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Cathepsin D and breast cancer: Useful?
Human PATHOLOGY VOLUME
25
September I994
NUMBER
9
Editorial Cathepsin D and Breast Cancer: Useful? endoproteinase that is glycosylated and appears in a 52kd precursor and a 4&kd intermediate form.-?.4 The 52kd procathepsin form was the antigen found by Westley and Rochefort.” Because cathepsin D is now known to have such a profound effect on the behavior of cancer cells, it has brought lysosomes, proteases, invasion, and metastases into the same conceptual scheme. The remaining question is whether the cathepsins also can serve as accurate prognostic markers. Rochefort and others have observed a consistent correlation between cathepsin D and prognosis.‘,3”‘0 Others, relying primarily on immunohistochemical techniques, have not.‘.“,” The study by Castiglioni et al’ perhaps mirrors the experience of many practicing pathologists who have tended to use a single dilution of a commercial antibody. They conclude that there is no significant correlation between the proportion of the cells staining and the known risk factors or the clinical outcome in their patient population. Castiglioni et al’ properly limit their conclusions to the antibodies, dilutions, and patient population in their study. In all fairness, it should be pointed out that the cathepsin D advocates always have said that the enzyme is an independent variable that does not correlate with the standard clinical markers.’ In this issue Roger et al” implicitly acknowledge that prognosis cannot be related to the simple presence or absence of stain in tumor cells. They suggest that both qualitative and quantitative issues may be the basis for the controversy. Their study is aimed at resolving some of the issues by establishing additional and more rigorous diagnostic criteria. They suggest, as have Castiglioni and others, that one explanation for the different results may be the fact that cathepsin also is expressed in the tumor stroma and in normal tissue.“’ Discrepancies could be explained by the differences between cytosolic assays of whole tissue samples and the more precise immunohistochemical analyses because of the relative contribution of the nontumor cells.“,’ Both studies present evidence that this is not the case. Both studies acknowledge that the cathepsins are expressed in normal and malignant mammary cells.
The current controversy over the value of cathepsin D as a prognostic marker in breast cancer is confusing to the practicing pathologist. A number of articles have appeared offering different opinions, creating a dilemma for practicing pathologists seeking good immunohistochemical markers to apply to their breast cancer cases. Is staining for cathepsin D or the other cathepsins useful or not? The contrasting viewpoints are highlighted in two independent studies in this issue of HUMAN PATHOL OGY.“” The article by Castiglioni et al’ introduces new polyclonal antibodies against cathepsins B and L to compare the immunohistochemical staining patterns for cathepsins D, B, and L, concluding that their results “do not support their prognostic value in carcinomas of the breast.” In contrast, the article from a team of French scientists, led by Henri Rochefort (Roger et al’), indicates that in their hands immunohistochemical staining for cathepsin D correlates with the cytosolic assays and, by implication, cathepsin D continues to be a valuable independent variable that can be used to estimate prognosis. Outside of the current clinical controversy, scientific interest in cathepsin D is the result of an interesting intersection of seemingly disparate research fields. Cathepsin D now appears to be a molecule central to the pathophysiology of cancer. It is a lysosomal enzyme that can act either directly by digesting the extracellular matrix or indirectly by initiating the proteolytic cascade that may be responsible for the breakdown of basement membranes.” It also appears to be capable of either releasing growth factors from the matrix or activating others.” Whatever the mechanism, cathepsin D can facilitate the invasive behavior of breast cancer cells. Therefore, the enzyme apparently plays a key role in the mechanism of invasion and metastases. In 1980 Bruce Westley and Henri Rochefort described an estrogen inducible 52-kd protein that was released into the media of MCF-7 cells.’ Six years later Rochefort recognized that the enzyme was the well known lysosomal protease cathepsin D and the convergence began.” Cathepsin D is a 34kd lysosomal aspartic
a47
HUMAN
PATHOLOGY
Volume 25, No. 9 (September
1994)
criteria for rating a tumor as positive.’ Castiglioni et al’ do not mention these structures. Because one of proposed pathological mechanisms requires that the enzyme is outside the cell, location of the enzyme may be significant. In sum, the apparent discrepancies and conflicting opinions about the relative value of cathepsin D are based on the use of different criteria, different techniques, different reagents, and different interpretations. It is clear that immunohistochemical stains for cathepsin D cannot be interpreted after casual application to breast cancer using single dilutions of uncharacterized antibodies. Until standardized techniques and criteria are established, pathologists using anti-cathepsin D should be careful to note which antibody and which techniques are being used before providing the clinician with a prognostic interpretation. The establishment of such standardized techniques and diagnostic criteria could be undertaken at a workshop similar to that held for the mucin panel, after which, using uniform criteria, techniques, and reagents, we can truly assess the value of staining for cathepsin D.
Roger et al’ suggest that breast cancers overexpress cathepsin D and find a correlation between the cytosolic assay and the quantitative image analysis that supports the viewpoint. If verified, the relative concentrations of the enzyme may be more important in determining malignant potential than the simple presence or absence of the enzyme. The dilution cutoff points would have to be carefully established to provide reliable prognostic interpretations based on immunohistochemical stains. Alternatively, seemingly trivial technical explanations might be the basis for differences in interpretation. For example, the cathepsin D molecule appears in at least three different forms and is glycosylated. The apparent differences in results could be based on the relative affinity of the different monoclonal and polyclonal antibodies against the different forms of the antigen. The relative concentration of the different forms also could be significant. Because the various investigators are using their own reagents, techniques, and criteria, it seems inevitable that they would get different results and controversy would emerge. The current controversy reminds this reviewer of the conflicting claims about the value of monoclonal antibodies against breast epithelial mucins. Like cathepsin D, the value of the antibodies was controversial among the experts and confusing to the practitioner. A group of responsible investigators established standard technical protocols and compared the different antibody preparations.” This approach proved to be very successful by providing a basis for interpreting the conflicting claims in the use of antimucins. The extensive collaboration established that everyone was right because the different antibody preparations had different affinities and were directed against different molecular configurations of the same molecule.14 The current articles demonstrate the difficulties in interpretation of techniques and reagents that have not been completely characterized or standardized. On the technical level Rochefort’s group2 used the monoclonal antibody MlG8, whereas the group led by Castiglioni et al’ used polyclonal rabbit antibodies against all three cathepsins. Isola et al9 used yet another monoclonal antibody. The two articles published here report different pretreatment techniques. Roger et al” state that “a short pronase digestion was necessary,” whereas Castiglioni et al’ declare that “the antisera . . . worked satisfactorily . . . without need of trypsinization on the sections.” Neither group used the microwave antigen retrieval technique that is currently popular in this country. Castiglioni et al’ used a subjective qualitative scoring system much like that on which the practicing pathologist relies. Roger et al’ used quantitative image analysis to relate the quantity of the stain in tumor cells to the cytosolic assay. Roger et al2 also describe large (>.5 pm) phagolysomes in many of the tumors, which probably correspond to those previously characterized as large acidic vesicles (LAVs) in cultured tumor cells. These large cathepsin D-containing vesicles, although relatively rare, seem to correlate with the level of the enzyme in the cytosolic assay. They suggest that the LAVs could be a
ROBERT D. CARDIFF,MD, PHD University of California, Davis Davis, CA
REFERENCES 1. Castiglioni T, Merino MJ, Elmer B, et al: Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer. HUM PATHOL25:857-862, 1994 2. Roger P, Montcourrier P, Maudelonde T, et al: Cathepsin D immunostaining in paraffmembedded breast cancer cells and macrophages: Correlation with cytosolic assay. HUM PATHOL 25:863-871, 1994 3. Rochefort H: Biological and clinical significance of cathepsin D in breast cancer. Acta Oncologica 31:125-130, 1992 4. Leto G, Gebbia N, Rausa L, et al: Cathepsin D in the malignant progression of neoplastic diseases (review). Anticancer Res 12:235 240, 1992 5. Westley B, Rochefort H: A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 20:353-362, 1980 6. Spyratos F, Maudelonde T, BrouilletJP, et al: Cathepsin D: An independent prognostic factor for metastasis of breast cancer. Lancet 2:1115-1118, 1989 7. Thorpe SM, Rochefort H, Garcia M, et al: Association between high concentrations of Mr 52,000 cathepsin D and poor prognosis in primary human breast cancer. Cancer ties 49:6008~014,‘1989 8. Tandon AK. Clark G, Chamness GC. et al: Catheosin D and prognosis in breast cancer. i Engl J Med 322:279-312, 1960 9. Isola J, Weitz S, Visakorpi T, et al: Cathepsin D expression detected by immunohistochemistry has independent prognostic value in axillary node-negative breast cancer. J Clin Oncol 11:36-43, 1993 10. Winstanley JHR, Leinster SJ, Cooke TG, et al: Prognostic significance of cathepsin D in patients with breast cancer. Br J Cancer 67:767-772, 1993 11. Henry JA, McCarthy AL, Angus B, et al: Prognostic significance of the estrogen-regulated protein, cathepsin D in breast cancer: An immunohistochemical study. Cancer 65:265-271, 1990 12. Kandalaft PL, Chang KL, Ahn CW, et al: Prognostic significance of immunohistochemical analysis of cathepsin D in low-stage breast cancer. Cancer 71:27562763, 1993 13. Taylor-Papadimitriou J: Report on the first international workshop on carcinoma associated mucins. Int J Cancer 49:1-5, 1991 14. Ceriani RL, Peterson JA, Blank EW, et al: Epitope expression on the breast epithelial mucin. Breast Cancer Res Treat 24:103-l 13, 1992
848
25
September I994
NUMBER
9
Editorial Cathepsin D and Breast Cancer: Useful? endoproteinase that is glycosylated and appears in a 52kd precursor and a 4&kd intermediate form.-?.4 The 52kd procathepsin form was the antigen found by Westley and Rochefort.” Because cathepsin D is now known to have such a profound effect on the behavior of cancer cells, it has brought lysosomes, proteases, invasion, and metastases into the same conceptual scheme. The remaining question is whether the cathepsins also can serve as accurate prognostic markers. Rochefort and others have observed a consistent correlation between cathepsin D and prognosis.‘,3”‘0 Others, relying primarily on immunohistochemical techniques, have not.‘.“,” The study by Castiglioni et al’ perhaps mirrors the experience of many practicing pathologists who have tended to use a single dilution of a commercial antibody. They conclude that there is no significant correlation between the proportion of the cells staining and the known risk factors or the clinical outcome in their patient population. Castiglioni et al’ properly limit their conclusions to the antibodies, dilutions, and patient population in their study. In all fairness, it should be pointed out that the cathepsin D advocates always have said that the enzyme is an independent variable that does not correlate with the standard clinical markers.’ In this issue Roger et al” implicitly acknowledge that prognosis cannot be related to the simple presence or absence of stain in tumor cells. They suggest that both qualitative and quantitative issues may be the basis for the controversy. Their study is aimed at resolving some of the issues by establishing additional and more rigorous diagnostic criteria. They suggest, as have Castiglioni and others, that one explanation for the different results may be the fact that cathepsin also is expressed in the tumor stroma and in normal tissue.“’ Discrepancies could be explained by the differences between cytosolic assays of whole tissue samples and the more precise immunohistochemical analyses because of the relative contribution of the nontumor cells.“,’ Both studies present evidence that this is not the case. Both studies acknowledge that the cathepsins are expressed in normal and malignant mammary cells.
The current controversy over the value of cathepsin D as a prognostic marker in breast cancer is confusing to the practicing pathologist. A number of articles have appeared offering different opinions, creating a dilemma for practicing pathologists seeking good immunohistochemical markers to apply to their breast cancer cases. Is staining for cathepsin D or the other cathepsins useful or not? The contrasting viewpoints are highlighted in two independent studies in this issue of HUMAN PATHOL OGY.“” The article by Castiglioni et al’ introduces new polyclonal antibodies against cathepsins B and L to compare the immunohistochemical staining patterns for cathepsins D, B, and L, concluding that their results “do not support their prognostic value in carcinomas of the breast.” In contrast, the article from a team of French scientists, led by Henri Rochefort (Roger et al’), indicates that in their hands immunohistochemical staining for cathepsin D correlates with the cytosolic assays and, by implication, cathepsin D continues to be a valuable independent variable that can be used to estimate prognosis. Outside of the current clinical controversy, scientific interest in cathepsin D is the result of an interesting intersection of seemingly disparate research fields. Cathepsin D now appears to be a molecule central to the pathophysiology of cancer. It is a lysosomal enzyme that can act either directly by digesting the extracellular matrix or indirectly by initiating the proteolytic cascade that may be responsible for the breakdown of basement membranes.” It also appears to be capable of either releasing growth factors from the matrix or activating others.” Whatever the mechanism, cathepsin D can facilitate the invasive behavior of breast cancer cells. Therefore, the enzyme apparently plays a key role in the mechanism of invasion and metastases. In 1980 Bruce Westley and Henri Rochefort described an estrogen inducible 52-kd protein that was released into the media of MCF-7 cells.’ Six years later Rochefort recognized that the enzyme was the well known lysosomal protease cathepsin D and the convergence began.” Cathepsin D is a 34kd lysosomal aspartic
a47
HUMAN
PATHOLOGY
Volume 25, No. 9 (September
1994)
criteria for rating a tumor as positive.’ Castiglioni et al’ do not mention these structures. Because one of proposed pathological mechanisms requires that the enzyme is outside the cell, location of the enzyme may be significant. In sum, the apparent discrepancies and conflicting opinions about the relative value of cathepsin D are based on the use of different criteria, different techniques, different reagents, and different interpretations. It is clear that immunohistochemical stains for cathepsin D cannot be interpreted after casual application to breast cancer using single dilutions of uncharacterized antibodies. Until standardized techniques and criteria are established, pathologists using anti-cathepsin D should be careful to note which antibody and which techniques are being used before providing the clinician with a prognostic interpretation. The establishment of such standardized techniques and diagnostic criteria could be undertaken at a workshop similar to that held for the mucin panel, after which, using uniform criteria, techniques, and reagents, we can truly assess the value of staining for cathepsin D.
Roger et al’ suggest that breast cancers overexpress cathepsin D and find a correlation between the cytosolic assay and the quantitative image analysis that supports the viewpoint. If verified, the relative concentrations of the enzyme may be more important in determining malignant potential than the simple presence or absence of the enzyme. The dilution cutoff points would have to be carefully established to provide reliable prognostic interpretations based on immunohistochemical stains. Alternatively, seemingly trivial technical explanations might be the basis for differences in interpretation. For example, the cathepsin D molecule appears in at least three different forms and is glycosylated. The apparent differences in results could be based on the relative affinity of the different monoclonal and polyclonal antibodies against the different forms of the antigen. The relative concentration of the different forms also could be significant. Because the various investigators are using their own reagents, techniques, and criteria, it seems inevitable that they would get different results and controversy would emerge. The current controversy reminds this reviewer of the conflicting claims about the value of monoclonal antibodies against breast epithelial mucins. Like cathepsin D, the value of the antibodies was controversial among the experts and confusing to the practitioner. A group of responsible investigators established standard technical protocols and compared the different antibody preparations.” This approach proved to be very successful by providing a basis for interpreting the conflicting claims in the use of antimucins. The extensive collaboration established that everyone was right because the different antibody preparations had different affinities and were directed against different molecular configurations of the same molecule.14 The current articles demonstrate the difficulties in interpretation of techniques and reagents that have not been completely characterized or standardized. On the technical level Rochefort’s group2 used the monoclonal antibody MlG8, whereas the group led by Castiglioni et al’ used polyclonal rabbit antibodies against all three cathepsins. Isola et al9 used yet another monoclonal antibody. The two articles published here report different pretreatment techniques. Roger et al” state that “a short pronase digestion was necessary,” whereas Castiglioni et al’ declare that “the antisera . . . worked satisfactorily . . . without need of trypsinization on the sections.” Neither group used the microwave antigen retrieval technique that is currently popular in this country. Castiglioni et al’ used a subjective qualitative scoring system much like that on which the practicing pathologist relies. Roger et al’ used quantitative image analysis to relate the quantity of the stain in tumor cells to the cytosolic assay. Roger et al2 also describe large (>.5 pm) phagolysomes in many of the tumors, which probably correspond to those previously characterized as large acidic vesicles (LAVs) in cultured tumor cells. These large cathepsin D-containing vesicles, although relatively rare, seem to correlate with the level of the enzyme in the cytosolic assay. They suggest that the LAVs could be a
ROBERT D. CARDIFF,MD, PHD University of California, Davis Davis, CA
REFERENCES 1. Castiglioni T, Merino MJ, Elmer B, et al: Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer. HUM PATHOL25:857-862, 1994 2. Roger P, Montcourrier P, Maudelonde T, et al: Cathepsin D immunostaining in paraffmembedded breast cancer cells and macrophages: Correlation with cytosolic assay. HUM PATHOL 25:863-871, 1994 3. Rochefort H: Biological and clinical significance of cathepsin D in breast cancer. Acta Oncologica 31:125-130, 1992 4. Leto G, Gebbia N, Rausa L, et al: Cathepsin D in the malignant progression of neoplastic diseases (review). Anticancer Res 12:235 240, 1992 5. Westley B, Rochefort H: A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 20:353-362, 1980 6. Spyratos F, Maudelonde T, BrouilletJP, et al: Cathepsin D: An independent prognostic factor for metastasis of breast cancer. Lancet 2:1115-1118, 1989 7. Thorpe SM, Rochefort H, Garcia M, et al: Association between high concentrations of Mr 52,000 cathepsin D and poor prognosis in primary human breast cancer. Cancer ties 49:6008~014,‘1989 8. Tandon AK. Clark G, Chamness GC. et al: Catheosin D and prognosis in breast cancer. i Engl J Med 322:279-312, 1960 9. Isola J, Weitz S, Visakorpi T, et al: Cathepsin D expression detected by immunohistochemistry has independent prognostic value in axillary node-negative breast cancer. J Clin Oncol 11:36-43, 1993 10. Winstanley JHR, Leinster SJ, Cooke TG, et al: Prognostic significance of cathepsin D in patients with breast cancer. Br J Cancer 67:767-772, 1993 11. Henry JA, McCarthy AL, Angus B, et al: Prognostic significance of the estrogen-regulated protein, cathepsin D in breast cancer: An immunohistochemical study. Cancer 65:265-271, 1990 12. Kandalaft PL, Chang KL, Ahn CW, et al: Prognostic significance of immunohistochemical analysis of cathepsin D in low-stage breast cancer. Cancer 71:27562763, 1993 13. Taylor-Papadimitriou J: Report on the first international workshop on carcinoma associated mucins. Int J Cancer 49:1-5, 1991 14. Ceriani RL, Peterson JA, Blank EW, et al: Epitope expression on the breast epithelial mucin. Breast Cancer Res Treat 24:103-l 13, 1992
848