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Angiogenesis in Squamous Cell Carcinomas of Canine Skin: An Immunohistochemical and Quantitative Analysis
J. Comp. Path. 2001, Vol. 125, 117–121 doi:10.1053/jcpa.2001.0485, available online at http://www.idealibrary.com on
Angiogenesis in Squamous Cell Carcinomas of Canine Skin: An Immunohistochemical and Quantitative Analysis P. Maiolino, S. Papparella, B. Restucci and G. De Vico∗ Dipartimento di Patologia e Sanita` Animale, Settore Anatomia Patologica, Facolta` di Medicina Veterinaria, Universita` degli Studi di Napoli Federico II, Via F. Delpino 1, 80137 Napoli and ∗Istituto di Patologia Generale e Anatomia Patologica Veterinaria, Facolta` di Medicina Veterinaria, Universita` di Messina, Italy
Summary In a number of recent papers, the intratumoral microvessel density (iMVD) has been described as a promising new prognostic factor. In this study, the angiogenic rate was evaluated immunohistochemically for platelet endothelial cell adhesion molecule (CD31) in 15 squamous cell carcinomas (SCCs) of canine skin. Computer image analysis was used to measure the iMVD, which increased progressively from differentiation grade I to IV. The iMVD was consistently and significantly greater in the poorly differentiated SCC cases. The correlation of angiogenesis data with differentiation grade of canine SCCs suggests that the iMVD value may provide an additional criterion for evaluating the intrinsic malignancy and growth potential of such tumours. 2001 Harcourt Publishers Ltd
Introduction Angiogenesis is the process by which new blood vessels develop from the endothelium of pre-existing vasculature (Folkman and Shing, 1992). The angiogenesis-inducing capacity of human solid tumours is regarded as an important factor influencing tumour growth and metastasis. Recent studies of human tumours [breast carcinoma, prostate carcinoma, squamous cell carcinoma, nonsmall cell lung carcinoma, malignant melanoma, gastrointestinal carcinoma, testicular germ cell tumours, multiple myeloma, central nervous system tumours, ovarian carcinoma, endometrial carcinoma, transitional-cell carcinoma of the bladder, vulvar carcinoma, nasopharyngeal carcinoma, laryngeal and oesophageal squamous carcinomas, and medullary thyroid carcinoma (Weidner, 1995, 1998, 1999; Fox, 1997)] have shown a statistically significant relationship between increased intratumoral microvessel density (iMVD) and tumour aggressiveness, metastatic risk and decreased patient survival. Little is known about the role 0021–9975/01/020117+05 $35.00
of angiogenesis in animal tumours (Graham and Myers, 1999; Restucci et al., 2000; Rosenthal, 2000). The aim of this study was to investigate the angiogenic rate in squamous cell carcinomas (SCCs) of canine skin in relation to their histological grading. Materials and Methods Animals and Tumour Samples SCC tissue samples from 15 dogs (12 male and three female) of different breeds and age were examined. One digital tumour had given rise to metastasis in the inguinal lymph nodes at the time of diagnosis. The 15 tumours were graded histologically according to the Broders grading system (Goldschmidt and Shofer, 1992): four were classified as well differentiated (grade I) because they had numerous “keratin pearls” and clearly evident intercellular bridges but minimal mitotic activity and nuclear pleomorphism; six were classified as moderately differentiated (grade II or III) because 2001 Harcourt Publishers Ltd
118
P. Maiolino et al. Table 1 Details of the 15 dogs with squamous cell carcinoma Dog no.
Sex
Age (years)
Breed
Site of tumour
Tumour grade∗
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
M F M F M M M F M M M M M M M
7 10 6 11 4 16 2 14 10 7 10 8 6 9 12
Mixed German shepherd German shepherd Mixed Bobtail Mixed Pit bull German shepherd Mixed Mixed Mixed Schnauzer German shepherd Chihuahua Schnauzer
Lip Neck Lip Back Cheek Shank Lip Coxa Head Neck Lip Toe Toe Toe Toe
I I I I II II II III III III IV IV IV IV IV
F, female; M, male; ∗ I, well differentiated; II and III, moderately differentiated; IV, poorly differentiated.
they had occasional “keratin pearls”, poorly defined intercellular bridges, and moderate mitotic activity and nuclear hyperchromatism; the remaining five, four of which were situated on the toe, were classified as poorly differentiated (grade IV) because they showed little squamous differentiation but marked mitotic activity, nuclear pleomorphism and hyperchromatism. Details of the animals and tumours are given in Table 1. Immunohistochemical Examination The specimens were fixed in 10% formalin and embedded in paraffin wax. Sections (4 m) were dewaxed in xylene, rehydrated and washed in 0·01 M phosphate-buffered saline (PBS), pH 7·2– 7·4. Endogenous peroxidase was blocked with hydrogen peroxide 0·3% in absolute methanol for 30 min. Before the immunohistochemical procedure [a streptavidin–biotin–peroxidase method with a commercial kit (LSAB Kit; Dako, Milan, Italy)], proteolytic treatment with pepsin 0·4% in 0·01 M HCl was applied for 30 min at 37°C. The primary antibody was a mouse monoclonal human anti-PECAM (Platelet Endothelial Cell Adhesion Molecule), also called CD31 (clone JC70) (Dako, Milan, Italy), diluted 1 in 20 in antibody diluent (Dako). The sections were incubated with antibody overnight at 4°C. The reaction was developed with diaminobenzidine (Dako) and haematoxylin was used as counterstain. Control slides were incubated with PBS instead of primary antibody.
Image Analysis The iMVD values were determined by two experienced pathologists by means of an automated (Image Pro-Plus 1) image analysis system (Sistema Mono; Immagini e Computer, Milan, Italy). The iMVD was assessed from areas of the tumour containing the highest number of immunolabelled vessels, i.e. angiogenic “hot spots” (Weidner et al., 1991). Briefly, a hot spot was identified by scanning the tumour at low magnification (×40 and ×100). The number of vessels within each selected hot spot was counted at a magnification of ×400. The images (10/tumour) were captured with a microscope (Nikon Eclipse E-400, Tokyo, Japan) coupled to a video camera ( JVC TK-C1380E, Japan), stored in the digital memory, and displayed on a monitor screen. Manual outlining of microvessels was performed and the number of vessels in the hot spot was then calculated. As suggested by Weidner et al. (1993) and Jacquemier et al. (1998), labelled endothelial cells, regardless of the presence or absence of a lumen and red blood cells, were considered to indicate individual vessels. All vessels with a diameter of more than 30 m and a thick muscular wall were excluded from the count. Statistical Analysis The numbers of vessels (means±SD) were correlated with the differentiation grades of canine skin SCCs by analysis of variance (ANOVA).
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Fig. 1. Squamous cell carcinoma, grade I. Immunolabelling for PECAM. Arrowheads indicate microvessels. Streptavidin– biotin–peroxidase method. ×200.
Fig. 2. Squamous cell carcinoma, grade III. Immunolabelling for PECAM. Arrowheads indicate microvessels. Streptavidin– biotin–peroxidase method. ×200.
Results Figures 1 and 2 illustrate the different microvessel densities in SCCs of different grade (I and III, respectively). In grade I SCC (n=4), the iMVD
values ranged from 16–28 (mean 20·5±5·2 SD). The corresponding values for grade II SCCs (n=3), grade III SCCs (n=3) and grade IV SCCs (n=5) were 22–34 (27·6±5·5), 34–39 (36·3±2·5) and 43–47 (45·6±1·6), respectively. One of the
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tumours (a grade IV SCC) showed clear evidence of metastasis. The iMVD values differed significantly between grade I and grades III and IV (P= 0·000006), grade II and grade IV (P=0·0003), and grade III and grade IV (P=0·0007), but not between grades I and II (P=0·14) or between grades II and III (P=0·06).
Discussion High tumour microvessel density was found to be associated with poor prognosis in many human studies (Weidner et al., 1993; Weidner, 1995, 1998, 1999; Fox, 1997), but this observation was not confirmed in several studies of breast cancer (Axelsson et al., 1995; Siitonen et al., 1995; Morphopoulos et al., 1996). In studies on SCC of the head and neck, Gasparini et al. (1993) found an association between tumour microvessel density and metastasis rate, but Dray et al. (1995) and Zatterstrom et al. (1995) failed to find an association between microvessel density and survival. In the same way, Tahan and Stein (1995) and Weninger et al. (1996), who studied SCCs of the human lip and skin, respectively, noted an increased density of microvessels but not a relationship with the risk of metastasis. The discrepancies regarding the proposed prognostic significance of angiogenesis in tumours may be explained by the fact that several antibodies can be used to label vessels in tissue sections, and the choice of antibody will influence the number of microvessels available for counting. In many studies, antibodies directed against Factor VIII-related antigen (Weidner et al., 1991, 1992, 1993; Siitonen et al., 1995; Weninger et al., 1996) or CD34 antigen (Siitonen et al., 1995) have been used; these identify only a proportion of the vascular endothelium and will therefore underestimate the true extent of the microvasculature. Moreover, antibody to Factor VIII-related antigen, which is not expressed on all endothelial cells, is present on lymphatic endothelium. We chose to use a monoclonal antibody ( JC70) that recognizes the membrane-bound glycoprotein CD31, since in our hands this is a more sensitive marker of endothelium than other endothelial markers and it does not react with lymphatic endothelium (Kuzu et al., 1992; Charpin et al., 1997). Qbend 10, an antiCD34 antibody, did not produce immunolabelling in formalin-fixed, paraffin-wax-embedded SCCs of canine skin (Maiolino, unpublished data). In the present study, the mean iMVD increased progressively from the SCCs of grade I to the poorly differentiated SCCs of grade IV. These results
accord with our previous findings on the quantification of Ag-NOR proteins and proliferating cell nuclear antigen (PCNA) and on the immunolabelling of vascular endothelial growth factor (VEGF) in canine SCCs (De Vico et al., 1994; Maiolino et al., 1995, 2000). They also confirm that iMVD assessment may provide an additional criterion for evaluating the intrinsic malignancy and growth potential of such tumours. Moreover, the fact that the iMVD values of grade I and II tumours differed significantly from those of grade IV tumours, the latter showing the highest degree of vascularization, suggests that angiogenesis may account for an invasive or aggressive phenotype in canine SCC. However, this requires confirmation by a study in which necropsy proves that apparently aggressive canine SCCs with high iMVD values do in fact have an increased tendency to metastasize. Acknowledgments This research was supported in part by grants from the Ministero dell’Universita`, della Ricerca Scientifica e Tecnologica (M.U.R.S.T.). We thank Mr R. Ilsami for technical assistance. References Axelsson, K., Ljiung, B. E., Moore, D. H., Thor, A. D., Chew, K. L., Edgerton, S. M., Smith, H. S. and Mayall, B. H. (1995). Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma. Journal of the National Cancer Institute, 87, 997–1008. Charpin, C., Garcia, S., Bouvier, C., Martini, F., Andrac, L., Bonnier, P., Lavaut, M. N. and Allasia, C. (1997). CD31/PECAM automated and quantitative immunocytochemistry in invasive ductal carcinomas. Correlation with patient follow-up. American Journal of Clinical Pathology, 107, 534–541. De Vico, G., Agrimi, U. and Maiolino, P. (1994). Nucleolar size and mitotic index in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) of canine skin. Journal of Veterinary Medicine A, 41, 76–79. Dray, T. G., Hardin, N. J. and Sofferman, R. A. (1995). Angiogenesis as a prognostic marker in early head and neck cancer. Annals of Otology, Rhinology and Laryngology, 104, 724–729. Folkman, J. and Shing, Y. (1992). Angiogenesis. Journal of Biological Chemistry, 267, 10931–10934. Fox, S. B. (1997). Tumour angiogenesis and prognosis. Histopathology, 30, 294–301. Gasparini, G., Weidner, N., Maluta, S., Pozza, F., Boracchi, P., Mezzetti, M., Testolin, A. and Bevilacqua, P. (1993). Intratumoral microvessel density and p53 protein: correlation with metastasis in head-and-neck squamous-cell carcinoma. International Journal of Cancer, 55, 739–744. Goldschmidt, M. H. and Shofer, F. S. (1992). Skin Tumors of the Dog and Cat. Pergamon Press, Oxford, pp. 16–49.
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Graham, J. C. and Myers, R. K. (1999). The prognostic significance of angiogenesis in canine mammary tumors. Journal of Veterinary Internal Medicine, 13, 416–418. Jacquemier, J. D., Penault-Llorca, F. M. and Bertucci, F. (1998). Angiogenesis as a prognostic marker in breast carcinoma and conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis. Journal of Pathology, 184, 130–135. Kuzu, I., Bicknell, R., Harris, A. L., Jones, M., Gatter, K. C. and Mason, D. Y. (1992). Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours. Journal of Clinical Pathology, 45, 143–148. Maiolino, P., De Vico, G. and Restucci, B. (2000). Expression of vascular endothelial growth factor in basal cell tumours and in squamous cell carcinomas of canine skin. Journal of Comparative Pathology, 123, 141–145. Maiolino, P., Restucci, B. and De Vico, G. (1995). Expression of proliferating cell nuclear antigen in basal cell carcinomas and in squamous cell carcinomas of canine skin: correlation with mitotic index and histological features. Journal of Veterinary Medicine A, 42, 339–343. Morphopoulos, G., Pearson, M., Ryder, W. D., Howell, A. and Harris, M. (1996). Tumour angiogenesis as a prognostic marker in infiltrating lobular carcinoma of the breast. Journal of Pathology, 180, 44–49. Restucci, B., De Vico, G. and Maiolino, P. (2000). Evaluation of angiogenesis for PECAM in canine mammary tumors by quantitative and immunohistochemical methods. Veterinary Pathology, 37, 297– 301. Rosenthal, R. C. (2000). The prognostic significance of angiogenesis in canine mammary tumors. Journal of Veterinary Internal Medicine, 14, 248–249. Siitonen, S. M., Haapasalo, H. K., Rantala, I. S., Helin, H. J. and Isola, J. J. (1995). Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas. Modern Pathology, 8, 745–752.
Tahan, S. R. and Stein, A. L. (1995). Angiogenesis in invasive squamous cell carcinoma of the lip: tumor vascularity is not an indicator of metastatic risk. Journal of Cutaneous Pathology, 22, 236–240. Weidner, N. (1995). Intratumoral microvessel density as a prognostic factor in cancer. American Journal of Pathology, 147, 9–19. Weidner, N. (1998). Tumoral vascularity as a prognostic factor in cancer patients: The evidence continues to grow. Journal of Pathology, 184, 119–122. Weidner, N. (1999). Tumour vascularity and proliferation: clear evidence of a close relationship. Journal of Pathology, 189, 297–299. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W. and Folkman, J. (1993). Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. American Journal of Pathology, 143, 401–409. Weidner, N., Folkman, J., Pozza, F., Bevilacqua, P., Allred, E. N., Moore, D. H., Meli, S. and Gasparini, G. (1992). Tumor angiogenesis: a new significant and independent prognostic indicator in early stage breast carcinoma. Journal of the National Cancer Institute, 84, 1875–1887. Weidner, N., Semple, J. P. and Welch, W. R. (1991). Tumour angiogenesis and metastasis—correlation in invasive breast carcinoma. New England Journal of Medicine, 324, 1–8. Weninger, W., Uthman, A., Pammer, J., Picheler, A., Ballaun, C., Lang, I. M., Plettenberg, A., Bankl, H. C., Stu¨rzl, M. and Tschachler, E. (1996). Vascular endothelial growth factor production in normal epidermis and in benign and malignant epithelial skin tumors. Laboratory Investigation, 75, 647–657. Zatterstrom, U. K., Brun, E., Willen, R., Kjellen, E. and Wennerberg, J. (1995). Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. Head and Neck, 17, 312–318.
Received, November 2nd, 2000 Accepted, April 12th, 2001
Angiogenesis in Squamous Cell Carcinomas of Canine Skin: An Immunohistochemical and Quantitative Analysis P. Maiolino, S. Papparella, B. Restucci and G. De Vico∗ Dipartimento di Patologia e Sanita` Animale, Settore Anatomia Patologica, Facolta` di Medicina Veterinaria, Universita` degli Studi di Napoli Federico II, Via F. Delpino 1, 80137 Napoli and ∗Istituto di Patologia Generale e Anatomia Patologica Veterinaria, Facolta` di Medicina Veterinaria, Universita` di Messina, Italy
Summary In a number of recent papers, the intratumoral microvessel density (iMVD) has been described as a promising new prognostic factor. In this study, the angiogenic rate was evaluated immunohistochemically for platelet endothelial cell adhesion molecule (CD31) in 15 squamous cell carcinomas (SCCs) of canine skin. Computer image analysis was used to measure the iMVD, which increased progressively from differentiation grade I to IV. The iMVD was consistently and significantly greater in the poorly differentiated SCC cases. The correlation of angiogenesis data with differentiation grade of canine SCCs suggests that the iMVD value may provide an additional criterion for evaluating the intrinsic malignancy and growth potential of such tumours. 2001 Harcourt Publishers Ltd
Introduction Angiogenesis is the process by which new blood vessels develop from the endothelium of pre-existing vasculature (Folkman and Shing, 1992). The angiogenesis-inducing capacity of human solid tumours is regarded as an important factor influencing tumour growth and metastasis. Recent studies of human tumours [breast carcinoma, prostate carcinoma, squamous cell carcinoma, nonsmall cell lung carcinoma, malignant melanoma, gastrointestinal carcinoma, testicular germ cell tumours, multiple myeloma, central nervous system tumours, ovarian carcinoma, endometrial carcinoma, transitional-cell carcinoma of the bladder, vulvar carcinoma, nasopharyngeal carcinoma, laryngeal and oesophageal squamous carcinomas, and medullary thyroid carcinoma (Weidner, 1995, 1998, 1999; Fox, 1997)] have shown a statistically significant relationship between increased intratumoral microvessel density (iMVD) and tumour aggressiveness, metastatic risk and decreased patient survival. Little is known about the role 0021–9975/01/020117+05 $35.00
of angiogenesis in animal tumours (Graham and Myers, 1999; Restucci et al., 2000; Rosenthal, 2000). The aim of this study was to investigate the angiogenic rate in squamous cell carcinomas (SCCs) of canine skin in relation to their histological grading. Materials and Methods Animals and Tumour Samples SCC tissue samples from 15 dogs (12 male and three female) of different breeds and age were examined. One digital tumour had given rise to metastasis in the inguinal lymph nodes at the time of diagnosis. The 15 tumours were graded histologically according to the Broders grading system (Goldschmidt and Shofer, 1992): four were classified as well differentiated (grade I) because they had numerous “keratin pearls” and clearly evident intercellular bridges but minimal mitotic activity and nuclear pleomorphism; six were classified as moderately differentiated (grade II or III) because 2001 Harcourt Publishers Ltd
118
P. Maiolino et al. Table 1 Details of the 15 dogs with squamous cell carcinoma Dog no.
Sex
Age (years)
Breed
Site of tumour
Tumour grade∗
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
M F M F M M M F M M M M M M M
7 10 6 11 4 16 2 14 10 7 10 8 6 9 12
Mixed German shepherd German shepherd Mixed Bobtail Mixed Pit bull German shepherd Mixed Mixed Mixed Schnauzer German shepherd Chihuahua Schnauzer
Lip Neck Lip Back Cheek Shank Lip Coxa Head Neck Lip Toe Toe Toe Toe
I I I I II II II III III III IV IV IV IV IV
F, female; M, male; ∗ I, well differentiated; II and III, moderately differentiated; IV, poorly differentiated.
they had occasional “keratin pearls”, poorly defined intercellular bridges, and moderate mitotic activity and nuclear hyperchromatism; the remaining five, four of which were situated on the toe, were classified as poorly differentiated (grade IV) because they showed little squamous differentiation but marked mitotic activity, nuclear pleomorphism and hyperchromatism. Details of the animals and tumours are given in Table 1. Immunohistochemical Examination The specimens were fixed in 10% formalin and embedded in paraffin wax. Sections (4 m) were dewaxed in xylene, rehydrated and washed in 0·01 M phosphate-buffered saline (PBS), pH 7·2– 7·4. Endogenous peroxidase was blocked with hydrogen peroxide 0·3% in absolute methanol for 30 min. Before the immunohistochemical procedure [a streptavidin–biotin–peroxidase method with a commercial kit (LSAB Kit; Dako, Milan, Italy)], proteolytic treatment with pepsin 0·4% in 0·01 M HCl was applied for 30 min at 37°C. The primary antibody was a mouse monoclonal human anti-PECAM (Platelet Endothelial Cell Adhesion Molecule), also called CD31 (clone JC70) (Dako, Milan, Italy), diluted 1 in 20 in antibody diluent (Dako). The sections were incubated with antibody overnight at 4°C. The reaction was developed with diaminobenzidine (Dako) and haematoxylin was used as counterstain. Control slides were incubated with PBS instead of primary antibody.
Image Analysis The iMVD values were determined by two experienced pathologists by means of an automated (Image Pro-Plus 1) image analysis system (Sistema Mono; Immagini e Computer, Milan, Italy). The iMVD was assessed from areas of the tumour containing the highest number of immunolabelled vessels, i.e. angiogenic “hot spots” (Weidner et al., 1991). Briefly, a hot spot was identified by scanning the tumour at low magnification (×40 and ×100). The number of vessels within each selected hot spot was counted at a magnification of ×400. The images (10/tumour) were captured with a microscope (Nikon Eclipse E-400, Tokyo, Japan) coupled to a video camera ( JVC TK-C1380E, Japan), stored in the digital memory, and displayed on a monitor screen. Manual outlining of microvessels was performed and the number of vessels in the hot spot was then calculated. As suggested by Weidner et al. (1993) and Jacquemier et al. (1998), labelled endothelial cells, regardless of the presence or absence of a lumen and red blood cells, were considered to indicate individual vessels. All vessels with a diameter of more than 30 m and a thick muscular wall were excluded from the count. Statistical Analysis The numbers of vessels (means±SD) were correlated with the differentiation grades of canine skin SCCs by analysis of variance (ANOVA).
Angiogenesis in Squamous Cell Carcinomas of Canine Skin
119
Fig. 1. Squamous cell carcinoma, grade I. Immunolabelling for PECAM. Arrowheads indicate microvessels. Streptavidin– biotin–peroxidase method. ×200.
Fig. 2. Squamous cell carcinoma, grade III. Immunolabelling for PECAM. Arrowheads indicate microvessels. Streptavidin– biotin–peroxidase method. ×200.
Results Figures 1 and 2 illustrate the different microvessel densities in SCCs of different grade (I and III, respectively). In grade I SCC (n=4), the iMVD
values ranged from 16–28 (mean 20·5±5·2 SD). The corresponding values for grade II SCCs (n=3), grade III SCCs (n=3) and grade IV SCCs (n=5) were 22–34 (27·6±5·5), 34–39 (36·3±2·5) and 43–47 (45·6±1·6), respectively. One of the
120
P. Maiolino et al.
tumours (a grade IV SCC) showed clear evidence of metastasis. The iMVD values differed significantly between grade I and grades III and IV (P= 0·000006), grade II and grade IV (P=0·0003), and grade III and grade IV (P=0·0007), but not between grades I and II (P=0·14) or between grades II and III (P=0·06).
Discussion High tumour microvessel density was found to be associated with poor prognosis in many human studies (Weidner et al., 1993; Weidner, 1995, 1998, 1999; Fox, 1997), but this observation was not confirmed in several studies of breast cancer (Axelsson et al., 1995; Siitonen et al., 1995; Morphopoulos et al., 1996). In studies on SCC of the head and neck, Gasparini et al. (1993) found an association between tumour microvessel density and metastasis rate, but Dray et al. (1995) and Zatterstrom et al. (1995) failed to find an association between microvessel density and survival. In the same way, Tahan and Stein (1995) and Weninger et al. (1996), who studied SCCs of the human lip and skin, respectively, noted an increased density of microvessels but not a relationship with the risk of metastasis. The discrepancies regarding the proposed prognostic significance of angiogenesis in tumours may be explained by the fact that several antibodies can be used to label vessels in tissue sections, and the choice of antibody will influence the number of microvessels available for counting. In many studies, antibodies directed against Factor VIII-related antigen (Weidner et al., 1991, 1992, 1993; Siitonen et al., 1995; Weninger et al., 1996) or CD34 antigen (Siitonen et al., 1995) have been used; these identify only a proportion of the vascular endothelium and will therefore underestimate the true extent of the microvasculature. Moreover, antibody to Factor VIII-related antigen, which is not expressed on all endothelial cells, is present on lymphatic endothelium. We chose to use a monoclonal antibody ( JC70) that recognizes the membrane-bound glycoprotein CD31, since in our hands this is a more sensitive marker of endothelium than other endothelial markers and it does not react with lymphatic endothelium (Kuzu et al., 1992; Charpin et al., 1997). Qbend 10, an antiCD34 antibody, did not produce immunolabelling in formalin-fixed, paraffin-wax-embedded SCCs of canine skin (Maiolino, unpublished data). In the present study, the mean iMVD increased progressively from the SCCs of grade I to the poorly differentiated SCCs of grade IV. These results
accord with our previous findings on the quantification of Ag-NOR proteins and proliferating cell nuclear antigen (PCNA) and on the immunolabelling of vascular endothelial growth factor (VEGF) in canine SCCs (De Vico et al., 1994; Maiolino et al., 1995, 2000). They also confirm that iMVD assessment may provide an additional criterion for evaluating the intrinsic malignancy and growth potential of such tumours. Moreover, the fact that the iMVD values of grade I and II tumours differed significantly from those of grade IV tumours, the latter showing the highest degree of vascularization, suggests that angiogenesis may account for an invasive or aggressive phenotype in canine SCC. However, this requires confirmation by a study in which necropsy proves that apparently aggressive canine SCCs with high iMVD values do in fact have an increased tendency to metastasize. Acknowledgments This research was supported in part by grants from the Ministero dell’Universita`, della Ricerca Scientifica e Tecnologica (M.U.R.S.T.). We thank Mr R. Ilsami for technical assistance. References Axelsson, K., Ljiung, B. E., Moore, D. H., Thor, A. D., Chew, K. L., Edgerton, S. M., Smith, H. S. and Mayall, B. H. (1995). Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma. Journal of the National Cancer Institute, 87, 997–1008. Charpin, C., Garcia, S., Bouvier, C., Martini, F., Andrac, L., Bonnier, P., Lavaut, M. N. and Allasia, C. (1997). CD31/PECAM automated and quantitative immunocytochemistry in invasive ductal carcinomas. Correlation with patient follow-up. American Journal of Clinical Pathology, 107, 534–541. De Vico, G., Agrimi, U. and Maiolino, P. (1994). Nucleolar size and mitotic index in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) of canine skin. Journal of Veterinary Medicine A, 41, 76–79. Dray, T. G., Hardin, N. J. and Sofferman, R. A. (1995). Angiogenesis as a prognostic marker in early head and neck cancer. Annals of Otology, Rhinology and Laryngology, 104, 724–729. Folkman, J. and Shing, Y. (1992). Angiogenesis. Journal of Biological Chemistry, 267, 10931–10934. Fox, S. B. (1997). Tumour angiogenesis and prognosis. Histopathology, 30, 294–301. Gasparini, G., Weidner, N., Maluta, S., Pozza, F., Boracchi, P., Mezzetti, M., Testolin, A. and Bevilacqua, P. (1993). Intratumoral microvessel density and p53 protein: correlation with metastasis in head-and-neck squamous-cell carcinoma. International Journal of Cancer, 55, 739–744. Goldschmidt, M. H. and Shofer, F. S. (1992). Skin Tumors of the Dog and Cat. Pergamon Press, Oxford, pp. 16–49.
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Angiogenesis in Squamous Cell Carcinomas of Canine Skin
Graham, J. C. and Myers, R. K. (1999). The prognostic significance of angiogenesis in canine mammary tumors. Journal of Veterinary Internal Medicine, 13, 416–418. Jacquemier, J. D., Penault-Llorca, F. M. and Bertucci, F. (1998). Angiogenesis as a prognostic marker in breast carcinoma and conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis. Journal of Pathology, 184, 130–135. Kuzu, I., Bicknell, R., Harris, A. L., Jones, M., Gatter, K. C. and Mason, D. Y. (1992). Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours. Journal of Clinical Pathology, 45, 143–148. Maiolino, P., De Vico, G. and Restucci, B. (2000). Expression of vascular endothelial growth factor in basal cell tumours and in squamous cell carcinomas of canine skin. Journal of Comparative Pathology, 123, 141–145. Maiolino, P., Restucci, B. and De Vico, G. (1995). Expression of proliferating cell nuclear antigen in basal cell carcinomas and in squamous cell carcinomas of canine skin: correlation with mitotic index and histological features. Journal of Veterinary Medicine A, 42, 339–343. Morphopoulos, G., Pearson, M., Ryder, W. D., Howell, A. and Harris, M. (1996). Tumour angiogenesis as a prognostic marker in infiltrating lobular carcinoma of the breast. Journal of Pathology, 180, 44–49. Restucci, B., De Vico, G. and Maiolino, P. (2000). Evaluation of angiogenesis for PECAM in canine mammary tumors by quantitative and immunohistochemical methods. Veterinary Pathology, 37, 297– 301. Rosenthal, R. C. (2000). The prognostic significance of angiogenesis in canine mammary tumors. Journal of Veterinary Internal Medicine, 14, 248–249. Siitonen, S. M., Haapasalo, H. K., Rantala, I. S., Helin, H. J. and Isola, J. J. (1995). Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas. Modern Pathology, 8, 745–752.
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Received, November 2nd, 2000 Accepted, April 12th, 2001