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Immunohistochemical detection of CD31 antigen in normal and neoplastic canine endothelial cells
j. Comp. Path. 1995 Vol, 112, 319-326
Irnrnunohistochemical D e t e c t i o n of CD31 Antigen in N o r m a l and N e o p l a s t i c Canine Endothelial Cells L. Ferrer, D. Fondevila, R. M. Rabanal and M. Vilafranca Department of Animal Pathology, Veterinary School, Universitat Aut~noma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Summary This report describes the immunohistochemical detection ofvon Willebrand's factor (vWf) and CO31 antigen in paraffin wax-embedded, formalin-fixed tissue sections of canine normal organs and vascular neoplasms. CO31 antigen and vWf were detected in endothelial cells of all organs examined, except for the endothelia of the renal glomeruli, which were negative for vWf. All haemangiomas examined (15) were positive for both markers. Eleven of 15 haemangiosarcomas were positive for vWf and all 15 expressed the CO31 antigen. All other neoplasms investigated (fibrosarcomas, schwannomas, haemangiopericytomas) were negative for both markers. It is concluded that immunohistochemical detection of CO31 antigen is of value for studying vascular disorders of the dog in routinely processed tissue.
Introduction Vascular endothelial cells have an important role in coagulation, inflammation, immunity and regulation of vascular tone, and in a wide variety of synthetic and metabolic functions (Fajardo, I989). Neoplastic growths, both benign and malignant, originating from endothelial cells, are reported frequently in animals (Pulley and Stannard, 1990; Hargis et al., 1992). The role of endothelial cells in health and disease is therefore of interest to histopathologists and, for this reason, it is important to be able to recognize these cells in routine tissue sections. Morphologically, endothelial cells may be difficult to distinguish from other cell types. Haemangiosarcomas, for instance, frequently pose a diagnostic problem for histopathologists as they are tumours with varying morphological patterns. Vascular differentiation may be absent and they may be mistaken for other soft tissue sarcomas or for undifferentiated carcinomas (Pulley and Stannard, 1990). Two different approaches have been used for the identification of endothelial ceils in tissue sections: histochemical examination with lectins, and the immunohistochemical detection of yon Willebrand's factor (vWf; previously named factor VIII-related antigen). Endothelial cells express, on their surface, glycoproteins that specifically bind lectins. For example, the lectin of Ulex europaeus is an excellent marker for human vascular-derived neoplasms (Alles and Bosslet, 1988). However, for the full characterization of a neoplasm it is necessary to investigate reactivity to several lectins. Thus, Augustin-Voss et al. (1990) investigated cell surface glycoconjugate expression in normal and 0021-9975/95/040319+08 $08.00/0
9 1995AcademicPressLimited
320
L. Fetter et al.
neoplastic canine endothelial cells with eight different lectins, concluding that lectins were not good markers for canine vascular neoplasia. The immunohistochemical detection of vWf is an excellent tool for the identification of endothelial cells in tissue sections and is the method of choice in many veterinary pathology laboratories for the definitive diagnosis of neoplasms of suspected vascular origin (von Beust et al., 1988; Miller et al., 1992). This technique, however, is associated with several difficulties. It is not clear whether vWf is present only on blood endothelial cells or on both lymphatic and blood endothelia. Furthermore, some endothelia, such as the renal glomeruli, apparently do not express vWf (Parums et al., 1990). Some poorly differentiated haemangiosarcomas are negative for vWf (von Beust et al., 1988) and some carcinomas and macrophages can aberrantly express vWf (Little et al., 1986; Millard et al., 1987). Recently, the presence of different adhesion molecules on the surface of endothelial cells has been reported (Kuzu et al., 1993). Modulation of leucocytevessel wall adhesion is thought to be mediated by constitutive or cytokineupregulated expression of some or all of the following vascular adhesion molecules: endothelial leucocyte adhesion molecule-1 (ELAM-1); vascular cell adhesion molecule (VCAM); intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM) (Page et al., 1992). PECAM, also called CD31, is a 100 kD glycoprotein which participates in the adhesion between platelets and endothelial cells. CD31 antigen is regarded in man as a useful marker for endothelial cells because (1) it is constantly expressed on all types of endothelial cell, (2) it is expressed only by endothelial cells, platelets and macrophages, and (3) it is conserved in neoplasia arising from endothelial cells. Moreover, a commercial monoclonal antibody (JC70) is available which reacts with CD31 antigen on formol-fixed, paraffin waxembedded tissue sections (Parums et al., 1990; Page et al., 1992; Kuzu et al., 1993). The aims of the present study were (1) to determine whether the antibody against human CD31 antigen also recognizes the canine antigen, (2) to investigate the presence of this antigen in canine haemangiomas and haemangiosarcomas, and (3) to evaluate the usefulness of this antigen as a specific marker for normal and neoplastic endothelial cells in comparison with the presently used marker, vWf. M a t e r i a l s and M e t h o d s
Tissues Examined
The presence of CD31 antigen and vWf was investigated on paraffin wax sections of normal canine organs and.of different neoplasms by means of immunohistochemical techniques. Sections of skin, myocardium, intestine, lung, liver, pancreas, kidney, nervous tissues, adrenal gland, thyroid, salivary gland, lymph node, spleen, tonsil, thymus, prostate, testis, penis, ovary and mammary gland from two healthy beagle dogs (a male and a female) were used as normal tissue controls. Tissue sections were prepared also from routine blocks of tumours stored in the Department of Animal Pathology at the Veterinary School of Barcelona, Spain. We selected 15 haemangiomas, 15 haemangiosarcomas, six fibrosarcomas, six schwannomas and six haemangiopericytomas. In all cases the diagnosis was made after clinical evaluation of the
321
CD31 i n N e o p l a s t i c Canine Endothelial C e l l s Table 1 I m m u n o r e a c t i v i t y o f vascular and spindle cell turnours with antibodies against von Willebrand's factor and CD-31 antigen Number* of tumours reacting with antibodies against Tumours
yon 144llebrand'sfactor
CD31
15/15 11 / 15 0/6 0/6 0/6
15/15 15/ 15 0/6 0/6 0/6
Haemangiomas Haemangiosarcomas Fibrosarcomas Schwannomas Haemangiopericytomas
* Numerator, nmnber giving positive result; denominator, number examined.
disease and histopathological study of tissue samples. Only cases with a definitive histopathological diagnosis, confirmed by clinical evaluation, were included in this study. All samples had been fixed in buffered 10% formol for 24 h (normal organs) or 1-7 days (neoplasms), embedded in paraffin wax and sectioned at 4 gm. Immunohistochemical Methods
Before labelling, paraffin wax sections were dewaxed, hydrated and incubated for 7 min in 0"1% pronase solution at room temperature. The immunohistochemical detection of vWf was done by the peroxidase-antiperoxidase method (von Beust et al., 1988). A commercial polyclonal antibody (Dako, Denmark) diluted 1 in 200, was used as primary antibody. For the demonstration ofCD31 antigen the avidin-biotin method was applied. As first antibody, the commercial monoclonal mouse antibody JC70 (Dako, Denmark), diluted 1 in 50, was used. As positive controls for both techniques, sections of human spleen and human lymph nodes were used. The negative controls were sections incubated in normal rabbit serum in place of the specific antibody.
Results As expected, the polyclonal antibody against vWf reacted with the endothelial cells of blood vessels of all calibres with the exception of the endothelial cells of the renal glomeruli. The reaction was specific, strong and diffuse in the cytoplasm of the endothelial cells. Lymphatic vessels were also positive, although the reaction was weaker. The endothelial cells of all 15 haemangiomas were clearly positive, as also were the endothelial cells in 1 l of the 15 haemangiosarcomas (Table 1). Two of the haemangiosarcomas were weakly positive and two were negative for vWf. These last two were poorly differentiated tumours, which displayed a solid pattern. In all tumours the endothelial cells of the normal or reactive blood vessels of the surrounding mesenchymal tissues reacted positively with the antibody against vWf. In some cases background staining was evident, especially in the serum present inside the vessels, making identification of the endothelial cells difficult. None of the remaining spindle cell turnouts gave a positive reaction with the antibody against vWf.
322
Fig. 1.
L . F e t t e r e t al.
Liver. Endothelial cells of the venules are clearly CD31-positive. x 430.
The monoclonal antibody J C 7 0 (anti-CD31) reacted specifically with endothelial cells in all tissues examined (Fig. 1), including renal glomeruli. The reaction was strong, especially at the periphery of the cytoplasm and on the cell membrane. Endothelial cells of lymphatic vessels were weakly positive. Background staining was absent. CD31 antibody gave a reaction that was stronger and less diffuse than the vWf reaction (Fig. 2). The endothelial cells of all haemangiomas (Fig. 3) and haemangiosarcomas (Fig. 4) gave an intense positive reaction with this antibody, even in cases which were negative with vWf antibody and in poorly differentiated haemangiosarcomas. All nonvascular tumours examined were negative (Table 1). Discussion
CD31 antigen participates in the adhesion of platelets to the endothelial cell (Parums et al., 1990). Endothelial cells from different anatomical sites display antigenic heterogeneity (Page et al., 1992) but CD31 antigen is expressed on all types of endothelial cells and also by neoplastic endothelial cells (Parums et al., 1990; Kuzu et al., 1993). These properties make this antigen one of the best markers of vascular differentiation. T h e monoclonal antibody JC70, raised against a m e m b r a n e preparation from a h u m a n spleen affected by hairy cell leukaemia, recognizes a fixationresistant epitope of the CD31 antigen molecule. In consequence it is useful
CD31 in Neoplastic Canine Endothelial Cells
Fig. 2.
323
Haemangiosarcoma. The immunoreaction with the anti-CD31 antibody (a) is stronger than the immunoreactivity with the antibody against vWf (b). x 100.
for the identification of endothelial cells in paraffin wax-embedded tissue sections (Parums et al., 1990). In this study it was demonstrated that JC70 also recognized the CD31 antigen on canine endothelial cells and, as in human beings, all canine endothelial cells expressed this antigen. The immunoreactivity was specific and intense and the background staining faint. Compared with the immunoreactivity of the vWf, a well-known marker of endothelial cells in the dog, the reaction was more intense and associated with less background staining. Endothelial cells of the renal glomeruli, which are negative for vWf, were CD31-positive. CD31 antigen was also detected in all neoplasms of vascular origin examined, proving to be an excellent marker for canine haemangiomas and haemangiosarcomas. Even cases of poorly differentiated haemangiosarcomas, which were negative for vWf, were clearly stained with JC70 antibody. Similar results have been reported in other animals and man. Kuzu et al. (1993) detected CD31 antigen in each of four human angiosarcomas examined, but only three were positive for vWf. Parums et aI. (1990) after studying 10 human angiosarcomas, also concluded that JC70 antibody stained malignant endothelial cells with more consistency than monoclonal or polyclonal antibodies
324
Fig. 3.
L. F e t t e r et al.
Cutaneous haemangioma. Immunoreactivity to CD31 in normal and neoplastic endothelial cells. x 100.
to vWf. von Beust et al. (1988) reported that four out of 36 canine haemangiosarcomas showed negative or equivocal staining with vWf. Because the JC70 antibody also reacted with normal lymphatic vessels, it is probably of only marginal value in distinguishing haemangiosarcomas and lymphangiosarcomas. The distinction between these two tumours should be based on ultrastructural studies (Swayne et al., 1989; Pulley and Stannard, 1990).
CD31 in Neoplastic Canine Endothelial Cells
325
Fig. 4. Cutaneous haemangiosarcoma. Intense immunoreaction for CD31 in the cytoplasm of the neoplastic endothelialcells. • 100. It is concluded that antibody JC70 is of value for studying benign and malignant vascular disorders of the dog in routinely processed tissue. Acknowledgments
This work was supported by the "Direcci6n General de Investigacidn Cientifica y T~cnica" (DGICYT), Project No. PS90-0169. References
Alles, J. U. and Bosslet, K. (1988). Immunocytochemistry of angiosarcomas. American Journal of Clinical Pathology, 89, 463-471. Augustin-Voss, H. G., Smith, C. A. and Lewis, R. M. (1990). Phenotypic characterization of normal and neoplastic canine endothelial cells by lectin histochemistry. VeterinaryPathology, 27, 103-109. Fajardo, L.J. (1989). The complexity of endothelial cells. American journal of Clinical Pathology, 92, 241-250. Hargis, A. M., Ihrke, P.J., Spangler, W. L. and Stannard, A. A. (1992). A retrospective clinicopathologic study of 212 dogs with cutaneous hemangiomas and hemangiosarcomas. ~terinary Pathology, 29, 316 328. Kuzu, I., Bicknell, R., Fletcher, C. D. M. and Gatter, K. C. (1993). Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors. Laboratory Investigations, 69, 322-328. Little, D., Said, J. W., Siegel, R. L., Fealy, M. and Fishbein, M. C. (1986). Endothelial cell markers in vascular neoplasms: an immunohistochemieal study comparing factor VIII related antigen, blood group specific antigens, 6-keto-PGF1 alpha and (flex europaeus I lectin. Journal of Pathology, 149, 89-95.
326
L. Ferrer
et
al.
Millard, P. R., Chaplin, A. J., Heryet, A. R. and McDougall, A. C. (1987). Factor VIII related antigen positive macrophages and acquired immune deficiency syndrome (AIDS): a problem of antibody specificity. Journal of Clinical Pathology, 40, 262-266. Miller, M. A., Ramos, J. A. and Kreeger, J. M. (1992). Cutaneous vascular neoplasia in i5 cats: clinical, morphologicai and immunohistochemical studies. Veterinary Pathology, 29, 329-336. Page, C., Rose, M., Yacoub, M. and Pigott, R. (1992). Antigenic heterogeneity of vascular endothelium. AmericanJournal of Pathology, 141,673-683. Parums, D. V., Cordell, J. L., Micklem, F., Heryet, A. R., Gatter, K. C. and Masons, D. Y. (1990).JC 70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. Journal of ClinicalPathology, 43, 752-757. Pulley, L. T. and Stannard, A. A. (1990). Tumors of the skin and soft tissues. In: Tumors of Domestic Animals, 3rd Edit.,J. Moulton, Ed., California University Press, Davis, pp. 23-87. Swayne, D. E., Mahaffey, E. A. and Haynes, S. G. (1989). Lymphangiosarcoma and haemangiosarcoma in a cat. Journal of Comparative Pathology, 100, 91-96. von Beust, B. R., Suter, M. M. and Summer, B. A. (1988). Factor VIII-related antigen in canine endothelial neoplasms: an immunohistochemical study. Veterinary Pathology, 25, 251=255.
]
Received, August 8th, 1994 Accepted, December 19th, 1994J
Irnrnunohistochemical D e t e c t i o n of CD31 Antigen in N o r m a l and N e o p l a s t i c Canine Endothelial Cells L. Ferrer, D. Fondevila, R. M. Rabanal and M. Vilafranca Department of Animal Pathology, Veterinary School, Universitat Aut~noma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Summary This report describes the immunohistochemical detection ofvon Willebrand's factor (vWf) and CO31 antigen in paraffin wax-embedded, formalin-fixed tissue sections of canine normal organs and vascular neoplasms. CO31 antigen and vWf were detected in endothelial cells of all organs examined, except for the endothelia of the renal glomeruli, which were negative for vWf. All haemangiomas examined (15) were positive for both markers. Eleven of 15 haemangiosarcomas were positive for vWf and all 15 expressed the CO31 antigen. All other neoplasms investigated (fibrosarcomas, schwannomas, haemangiopericytomas) were negative for both markers. It is concluded that immunohistochemical detection of CO31 antigen is of value for studying vascular disorders of the dog in routinely processed tissue.
Introduction Vascular endothelial cells have an important role in coagulation, inflammation, immunity and regulation of vascular tone, and in a wide variety of synthetic and metabolic functions (Fajardo, I989). Neoplastic growths, both benign and malignant, originating from endothelial cells, are reported frequently in animals (Pulley and Stannard, 1990; Hargis et al., 1992). The role of endothelial cells in health and disease is therefore of interest to histopathologists and, for this reason, it is important to be able to recognize these cells in routine tissue sections. Morphologically, endothelial cells may be difficult to distinguish from other cell types. Haemangiosarcomas, for instance, frequently pose a diagnostic problem for histopathologists as they are tumours with varying morphological patterns. Vascular differentiation may be absent and they may be mistaken for other soft tissue sarcomas or for undifferentiated carcinomas (Pulley and Stannard, 1990). Two different approaches have been used for the identification of endothelial ceils in tissue sections: histochemical examination with lectins, and the immunohistochemical detection of yon Willebrand's factor (vWf; previously named factor VIII-related antigen). Endothelial cells express, on their surface, glycoproteins that specifically bind lectins. For example, the lectin of Ulex europaeus is an excellent marker for human vascular-derived neoplasms (Alles and Bosslet, 1988). However, for the full characterization of a neoplasm it is necessary to investigate reactivity to several lectins. Thus, Augustin-Voss et al. (1990) investigated cell surface glycoconjugate expression in normal and 0021-9975/95/040319+08 $08.00/0
9 1995AcademicPressLimited
320
L. Fetter et al.
neoplastic canine endothelial cells with eight different lectins, concluding that lectins were not good markers for canine vascular neoplasia. The immunohistochemical detection of vWf is an excellent tool for the identification of endothelial cells in tissue sections and is the method of choice in many veterinary pathology laboratories for the definitive diagnosis of neoplasms of suspected vascular origin (von Beust et al., 1988; Miller et al., 1992). This technique, however, is associated with several difficulties. It is not clear whether vWf is present only on blood endothelial cells or on both lymphatic and blood endothelia. Furthermore, some endothelia, such as the renal glomeruli, apparently do not express vWf (Parums et al., 1990). Some poorly differentiated haemangiosarcomas are negative for vWf (von Beust et al., 1988) and some carcinomas and macrophages can aberrantly express vWf (Little et al., 1986; Millard et al., 1987). Recently, the presence of different adhesion molecules on the surface of endothelial cells has been reported (Kuzu et al., 1993). Modulation of leucocytevessel wall adhesion is thought to be mediated by constitutive or cytokineupregulated expression of some or all of the following vascular adhesion molecules: endothelial leucocyte adhesion molecule-1 (ELAM-1); vascular cell adhesion molecule (VCAM); intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM) (Page et al., 1992). PECAM, also called CD31, is a 100 kD glycoprotein which participates in the adhesion between platelets and endothelial cells. CD31 antigen is regarded in man as a useful marker for endothelial cells because (1) it is constantly expressed on all types of endothelial cell, (2) it is expressed only by endothelial cells, platelets and macrophages, and (3) it is conserved in neoplasia arising from endothelial cells. Moreover, a commercial monoclonal antibody (JC70) is available which reacts with CD31 antigen on formol-fixed, paraffin waxembedded tissue sections (Parums et al., 1990; Page et al., 1992; Kuzu et al., 1993). The aims of the present study were (1) to determine whether the antibody against human CD31 antigen also recognizes the canine antigen, (2) to investigate the presence of this antigen in canine haemangiomas and haemangiosarcomas, and (3) to evaluate the usefulness of this antigen as a specific marker for normal and neoplastic endothelial cells in comparison with the presently used marker, vWf. M a t e r i a l s and M e t h o d s
Tissues Examined
The presence of CD31 antigen and vWf was investigated on paraffin wax sections of normal canine organs and.of different neoplasms by means of immunohistochemical techniques. Sections of skin, myocardium, intestine, lung, liver, pancreas, kidney, nervous tissues, adrenal gland, thyroid, salivary gland, lymph node, spleen, tonsil, thymus, prostate, testis, penis, ovary and mammary gland from two healthy beagle dogs (a male and a female) were used as normal tissue controls. Tissue sections were prepared also from routine blocks of tumours stored in the Department of Animal Pathology at the Veterinary School of Barcelona, Spain. We selected 15 haemangiomas, 15 haemangiosarcomas, six fibrosarcomas, six schwannomas and six haemangiopericytomas. In all cases the diagnosis was made after clinical evaluation of the
321
CD31 i n N e o p l a s t i c Canine Endothelial C e l l s Table 1 I m m u n o r e a c t i v i t y o f vascular and spindle cell turnours with antibodies against von Willebrand's factor and CD-31 antigen Number* of tumours reacting with antibodies against Tumours
yon 144llebrand'sfactor
CD31
15/15 11 / 15 0/6 0/6 0/6
15/15 15/ 15 0/6 0/6 0/6
Haemangiomas Haemangiosarcomas Fibrosarcomas Schwannomas Haemangiopericytomas
* Numerator, nmnber giving positive result; denominator, number examined.
disease and histopathological study of tissue samples. Only cases with a definitive histopathological diagnosis, confirmed by clinical evaluation, were included in this study. All samples had been fixed in buffered 10% formol for 24 h (normal organs) or 1-7 days (neoplasms), embedded in paraffin wax and sectioned at 4 gm. Immunohistochemical Methods
Before labelling, paraffin wax sections were dewaxed, hydrated and incubated for 7 min in 0"1% pronase solution at room temperature. The immunohistochemical detection of vWf was done by the peroxidase-antiperoxidase method (von Beust et al., 1988). A commercial polyclonal antibody (Dako, Denmark) diluted 1 in 200, was used as primary antibody. For the demonstration ofCD31 antigen the avidin-biotin method was applied. As first antibody, the commercial monoclonal mouse antibody JC70 (Dako, Denmark), diluted 1 in 50, was used. As positive controls for both techniques, sections of human spleen and human lymph nodes were used. The negative controls were sections incubated in normal rabbit serum in place of the specific antibody.
Results As expected, the polyclonal antibody against vWf reacted with the endothelial cells of blood vessels of all calibres with the exception of the endothelial cells of the renal glomeruli. The reaction was specific, strong and diffuse in the cytoplasm of the endothelial cells. Lymphatic vessels were also positive, although the reaction was weaker. The endothelial cells of all 15 haemangiomas were clearly positive, as also were the endothelial cells in 1 l of the 15 haemangiosarcomas (Table 1). Two of the haemangiosarcomas were weakly positive and two were negative for vWf. These last two were poorly differentiated tumours, which displayed a solid pattern. In all tumours the endothelial cells of the normal or reactive blood vessels of the surrounding mesenchymal tissues reacted positively with the antibody against vWf. In some cases background staining was evident, especially in the serum present inside the vessels, making identification of the endothelial cells difficult. None of the remaining spindle cell turnouts gave a positive reaction with the antibody against vWf.
322
Fig. 1.
L . F e t t e r e t al.
Liver. Endothelial cells of the venules are clearly CD31-positive. x 430.
The monoclonal antibody J C 7 0 (anti-CD31) reacted specifically with endothelial cells in all tissues examined (Fig. 1), including renal glomeruli. The reaction was strong, especially at the periphery of the cytoplasm and on the cell membrane. Endothelial cells of lymphatic vessels were weakly positive. Background staining was absent. CD31 antibody gave a reaction that was stronger and less diffuse than the vWf reaction (Fig. 2). The endothelial cells of all haemangiomas (Fig. 3) and haemangiosarcomas (Fig. 4) gave an intense positive reaction with this antibody, even in cases which were negative with vWf antibody and in poorly differentiated haemangiosarcomas. All nonvascular tumours examined were negative (Table 1). Discussion
CD31 antigen participates in the adhesion of platelets to the endothelial cell (Parums et al., 1990). Endothelial cells from different anatomical sites display antigenic heterogeneity (Page et al., 1992) but CD31 antigen is expressed on all types of endothelial cells and also by neoplastic endothelial cells (Parums et al., 1990; Kuzu et al., 1993). These properties make this antigen one of the best markers of vascular differentiation. T h e monoclonal antibody JC70, raised against a m e m b r a n e preparation from a h u m a n spleen affected by hairy cell leukaemia, recognizes a fixationresistant epitope of the CD31 antigen molecule. In consequence it is useful
CD31 in Neoplastic Canine Endothelial Cells
Fig. 2.
323
Haemangiosarcoma. The immunoreaction with the anti-CD31 antibody (a) is stronger than the immunoreactivity with the antibody against vWf (b). x 100.
for the identification of endothelial cells in paraffin wax-embedded tissue sections (Parums et al., 1990). In this study it was demonstrated that JC70 also recognized the CD31 antigen on canine endothelial cells and, as in human beings, all canine endothelial cells expressed this antigen. The immunoreactivity was specific and intense and the background staining faint. Compared with the immunoreactivity of the vWf, a well-known marker of endothelial cells in the dog, the reaction was more intense and associated with less background staining. Endothelial cells of the renal glomeruli, which are negative for vWf, were CD31-positive. CD31 antigen was also detected in all neoplasms of vascular origin examined, proving to be an excellent marker for canine haemangiomas and haemangiosarcomas. Even cases of poorly differentiated haemangiosarcomas, which were negative for vWf, were clearly stained with JC70 antibody. Similar results have been reported in other animals and man. Kuzu et al. (1993) detected CD31 antigen in each of four human angiosarcomas examined, but only three were positive for vWf. Parums et aI. (1990) after studying 10 human angiosarcomas, also concluded that JC70 antibody stained malignant endothelial cells with more consistency than monoclonal or polyclonal antibodies
324
Fig. 3.
L. F e t t e r et al.
Cutaneous haemangioma. Immunoreactivity to CD31 in normal and neoplastic endothelial cells. x 100.
to vWf. von Beust et al. (1988) reported that four out of 36 canine haemangiosarcomas showed negative or equivocal staining with vWf. Because the JC70 antibody also reacted with normal lymphatic vessels, it is probably of only marginal value in distinguishing haemangiosarcomas and lymphangiosarcomas. The distinction between these two tumours should be based on ultrastructural studies (Swayne et al., 1989; Pulley and Stannard, 1990).
CD31 in Neoplastic Canine Endothelial Cells
325
Fig. 4. Cutaneous haemangiosarcoma. Intense immunoreaction for CD31 in the cytoplasm of the neoplastic endothelialcells. • 100. It is concluded that antibody JC70 is of value for studying benign and malignant vascular disorders of the dog in routinely processed tissue. Acknowledgments
This work was supported by the "Direcci6n General de Investigacidn Cientifica y T~cnica" (DGICYT), Project No. PS90-0169. References
Alles, J. U. and Bosslet, K. (1988). Immunocytochemistry of angiosarcomas. American Journal of Clinical Pathology, 89, 463-471. Augustin-Voss, H. G., Smith, C. A. and Lewis, R. M. (1990). Phenotypic characterization of normal and neoplastic canine endothelial cells by lectin histochemistry. VeterinaryPathology, 27, 103-109. Fajardo, L.J. (1989). The complexity of endothelial cells. American journal of Clinical Pathology, 92, 241-250. Hargis, A. M., Ihrke, P.J., Spangler, W. L. and Stannard, A. A. (1992). A retrospective clinicopathologic study of 212 dogs with cutaneous hemangiomas and hemangiosarcomas. ~terinary Pathology, 29, 316 328. Kuzu, I., Bicknell, R., Fletcher, C. D. M. and Gatter, K. C. (1993). Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors. Laboratory Investigations, 69, 322-328. Little, D., Said, J. W., Siegel, R. L., Fealy, M. and Fishbein, M. C. (1986). Endothelial cell markers in vascular neoplasms: an immunohistochemieal study comparing factor VIII related antigen, blood group specific antigens, 6-keto-PGF1 alpha and (flex europaeus I lectin. Journal of Pathology, 149, 89-95.
326
L. Ferrer
et
al.
Millard, P. R., Chaplin, A. J., Heryet, A. R. and McDougall, A. C. (1987). Factor VIII related antigen positive macrophages and acquired immune deficiency syndrome (AIDS): a problem of antibody specificity. Journal of Clinical Pathology, 40, 262-266. Miller, M. A., Ramos, J. A. and Kreeger, J. M. (1992). Cutaneous vascular neoplasia in i5 cats: clinical, morphologicai and immunohistochemical studies. Veterinary Pathology, 29, 329-336. Page, C., Rose, M., Yacoub, M. and Pigott, R. (1992). Antigenic heterogeneity of vascular endothelium. AmericanJournal of Pathology, 141,673-683. Parums, D. V., Cordell, J. L., Micklem, F., Heryet, A. R., Gatter, K. C. and Masons, D. Y. (1990).JC 70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. Journal of ClinicalPathology, 43, 752-757. Pulley, L. T. and Stannard, A. A. (1990). Tumors of the skin and soft tissues. In: Tumors of Domestic Animals, 3rd Edit.,J. Moulton, Ed., California University Press, Davis, pp. 23-87. Swayne, D. E., Mahaffey, E. A. and Haynes, S. G. (1989). Lymphangiosarcoma and haemangiosarcoma in a cat. Journal of Comparative Pathology, 100, 91-96. von Beust, B. R., Suter, M. M. and Summer, B. A. (1988). Factor VIII-related antigen in canine endothelial neoplasms: an immunohistochemical study. Veterinary Pathology, 25, 251=255.
]
Received, August 8th, 1994 Accepted, December 19th, 1994J