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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.

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.

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.

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.

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.

Two types of crystalloids in salivary gland pleomorphic adenomas were studied by light microscopy and electron microscopy. The first type of crystalloid, the previously described tyrosine-rich crystalloid, was identified in three (1.5%) of 205 cases. The crystalloids by light microscopy assumed a radial configuration, resulting in the characteristic petal-shaped morphology. Transmission electron microscopy revealed them to be electron-dense, lobular projections without internal structure. Scanning electron microscopy demonstrated a range of morphology from rounded and intact doughnutlike structures to aggregates of irregular, loosely cohesive plates. The crystalloids were backscatter positive by backscattered electron imaging, and by x-ray microanalysis exhibited prominent calcium, phosphorus, and magnesium peaks that were not present in the adjacent tumor tissue; these three elements may be important in the formation and structure of tyrosine-rich crystalloids. The second type of crystalloid was intraductal and birefringent and was identified in 26 (12.7%) of 205 cases. In 21 of these 26 cases the crystalloids were lost on 10% formaldehyde fixation and paraffin embedding. Histochemical stains and x-ray microanalysis did not reveal a definite chemical composition, but did suggest a predominantly organic nature.

Tyrosine crystals were seen in 24 of 113 salivary gland pleomorphic adenomas from Malawi and one of six carcinomas arising in a pleomorphic adenoma; they were present in tumours from all common anatomical sites. They were not found in 71 other salivary tumours of varying histological type. Similar crystals were found in one out of 56 pleomorphic adenomas from patients operated on at St Thomas's Hospital. No excess tyrosine was demonstrated in a tumour containing many crystals. Their great frequency in black patients is confirmed but an adequate explanation for this phenomenon has not been found.

A total of 27 cases of salivary gland adenocarcinomas were studied from clinicopathological view point. Adenocarcinomas of the salivary gland were microscopically subclassified into 3 groups according to Luna's classification: Salivary duct carcinomas histologically resembled the ductal carcinoma of the breast, displayed nuclear atypia and had poorer prognosis than the other subclasses of salivary gland adenocarcinomas. Terminal duct carcinomas lacked in nuclear atypia and displayed a variety of growth patterns, including papillary, cribriform, tubular, and solid. Some terminal duct carcinomas showed prominent mucin-production. Epithelial-myoepithelial carcinomas had clear cytoplasms and exuberant glycogen. In addition to the clinicopathological study, nuclear areas of the tumor cells were measured in each of the 27 salivary gland adenocarcinomas, and mean nuclear area (MMA) and standard deviation (SD) were calculated. The group with more than 50 microns 2 of MNA had poorer prognosis than the group with 50 microns 2 or less of MNA, and the group with more than 13 microns 2 of SD had poorer prognosis than the group with 13 microns 2 or less of SD. Finally, immunohistochemical study was performed against various markers including keratin, epithelial membrane antigen, lactoferrin, S-100 protein, CEA, etc., using the Avidin-biotin-peroxidase complex method. Lactoferrin was present in most of the salivary duct carcinomas, on the other hand, S-100 protein was detected in all of the five cases of the terminal duct carcinoma investigated. But immunohistochemical study is not especially useful in distinguishing subclasses of salivary gland adenocarcinomas or investigating the origin of tumor cells.

A clonal cell with an acinar cell phenotype, which was induced by 5-azacytidine treatment of a neoplastic human salivary intercalated duct cell line, was cultivated in the presence of 1 alpha,25-dihydroxyvitamin D3. Morphological changes occurred; large cells that were polygonal or round in shape and had numerous vacuoles in their cytoplasm appeared in the treated cells, whereas the same concentration of 1 alpha,25-dihydroxyvitamin D3 did not affect the morphology of the parental cells. Major alterations, such as expression of type II collagen, alpha and beta chains of S-100 protein, and sulfated proteoglycans, were observed in these cells with a phenotype similar to chondrocytes. After the removal of 1 alpha,25-dihydroxyvitamin D3 from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that the reversible differentiation into chondrocyte-like cells of a human salivary acinar cell line occurs in growth medium containing 1 alpha,25-dihydroxyvitamin D3.

The adenocarcinoma cell line HSG from human salivary gland, which proliferates in vitro or in nude mice, was examined by the immunoperoxidase method for the expression of three different types of intermediate-sized filaments (IFs) and of specific antigens such as carcinoembryonic antigen, S-100 protein, secretory component, lactoferrin, myosin, tropomyosin, and actin. The cultured HSG cells were found to express three different types of IFs defined by antibodies to keratin, vimentin, and desmin. In HSG cells proliferating in vitro at 34 degrees C and 37 degrees C but not at 39 degrees C, the expression of tropomyosin and carcinoembryonic antigen was observed, although myosin and S-100 protein were not detected. The expressions of actin, lactoferrin, and secretory component were restricted to cultured HSG cells at 39 degrees C and 37 degrees C, respectively. Transplantation of HSG cells into nude mice resulted in the establishment of a nude mouse system with malignant characteristics such as invasion and metastasis. The expression of IFs in the primary tumors was restricted to keratin and desmin IFs, whereas coexpression of keratin, vimentin, and desmin IFs was observed in some neoplastic cells present in the metastatic tumors in regional lymph nodes and lung. In addition, expression of actin, myosin, tropomyosin, and S-100 protein was found in the metastatic tumors, whereas myosin and S-100 protein were not detected in the primary tumors. Moreover, the metastatic tumors were almost occupied by the neoplastic cells with oncocytic changes, although oncocytic change was not found in the cultured HSG cells and their primary tumors.

Major salivary gland tumours were studied for the presence of hormone receptors for oestrogen and progesterone. Of the eight salivary gland tumours exhibiting varied histology, none showed high affinity receptors for oestrogen or progesterone. Salivary tissue from four patients with non-neoplastic salivary gland disease was also studied and found not to contain high affinity receptor sites. The absence of hormone receptors in these glands suggests that such tumours are not dependent on endocrine function.

The distribution of fibronectin (FN) and collagen type III (IIIC) have been compared in a series of epithelial neoplasms from the gastrointestinal tract and salivary gland. The difficulty of distinguishing between FN of epithelial and fibroblastic origin is emphasised and evidence is presented for the validity of this distinction. In carcinomas FN was sometimes, but not invariably, lost from epithelial cell surfaces. Both FN and IIIC were increased in reactive connective tissue stroma. It is concluded that loss of cell surface FN is unlikely to be a useful diagnostic marker for malignancy, but that the occurrence of this phenomenon in vivo as in vitro indicates that it is biologically significant.