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1 Vet Pathol 4: (29) BRIEF COMMUNICATIONS Tumor-associated Carbohydrate Antigens: Sialyl Le a and T/Tn Antigens in Canine Mammary Tumors M. NOWAK, J. MADEJ, P. DZIĘGIEL, W. ŁOPUSZYŃSKI, A. RODO, AND M. UGORSKI Department of Pathological Anatomy, Pathophysiology, Microbiology, and Forensic Veterinary Medicine (MN, JM) and Department of Biochemistry, Pharmacology, and Toxicology (MU), Faculty of Veterinary Medicine, University of Environmental and Life Sciences, ul. Norwida 3, Wrocław; Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 2, 53-4 Wrocław (MU); Department of Histology and Embryology, Medical University, ul. Chałubińskiego a, 5-38 Wrocław (PD); Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Life Sciences, ul. Głęboka 3, 2-2 Lublin (WŁ); and Department of Clinical Sciences, Faculty of Veterinary Medicine, ul. Nowoursynowska 59c, 2-7 Warsaw, Poland (AR) Abstract. Twenty-eight canine mammary tubulopapillary carcinomas and 4 simple adenomas were studied by immunohistochemistry for the expressions of the tumor-associated carbohydrate antigens. Sialyl Le a was detected in 7.42% of the malignant and 92.84% of the benign tumors. Staining with anti-t and anti-tn monoclonal antibodies revealed that 85.7% of the tubulopapillary carcinomas expressed T and Tn antigens. In contrast, 5% of the adenomas did not express T antigen, and 42.85% of them were only weakly stained for this carbohydrate structure. In the case of Tn antigen, the majority (57.4%) of samples was weakly stained, and no binding was observed in 35.7% of the analyzed specimens. Comparison of average values of reaction intensity (IRS) scale for malignant versus benign tumors by the Mann-Whitney U-test revealed a significant relationship between T and Tn antigens expression and type (malignant vs. benign) mammary tumors. Based on the results obtained, it is suggested that each of the studied antigens can be treated as a tumorassociated antigen of canine mammary tumors. However, only the T and Tn antigens seem to be associated with malignant transformation of mammary gland cells and to be of potential value as diagnostic markers. Key words: Dogs; mammary gland; tumor; tumor-associated carbohydrate antigens. Mammary tumors are the most frequent type of neoplasia in female dogs, accounting for approximately 5% of all tumors, which is similar to humans in that breast cancer is the most common type of tumor in women. It is well established that many human tumorassociated antigens are the result of aberrant glycosylation of cell-surface glycoproteins and/or glycosphingolipids. These so-called tumor-associated carbohydrate antigens (TACAs) can be divided in 2 major groups: ) modified lacto-series type or type 2 chains, represented by sialyl Le a and sialyl Le x structures and 2) core carbohydrate structures of O-linked mucin type, represented by T and Tn antigens. 4 Sialyl Le a, a tetrasaccharide with the structure: Neu5Aca2 3Galb 3GlcNAcb 3Galb 4Glcb Cer 4 j Fuca is a well-known tumor-associated antigen of human gastrointestinal cancer. 4 Using immunohistochemistry, increased levels of sialyl Le a antigen were also found in breast carcinomas. 9 It was shown that the level of this carbohydrate structure increased during mammary tumor progression, with the highest level observed in tumor cells from metastatic lymph node lesions. 5 However, opposite results were obtained by Nakagoe et al. (998), who found reduced amounts of the sialyl Le a structure in metastatic lymph node compared with primary tumor. T antigen (known as Thomsen- Friedenreich or TF antigen) is a simple disaccharide Galb-3GalNAca-O-, and its precursor Tn antigen is just a GalNAc residue bound covalently to serine/ threonine of the polypeptide chain. Both structures were found in different types of cancer, including breast cancer. 4 In breast cancer patients, increased expression of T and Tn antigen has been correlated with tumor progression and metastatic spread. 5,9 Tn-antigen expression was statistically significantly associated with shortened 5-year disease-free interval, higher ptnm 222

2 Vet Pathol 4:2, 29 Brief Communications 223 Table. Semiquantitative IRS scale taking into account both the percentage of positive cells (A) and the intensity of the reaction color (B), with the final score representing the product of the two variables (A 3 B). A pts no cells with positive reaction pts no color reaction pt to % of cells with positive reaction pt low intensity of color reaction 2 pts 5% of cells with positive reaction 2 pts moderate intensity of color reaction 3 pts 5 8% of cells with positive reaction 3 pts intense color reaction 4 pts.8% of cells with positive reaction 3 pts intense color reaction B stages, positive lymph node status, and increased combined histologic grades. 8 On the other hand, it was recently shown that patients with T-positive tumors had significantly better prognosis. 3 Sialyl Le a is a ligand for P- and E-selectins and plays an important role in the binding of several types of cancer cells to vascular endothelium and in the subsequent formation of metastases. 7 Similarly, it was suggested that T antigen supports the binding of cancer cells to vascular endothelium by its interaction with galectin-3, facilitating metastatic spread. 3 Of the tumor-associated carbohydrate antigens, only the expression of sialyl Le x has been studied so far in canine breast cancer cells. In study, the presence of sialyl Le x was found in 47.% of adenomas and.7% of adnocarcinomas. 7 There was no correlation between the expression of this carbohydrate structure and clinicopathologic features and prognosis. 7,8 A recent study showed that sialyl Le x antigen was present in all analyzed carcinomas, regardless of histologic type. A strong correlation between the level of sialyl Le a expression and the presence of lymph node metastases was found. Therefore, this study was undertaken to analyze the expressions of 3 other tumor-associated carbohydrate antigens, that is, sialyl Le a, T, and Tn antigens, in canine mammary gland tumors. Tissue specimens were obtained from 42 female dogs of different pure and mixed breeds, aged to years, undergoing surgery at the Department of Surgery, Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wrocław, Poland, the Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland, and the Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw, Poland. The tumors were sampled with a margin of approximately 2 cm of healthy tissues. For histologic studies the tissue specimens were fixed in % neutralbuffer formalin and embedded in paraffin. Paraffin sections, mounted on Superfrost slides, were dehydrated and stained with hematoxylin and eosin. The tumors were classified on the basis of the diagnostic criteria of the World Health Organization. Maligant tumors were graded according to the classification of Bloom- Richardson in the modification of Elston and Ellis. 2 For immunohistochemical (IHC) detection, 4-mmthick paraffin sections were deparaffinized in xylene and gradually rehydrated with the use of ethanol. Endogenous peroxidase activity was blocked by 5 minutes of exposure to 3% H 2 O 2. Antigen retrieval was done by exposure of the tissue sections to boiling Antigen Retrieval Solution (DakoCytomation, Denmark) in a microwave oven (25 W) for 5 minutes. Murine monoclonal antibody (mab) H4 specific to sialyl Le a was purchased from Seikagaku Corporation (Japan). Murine mabs 22.9 and Tn5 directed, respectively, against T and Tn antigens were used as culture supernatants (the respective hybridoma were from the Collection of the Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Wrocław, Poland). The antibodies were diluted with Background Reducing Antibody Diluent (DakoCytomation) (anti-sialyl Le a :, anti-t :, anti-tn : 5) and incubated with the sections for hour at room temperature. Subsequently, the slides were rinsed in PBS ( mm sodium phosphate buffer ph 7.2, 5 mm NaCl), incubated with biotin-conjugated swine antibodies directed against mouse and rabbit immunoglobulins for 5 minutes at room temperature, rinsed again, and incubated with streptavidin-biotin peroxidase complex (LSAB2, HRP, DakoCytomation) for another 5 minutes at room temperature. 3,3-diaminobenzidine tetrahydrochloride (DAB, DakoCytomation) was used as a chromogen (7 minutes, room temperature). All sections were counterstained with Mayer s hematoxylin counterstain. The immunoglobulin fraction of serum from nonimmunized rabbits instead of primary antibody (Primary Negative Control, DakoCytomation) was used as the negative control. The obtained photomicrographs were subjected to computer-assisted image analysis via a computer coupled to an Axiophot optical microscope (Carl Zeiss, Germany) using MultiScanBase V 4.2 software (Computer Scanning Systems, Warsaw, Poland) The degree of TACA expression was ranked using the modified semiquantitative Immunoreactive Remmele Score (IRS) according to Remmele and Stegner (Table ). 2 Correlation between TACA expression and the histopathologic malignancy grade was made by Spearman s correlation analysis. Mann-Whitney U-test for independent samples was used to compare the expressions of the TACAs in malignant versus benign tumors. The results were considered statistically significant when P,.5 in both analyses. Software Statistica 7. (STATSoft, Kraków, Poland) was used for all analyses. Based on histologic examination, 28 mammary tubulopapillary carcinomas and 4 simple adenomas

3 224 Brief Communications Vet Pathol 4:2, 29 Table 2. Expression of tumor-associated carbohydrate antigens (TACAs): sialyl Lea, T, and Tn antigens in canine mammary adenocarcinomas. No. of TACA-Positive Cases* Reaction Intensity Tubulopapillary Carcinoma Simple Adenoma No reaction Low Moderate Intense No reaction Low Moderate Intense Sialyl Le 8 2 a (35.7%) (28.57%) T 8 7 (4.28%) (2.42%) (5.%) Tn (35.7%) (35.7%) (57.4%) * Total number of tubulopapillary carcinomas: 28; total number of simple adenomas: 4. were included in this study. The tubulopapillary carcinomas were classified, according to the BloomRichardson scale in the modification of Elston and Ellis,2 as malignancy grades, that is, G (8 cases), G2 ( cases), and G3 (4 cases). The expressions of sialyl Lea in the canine mammary tubulopapillary carcinoma specimens and normal mammary epithelium from the same animals as well as simple adenoma specimens were analyzed using the immunoperoxidase method. Sialyl Lea was detected with different intensities in 7.42% of the mammary malignant tumors and 92.84% of the benign tumors (Table 2). Therefore, no significant difference was found in the Fig.. Expression of sialyl Lea, T, and Tn antigens in canine mammary tubulopapillary carcinomas (n 5 28) and simple adenomas (n 5 4). Reaction intensities for each TACA were calculated based on the semiquantitative IRS scale of Remmele and Stegner8 and are presented as means. *P,. for Tn-expressing tubulopapillary carcinomas compared with Tn-expressing adenomas and **P,.5 for T-expressing tubulopapillary carcinomas compared with T-expressing adenomas (Mann-Whitney). expression of sialyl Lea between malignant tubulopapillary carcinomas and benign adenomas (Fig. ). No staining with anti-sialyl Lea mab H4 was observed in normal tissue sections distant from the carcinoma Fig. 2. Immunohistochemical staining of canine mammary tubulopapillary carcinomas with (A) antisialyl Lea mab H4, (C) anti-t mab 22.9, (E) anti-tn mab Tn5, and simple adenomas with (B) anti-sialyl Lea mab, (D) anti-t mab 22.9, and (F) anti-tn mab Tn5. 43.

4 Vet Pathol 4:2, 29 Brief Communications 225 lesions. Sialyl Le a antigen was present not only on the surface of tumor cells, but also in the cytoplasm as well as extracellularly in secreted form (Fig. 2A, B). No correlation between sialyl Le a expression and the histopathologic malignancy grade was found when the data were subjected to Spearman s correlation analysis. Of the 28 tubulopapillary carcinomas that were subjected to IHC analysis with anti-t and anti-tn mab, the majority of the specimens expressed T and/or Tn antigens. For T antigen, 24/28 (85.7%) of the malignant tumor sections were stained with mab In contrast, 5% of the adenomas did not express T antigen, and 42.85% of them were only weakly stained for this carbohydrate structure (Table 2). Comparison of the average values of the reaction intensity (IRS scale) for malignant versus benign tumors by the Mann- Whitney U-test revealed a significant relationship between T-antigen expression and type (malignant vs. benign) of mammary tumor (Fig. ). In tubulopapillary carcinomas, the presence of Tn antigen, detected by binding of mab Tn5, was also observed in 85.7% of samples. However, when the adenomas were stained with anti-tn monoclonal antibody, the majority (57.4%) of samples was weakly stained, and no binding was observed in 35.7% of the analyzed specimens (Table 2). Again, using the similar statistical approach as was used for T antigen, significant relationship between Tn-antigen expression and type (malignant vs. benign) of tumor was found (Fig. ). Spearman s rank correlation analysis showed no correlation between the expression of T/Tn antigens and histologic grade of the analyzed malignant tumors. No positive staining was found with normal mammary tissues from the same animal. As in the case of sialyl Le a antigen, the staining was not limited to the cell surface, but the antigens also were found in the cytoplasm and in secreted form (Fig. 2C, D, E, F). In humans, malignant transformation is invariably associated with alterations in the expressions of cellular glycoconjugates and with structural changes of their carbohydrate moieties. These so-called TACAs are represented by sialyl Le a /sialyl Le x and T/Tn antigens. 4 In contrast to human cancers, including breast cancer, very little is known about TACA expression in canine mammary tumors. So far, only the presence of sialyl Le x antigen has been analyzed in tumors obtained by surgical resection and in established in vitro cell lines derived from adenocarcinoma. 7,8, In the present study, we showed that canine mammary gland tumors express 3 other human TACAs: sialyl Le a, T, and Tn antigens. Similarly to sialyl Le x, the carbohydrate antigen sialyl Le a was present in the majority of malignant carcinomas (7.42%), but no correlation between the level of expression of this TACA and clinical grade of tumors was found. Even more adenomas were stained with antisialyl Le a antibody, and the expression of this carbohydrate structure was confirmed in more than 9% of the analyzed cases. These data suggest that the expression of sialyl Le a is not directly related to malignant transformation of mammary cells, but rather is an early event associated with a precancerous stage. In contrast, 2 other analyzed TACAs, T and Tn antigens, were absent or only weakly stained in the majority of the adenomas and expressed in about 85% of the tubulopapillary carcinomas. Therefore, these carbohydrate structures seem to be directly associated with mammary carcinoma and can represent new potential markers of malignant transformation in dogs. In summary, based on the results obtained we suggest that each of the studied carbohydrate structures can be treated as a tumor-associated antigen of canine mammary tumors. However, only T and Tn antigens seem to be associated with malignant transformation of mammary gland cells and to be of a potential value as diagnostic markers. References Duk M, Steuden I, Duś D, Radzikowski C, Lisowska E: Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-tn monoclonal antibodies. Glycoconjugate J 9:48 53, Elston CW, Ellis IO: Pathological prognostic factors in breast cancer: experience from a large study with long-term follow-up. Histopathology 9:43 4, 99 3 Glinsky VV, Glinsky GV, Rittenhouse-Olson K, Huflejt ME, Glinskii OV, Deutscher SL, Quinn TP: The role of Thomsen-Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium. Cancer Res : , 2 4 Hakomori S: Possible functions of tumor-associated carbohydrate antigens. Curr Opin Immunol 3:4 53, 99 5 Jeschke U, Mylonas I, Shabani N, Kunert-Keil C, Schindlbeck C, Gerber B, Friese K: Expression of sialyl Lewis X, sialyl Lewis A, E-cadherin and cathepsin-d in human breast cancer: immunhistochemical analysis in mammary carcinoma in situ, invasive carcinomas and their lymph node metastasis. Anticancer Res 25:5 22, 25 Misdorp W, Else RW, Hellmen E, Lipscomb TP: Histological classification of the mammary tumors of the dog and the cat. In: World Health Organization International Histological Classification of Tumors of Domestic Animals, 2nd series, vol. 7, pp. 59. AFIP, Washington, DC, Nakagawa T, Uyama R, Ohashi E, Takahashi T, Hong SH, Mochizuki M, Matsunaga S, Nishimura R, Sasaki N: The expression of sialyl Lewis X in canine and feline mammary gland tumors. J Vet Med Sci 4: , 22 8 Nakagawa T, Watanabe M, Ohashi E, Uyama R, Takauji S, Mochizuki M, Nishimura R, Ogawa R, Sugano S, Sasaki N: Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumors. Res Vet Sci 8:37 323, 2

5 22 Brief Communications Vet Pathol 4:2, 29 9 Nakagoe T, Fukushima K, Hirota M, Kusano H, Kawahara K, Ayabe H, Tomita M, Kamihira S: Immunohistochemical expression of blood group substances and related carbohydrate antigens in breast carcinoma. Jpn J Cancer Res 82:559 58, 99 Nakagoe T, Fukushima K, Tuji T, Sawai T, Nanashima A, Yamaguchi H, Yasutake T, Hara S, Ayabe H, Matuo T, Kamihira S: Immunohistochemical expression of ABH/Lewis-related antigens in primary breast carcinomas and metastatic lymph node lesions. Cancer Detect Preve 22:499 55, 998 Pinho SS, Matos AJF, Lopes C, Marcos NT, Carvalheira J, Reis CA, Gartner F: Sialyl Lewis x expression in canine malignant mammary tumours: correlation with clinicopathological features and E- cadherin. BMC Cancer 7:24 34, 27 2 Remmele W, Stegner HE: Vorschlag zur einheitlichen Definition eines immunreaktiven Score (IRS) fuer den immunohistochemischen Ostrogenrezeptor Nachweis (ER-ICA) im Mammakarzinomgewebe. Pathologe 8:38 4, 987 [In German] 3 Schindlbeck C, Jeschke U, Schulze S, Karsten U, Janni W, Rack B, Krajewski S, Sommer H, Friese K: Prognostic impact of Thomsen-Friedenreich tumor antigen and disseminated tumor cells in the bone marrow of breast cancer patients. Breast Cancer Res Treat :7 25, 27 4 Springer GF: T and Tn, general carcinoma autoantigens. Science 224:98 2, Springer GF: Tn epitope (N-acetyl-D-galactosamine a-o-serine/threonine) density in primary breast carcinoma: a functional predictor of aggressiveness. Mol Immunol 2: 5, 989 Steuden I, Duk M, Czerwiński M, Radzikowski C, Lisowska E: The monoclonal antibody, anti-asialoglycophorin from human erythrocytes, specific for b-d-gal--3-a-d-galnac-chains (Thompsen-Friedenreich receptors). Glycoconjugate J 2:33 34, Ugorski M, Laskowska A: Sialyl Lewis a : a tumourassociated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells. Acta Biochim Polon 49:33 3, 22 8 Wang BL, Springer GF, Carlstedt SC: Quantitative computerized image analysis of Tn and T (Thomsen- Friedenreich) epitopes in prognostication of human breast carcinoma. J Histochem Cytochem 45:393 4, Wolf MF, Ludwig A, Fritz P, Schumacher K: Increased expression of Thomsen-Friedenreich antigens during tumor progression in breast cancer patients. Tumor Biol 9:9 94, 988 Request reprints from Dr. M. Ugorski, Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Environmental and Life Sciences, ul. Norwida 3, Wrocław (Poland). ugorski@ozi.ar.wroc.pl.