Ultrastructural and immunohistochemical observations of a true malignant mixed tumor (carcinosarcoma) of the tongue.

True malignant mixed tumor (TMMT) of salivary glands, with both carcinomatous and sarcomatous components, is exceedingly rare. We offer a case of TMMT in a 79-yr-old man, which may represent the first report example of this unusual neoplasm arising in the tongue. The carcinomatous component was mainly of solid basaloid carcinoma with focal glandular differentiation, while the sarcomatous component was composed of pleomorphic elements such as chondrosarcoma, myxosarcoma and fibrosarcoma. Carcinoma cells at the periphery of solid nests occasionally merged into these sarcomatous elements. Immunohistochemically, basaloid carcinoma cells showed positive reaction for both low molecular weight cytokeratin and S-100 protein, whereas carcinoma cells lining ductal spaces were positive for a wide spectrum of keratin and EMA. The sarcomatous elements revealed the presence of vimentin and S-100 protein. Ultrastructurally, basal lamina-like material and/or mucoid precipitates often accumulated separating the tumor cells from each other singly or into a few cell group. Some sarcomatous cells assumed the myoepithelial features, such as the presence of microfilament bundles with dense bodies and pinocytotic vesicles along the cell periphery. These findings may indicate that TMMT shares a common histogenesis with pleomorphic adenoma.

Immunolocalization of basement membrane molecules in the stroma of salivary gland pleomorphic adenoma.

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.

Immunohistochemical localization of carcinoembryonic antigen (CEA) in salivary gland tumors.

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.

True malignant mixed tumor of the submandibular gland.

A case of true malignant mixed tumor of the submandibular gland is reported. The submandibular tumor, occurring in a 52-year-old man, started to grow rapidly after a long history without any change in size. Surgical resection was carried out and the resected tumor measured 5.5 cm with a cut surface showing mixed solid structures. Microscopically, the tumor had both carcinomatous and sarcomatous elements, the former consisting of poorly differentiated adenocarcinoma with squamous cell differentiation and the latter consisting of osteosarcoma with chondrosarcomatous and fibrosarcomatous elements. A remnant of benign pleomorphic adenoma could also be identified. Immunohistochemical study demonstrated keratin and epithelial membrane antigen in the carcinoma cells and vimentin in all elements of the osteosarcoma. It is assumed from these clinical and histological findings that the tumor had transformed from a pre-existing benign pleomorphic adenoma.

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

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.

Two-colour immunofluorescence marker study of pleomorphic adenomas.

Extensive use of two-colour immunofluorescence staining for various cell markers in pleomorphic adenoma, revealed three consistent phenotypic features: (1) keratin polypeptide No. 14, which was virtually restricted to myoepithelial cells (MEC) in normal salivary glands, appeared in a large fraction of the tumour cells, suggesting that the principal neoplastic element is derived from MEC or their immediate precursors; (2) a complex co-expression pattern of various cell markers was found, with extensive concurrence of keratin and vimentin in strands of MEC-like and myxoid tumour cells, probably reflecting different degrees of tumour cell differentiation; and (3) two phenotypically distinctive dendritic cell populations were identified, one consisting of keratin positive tumour cells and the other of HLA-DR positive but keratin negative stromal cells. The significance of these findings with regard to the histogenesis and complex morphology of pleomorphic adenoma is discussed.

[Ultrastructural analysis of amyloid deposits in pleomorphic adenoma of the salivary glands by topo-optical studies]. / Amyloid lerakódások ultrastrukturális elemzése nyálmirigy pleomorph adenomákban topo-optikai vizsgálatok alapján.

It is well established that the stroma of some tumours contains amyloid. Authors have studied in pleomorphic tumours the occurrence of amyloid. Out of 40 salivary gland tumours 5 contained amyloid in their stroma between tumour-cell cords. Amyloid of salivary gland tumours had a resistant structure. The possible pathomechanisms of amyloid deposition are discussed.

Crystalloids in salivary gland pleomorphic adenomas.

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.

Coexpression of keratin and vimentin in salivary pleomorphic adenomas.

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.

Discrimination of benign versus malignant mixed tumors of the salivary gland using digital image analysis.

Seven benign and four malignant mixed tumors of the salivary gland, biopsied using fine-needle aspiration, were analyzed using digital image analysis. Mean nuclear form factor, perimeter, and area were significantly increased in malignant cases. Better separation between diagnostic categories, however, was achieved by utilizing the coefficient of variation (CV) within a case rather than mean value. Form factor CV alone divided cases into nonoverlapping diagnostic categories. This quantitative analog of "pleomorphism" provided a useful marker for malignancy in mixed tumors.

[Pleomorphic adenoma of the salivary glands. Impact of the myxoid component on the prognosis]. / Adénomes pléomorphes des glandes salivaires. Incidence de la composante myxoïde sur le pronostic.

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 localization of the alpha and beta subunits of S-100 protein in pleomorphic adenoma of the salivary glands.

The immunohistochemical expression of the alpha and beta subunits of S-100 protein in reactive, modified and transformed of myoepithelial cells, salivary pleomorphic was investigated using monoclonal antibodies. With S-100 alpha, normal salivary glands showed strong staining in serous acinar cells and moderate to slight staining in ductal segments, and with S-100 beta staining was slight or negative in acinar cells, but strong in nerve fibres. In pleomorphic salivary adenomas, the immunohistochemical distribution of S-100 alpha and beta proteins indicated great variation in the tumour cells. Some neoplastic cells gave similar staining for both S-100 alpha and beta, others were strongly positive for S-100 alpha and stained only slightly for S-100 beta, or vice versa. Yet other cells were positive for S-100 alpha and negative for S-100 beta, or vice versa. Pleomorphic salivary adenomas were classified both by histopathological criteria and by their staining pattern for S-100 alpha and beta proteins. Great heterogeneity in S-100 alpha and beta protein expression was found in individual tumour cells of both ductal and myoepithelial origin, and no regular pattern was identified. The cellular origin of salivary pleomorphic adenomas is discussed in terms of S-100 alpha and beta protein immunohistochemistry. Pleomorphic adenoma cells may be transformed from reserve cells into tumour cells displaying biologic properties of myoepithelial cells, ductal cells, or a mixture of both.

Tyrosine-rich crystalloids in pleomorphic adenoma: SEM findings and partial biochemical characterization.

The rare tyrosine-rich crystalloids (TRC) of salivary gland pleomorphic adenoma (PA) give a positive Million reaction indicating the presence of tyrosine. Their varied histochemical reactions, however, suggest a more complex composition. Two cases of TRC were encountered in a series of 144 PA (1.4%). Both were studied with several histochemical stains, and one tumor particularly rich in TRC was further examined with transmission and scanning electron microscopy and subjected to biochemical analysis using fresh-frozen tissue. TEM showed amorphous, electron-dense masses with no discernable internal structure. SEM revealed a geodelike structure of radially arranged, interlocking plates. Amino acid analysis of normal parotid, tumor with TRC, and a similar tumor without TRC indicated a slightly elevated level of tyrosine and arginine in the tumor with TRC. Polyacrylamide gel electrophoresis of tissue dissolved in sodium dodecyl sulfate revealed dense banding corresponding to polypeptides of a relative molecular weight of approximately 17,000 only in the TRC-rich sample. These bands on further analysis contained relatively large amounts of arginine. Tyrosine was present in only small amounts. TRC appear to be small proteins containing some tyrosine but rich in arginine.

S-100 protein localization in minor salivary gland tumours: an aid to diagnosis.

The clinician is almost entirely dependent on the histopathologist to accurately diagnose minor salivary gland tumours, but in some cases the histological interpretation of the specimen is very difficult. Recently it has been demonstrated using immunohistochemical techniques that S-100 protein is present in some salivary gland tissues and its localization has been used as an aid in the differentiation of major salivary gland tumours. To assess its value in the diagnosis of minor salivary gland tumours it was localized in sections from 15 such tumours using both a standard peroxidase-antiperoxidase (PAP) and a newly developed immunogold-silver staining sequence (IGSS) technique. Strong staining for S-100 protein was seen in the nuclei and cytoplasm of the cellular areas and also in the cells in the chondroid and myxoid areas of pleomorphic adenomas. Generally the staining was more intense and widespread with the IGSS method. No staining was observed in any of the other tumour types. We conclude that S-100 protein localization is a valuable aid in the differentiation of minor salivary gland tumours. Furthermore, the IGSS method enables more sensitive 'reading' of the staining reaction.

Coexpression of glial fibrillary acid protein, keratin and vimentin. A unique feature useful in the diagnosis of pleomorphic adenoma of the salivary gland in fine needle aspiration biopsy smears.

Positive staining for glial fibrillary acidic protein (GFAP) of tumor cells in fine needle aspirates of 11 of 12 pleomorphic adenomas of the parotid gland is reported. Tumor cells in these neoplasms also coexpressed keratin and vimentin to varying extents. Coexpression of GFAP, keratin and vimentin in tumor cells in aspirates is an unusual feature, so far demonstrated only in pleomorphic adenomas. Thus, intermediate filament typing may help to distinguish: (1) pleomorphic adenomas of the salivary glands from head and neck tumors of nonsalivary gland origin; (2) intracranial metastases of malignant mixed tumors of the salivary gland from gliomas; and (3) pleomorphic adenomas from extracranial gliomas.

Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands.

We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and alpha-smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against alpha-smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti-alpha-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for alpha-smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.

Immunohistochemical localization by monoclonal antibody of human epidermal growth factor in mixed tumours of the skin.

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.