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2 Table 1 List of immuno and mucin histochemistry parameters

3 Table 2 Subtypes of adenoid cystic carcinoma of the salivary glands Table 3 Average score of proteins expressed in either epithelial or myoepithelial cell types in adenoid cystic carcinomas of the salivary glands

4 examinations of specimens obtained by fine needle aspiration cytology. Study Design: Immunohytochemical and cytochemical anaylyses were performed for 24 ACC specimens using antibodies against cytokeratin (CK), epithelial membrane antigen, carcinoembryonic antigen, p63, a -smooth muscle actin, S100 protein, CD44 variant 6, maspin, c-kit, glial fibrillary acidic protein, and vimentin. Ki-67 was utilized to evaluate cell proliferation. Periodic acid-schiff and Alcian blue staining were performed to assess mucin production. Results: The combination of CK and p63 antibodies produced the best results in terms of both specificity and sensitivity for the visualization of epithelial and myoepithelial cells, respectively. CK positive (+) luminal cells were localized in areas of tubular morphology, whereas p63 +. myoepithelial cells faced pseudoglands in cribriform lesions. Luminal cells tended to disappear during progression toward solid type ACCs. Immunocytochemical analysis revealed myoepithelial cells on hyaline spherical globules surrounded by epithelial cells in cribriform ACCs. In the tubular type, two types of cells were mixed at various ratios; CK + epithelial cells that tended to form small clusters and scattered p63 + myoepithelial cells with scant cytoplasm. Conclusion: CK and p63 antibodies enable epithelial and myoepithelial cells tobe clearly differentiated in ACCs and are good candi- date markers for the diagnosis of salivary gland tumors. 2) Foote, F.W.J., Frazell, E.L. Tumors of the major salivary glands. Cancer 1953; 6: 1065 ` ) Ellis, G.L., Auclair, P.L. Tumor of the salivary glands. Vol. 17. Washington, D. C.: Armed Forces Institute of Pathology, ) Rosai, J. Major and minor salivary glands. In: Ackerman's surgical pathology, 8th edition, St. Louis, Missouri: Mosby- Year Book, Inc., 1996: p ) Chen, J.C., Gnepp, D.R., Bedrossian, C.W. Adenoid cystic carcinoma of the salivary glands: an immunohistochemical analysis. Oral Surg Oral Med Oral Pathol 1988; 65: ) Reis-Filho, J.S., Schmitt, F.C. Taking advantage of basic research: p63 is a reliable myoepithelial and stem cell marker. Abstract Objective: Epithelial and myoepithelial markers used for the diagnosis of adenoid cystic carcinomas (ACC) of the salivary gland were evaluated utilizing cytopathological and immunohistochemical Adv Anat Pathol 2002; 9: 280 `289. 8) Bilal, H., Handra-Luca, A., Bertrand, J.C., Fouret, P.J. P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues. J Histochem Cytochem 2003; 51: 133 `139.

5 9) Sternlicht, M.D., Barsky, S.H. The myoepithelial defense: a host defense against cancer. Med Hypotheses 1997; 48: `4 10) Fonseca, I., Moura Nunes, J. F., Soares, J. Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study. Histochem Cell Biol 2000; 114: ) Penner, C. R., Folpe, A. L., Budnick, S. D. C-kit expression distinguishes salivary gland adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma. Mod Pathol 2002; 15: ) Leong, A. S.-Y., Cooper, K., Leong, F. J. W.-M. Manual of Diagnostic Antibodies for Immunohistochemistry. London: Greennwich Medical Media Ltd, ) Schmitt, F.C., Zelandi Filho, C., Bacchi, M.M., Castilho, E.D., Bacchi, C. E. Adenoid cystic carcinoma of trachea metastatic to the placenta. Hum Pathol 1989; 20: ) Morinaga, S., Nakajima, T., Shimosato, Y. Normal and neoplastic myoepithelial cells in salivary glands : an immunohistochemical study. Hum Pathol 1987; 18: ) Orell, S. R., Sterrett, G. F., Walters, M. N.-I., Whitaker, D. Head and neck ; salivary glands. In: Manual and Atlas of Fine Needle Aspiration Cytology, 3 rd edition. London: Churchill Livingstone, 1999: p ) Young, J. A., Warfield, A. T. The salivary glands. In: Gray, W., McKee, G. T., editors. Diagnostic Cytopathology, edition. London: Churchill Livingstone, 2003: p nd 17) Perzin, K. H., Gullane, P., Clairmont, A. C. Adenoid cystic carcinomas arising in salivary glands: a correlation of histologic features and clinical course. Cancer 1978; 42: ) Szanto, P. A., Luna, M. A., Tortoledo, M. E., White, R. A. Histologic grading of adenoid cystic carcinoma of the salivary glands. Cancer 1984; 54: ) Frierson, H. F., Jr., El-Naggar, A. K., Welsh, J. B., Sapinoso, L. M., Su, A. I., Cheng, J., et al. Large scale molecular analysis identifies genes with altered expression in salivary adenoid cystic carcinoma. Am J Pathol 2002; 161: Photo. 1 Histopathological and immunohistochemical analysis of adenoid cystic carcinomas. Tubular (A, B, and C) cribriform (D, E, and F), and solid (G, H, and I) types. H&E staining (A, D, and G). Immuohistochemistr3 for cytokeratin (CK) (AE1/AE3) (B, E, and H) and p63 (C, F, and I). Original magnification, X200. Photo. 2 Cytological findings for the cribriform type of adenoid cystic carcinoma. Myoepithelial cells are attached tc the surface of hyaline globules and are usually surrounded by epithelial cells. (A) Papanicolaou staining Photo. 3 (B) May-Giemsa staining. (C and E) CK immunohistochemistry counterstained with hematoxylin and Al cian blue. (D and F) p63 immunohistochemistry counterstained with hematoxylin. Original magnification: x 320 (B), x400 (A, C, and D), orx1, 000 (E and F). Cytological findings for the tubular type of adenoid cystic carcinoma. Epithelial cells harboring relatively abundant cytoplasm are clustered (red arrows), whereas myoepithelial cells with little cytoplasm are scattered around them. (A) Papanicolaou staining. (B) May-Giemsa staining. (C) CK immunohistochemistry counterstained with hematoxylin and Alcian blue. (D) p63 immunohistochemistry counterstained with hematoxylin. Original magnification : x640.

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