Expression of Cathepsin D in Colorectal Adenocarcinomas: Correlation with Clinicopathologic Features

Abstract

Brief ReportsExpression of Cathepsin D in Colorectal Adenocarcinomas: Correlation with Clinicopathologic Features Fahri Yilmaz, MD Ali Kemal Uzunlar, MD Nihal Kilinc, and MD H. Gülsen YilmazMD Fahri Yilmaz From the Department of Pathology and surgery, Faculty of Medicine, Dicle Universitesi, Patoloji, Diyarbakir, Turke Search for more papers by this author , Ali Kemal Uzunlar From the Department of Pathology and surgery, Faculty of Medicine, Dicle Universitesi, Patoloji, Diyarbakir, Turke Search for more papers by this author , Nihal Kilinc From the Department of Pathology and surgery, Faculty of Medicine, Dicle Universitesi, Patoloji, Diyarbakir, Turke Search for more papers by this author , and H. Gülsen Yilmaz Correspondence to: Dr. F. Yilmaz Dicle Universitesi Tip Fakultesi Patoloji AD 21280 Diyarbakir Turkey From the Department of Pathology and surgery, Faculty of Medicine, Dicle Universitesi, Patoloji, Diyarbakir, Turke Search for more papers by this author Published Online:1 May 2003https://doi.org/10.5144/0256-4947.2003.208SectionsPDF ToolsAdd to favoritesDownload citationTrack citations ShareShare onFacebookTwitterLinked InRedditEmail AboutIntroductionColorectal carcinoma is the second most frequent carcinoma, after carcinoma of the lung, in Western countries.1 The prognosis for patients with colorectal carcinoma seems to be related mainly to the control of both progression and metastasis of the malignant tumor. In metastasis, proteolytic enzymes play an important role in mediating passage of the malignant cell through the cell membrane.2 Proteases are thought to be important factors for tumor invasion and metastasis because they can degrade extracellular matrices and basement membranes,3,4 which play a defensive role for tumor invasion and metastasis.5 Cathepsin D (CD) is known to be a lysosomal acid protease that is mainly included in intracellular protein catabolism and is inducible by estrogens.6 Therefore, many investigators have studied intensively the role of CD in breast carcinomas. These studies have suggested that the expression of CD is correlated with the invasion and metastasis of breast carcinomas,7–9 but the role of CD expression in predicting prognosis or invasive potential in colorectal carcinoma is unknown.1In this study, we evaluated the expression of CD in a series of colorectal carcinomas and its correlation with conventional clinicopathologic features such as age, gender, tumor size, tumor grade and Dukes’ stage.PATIENTS AND METHODSTissue samples were obtained from the surgical specimens of 100 primary colorectal adenocarcinomas. All specimens were collected at the Department of Pathology, Dicle University Hospital, Diyarbakir, Turkey between 1995 and 2000. The clinical data were obtained from pathological reports. The specimens were fixed in formalin and embedded in paraffin. Consecutive 4-mm-thick sections were stained with hematoxylin and eosin for histological diagnosis.Histologically, all colorectal carcinomas were adenocarcinomas. The grade of differentiation was high in 17, moderate in 58, and low in 25 cases. Staging of cases was pathological only (Dukes’ stage A, B and C). There were 10 cases of stage A, 31 cases of stage B and 59 cases of stage C. The mean age of the patients was 53.7 years (range,24 to 80 years), and the male/female ratio was 1.3 (56/44). Most adenocarcinomas were located in the rectosigmoid (n=65).One or two blocks from each tumor were stained for immunohistochemical analysis using the Avidin-Biotin and immunoperoxidase methods. Additional 4-mm-thick sections were cut from paraffin blocks. After blockage of endogenous peroxidase with H2O2 in methanol for 30 minutes, sections were immersed in citrate buffer (pH=6.0) in a microwave-resistant container. Subsequently, the sections were incubated with a protein-blocking solution for five minutes and then incubated with a mouse anti-human cathepsin D monoclonal antibody (Dako, Carpinteria, USA). Immunoperoxidase detection was employed, using the diaminobenzidine substrate (DAB). Counter staining was performed with hematoxylin.All immunostained sections were analyzed by two different pathologists who had no knowledge of the patients’ clinical and pathological status. For each section, at least ten high power fields were examined under light microscopy, individual cells were counted, and the mean percentage of CD positive cancer cells was calculated among all the malignant cells observed within the primary lesion. CD immunohistochemical expression in nonneoplastic stromal cells was considered positive if CD was expressed in <10% of the stromal and tumor cells. Overexpression of CD was considered present when >10% of stromal and tumor cells were stained. The results were evaluated quantitatively and divided into several groups (Table 1).Table 1. Correlation of cancer cell CD expression and clinicopathologic features in colorectal cancer.Table 1. Correlation of cancer cell CD expression and clinicopathologic features in colorectal cancer.For statistical analysis, Chi-square tests were applied. All results were considered significant at the 5% level. Data were expressed as the mean ± SD and range.RESULTSImmunoreactivity for cathepsin D (CD) was seen as brown, fine to coarse granular heterogeneous cytoplasmic staining in neoplastic cells of the primary tumor (Figure 1). Normal colorectal epithelium adjacent to carcinoma was negative for CD in the cytoplasm of epithelial cells. Macrophages and polymorphonuclear leukocytes that infiltrated in and around the carcinoma tissue were intensively stained for CD (Figure 2).Figure 1. Heterogenous cytoplasmic staining of CD in neoplastic and stromal cells.Download FigureFigure 2. Staining of CD in macrophages, polymorphonuclear leukocytes and stromal cells around the carcinoma tissue.Download FigureThere were different patterns of CD distribution. In some cases, the expression was polarized to the luminal surface inmost of the cells. In other cases, the CD was polarized to the base of the cancer cells, giving a linear impression (Figure 2). These two patterns were usually observed in well-differentiated carcinomas with ductal architecture. In more aggressive tumors, there was a random cytoplasmic contribution of CD expression (Figure 4).Figure 3. CD staining in base of the cancer cells in a well-differentiated carcinomas with ductal architicture.Download FigureFigure 4. Random cytoplasmic expression of CD in a high-grade colorectal carcinoma.Download FigureCD expression in carcinoma cells was present in 68 of the 100 evaluated cases (68%). CD positivity in cancer cells was 56% in grade 1, 69% in grade 2, and 82% in grade 3 tumors. The frequency of CD immunopositivity in cancer cells occurred in 5 of 10 cases of stage A tumors and in 19 of 31 cases of stage B tumors (50% and 61%, respectively). Stage C and grade 3 tumors demonstrated the highest incidence of cancer cell CD positivity (75% and 82%, respectively). However, there was not a statisticallysignificant difference between Dukes’ stage and grade of tumor cells (Table 1) and clinical features.Stromal cells showed positive immunostaining (any degree of CD positivity) in 65 cases (65%) (Figure 1, 2, and 4). According to Dukes’ stage, the stromal cells stained were positive in 5 (50%) stage A, 20 (64%) stage B (64%) and 39 (66%) stage C cases. CD expression was seen in 12 of 17 cases (71%) of grade 3 tumors. Overexpression of CD in stromal cells (>10% positive) was observed in 25 of 59 cases (42%) with Dukes’ C adenocarcinoma (Table 2). There was not a statistical significant correlation between neoplastic and stromal cell CD positivity in colorectal carcinomas (p=0.153).Table 2. Correlation of stromal cell CD expression and grades and Dukes’ stage in colorectal cancer.Table 2. Correlation of stromal cell CD expression and grades and Dukes’ stage in colorectal cancer.DISCUSSIONCarcinoma cells must penetrate into the epithelial basement membrane and gain access to lymphatics or blood vessels to initiate the metastatic process. The mechanism of invasion by tumor cells through tissue barriers and into blood and lymphatic vessels appears to involve both mechanical and enzymatic processes. Tumors are known to contain and secrete various tissue proteolytic enzymes, and many malignant neoplasms produce higher levels of proteolytic enzymes than benign tumors or normal tissues.10 Cathepsin D may play an important role in this process, because it can degrade extracellular matrices and basement membranes.3,11 It also has mitogenicproperties12 and facilitates the releaseof angiogenic factor and basic fibroblast growth factor, stimulating neovascularization.13Galanduiket al.,14 using immunoassay, showed increased CD levels in neoplastic colon tissues with respect to the normal colon, regardless of whether cells expressed this protease. Theodoropouloset al.,1 performing immunohistochemistry with a monoclonal antibody, detected CD in tumor and stromal cells in colon cancer specimens. Our findings are consistent with those of Theodoropoulos and other immunohistochemical studies that show expression of CD in stromal cells.1,3,10,15,16 The immunohistochemical approach has the advantage of precise and selective tissue localization of the antigen and can easily discriminate the CD expression in stromal cells from that in cancer cells.1 Immunostaining seems more suitable for clarifying whether CD in cancer cells is correlated with tumor invasion and metastasis. However, the correlation between CD expression and clinicopathologic factors is not conclusive, even by use of immunostaining methods.3Although several investigators have studied the correlation between CD expression and tumor progression, invasion, and metastasis in colorectal carcinomas, their data are conflicting.15,17–19 This finding may be caused by differences in methods used to estimate CD expression in colorectal tissue. In Valentini’sstudy,15 8 of 46 carcinomas (17%) were cathepsin D positive both in cancer and stromal cells. In the remaining 38 carcinomas, no staining was seen either in cancer or stromal cells. However, in our study, we found more CD expression in both stromal and epithelial neoplastic cells than Valentini’s study.The less aggressive, lower stage colorectal tumors, which show better biological behavior and phenotype than more aggressive tumors, may preserve the functional integrity of the biochemical pathways and synthesizing systems that are responsible for CD expression. These biological properties may be preserved only in tumor clones comprising the invasion margin of the advanced stage tumors.1,20 Although a decreased frequency of CD expression in tumor cells was observed in the advanced stages of colorectal carcinomas, the intensely stained malignant cells were mainly located at the advancing margin of the invasion front in those specimens. A prominent finding in this study was the increased incidence of CD immunopositivity in the stromal cells of advanced Dukes’ stage and in grade 3 cancer cells, as well as its correlation with a worse prognosis. In well-differentiated carcinomas with ductal architecture, CD was expressed on the basal and luminal surface of the cancer cells, giving a linear impression.Some clinical studies21–23 suggest that increased CD activity in stromal components, such as infiltrative inflammatory cells, might have a significant biological role. Although it is known that inflammatory cell infiltration at the border of the invasive front acts as a defense mechanismagainst tumor invasion, inflammatory cells of the stroma inside and adjacent to the tumor mass contain various enzymes, including CD, that could destroy the tissue architecture and facilitate tumor spread.1 We detected more CD immunoreactivity in inflammatory cells in advanced stage and grade 3 tumors. Many studies reported that the normal epithelium adjacent to the tumor was negative for CD; some stained stromal cells were observed around the epithelium.1,3,10,15 These studies and our study showed that the absence of immunoperoxidase staining in the normal mucosa might be related to significantly lower cytosol levels of CD in the normal colon.The high percentage of CD positivity in the reactive stromal and cancer cells of colorectal carcinomas supports the hypothesis that this marker may facilitate invasion of the surrounding stroma. Our results show a closer association between the expression of CD in stromal cells and that in cancer cells, probably because factors produced by cancer cells stimulate CD production in stromal and cancer cells.We believe there is an apparent correlation between the percentage of CD expression and the grade/stage of the colorectal carcinoma; however, the correlation did not reach statistical significance in our study, possibly because of the sample size.ARTICLE REFERENCES:1. Theodoropoulos GE, Panoussopoulos D, Larazis ACH, Golematis BCH. "Evaluation of Cathepsin D Immunostaining in Colorectal Adenocarcinoma" . J Surg Oncol. 1997; 65:242–248. Google Scholar2. Schwartz MK. "Tissue cathepsins as tumor markers" . Clin Chim Acta. 1995; 237:67–78. Google Scholar3. Aroa J, Fukui H, Ono Y, Ueda Y, Chiba T, Fujimori T. "Immunohistochemical localization of cathepsin D in colorectal tumors" . Dis Colon Rectum. 2000; 43:396–401. Google Scholar4. Montcourrier P, Mangeat PH, Salazar G, Morisset M, Sahuquet A, Rochefort H. "Cathepsin D in breast cancer cells can digest extracellular matrix in large vesicles" . Cancer Res. 1990; 50:6045–54. Google Scholar5. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S. "Metastatic potential correlates with enzymatic degradation of basement membrane collagen" . Nature. 1980; 248:67–8. Google Scholar6. Westley B, Rochefort H. "A secreted glycoprotein induced by estrogen in human breast cancer cell lines" . Cell. 1980; 20:353–62. Google Scholar7. Thorpe SM, Rochefort H, Garcia M, Freiss G, Christensen IJ, Khalaf S, et al.. "Association between high concentrations of Mr 52.000 cathepsin D and poor prognosis in primary human breast cancer" . Cancer Res. 1989; 49:6008–14. Google Scholar8. Spyratos F, Maudelonde T, Brouillet GB, Brunet M, Defrenne A, Andrieu C, et al.. "Cathepsin D: An independent prognostic factor for metastasis of breast cancer" . Lancet. 1989; 2:1115–8. Google Scholar9. Kute TE, Shao ZM, Sugg NK, Long RT, Russell GB, Case LD. "Cathepsin D as a prognostic indicator for node-negative breast cancer patients using both immunoassay and enzymatic assays" . Cancer Res. 1992; 52:5198–203. Google Scholar10. loachim EE, Goussia AC, Machera M, Tsianos EV, Kappas AM, Agnantis NJ. "Immunohistochemical evaluation of cathepsin D in colorectal tumours: a correlation with extracellular matrix components, p53, pRb, bcl-2, c-erbB-2, EGFR and proliferation indices" . Anticancer Research. 1999; 19:2147–56. Google Scholar11. Agarwal S. "Protease cathepsins-A view" . Biochem Educ. 1990; 18:67–72. Google Scholar12. Briozzo P, Morriset M, Capony F, Rougeot C, Rochefort H. "In vitro degradation of extracellular matrix with Mr 52.000 Cathepsin D secreted by breast cancer cells" . Cancer Res. 1988; 48:3388–92. Google Scholar13. Briozzo P, Badet J, Capony F, Piery I, Montcourrer P, Barritault D, et al.. "MCF7mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D" . Exp Cell Res. 1991; 194:252–59. Google Scholar14. Galandiuk S, Mireljic S, Yang AR, Early M, McCoy MD, Wittliff JL. "Expression of hormone receptors, cathepsin D, and HER-2/neu oncoprotein in normal colon and colonic disease" . Arch Surg. 1993;128–637-42. Google Scholar15. Valentini AM, Pirelli H, Armentano R, Caruso ML. "The immunohistochemical expression of cathepsin D in colorectal cancer" . Anticancer Res. 1996; 16:77–80. Google Scholar16. Valentini AM, Amati L, Pirelli M, Armentano R, Caruso ML. "Immunohistochemical expression of pS2 and cathepsin D in breast cancer relationship with ER, PgR immunostaining and clinicopathologic aspects" . Oncol Rep. 1994; 1:585–90. Google Scholar17. Sheahan K, Shuja S, Murnane MJ. "Cystein protease activities and tumor development in human colorectal carcinoma" . Cancer Res. 1989; 49:3809–14. Google Scholar18. Tumminello FM, Gebbia N, Pizzolanti G, Russo A, Bazan V, Leto G. "Cathepsin D content in colorectal cancer" . Oncology. 1995; 52:237–42. Google Scholar19. Mayer A, Fritz E, Fortelny R, Kofier K, Ludwig H. "Immunohistochemical evaluation of cathepsin D expression in colorectal cancer" . Cancer Invest. 1997; 15:106–10. Google Scholar20. Augereau P, Garcia M, Mattei MG, Cavailles V, Depadova F, Derocq D, et al.. "Cloning and sequencing of the 52 K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer and mapping on chromosome 11" . Mol Endocrinol. 1988; 2:186–92. Google Scholar21. Theodoropoulos GE, Lazaris AC, Panoussopoulos D, Davaris P, Golematis BC. "Significance of estrogen receptors and cathepsin D tissue detection in gastric adenocarcinoma" . J Surg Oncol. 1995; 58:176–83. Google Scholar22. Henry JA, McCarthy AL, Angus B, Westley BR, May FE, Nicholson S, et al.. "Prognostic significance of the estrogen-regulated protein, cathepsin D, in breast cancer: An immunohistochemical study" . Cancer. 1990; 65:265–71. Google Scholar23. Rogers S, Day CA, Fox SB. "Expression of cathepsin D and estrogen receptor in male breast carcinoma" . Hum Pathol. 1993; 24:148–51. Google Scholar Previous article Next article FiguresReferencesRelatedDetails Volume 23, Issue 3-4May-July 2003 Metrics History Accepted1 December 2002Published online1 May 2003 InformationCopyright © 2003, Annals of Saudi MedicinePDF download