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.     

Coexpression of Vimentin, Cytokeratin and S-100 In Monomorphic Adenoma of Salivary Gland; Value of Marker Studies In the Differential Diagnosis of Salivary Gland Tumours

An unusual coexpression of glial fibrillary acid protein (GFAP), keratin and vimentin occurs in pleomorphic adenoma of salivary gland. We designed this study to see if coexpression of the markers was also present in monomorphic adenoma of the salivary gland and whether monomorphic adenoma could be distinguished from other salivary gland tumours by marker studies. Immunocytochemical markers were used on fine needle aspiration samples from four cases of monomorphic adenoma, two cases of oncocytic adenoma, three cases of adenoid cystic carcinoma and four cases of pleomorphic adenoma. While positivity for cytokeratin, vimentin and S-100 was consistently found in all cases of monomorphic adenoma, only cytokeratin was expressed in adenoid cystic carcinoma. In pleomorphic adenoma, GFAP, cytokeratin and vimentin were coexpressed while in cases of oncocytic adenoma none of the markers was localized. Thus, it appears that by using a combination of GFAP, cytokeratin, vimentin and S-100 a distinction between these neoplasms may be possible. However, a larger study is needed to establish the usefulness of this approach.     

Role of fine needle aspiration cytology in diagnosis of pleomorphic adenomas

Role of fine needle aspiration cytology in diagnosis of pleomorphic adenomas This retrospective study was carried out to review the cases diagnosed as pleomorphic adenoma in major or minor salivary glands and determine the difficulties encountered on typing this tumour on fine needle aspiration cytology (FNAC). Over a 19‐year period (1982–2000) 488 pleomorphic adenomas were diagnosed on FNAC from different sites (parotid – 372 cases, submandibular – 95 cases; oral cavity – 21 cases). Histology was available in 232 cases. Twenty‐nine cases where a histological diagnosis of pleomorphic adenoma was made but the cytological diagnosis was variable were also reviewed. In 216 of the 232 cases a good cytohistological correlation was available. On review only 4 of the 16 cases initially diagnosed as pleomorphic adenoma on FNAC where the histology revealed a different tumour were categorized as pleomorphic adenoma, while 3 each were classified as adenoid cystic carcinoma and benign tumour ?type, and 2 each were diagnosed to be muco‐epidermoid carcinoma, monomorphic adenoma and acinic cell carcinoma. On review of the FNAC smears from 29 cases where a histological diagnosis of pleomorphic adenoma was available while the cytological diagnosis was variable, only 11 (38%) were categorized as pleomorphic adenoma. In the majority of the remaining cases the cytological diagnosis did not alter markedly, 7 of 10 cases where the tumour could not be typed on cytology initially could not be typed even on review. In conclusion, FNAC is an ideal, fairly accurate preoperative procedure for the diagnosis of pleomorphic adenomas. Certain diagnostic problems occur in differentiating pleomorphic adenomas from adenoid cystic carcinoma, monomorphic adenoma and mucoepidermoid carcinoma. Carcinoma ex‐pleomorphic adenoma is difficult to identify on FNAC and in our series all 4 such cases on histology were considered benign on cytology.     

Glial fibrillary acidic protein and desmin in salivary neoplasms. Expression of four different types of intermediate filament proteins within the same cell type

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Abundant dendritic cells express HLA-DR in pleomorphic adenomas

Most pleomorphic adenomas were found to contain abundant dendritic cells (DC) with major histocompatibility complex (MHC) class II (HLA-DR) expression. Their immunohistochemical staining features were suggestive of dendritic histiocytic cells. Extensive phenotypic characterization by two-colour immunofluorescence staining for various cell markers was performed. The DC expressed both HLA class I and II determinants, vimentin, S-100 protein, and various monocyte-related markers (10G11, 3D10, 7G5 or CD11a, 8C2) but were negative for leucocyte common antigen (CD45), Leu-6 (CD1), and the myelomonocytic L1 antigen. Characterization of HLA-DR positive DC isolated by an immunomagnetic bead method confirmed the immunohistochemical staining pattern that corresponds to the phenotype of interdigitating cells. Morphological and immunological implications of the abundant presence of these cells in pleomorphic adenomas are discussed.     

Fine needle aspiration cytology of basal cell adenoma of the salivary gland: a cytohistological correlation study of 35 cases

B. Vicandi, J.A. Jiménez‐Heffernan, P. López‐Ferrer, P. González‐Peramato, M. Patrón and J.M. Viguer
Fine needle aspiration cytology of basal cell adenoma of the salivary gland: a cytohistological correlation study of 35 cases Objective: In order to evaluate the possibility of a specific cytological recognition of basal cell adenoma (BCA) we reviewed our experience with 35 histologically proven cases. Few series describing cytological features of BCA are available and diagnostic cytological criteria are not well established. Methods: This study was based on 41 cytology samples from 35 patients with BCA. Thirty‐five aspiration procedures were performed pre‐operatively and six on tumour recurrence. Nineteen of the 35 patients were men and 16 women. The mean age at diagnosis was 55 years old (range 24–92). The series includes one non‐representative case. Except for one tumour located in the upper lip, all of them involved the parotid gland. Results: Aspirates were cellular, showing groups with dense, homogeneous metachromatic stroma and single cells. Relevant features were the trident‐like configuration of groups, intimate relationship between neoplastic cells and stroma and cellular polymorphism. In approximately half of the cases a precise diagnosis was given. Most of the remaining tumours were diagnosed as benign but they were difficult to differentiate from pleomorphic adenoma. Regarding malignancy, there were two misdiagnoses of acinic cell carcinoma, due to high epithelial cellularity along with scarcity of stroma, and one case was considered to be suspicious of malignancy. Conclusion: BCA shows characteristic cytological features that allow a precise diagnosis. The main differential diagnosis is epithelial‐rich pleomorphic adenoma, while acinic cell carcinoma is a potential false positive.     

Proliferative activity as detected by immunostaining with Ki-67 and proliferating cell nuclear antigen in benign and malignant epithelial lesions of the human parotid gland

OBJECTIVE: A retrospective immunohistochemical study of parotid gland lesions was designed to evaluate the diagnostic and prognostic value of the proliferating cell nuclear antigen (PCNA) and Ki-67 with monoclonal antibodies PC 10 and MIB-1, respectively. STUDY DESIGN: Tissue samples comprised normal parotid gland (N, n = 10), chronic sialadenitis (CS, n = 8), Warthin's tumor (W, n = 10), benign pleomorphic adenoma (BPA, n = 8), mucoepidermoid carcinoma (MEC, n = 13), carcinoma in pleomorphic adenoma (CPA, n = 8) and adenoid cystic carcinoma (ACC, n = 12). The morphometric parameters for PCNA and MIB-1 comprised the PI and MI labelling indices (the numerical percentage of positive nuclei), NAP and NAM (the numerical density of positive nuclei), and NPI and NMI (volume corrected index). RESULTS: The values of MIB-1 parameters increased progressively in benign lesions in comparison with the N group and in malignant neoplasms in comparison with nonneoplastic groups and benign lesions. Values for all parameters in BPA were significantly lower than those in malignant groups. Spearman rank correlation analysis showed a highly positive correlation between the morphometric parameters and severity of the lesions. The mean values of MI and NMI were significantly higher in patients who died of the malignant tumors than in those who survived. The same quantitative parameters for PCNA did not differ significantly from those obtained for MIB-1 and showed similar trends. CONCLUSION: PCNA and MIB-1 indices are reliable markers for discriminating between benign and malignant tumors of the parotid gland, and the parameters PI, MI, NPI and NMI may have prognostic applications.     

Diagnostic significance of coexpression of intermediate filaments in fine needle aspirates of human tumors

A study was undertaken of the diagnostic significance of the coexpression of intermediate filaments in fine needle aspirates of human tumors. Three types of coexpression were found: (1) true coexpression, in which tumor cells simultaneously express more than one intermediate filament protein; (2) pseudocoexpression, in which various tumor cell types from histogenetically different parts of a complex tumor show different results; and (3) false coexpression, in which tumor cells with one or two types of intermediate filaments are present together with benign cells expressing a different filament type. True coexpression of vimentin and keratin was documented in renal cell carcinomas, endometrial carcinomas, certain thyroid carcinomas and Hürthle cell adenomas. Coexpression of keratin and neurofilaments was seen in Merkel cell carcinomas, and coexpression of desmin and vimentin was found in leiomyosarcomas. Keratin, vimentin and neurofilament expression was seen in medullary thyroid carcinomas, and keratin, vimentin and glial fibrillary acidic protein expression was observed in pleomorphic adenomas of the salivary gland. Pseudocoexpression was noted in synovial sarcoma, epithelioid sarcoma, benign cystosarcoma phyllodes of the breast, teratocarcinoma, malignant granular cell tumor, progonoma, Wilms' tumor and triton tumor. Sources of false coexpression are also discussed.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin''s tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin''s tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin''s tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.     

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted s.c. in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation.     

A cell line from pleomorphic adenoma of the rat salivary gland

Pleomorphic adenoma of rat salivary gland is extremely rare, and culture of cells obtained from rat salivary gland tumors has not been reported. We have established a long-term cell line from a pleomorphic adenoma of a Sprague-Dawley rat submaxillary gland. The pleomorphic adenoma was composed of oval or spindle-shaped cells occasionally forming a small duct. Alcian blue-positive intercellular matrices, consisting mainly of glycosaminoglycans, were abundant. The cultured cells showed characteristics similar to those of the original tumor. This cell line should be useful for biological and biochemical studies of glycosaminoglycan-synthesis of pleomorphic adenoma cells.     

Immunohistochemical localization of the α and β 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.     

Metaplastic carcinomas of the breast—fine needle aspiration (FNA) cytology findings

nogueira m., andré s. and mendonça e. . (1998) Cytopathology 9, 291–300
Metaplastic carcinomas of the breast—fine needle aspiration (FNA) cytology findings
Metaplastic carcinomas of the breast are defined by mesenchymal and/or squamous cell components associated with ductal carcinoma and may raise diagnostic problems in FNA cytology. We reviewed FNA smears of a series of nine cases; seven were compared with histological sections and two with cell-block sections. The cytological pattern was diagnostic of carcinoma in six cases; in two cases a diagnosis of sarcoma/phyllodes tumour was considered, as cells were predominantly spindle-shaped. One case had a pleomorphic adenoma type pattern. The cytological findings suggesting a diagnosis of metaplastic carcinoma include a liquid aspirate, a proteinaceous or chondromyxoid background and a poorly differentiated tumour with multinucleated giant cells, neoplastic or histiocytic. A definite diagnosis requires the presence of both carcinomatous and metaplastic (squamous/mesenchymal) components.     

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.     

Diagnosis of pleomorphic adenoma in a heterotopic salivary gland: a case report

BACKGROUND: The histologic diversity encountered in pleomorphic adenoma may cause diagnostic difficulty in fine needle aspiration cytology (FNA) due to limited and selective sampling. CASE: A 40-year-old woman presented with a mass in the anterior aspect of the neck along the sternocleidomastoid muscle. FNA revealed a cellular tumor with a chondrimyxoid background and epithelial cells intermingled with a few mesenchymal cells. The diagnosis of pleomorphic adenoma was confirmed on histopathology. CONCLUSION: Primary ectopic pleomorphic adenoma can confidently be diagnosed by FNA. This technique is a useful tool in the initial assessment of the tumor.     

Structural organization of the toe pads in the amphibian Philautus annandalii (Boulenger, 1906)

We describe the morphology of toe pads in the Himalayan tree frog Philautus annandalii. These are expanded tips of digits and show modifications of their ventral epidermis for adhesion. The outer cells of toe pad epidermis (TPE) bear surface microstructures (0.7 × 0.2 μm), which are keratinized. Their cytoplasm contains no organelles, but pleomorphic nuclei and mucous granules (0.4–0.5 μm) that glue the keratin filaments. In the intermediate cell layer of TPE, similar keratinized microstructures as in the outer cells are present, so that when the outer layer is shed, it is ready with features for adhesion. These cells contain more keratin than the outer cells. The basal cell layer contains thin keratin bundles and usual cell organelles. The dermis contains mucous‐secreting glands, whose ducts open in the outer epidermal cell layer in channels. The dorsal epidermal cells lack surface microstructures and keratin bundles. Ultrastructural features suggest that toe pads utilize the surface microstructures for adhesion aided by mucus, in which the intermediate cell layer seems to bear the shear stress generated during locomotion. Further, TPE can expand and fit into an increased contact area of the substrate. The long, surface microstructures may also help in mechanical interlocking with rough surfaces on plants.     

Characterization of the epithelial components in pleomorphic adenoma of the salivary gland

OBJECTIVE: To clarify the cytomorphologic characteristics of luminal epithelial cells (LEC) and neoplastic myoepithelial cells (NMC) in pleomorphic adenoma (PA). STUDY DESIGN: Imprint cytologic smears stained with Papanicolaou stain were examined in 20 patients with PA (including recurrent cases). Immunocytochemistry was performed using the antibodies of epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA); cells positive for both CEA and EMA were interpreted as LEC and those negative as NMC. RESULTS: LEC were found in 9 of 20 cases as cell clusters in various shapes or as isolated cells with ample cytoplasm. NMC were classified into four types according to their visual patterns and cytoplasmic features: type A, isolated cells with ample cytoplasm; type B, isolated naked cells; type C, cluster of cells with ample cytoplasm; and type D, cluster of cells with scant cytoplasm. NMC were found in all 20 cases, with an absolute incidence of 100%, 90%, 65% and 50%, respectively. CONCLUSION: The different features of NMC (Types A-D) are essential to a specific differential diagnosis. This classification was useful to discriminate PA from other salivary gland tumors with NMC.     

Phenotypic analysis of cultured melanoma cells : Expression of cytokeratin-type intermediate filaments by the M5 human melanoma cell line

Expression of intermediate filament (IF) isotypes was studied in six human and two murine melanoma cell lines. With one exception, these lines expressed IFs only of the vimentin type; neurofilament peptides, desmin and GFAP were not detected. However, the M5 human melanoma line also expressed extensive cytokeratin tonofilament arrays, as visualized by immunofluorescence with a panel of eleven monoclonal antibodies and hetero-antisera to cytokeratins; only the keratin 19-specific antibody BA16 did not react. By 2 D gel electrophoresis, five major keratin peptides were detected (keratins 7, 8, 13, 17 and 18), and an additional 57 kD peptide was detected on immunoblots with several antikeratin antibodies. Also observed in M5 cells was focal collapse of tonofilament arrays in mitotic cells. All the melanoma lines tested were positive for S100; M5 and two other cell lines were also positive for the 220-240 kD neuroectoderm-associated cell-surface differentiation antigen defined by monoclonal antibody UJ 127:11. In all the melanoma cell lines, secretion of extracellular matrix proteins (fibronectin, laminin and collagen type IV) was sparse or absent, and all were negative for the epithelial cell markers HMG-1 and HMG-2. Co-expression of keratin and vimentin by a melanoma cell line is discussed in the light of recent controversy concerning expression of cytokeratins by other neoplasms of putative neuroectodermal origins.