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Two cell lines (ACCS and ACCY) were isolated from two individuals with adenoid cystic carcinoma (AdCC) using tissue culture techniques. Both cell lines have similar morphology, i.e., elongated and flattened cells with slender cytoplasmic processes. The two cell lines tend to form pseudocysts, which are a specific architectural feature of AdCC. Coexpression of cytokeratin and vimentin was found in the two cell lines, which occasionally also contained S-100 protein and lactoferrin or lysozyme immunoreactivity. Moreover, ACCS and ACCY displayed potential for the production of a large amount of extracellular matrix including basal lamina components such as fibronectin, laminin, and type IV collagen and glycosaminoglycans which are also part of the basal lamina. These findings suggest that the tumor cells, probably basal or myoepithelial like cells, are responsible for the formation of the peculiar stroma of AdCC consisting of a large amount of collagen-like fibers, basal lamina components, and mucopolysaccharides.

Thirty-two cases of adenoid cystic carcinoma (ACC) of the salivary glands were examined immunohistochemically by monoclonal antibodies to proteoglycans (PG) such as chondroitin 6 sulfate (C6SPG), chondrotin 4 sulfate (C4SPG), dermatan sulfate (DSPG), heparan sulfate (HSPG) and keratan sulfate (KSPG) in conjunction with specific enzymatic digestion. The cribriform structure of ACC consisted of basaloid, myoepithelium-derived, and luminal tumor cells. The myoepithelial tumor cells were positive for PG, whereas luminal tumor cells were unstained. Occasional pseudocysts also gave positive staining for PG. Tubular structures consisting of modified myoepithelial cells indicated a high intensity reaction for C6SPG and C4SPG, and a slight one for DSPG, HSPG, and KSPG. Immunodepositions in solid and cluster structures were comparatively light in terms of PG. Basement membrane in ACC stained strongly for C4SPG, slightly for C6SPG, and very slightly for DSPG, HSPG, and KSPG. In ACC, immunohistochemical staining of PG was regularly positive in myoepithelium-derived tumor cells, but was irregular in other types of tumor cells.

Previous immunocytochemical studies of pleomorphic adenomas have demonstrated consistent labeling with glial fibrillary acidic protein (GFAP). Cross-reactivity with other intermediate filaments of similar structure and chemical composition has been suggested to account for this seemingly inappropriate pattern of immunoreactivity. To investigate further this phenomenon, we examined five pleomorphic adenomas by immunoelectron microscopy. Ultrastructural features were similar to those described by other investigators, with ductal epithelium being surrounded by myoepithelial cells and modified cells becoming detached to form the isolated stellate and spindle cells of the stroma. As part of this process, many neoplastic myoepithelial cells appeared to lose their specialized ultrastructural features, assuming a rather undifferentiated appearance. Single and double immunoelectron microscopic labeling showed vimentin filaments in all these neoplastic myoepithelial cells. In contrast, GFAP filaments were identified only in the most undifferentiated cells. Such restriction of GFAP filaments to an ultrastructurally definable subset of neoplastic cells provides strong evidence against nonspecific staining due to cross-reactivity. Given the previously described coexpression of vimentin and GFAP by neoplastic cartilage, it appears likely that this immunophenotype in neoplastic myoepithelial cells reflects early chondroid differentiation.

In order to determine the participation of basement membrane molecules in formation of its characteristic stroma, 30 cases of salivary gland pleomorphic adenoma were examined by immunohistochemical staining for type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. The stroma was histopathologically classified into four types: hyaline, fibrous, myxoid, and chondroid. Immunohistochemically, type IV collagen and laminin were more intensively localized in hyaline, fibrous and chondroid types of stroma, whereas heparan sulfate proteoglycan was more prominent in myxoid areas. The results suggest that the stroma contains these basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that histological variety of the stroma depends on proportion of local contents of each basement membrane molecule.

Fifteen acinic cell carcinomas from an equal number of patients were analysed for their DNA content and proliferative (S-phase) index by flow cytometry from archival tissues. Seven of the carcinomas manifested a diploid DNA content. None of the patients with diploid acinic cell carcinomas died of their carcinomas and none developed metastases in follow-up periods extending for 10 or more years. Four of eight patients with aneuploid acinic cell carcinomas have died because of their malignancies within a 10 year period after the first surgical removal of the carcinoma. Five of the eight patients exhibited metastases. Although the number of cases does not permit strong correlations between histopathological features, abnormalities in DNA content and outcome of patients, it was noted that carcinomas with prominent necrosis, tubuloductal differentiation and 'dedifferentiated' areas displayed more aggressive biological courses.

Carcinoembryonic antigen (CEA) was first isolated from colonic carcinoma and has been used as a diagnostic marker. CEA has also been observed in a variety of epithelial tumors and normal tissues. In this study, CEA was localized by means of immunohistochemical procedures in benign and malignant salivary gland tumors, as well as in normal parotid gland, indicating that CEA is not a reliable marker for differentiation between benign and malignant salivary gland neoplasms.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.

The neoplastic epithelial duct cells human salivary gland (HSG) and myoepithelial cells human pleomorphic adenoma (HPA) established from human salivary gland were examined by the immunoperoxidase method for the presence of specific antigens such as carcinoembryonic antigen (CEA), S-100 protein, secretory component (SC), lactoferrin (LF), and myosin. Isolation of the cells and their morphologic features were reported previously. Consequently, the presence of CEA, SC, and LF in the HSG cells was demonstrated. The HPA cells were identified to express the specific antigens reactive to anti-S-100 protein, anti-myosin and anti-CEA sera in addition to the presence of oxytocin receptor. When the two cell lines were co-cultured in monolayer culture or within the sponge matrix, a large number of ductlike or tubular structures were formed in an optimal ratio of 1:2 in HSG and HPA cells, whereas the cultures of HSG cells only grew with occasional formation of ductlike structure. In addition, in HSG and HPA cells in an area with their contact in the mixed cultures, CEA staining was intensified as compared with the culture of HSG or HPA cells only and further S-100 protein was detected in HSG cells, whereas S-100 protein was not detected in the culture of HSG cells only. These findings strongly suggest that the intercalated duct and myoepithelial cells from human salivary gland propagate with their interaction together in the expression of specific antigens such as CEA and S-100 protein or in the morphogenesis of salivary gland epithelial cells.

A detailed electronmicroscopic study on glycogen-rich tumor is presented. The neoplasm originated from the minor salivary glands on the ventral surface of the tongue. The role of myoepithelial cells in the histogenesis of this lesion is not supported. It is proposed that the tumor arises from "undifferentiated" stem cells analogous to cells in the "end bud" stage of salivary gland morphogenesis. The presence of a large amount of glycogen is secondary to defective carbohydrate metabolism within the tumor cells.

Antisera of several secretory products of the salivary gland were used to investigate the histogenesis of acinic cell tumors and mixed salivary gland tumors for comparison. Amylase, lactoferrin, secretory piece, and proline-rich protein (PRP) immunoreactivity was detected in the majority of acinic cell tumors; staining was focal, except for PRP, which was diffuse. Lysozyme immunoreactivity was rare. There was discordance for immunoreactivity with several antisera in identifiable tumor lobules of half of the neoplasms. An antikeratin serum outlined microcystic and follicular areas but rarely solid foci. These findings support the contention that acinic cell tumors derive from a tubular type stem cell. Lactoferrin and secretory piece immunoreactivity was not common in mixed tumors and was confined to scattered ductal cells and luminal contents. Rare small foci of amylase and PRP immunoreactivity were found in two mixed tumors only.

Actomyosin-containing cells in both non-neoplastic and neoplastic tissues of the salivary gland, lung, breast and some other organs were studied by immunofluorescent microscopy using antiactomyosin rabbit serum. In the breast, myoepithelial-like cells with positive immunofluorescence in the cytoplasm were observed not only in sclerosing adenosis and fibroadenoma but also in scirrhous and medullary-tubular duct carcinomas. No positive cells were observed in medullary carcinomas with lymphoid infiltration. The actomyosin positive cells were also seen at the outer layer of tubules of "mixed tumors" and of cell nests in adenoid cystic carcinoma and in myoepithelioma of the salivary gland, but not in the metaplastic squamous cells or in the cells of myxomatous and chondroid areas of "mixed tumor". In carcinoma of the lung, actomyosin-positive cells were observed in adenoid cystic carcinomas and adenocarcinoma of the bronchial gland type, but they were not seen in squamous cell carcinomas or papillary adenocarcinomas. It was concluded that the actomysoin-containing cells with structural appearances of myoepithelial cells in a variety of tumors were neoplastic myoepithelial cells.

A peroxidase-antiperoxidase technique for S-100 protein has been applied to 68 salivary glands. These included 17 pleomorphic adenomas, seven adenoid cystic carcinomas, 23 adenolymphomas and a number of other neoplasms. In addition, five specimens of myoepithelial sialadenitis ('benign lymphoepithelial lesion') and five normal parotid glands were included. Consistent results were obtained, myoepithelial cells and cells in myxoid and chondroid areas in pleomorphic adenomas staining intensely. In adenoid cystic carcinoma, on the other hand, there was no staining. The adenolymphomas possessed intensely S-100 protein-positive cells in the interfollicular lymphoid areas; these were probably interdigitating reticulum cells. In addition, branching structures, probably corresponding to Langerhans' cells, were observed in the epithelium of adenolymphomas.

Immunohistochemical distribution of human epidermal growth factor (hEGF) was described in 17 cases of mixed tumour of the skin with monoclonal antibody. In normal sweat glands, epithelial cells in the secretory portion and in the transitional area between secretory portion and duct showed prominent staining for hEGF. In the salivary pleomorphic adenoma type of mixed tumour of the skin, luminal tumour cells of tubular and duct-like structures gave a very characteristic hEGF staining reaction. The tumour cells showing strong staining for hEGF were scattered throughout the solid foci in this type of mixed tumour. Tubular epithelial cells in the clear cell adenoma type also displayed a positive hEGF reaction. And apocrine mixed tumours strong staining for hEGF occurred on the apical side of tubular and ductal tumour cells. In view of the immunohistochemical staining patterns for hEGF, the histologic origin of mixed tumours of the skin is suggested to be cells in the secretory portion and those in the transitional portion between secretory portion and duct of the sweat gland.

Glycogen-rich clear cell carcinoma arising from minor salivary glands of the uvula in a 35-year-old woman is reported. This tumor was composed of nests and cords of clear cells containing cytoplasmic glycogen but no mucin. The epithelial nature of this lesion was obvious because of positive immunoreactivity for cytokeratin and epithelial membrane antigen, and the presence of immature lumina and intercellular spaces lined by many microvilli, associated with desmosomal junctions and basal lamina, as revealed by ultrastructural study. However, no myoepithelial cells could be detected. From these findings, it may be concluded that this tumor corresponds to glycogen-rich clear cell carcinoma (a variant of clear cell tumor), revealing glandular differentiation.

The association between malignant transformation and the loss of ABO(H) blood group antigens was documented by several authors. Most of the work was done on paraffin sections, though a small portion was performed on fresh frozen tissues. We suggest that the specific red cell adherance (SRCA) test can be applied to tissues cultivated in tissue culture for determination of malignant transformation. This study supports this assumption.

Lipofuscin granules were observed in normal salivary glands (parotid, submandibular, and minor salivary glands). The pigment was confined mainly to the epithelial cells of the intralobular ducts, but isolated granules were also found in acinar cells and myoepithelial cells. In chronic sialadenitis pigment granules were found in the intralobular epithelial cells and in macrophages in the surrounding ionnective tissue. In benign epithelial tumors pigment granules were observed within neoplastic epithelial cells and in macrophages in the stroma, while malignant tumors displayed pigmented granules only in macrophages in the stroma.

The coexpression of keratin and vimentin is described in 45 pleomorphic adenomas using an immunoperoxidase MAb method. Histopathologically, the outer layer of tubuloductal structures and peripheral tumor cells in solid masses, including modified or neoplastic myoepithelial cells, showed positive staining with monoclonal keratin antibody K8.12 and vimentin. This staining was found in the ratio of 10/26 (38.5%) in tubuloductal structures, 2/7 (28.6%) in peripheral tumor cells and 8/12 (66.7%) in modified myoepithelial cells. Concomitant staining of other keratin antibodies (PKK1, KL1) and vimentin did not exist. In addition, the ductal basal cells of normal salivary glands showed positive K8.12 labelling. The histogenesis of pleomorphic adenoma is discussed in relation to the differentiation of either ductal basal cells or ductal luminal cells from a single stem cell origin or the direct transformation of ductal basal cells to outer tumor cells and/or modified myoepithelial cells, both coexpressing K8.12 and vimentin.

A silver colloid technique used to identify nucleolar organizer regions associated protein (Ag-NORs) has been applied to 20 salivary gland tumors. The method was readily applicable to the preparations of paraffin-embedded sections and the Ag-NORs were enumerated with ease. A significant difference was found between the numbers of Ag-NORs in the nuclei of malignant salivary gland tumors, such as adenoid cystic carcinoma, mucoepidermoid tumor and adenocarcinoma (with a mean of from 2.05 to 2.78 per nucleus) and those of benign salivary gland, such as pleomorphic adenoma, adenolymphoma (Wartin tumor) and clear cell adenoma (with a mean of from 1.47 to 1.72 per nucleus). It is proposed that the Ag-NORs technique, which is rapid, simple, and inexpensive, may be useful in the differential diagnosis of malignant and benign salivary gland tumors.

Five cases of adenoid cystic carcinoma of minor salivary glands were studied. The mucoid material in the characteristic cystlike space of this neoplasm was distaseresistant periodic acid-Schiff (PAS)--positive, alcain blue-positive, toluidine blue-positive, and mucicarmine-positive. Verhoeff-Van Gieson's method and Weighert's method did not reveal elastic tissue in the cystlike spaces. Mallory's method revealed that a central core in cystlike spaces was similar in stainability to collagen. Wilder's method did not reveal reticular fibers in these spaces. Electron microscopy revealed three readily recognizable zones: a juxtacellular zone of a network of replicated basal lamina, and intermediate zone of stellate granules of mucoid material, and a central core of densely packed aperiodic filaments or collagen fibrils. The histogenesis of cystlike spaces and their realtionship with biologic behaviors of the neoplasm were discussed.

Polymorphous low-grade adenocarcinoma (PLGA) is a minor salivary gland neoplasm that is characterized by morphologic variability, cytologic uniformity, and an infiltrating growth pattern. To date, this entity has been identified either within the confines of the oral cavity or, rarely, in the nasal cavity. The authors report a case of PLGA that arose in the nasopharynx of a 44-year-old female, representing the first documented occurrence of this tumor outside the oral cavity or the sinonasal tract. The histopathologic and immunohistochemical differentiation from pleomorphic adenoma and adenoid cystic carcinoma is discussed.

Immunohistochemical expression of 8 cases of mucoepidermoid carcinomas (G-I, 3 cases; G-II, 2 cases; and G-III, 3 cases) revealed marked heterogeneity of the proteins examined. Immunohistochemically detectable keratins (TK, KL1, and PKK1) were distributed in epidermoid cells, but were absent in mucous secreting cells. Strongly positive deposits of keratin proteins were detected in squamoid tumor cells in the G-I tumors. The tumor cells displayed positive staining for S-100 alpha, but did not stain with polyclonal S-100 antiserum or with monoclonal S-100 beta. The cells showing highest reactivity for S-100 protein were scattered in neoplastic foci and were probably Langerhans cells. Lactoferrin and lysozyme reactions were generally negative in tumor foci; but a positive reaction for lactoferrin was found in luminal tumor cells although rarely, and lysozyme staining was occasionally noted in histiocytes in the stroma. Amylase activity was usually absent in the tumor cells, with the exception of one case in which it was confined to the tumor cells. Mucoepidermoid carcinomas of various grades indicated marked heterogeneity in terms of various immunohistochemically detectable proteins.

Dibutyryl cyclic AMP (dB-cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture. A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB-cAMP. Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S-100 protein, were observed in those cells with a phenotype similar to myoepithelial cells. Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of dB-cAMP. After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Undoubtedly, electron microscopy has a specific role to play in the diagnosis of a select group of salivary gland neoplasms. However, this tool has a current central role, along with immunohistochemical techniques, in elucidating morphogenetic processes in salivary gland tumors. New information gained from ultrastructural surveys of these tumors can be applied to improving classification and the diagnostic problems that are not infrequent for the surgical pathologist with salivary gland lesions.

Antibodies against different fractions of keratins can be helpful in various fields of special pathology. Antibodies against "small" and "large" keratins permit to evaluate epithelial maturation in skin and oral mucosa. In addition, disturbances of keratinization during inflammatory processes and malignant transformation can be analyzed. The main application of antibodies against the entire fractions of keratins is the detection of the epithelial nature of a neoplasm. By this tool, particular problems in surgical pathology concerning differential diagnosis can be handled in an easier way. Among the different tissues and their neoplasms, examples of the analysis of thymus tumours and salivary gland tumours are presented. Immunoreactivity with keratin antibodies depends crucially on tissue processing. In the normal diagnostic procedure, good results are regularly obtained if cryostat or Bouin-fixed paraffin-embedded sections are used.

A malignant glycogen-rich adenocarcinoma of palatal salivary glands is reported. Histopathology revealed nonencapsulated nests and cords of polyhedral cells showing voluminous clear cytoplasms and cellular pleomorphisms, separated by fine vascular septae. Small and large ducts were infrequently seen showing apparent transition of large ducts into clear cells. The tumor cells were PAS- and Best-carmine positive, diastase soluble, and mucicarmine and Alcian-blue negative. Ultrastructurally, the tumor cells were arranged in solid nests and cords of electron-lucent cells surrounding single or multiple lumina, and surrounded by basement lamina. Occasional fusiform electron-dense cell processes were present beneath the basement lamina. The electron-lucent cells were joined by junctional complexes, contained intracytoplasmic canals, and were filled with massive accumulations of beta glycogen particles. The electron-dense processes contained interlacing whorls of fine filaments and exhibited peripheral focal densities. The findings suggest that this glycogen-rich malignant tumor is of epithelial origin most probably of ductal cells.

Nuclear DNA ploidy studies were performed by flow cytometry on extracted nuclei from 12 oncocytic tumours of the salivary gland, 65 oncocytic tumours of the kidney, and 37 oncocytic tumours of the thyroid gland from the pathology archives of the Mayo Clinic. In order to provide an interesting clinical spectrum, three different classes of well-differentiated oncocytic tumours were selected for examination. Salivary gland oncocytic tumours were chosen for their generally benign behaviour. Oncocytic thyroid cancers exhibiting malignant potential because of local invasion, were thought to represent the opposite extreme of aggressiveness. Renal oncocytic tumours were known to demonstrate an intermediate degree of malignancy. All of the oncocytic salivary gland tumours showed a 'normal' DNA histogram and had a benign clinical course. For the oncocytic tumours of the kidney, 45% of DNA histograms were normal, 40% exhibited a significant increase in the DNA tetraploid/polyploid (4C) peak, and 15% showed a DNA aneuploid peak. Three patients with a DNA tetraploid pattern developed tumour metastasis and two have died from metastatic renal cancer. Among the oncocytic thyroid cancers, 27% were normal, 22% exhibited an increased DNA tetraploid peak, and 51% had a distinct DNA aneuploid peak. None of the thyroid tumour patients with a normal DNA pattern or with an increased DNA tetraploid peak died as a result of thyroid malignancy. In contrast, 58% of patients whose thyroid tumours showed a DNA aneuploid peak subsequently died from thyroid cancer.

In a series of 570 pleomorphic adenomas of main and accessory salivary glands, we found 48 recurrences (8.04%). These recurrences occurred between several months and 19 years after surgical treatment. They were more frequently noted in parotid tumors. Comparatively to a control series, in recurrent tumors myxoid structures were more frequent than epithelial structures, as demonstrated by immuno-markings (anti-protein S 100 and anti-vimentin sera). Besides, in these tumors, extracapsular myxoid expansions were commonly seen. The proliferative potential of myoepithelial cells which are the main cellular component of myxoid areas, is thus suggested as a mainly factor of recurrence of pleomorphic adenomas.

Immunohistochemical identification of human epidermal growth factor (hEGF) was carried out in a total of 152 cases of salivary gland tumours, consisting 107 pleomorphic adenomas and their variants, 13 adenolymphomas and 32 adenoid cystic carcinomas. A high percentage of pleomorphic adenomas revealed markedly positive hEGF staining of the luminal surface cells of tubuloductal structures and of modified or neoplastic myoepithelial cells. Clear cells of the tumour showed various reactivities from very slight to strong. Eosinophilic epithelial cells of adenolymphoma gave a positive reaction for hEGF in all the cases, whereas most adenoid cystic adenoma lacked hEGF staining; however some cases showed positive staining of the tumour cells. The immunohistochemical detection of hEGF in most salivary gland tumours suggests this factor to be a possible new marker of salivary glands tumours, and to have a biological role in tumour proliferation.

The expression and distribution of cytokeratins and vimentin in fifteen malignant salivary neoplasms were examined by immunocytochemical techniques using, five monoclonal antibodies (mAbs) against different epitopes of Cytokeratins (CKs) (mAbs PKK1, PKK2, and PKK3, identifying CKs 8, 18 and 19, CKs 7, 17 and 19, and CK 18, respectively) and Vimentin (mAbs V9 and V24). Antibody PKK1 gave strong reactions in all neoplasms showing the similarity of these tumours to other digestive system adenocarcinomas. Three general staining patterns of the neoplasms were recognized with respect to the reactivity of mAbs PKK2, PKK3, and V9. Mucoepidermoid cancer, salivary duct carcinoma and a clear cell carcinoma had a higher relative content of CKs 7, 17 and 19 than of CK 18. Adenoid cystic carcinoma showed the same CK pattern but in the periphery of the tumour cords vimentin was readily detected. In two acinic cell carcinomas, the relative content of CK 18 was higher than that of CKs 7, 17 and 19. Furthermore vimentin was expressed in the tumour cells. However, one mucoepidermoid carcinoma showed vimentin expression and two acinic cell carcinomas were vimentin negative and more reactive for PKK2 than PKK3. Pecularities in CK expression were seen: squamous areas of mucoepidermoid carcinomas were stained by mAb PKK3 although CK 18 is not present in normal squamous epithelia or in squamous cell carcinomas of tongue and skin. In conclusion, the different salivary neoplasms can be distinguished on basis of IFP content. Such a differentiation fits with current theories of histogenesis, i.e. vimentin is seen in tumours presumed to arise from intercalated duct reserve cells, whilst the vimentin negative neoplasms would be expected to arise in excretory duct reserve cells.

Biopsy specimens from nasopharyngeal carcinomas (NPC) or salivary-gland carcinomas (SGC) in Greenland Eskimoes were examined for the presence of Epstein-Barr virus (EBV) DNA and sera from the patients were tested for EBV-specific antibody titres. Six out of 7 NPCs and one from an undifferentiated SGG were positive for EBV DNA. The EBV-specific antibody spectra and titres of the patients with NPC or undifferentiated SGG conformed to the results of earlier studies in other high-incidence areas.

An immunohistochemical study of 34 pleomorphic adenomas of the major salivary glands demonstrated phenotypic differences among the various morphologic regions in these tumors. The phenotypes expressed were comparable to those of normal salivary gland cells. In the normal glands, myoepithelial cells were immunoreactive for glial fibrillary acidic protein (GFAP), S-100 protein, and keratin; acinic cells exhibited strong, predominantly nuclear S-100 staining and weaker keratin staining; intercalated ducts had both cytoplasmic and nuclear S-100 positivity; and several epithelial antigens were observed throughout the ductal system. In the tumors, the presence of classic epithelial markers (including carcinoembryonic antigen, epithelial membrane antigen, secretory component, and keratin) in the luminal cells of ducts and the intense immunoreactivity with GFAP (with weaker keratin and S-100 staining) in periductal and stromal cells indicated distinct epithelial and myoepithelial differentiation. Solid epithelioid areas consisted phenotypically of intercalated duct/acinic cells and/or myoepithelial cells, the former exhibiting predominant nuclear S-100 positivity. The presence of GFAP-like immunoreactivity in normal myoepithelial cells strongly supports the extensive involvement of this cell in pleomorphic adenomas. The spectrum of phenotypes expressed adds weight to existing evidence for pleomorphism rather than a mixed origin of this tumor. The combination of keratin, S-100, and GFAP immunostaining is particularly useful in identifying the component cells in pleomorphic adenomas of the salivary glands.

Cytophometric analysis of nuclear DNA content was performed in a series of mucoepidermoid carcinomas with an extreme difference in the clinical course, which was observed during a follow-up period of 6-13 years. The prerequisite for such a study was the development of a method, which made it possible to study the nuclear DNA content in tumour cells obtained at the time of diagnosis, i.e. 6-13 years ago in the present material. A special cytochemical procedure with destaining of the original Giemsa stain, refixation and subsequent Feulgen staining of the smears of aspirates in the original cytological material was developed. The cytophotometric nuclear DNA analysis of tumour cells in smears of aspirate from mucoepidermoid carcinomas showed that higher ploidy tumours (near-triploid) had a worse prognosis when compared with near-diploid tumours. These data are supported by our previous findings that a shift from a near-diploid to a near-triploid DNA content of the tumour cell nuclei was associated with the prognostic important property of invasive growth. Thus, the cytophotometric nuclear DNA analysis of aspirated tumour cells seems to give valuable information about the malignancy degree of the individual tumour, which is not possible to obtain only from the cytomorphological features.

Tyrosine-rich crystalloids were identified in three benign mixed tumors of the parotid gland, one terminal duct adenocarcinoma of a minor salivary gland, and the fibrous connective tissue of two laryngectomy specimens. Light and electron microscopic studies showed the crystalloids to be composed of irregular deposits of amorphous electron-dense material. In the salivary gland tumors this material was commonly associated with interstitial collagen and was found in greatest abundance near myoepithelial cells. This proximity suggests that the tyrosine-rich crystalloids result from the precipitation on stromal collagen of products secreted by neoplastic myoepithelial cells.

We have found that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line (HSG) occurs in growth medium containing dibutyryl cAMP (dB-cAMP). In the current study, the relationship between intracellular cAMP levels and anchorage-dependent and -independent growth or ras oncogene of p21 levels was analyzed in the differentiation process toward myoepithelial cells of HSG cells cultured in the presence of dB-cAMP. Correlation between the concentrations of dB-cAMP and intracellular cAMP in the HSG cells was statistically significant. There was a significant inverse correlation between the concentrations of dB-cAMP and colony-forming ability of the cells in semisolid agar or on a plastic surface. We have found the expression of ras p21 protein in HSG cells. When HSG cells were cultured in the presence of dB-cAMP and were committed to differentiate into myoepithelial cells, it was shown by double-antibody labeling technique and/or immunoblotting that the committed cells expressed myosin with a concomitant decrease of ras p21 protein. Moreover, intracellular cAMP levels were found to be inversely associated with ras p21 content of the cells. These findings indicate that the intracellular cAMP levels regulate significantly cell proliferation and ras p21 expression in HSG cells.

Amylase (Am), lactoferrin (Lf), lysozyme (Ly), secretory component (SC), epithelial IgA, and epithelial IgM were traced by paired immunofluorescence staining in ethanol-fixed specimens from 15 pleomorphic adenomas of the parotid gland. Epithelial elements positive for some of the markers were detected in a variable number of the specimens (Am, 0; Lf, 11, Ly, 2; CEA, 6; SC, 11; IgA, 9; and IgM, 6); their expression seemed to depend on a certain degree of glandular differentiation. Variable co-expression of secretory epithelial markers probably reflected different degrees of differentiation, indicating that clonal diversification may explain the histological complexity of pleomorphic adenomas. The most consistent expression (in almost 75% of the specimens) shown by Lf and SC might further reflect histogenetic relationship to intercalated ducts in which these antigens are normally found in largest amounts.

The purpose of the present investigation was to study the histogenesis of the mucoepidermoid carcinoma of the salivary glands. Eleven cases of mucoepidermoid carcinoma of the minor salivary glands and five of the major glands were extensively studied employing immunohistochemical and fluorescent microscopic techniques. Both the intermediate cells and the duct cells showed a rather similar pattern of reactivity for vimentin, actin and EMA. Also, the intermediate cells and the myoepithelial cells showed a similar reaction pattern for keratin and UGA-1. The intermediate, myoepithelial and duct cells shared a similar reaction pattern for desmin, myosin, CEA, and S-100 protein. However, the rest of the tumor markers studied (AFP, PNA and WGA) were found to be non contributary. We also found that the intermediate and to some extent the epidermoid tumor cells showed a positive reaction with Azophloxine GA, which is a selective stain for myoepithelial cells in the normal glands. Based on these findings, the duct cells, the myoepithelial cell in the normal glands and the intermediate cells of the mucoepidermoid carcinoma share certain similar characteristics. The intermediate cells may actually be a mixed population, some having characteristics of the myopithelial cells and others of duct cells. These findings are relevant to the possible role of the intermediate cell in the histogenesis of the mucoepidermoid carcinoma.

Immunohistochemical localization of carcinoembryonic antigen (CEA) with conventional antibody to CEA (anti-CEA), nonspecific crossreacting antigen (NCA)-absorbed polyclonal antibody to CEA (NCAa-CEA), and monoclonal antibody to CEA (Mono-CEA) have been compared in obstructive lesions and salivary gland tumors. Normal salivary glands gave strong staining of the luminal borders of acinar cells with anti-CEA, whereas no staining occurred with Mono-CEA. Obstructive lesions showed occasionally marked staining with anti-CEA in some acinar cells, but there was no reaction with Mono-CEA. Of 69 pleomorphic adenomas examined, 34 were positively stained with anti-CEA, 18 with NCAa-CEA and 8 with Mono-CEA along the luminal borders of the tumor cells. The frequency of positive staining of material within tubular lumina was similar with all three immunoreagents. Neoplastic cells were positive with Mono-CEA in only three cases, while eight cases were positive with NCAa-CEA and 11 cases with anti-CEA. In salivary gland tumors true CEA is be found mainly at the border of tumor cells, but the frequency of positive reactions is low.

A neoplastic human salivary intercalated duct cell clone was cultured in 5 microM 5-azacytidine for 5 days at 37 degrees C; then the cells were trypsinized and subcultured in growth medium without 5-azacytidine. Thereafter, subclones were cloned from the subculture. Of 12 subclones isolated, 7 clonal cell lines were established and characterized. The two subclones composed of cells which were spindle shaped or stellate exhibited phenotypes similar to those of myoepithelial cells such as microfibrils and myosin and formed a myoepithelioma upon transplantation of the cells into nude mice. The other five subclones were composed of polygonal cells with numerous secretory granules in their cytoplasm and containing amylase that seems to be specific to acinar cells; transplantation of these cells into nude mice resulted in production of acinic cell carcinoma. These findings indicate that a neoplastic human salivary intercalated duct cell is capable of at least bidirectional differentiation.

Immunohistochemical studies were performed for the presence of S-100b protein in non-neoplastic and neoplastic salivary gland tissues by the peroxidase anti-peroxidase (PAP) method. Some cases of pleomorphic adenoma were investigated by immuno-electron microscopy. S-100b protein could not be detected in epithelial cells of intercalated ducts, acini, striated ducts and excretory ducts of non-neoplastic salivary gland. However, myoepithelial cells surrounding the acini and intercalated ducts were specifically stained by S-100b protein. In pleomorphic adenomas, S-100b protein-positive cells could be mostly observed in the myxoid and chondroid areas, and the basal layer cells of the double-layered ductal cells were also positive. In clear cell adenoma, the clear cells were also S-100b protein positive. In adenoid cystic carcinomas, S-100b protein-positive cells could be found in trabecular areas, but not in tumor cells showing cribriform-pattern. In other tumors (Warthin's tumor, oxyphilic adenoma, basal cell adenoma, mucoepidermoid tumor and acinar cell carcinoma), S-100b protein positive cells were seldom observed. Immuno-electron microscopically, S-100b protein was diffusely distributed in the cytoplasm of myoepithelial cells as well as of tumor cells of pleomorphic adenoma, being distributed especially on the membrane of endoplasmic reticulum and the outer nuclear membrane.

Pleomorphic adenoma of the salivary glands was studied by the peroxidase-antiperoxidase method and Ouchterlony's double diffusion for the presence of so-called nervous system-specific proteins, S-100 protein, glial fibrillary acidic protein (GFAP), and astroprotein. Positive immunostaining for S-100 protein was observed in tumor cells of epithelial, myxomatous, and chondroid areas. An immunodiffusion test confirmed its presence in the tumor. Normal salivary glands were also stained with anti-S-100 serum, but the reaction was less intense than that of tumor. Specific immunostaining for GFAP and astroprotein was found in a small number of cells of myxomatous and chondroid areas and occasionally in cells in epithelial areas. An immunodiffusion test suggests that the GFAP-related antigen of the pleomorphic adenoma showing a minor heterogeneity of antigenic determinants is almost identical with GFAP. Normal salivary glands did not stain with GFAP or astroprotein antisera, nor did they react with anti-GFAP serum by immunodiffusion. Thus, the S-100 protein and GFAP-related antigen may be actively synthesized in adenoma cells during the course of tumor development. In addition, the GFAP-related antigen is considered to be a tumor-associated antigen of pleomorphic adenoma of the salivary glands.