CPP32 staining with advanced stage. The short follow-up periods in these cases precluded a meaningful analysis of outcome and CPP32 expression.

COMMENT

The assessment of clinical behavior and outcome in prostate adenocarcinoma has been studied extensively by a variety of morphology-driven measures and molecular markers.11^17 The morphologic assessment includes tumor grade, volume, tumor type, perineural invasion, vascular invasion, and pathologic stage. Various molecular markers have also been studied for their potential usefulness for prognosis assessment. These include p21, p27, p53, Bcl-2, cyclin Dl, HER-2/neu, E-cadherin, matrix metalloproteases, GST-[pi], telomerase, NF[kappa]B, I[kappa]B[alpha], and caspases.11-17 CPP32, also called Yama, apopain, and caspase-3, has been shown to have a significant correlation with apoptosis and is thought to play a crucial role in programmed cell death. The role of caspases has also been suggested in nonneoplastic conditions. CPP32 has been thought to contribute to the delayed cell death and brain injury found in neonatal hypoxia/ischemia in the developing brain.18 Caspase-3-activated deoxyribonuclease (CAD), a molecule that can cause DNA degradation in nuclei after being activated by caspase-3, recently has been cloned.19,20 Caspase-3-activated deoxyribonuclease inhibitor (ICAD) is an inhibitor of CAD found in the cytoplasm of growing cells.20 Overexpression of ICAD results in enhanced tumor progression by causing resistance to therapy-induced apoptosis in renal cell carcinoma.21 These findings have generated a lot of interest in caspases and their possible role in prostatic adenocarcinoma progression.

Some studies have reported reduced CPP32 expression in moderately and poorly differentiated prostatic adenocarcinoma, as compared with well-differentiated PAC and the normal prostate.9 Winter et al9 studied 42 radical prostatectomy specimens for localized primary adenocarcinoma and 6 normal prostates from age-matched patients undergoing cystoprostatectomy for bladder cancer. Expression for caspase-1, caspase-3, and caspase-9 was studied in both normal and malignant prostate tissue. Ki-67 immunostaining to determine the proliferative index was used. A semiquantitative reverse transcription-polymerase chain reaction analysis was performed on normal prostate tissue, primary adenocarcinoma, lymph nodes both with and without metastatic disease, and cases with benign prostatic hyperplasia (BPH). Diffuse, homogenous cytoplasmic staining for caspase-1 and caspase-3 was observed in normal epithelial cells. Eighty percent of the prostate cancer cases showed complete lack of caspase-1 expression, while the remainder had a marked reduction in expression when compared with normal prostate. The normal prostate and well-differentiated prostate cancer showed high expression of caspase-3 as compared to diminished expression seen in moderately and poorly differentiated adenocarcinoma (P = .001). Benign prostatic hyperplasia showed significantly reduced caspase-1 expression in epithelial cells. The correlation between loss of caspase-1 and caspase-3 expression and apoptotic index of malignant cells was not found by linear regression analysis. Furthermore, no correlation was seen between the Gleason score and caspase-1 or caspase-3 expression. However, the authors suggested that study of a large number of cases with both low- and high-grade tumors may yield statistically significant results.9

Others have seen variable immunoreactivity of CPP32 and no significant correlation between CPP32 expression and the frequency of apoptosis and Gleason score.2 Sohn et al2 studied 40 cases of BPH and 40 cases of PAC for expression of CPP32 and apoptotic index by in situ labeling and immunohistochemistry. Transurethral resection specimens were used for this study. The cases of PAC were divided into 3 broad categories: PAC I, Gleason scores 2 through 4; PAC II, Gleason scores 5 through 7; and PAC III, Gleason scores 8 through 10. In this study, the average number of apoptoses seen in cases with BPH was not different from that of PAC I. However, the cases with PAC II and PAC III showed a significantly higher number of apoptoses than BPH. No correlation was seen in the expression of CPP32 and the apoptotic index. Also, CPP32 expression and Gleason score were not in correlation.2 Still others have seen high CPP32 expression in BPH with equal staining of both basal and secretory epithelial cells, loss of expression in increasing grades of prostatic cancer, and little staining in epithelial cells of high-grade PAC.22 O'Neill et al22 studied 6 cases of BPH, 6 cases of intermediate-grade adenocarcinoma (Gleason scores 5-7), and 10 cases of high-grade adenocarcinoma (Gleason scores 810) for CPP32 expression. CPP32 was overexpressed in BPH. Both basal cells and secretory epithelial cells showed staining. Significant loss of expression was observed in the secretory epithelial cells of intermediate-grade adenocarcinoma, and rare staining was observed in cells of highgrade adenocarcinoma.22

Our results indicated that high CPP32 expression correlated with high tumor grade (Gleason score 7 and higher). The correlation between high expression of CPP32 and high tumor grade suggests a CPP32-related high cell turnover rate in high-grade primary prostatic adenocarcinoma. Furthermore, this strong correlation with Gleason score, which is a powerful predictor of disease outcome, suggests the potential usefulness of CPP32 as a diagnostic and prognostic factor for high-grade disease. Kadkol et al17 found a similar correlation between CPP32 expression and intermediate- and high-grade prostate cancer by in situ hybridization. Most importantly, numerous studies have shown the inhibition of prostate cancer growth through CPP32-dependent mechanisms. Both radiation and certain cardiac glycosides share a CPP32-dependent mechanism of apoptosis in PC-3 human prostate cells.23 Adenovirusmediated, caspase-based gene therapy studies performed in both human and murine prostate cancer cell lines show inhibition in the growth of prostate cancer through induction of cell death in malignant cells, while sparing normal tissues.24 Another novel gene product, pHyde, which was cloned from Dunning rat prostate cancer cells, involves CPP32 for the induction of apoptosis and leads to inhibition of human prostate cancer cell growth.23 The high expression of CPP32 in high-grade PAC and its potential use in treatment make it an important marker for prostate cancer. Our findings are similar to those reported by Kadkol et al.17 The reason for the difference in staining patterns observed by some other authors is not clear. To gain a better understanding of this variation, further studies are warranted.

References

l. Krajewska M, Wang HG, Krajewski S, et al. lmmunohistochemical analysis of in vivo patterns of expression of CPP32 (caspase-3), a cell death protease. Cancer Res. 1997;57:1605-1613.

2. Sohn JH, Kim DH, Choi NG, Park YE, Ro JY. Caspase-3/CPP32 immunoreactivity and its correlation with frequency of apoptotic bodies in human prostatic carcinomas and benign nodular hyperplasias. Histopathology. 2000:37:555-560.

3. Hoshi T, Sasano H, Kato K, et al. lmmunohistochemistry of caspase-3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis. Anticanccr Res. 1998;18:4347-4354.

4. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science. 1995;267:1456-1462.

5. Fernandes-Alnemri T, Litwack G, Alnemri ES. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J BIoI Chem. 1994;269: 30761-30764.

6. Nakopoulou L, Alexandrou P, Stefanaki K, Panayotopoulou E, Lazaris AC, Davaris PS. lmmunohistochemical expression of caspase-3 as an adverse indicator of the clinical outcome in human breast cancer. Pathobiology. 2001,169: 266-273.

7. Schimmer AD, Pedersen IM, Kitada S, et al. Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy. Cancer Res. 2003:63:1242-1248.

8. Chhanabhai M, Krajewski S, Krajewska M, Wang HG, Reed JC, Gascoyne RD. lmmunohistochemical analysis of interleukin-1 beta-converting enzyme/Ced-3 family protease, CPP32/Yama/caspase-3, in Hodgkin's disease. Blood. 1997;90: 2451-2455.

9. Winter RN, Kramer A, Borkowski A, Kyprianou N. Loss of caspase-1 and caspase-3 protein expression in human prostate cancer. Cancer Res. 2001:61: 1227-1232.

10. Hamilton LC. Regression With Graphics. Pacific Grove, Calif: Brooks/Cole Publishing Co: 1992.

11. Isaacs JT. Molecular markers for prostate cancer metastasis. Am J Pathol. 1997:150:1511-1521.

12. Bostwick DG, Grignon DJ, Hammond ME, et al. Prognostic factors in prostate cancer: College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med. 2000; 124:995-1000.

13. Koch MO, Foster RS, Bell B, et al. Characterization and predictors of prostate specific antigen progression rates after radical retropubic prostatectomy. J Urol. 2000:164:749-753.

14. Alers JC, Rochat J, Krijtenburg PJ, et al. Identification of genetic markers for prostatic cancer progression. Lab Invest. 2000;80:931-942.

15. Ross JS, Sheehan CE, Fisher HAG, Kauffman RA, Dolen EM, Kallakury BVS. Prognostic markers in prostate cancer. Expert Rev MoI Diagn. 2002:2:129-142.

16. Ross JS, Kallakury BVS, Sheehan CE, et al. Expression of NF[kappa]B and I[kappa]B[alpha] proteins in prostatic adenocarcinomas: correlation of NF[kappa]B immunoreactivity with disease recurrence. Mod Pathol. 2003;16:156A.

1 7. Kadkol SS, Brody JR, Epstein Jl, Kuhajda FP, Pasternack GR. Novel nuclear phosphoprotein pp32 is highly expressed in intermediate- and high-grade prostate cancer. Prostate. 1998;34:23l-237.

18. Byung HH, Daigen X, junjeong C, et al. Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem. 20022277:30128-30136.

19. Enari M, Sakahira H, Yokoyama H. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998:391:43-50.

20. Sakahira H, Anari H. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998;391:96-99.

21. Hara S, Myake H, Arakawa S, Kamidono S, Hara 1. Overexpression of inhibitor of caspase-3 activated deoxyribonuclease in human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy in vivo, j Urol. 2001:166:2491-2494.

22. O'Neill AJ, Boran SA, O'Keane C, et al. Caspase 3 expression in benign prostatic hyperplasia and prostate carcinoma. Prostate. 2001 ;47:183-188.

23. Nasu S, Milas L, Kawabe S, RaJu U, Newman R. Enhancement of radiotherapy by oleandrin is a caspase-3 dependent process. Cancer Lett. 2002;185: 145-151.

24. Shariat SF, Desai S, Song W, et al. Adenovirus-mediated transfer of inducible caspases: a novel "death switch" gene therapeutic approach to prostate cancer. Cancer Res. 2001;61:2562-2571.

25. Zhang X, Steiner MS, Rinaldy A, Lu Y. Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3. Oncogene. 2001;20:5982-5990.

Shahgul Anwar, MD; Robert A. Ambros, MD; Timothy A. Jennings, MD; Jeffrey S. Ross, MD; Anton Beza; Badar Mian, MD; Tipu Nazeer, MD

Accepted for publication January 28, 2004.

From the Department of Pathology and Laboratory Medicine (Drs Anwar, Ambros, ]ennings, Ross, and Nazeer, and Mr Beza) and Department of Surgery, Division of Urology (Dr Mian), Albany Medical College, Albany, NY.

Presented in part at the 92nd Annual Meeting of the United States and Canadian Academy of Pathology, Washington, DC, March 2003.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Tipu Nazeer, MD, Department of Pathology, Mail Code 81, Albany Medical College, 47 New Scotland Ave, Albany, NY 12208 (e-mail: nazeert@mail.amc.edu).

Copyright College of American Pathologists Jun 2004
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