Myofilament Localization and Immunoelectron Microscopic Detection of Muscle-Specific Actin in Neoplastic Myoepithelial Cells in Pleomorphic Adenomas and Myoepitheliomas

Elucidating the cellular characteristics of the nonluminal or myoepithelial cells of pleomorphic adenomas is one approach to establishing the diagnostic criteria for myoepitheliomas. Ultrastructural features of nonluminal tumor cells in 22 pleomorphic adenomas and of tumor cells in 9 myoepitheliomas were assessed from micrographs of routinely fixed and epoxy resin-embedded samples. Recognizable myofilaments were only moderately prominent in 1 myoepithelioma. In the rest of the cases, irrespective of whether nonluminal cells of pleomorphic adenomas or tumor cells of myoepitheliomas were spindle, angular, round, or plasmacytoid in form, myofilaments were noted only in one third of the cases and were present even in these in a small proportion of the tumor cells. Intermediate filament accumulations and basal lamina were more frequent findings associated with nonluminal tumor cells. Six pleomorphic adenomas and 2 myoepitheliomas had been fixed in half-strength glutaraldehyde and embedded in LR White resin for immunoelectron microscopic detection of muscle-specific actin. In 3 (2 pleomorphic adenomas and myoepitheliomas) of these 8 cases, readily visualized bands of filaments in many tumor cells were strongly labeled by the colloidal gold probe detecting muscle-specific actin even when myofilaments were minimal and infrequent in 2 cases and undetectable in the third by routine transmission electron microscopy. Lack of myofilament detection by immunocyto-chemistry or routine electron microscopy does not exclude a diagnosis of pleomorphic adenoma or myoepithelioma when growth patterns and cytology indicate such diagnoses. Immunoelectron microscopy, in fact, shows that muscle-specific actin can be detected even when myofilaments or muscle actin are apparently absent or minimal by routine electron microscopy or immunohistochemistry, respectively. Because examples of pleomorphic adenoma and myoepithelioma each with similar histologic and cytologic features of the myoepithelio-matous cells can have variable degrees or complete absence of expression of myofilaments or muscle-specific actin, the time-honored term myoepithelial for the nonluminal cells of pleomorphic adenomas and the term myoepithelioma are legitimate even in the absence of those markers that are specific for normal myoepithelial cells.     

Myofilament Localization and Immunoelectron Microscopic Detection of Muscle-Specific Actin in Neoplastic Myoepithelial Cells in Pleomorphic Adenomas and Myoepitheliomas

Elucidating the cellular characteristics of the nonluminal or myoepithelial cells of pleomorphic adenomas is one approach to establishing the diagnostic criteria for myoepitheliomas. Ultrastructural features of nonluminal tumor cells in 22 pleomorphic adenomas and of tumor cells in 9 myoepitheliomas were assessed from micrographs of routinely fixed and epoxy resin-embedded samples. Recognizable myofilaments were only moderately prominent in 1 myoepithelioma. In the rest of the cases, irrespective of whether nonluminal cells of pleomorphic adenomas or tumor cells of myoepitheliomas were spindle, angular, round, or plasmacytoid in form, myofilaments were noted only in one third of the cases and were present even in these in a small proportion of the tumor cells. Intermediate filament accumulations and basal lamina were more frequent findings associated with nonluminal tumor cells. Six pleomorphic adenomas and 2 myoepitheliomas had been fixed in half-strength glutaraldehyde and embedded in LR White resin for immunoelectron microscopic detection of muscle-specific actin. In 3 (2 pleomorphic adenomas and myoepitheliomas) of these 8 cases, readily visualized bands of filaments in many tumor cells were strongly labeled by the colloidal gold probe detecting muscle-specific actin even when myofilaments were minimal and infrequent in 2 cases and undetectable in the third by routine transmission electron microscopy. Lack of myofilament detection by immunocyto-chemistry or routine electron microscopy does not exclude a diagnosis of pleomorphic adenoma or myoepithelioma when growth patterns and cytology indicate such diagnoses. Immunoelectron microscopy, in fact, shows that muscle-specific actin can be detected even when myofilaments or muscle actin are apparently absent or minimal by routine electron microscopy or immunohistochemistry, respectively. Because examples of pleomorphic adenoma and myoepithelioma each with similar histologic and cytologic features of the myoepithelio-matous cells can have variable degrees or complete absence of expression of myofilaments or muscle-specific actin, the time-honored term myoepithelial for the nonluminal cells of pleomorphic adenomas and the term myoepithelioma are legitimate even in the absence of those markers that are specific for normal myoepithelial cells.     

Myoepithelioma: Definitions and Diagnostic Criteria

Due to their infrequency and multiplicity of histopathology, myoepitheliomas present difficulties in diagnosis and classification. Cellular varieties can be misdiagnosed as malignancies. Improvements in and clarification of diagnostic criteria are, therefore, required. A key to determining diagnostic criteria for myoepitheliomas is to study cellular morphology, cytoplasmic filament expression, and ultrastructural features of the nonluminal, i.e., neoplastic myoepithelial/basal, tumor cells of pleomorphic adenomas, and apply this information to defining myoepitheliomas. Cytologic and growth patterns of nonluminal cells in pleomorphic adenomas, including plasmacytoid cells, are reflected in myoepitheliomas. Results also indicate that muscle-specific actin and myofilaments are expressed only in a proportion of cases, and generally in not more than 60-70% of nonluminal cells in pleomorphic adenoma; this also applies to benign and malignant myoepitheliomas. The absence of these markers does not exclude a diagnosis of myoepithelioma. Vimentin and glial acidic fibrillary protein, however, are strongly and diffusely expressed in the majority of pleomorphic adenomas and myoepitheliomas and are more reliable markers for these tumors than muscle-specific actin. Like so many other salivary gland tumors, myoepitheliomas present an equally complex histomorphology and variable expression of antigenic markers, only some of which are associated with myoepithelial and basal cells of the acini and ducts of the normal salivary gland.     

Immunophenotypical profiles of salivary gland tumours: a new evidence for their histogenetic origin

The histogenetic origin of salivary gland tumours is not clear. In normal tissues smooth muscle actin (SMA) is expressed in myoepithelial cells, CK14 immunoreactivity is seen in myoepithelial and basal cells and CK10 in keratinized squamous epithelium. In this study, we examine the immunophenotypic properties of salivary gland tumours in order to obtain further insight into their histogenesis. 30 cases of salivary gland tumours (18 pleomorphic adenomas, 8 Warthin's tumours, 2 basal cell adenomas, 2 acinic cell carcinomas) were included in our study. Cytokeratin (CK) 10, CKI4, CKI7, CK18, CK 19, and smooth muscle actin (SMA) immunostains were applied to the sections. Immunoreactivities were detected and the statistical significance was evaluated by chi square test. SMA was not detected in Warthin's tumour (p < 0.0001). CK14 was found in all tumours except acinic cell carcinomas (p < 0.0001). CK10 immunoreactivity was observed in 5 Warthin's tumour. In conclusion, pleomorphic adenomas and basal cells adenomas originate from stem cells. Immunophenotypic profile of Warthin's tumour is suggestive of an embryological remnant origin.     

Muscle-specific protein expression in normal salivary glands and pleomorphic adenomas: an immunocytochemical study with biochemical confirmation.

Normal salivary gland myoepithelia are contractile cells with hybrid epithelial/myogenic ultrastructural features. It is known that these cells co-express the intermediate filaments cytokeratin, vimentin, and occasionally GFAP. This complex cytoskeletal immunophenotype is also reflected in multiple morphologic cell types of pleomorphic adenoma. At present, the myofilament complement of normal and neoplastic myoepithelium is not well defined. We have evaluated the expression of desmin and smooth and sarcomeric muscle actins in 11 normal salivary glands (six snap-frozen and five methacarn fixed) and 26 pleomorphic adenomas (11 snap-frozen and 15 methacarn fixed) by ABC-immunoperoxidase method. Two of 11 frozen pleomorphic adenomas contained the muscle-specific intermediate filament desmin, which is not found in the normal glands. This novel finding was confirmed by gel electrophoresis and immunoblot. Using specific antibodies, normal gland myoepithelial cells consistently contained muscle actin isoforms of the smooth muscle type but not sarcomeric muscle actins. Muscle actin expression by the neoplastic cells of pleomorphic adenoma was found in 13 of 26 tumors (six of 11 frozen tumors (desmin negative) and seven of 15 methacarn fixed tumors). In comparison to the normal myoepithelial cell, the transformed myoepithelial-like cells of pleomorphic adenoma are not always characterized by a muscle actin cytoskeleton. Expression of desmin intermediate filaments in pleomorphic adenomas appears to be a rare event that is independent of a muscle actin cytoskeleton.     

Oncocytic myoepithelioma and pleomorphic adenoma of the salivary glands

 Twenty oncocytic myoepitheliomas (MEs) and pleomorphic adenomas (PAs) were composed of interlacing fascicles of swollen spindle-shaped or/and epithelioid oncocytic myoepithelial cells showing intense finely granular immunoreactivity with anti-mitochondrial antibody. Focal vacuolation of the cytoplasm of oncocytic myoepithelial cells and their gradual transition into sebaceous metaplasia were observed in 3 cases. Another unusual feature found in 5 cases was the presence of slit-like adenomatoid spaces lined with double-layered oncocytic myoepithelium closely resembling Warthin’s tumour. The nuclei of oncocytic cells were characterized by enlargement, hyperchromasia and polymorphism, which should not be confused with malignancy. Oncocytic change in myoepithelial cells in MEs and PAs can cause pitfalls in the differential diagnosis of salivary gland tumours. We describe some unusual histological features associated with onococytic metaplasia in benign myoepithelial cell-derived salivary gland tumours, hoping to help to avoid the overdiagnosis of malignancy. Received: 24 September 1998 / Accepted: 31 January 1999     

Malignant myoepithelioma of salivary glands: Clinicopathological features of ten cases

Malignant myoepithelioma of the salivary gland is discussed in terms of its clinical behaviour, morphological features and the frequent pre-existence of a pleomorphic adenoma. The study comprised six female and four male patients aged 14–63 years (mean age 38.9 years). Two tumours presented as intraoral lesions and eight were located in the parotid gland. Tumour cells displayed a morphological spectrum ranging from round epithelioid cells to spindle-shaped and stellate cells. Most cells displayed reactivity for high molecular weight keratins and in four tumours there was strong immunoreactivity for smooth muscle actin. Malignant myoepithelioma seems to arise in two different clinical settings: either de novo or in a recurrent pleomorphic adenoma. De novo malignant myoepitheliomas arise in normal salivary gland, tend to be more aggressive and have a short clinical history. Recurrences may not develop or may occur as a single event within a short time interval, and métastases develop in the lungs. Malignant myoepitheliomas arising in recurrent pleomorphic adenomas have a long clinical history, are characterized by multiple recurrences and have to be distinguished from aggressive carcinomas arising in these adenomas. In contrast, the tumours described in the present series arising in pleomorphic adenomas showed local aggressiveness and metastases did not occur until decades after the first treatment. The general opinion that all malignant tumours that arise from pleomorphic adenomas are highly aggressive is not confirmed by the present study.     

Spindle cell myoepithelial tumours of the parotid gland with extensive lipomatous metaplasia

We report four cases of parotid gland tumours composed predominantly of spindle-shaped myoepithelial cells and mature adipocytes. The central portion of one tumour showed extensive adipose differentiation, whereas in the peripheral parts there were small foci of ductal epithelium arranged in cords and tubules within an abundant myxoid stroma. The other cases were adipose spindle cell myoepitheliomas without an obvious glandular component. Under high-power examination, a transition between modified spindle-shaped myoepithelial cells and adipocytes was observed, and this was confirmed with immunohistochemistry. Ultrastructurally, the modified myoepithelial cells showed intracytoplasmic tonofilaments, bundles of actin microfilaments and lipid droplets. A possible pathogenesis is proposed of true metaplastic transformation of myoepithelial cells to adipocytes. This lesion is important to identify correctly, as inadequate surgery can lead to recurrence.     

Basal cell hyperplasia, adenoid basal cell tumor, and adenoid cystic carcinoma of the prostate gland: An immunohistochemical study

Basal cell hyperplasia (BCH) is an uncommon proliferative lesion of the prostate gland. We studied ten cases of BCH, one case of an unusual adenoid basal cell tumor (ABT), and one case of a prostatic adenoid cystic carcinoma (ACC), using a panel of antibodies to define the histogenesis of these lesions. Monoclonal antibodies (MoAb) directed against a cytokeratin, which selectively stains basal cells (34 beta E12), and against muscle-specific actin, which stains myoepithelial cells (HHF35), were used. In addition, antibodies directed against prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), S-100 protein, and vimentin were used. In the normal prostate, epithelial cells reacted positively with 34 beta E12, PAP, and PSA, and negatively with the actin, S-100 protein, and vimentin antibodies. In BCH, positive staining was seen for 34 beta E12, PSA, and PAP, with no reactivity for actin, S-100 protein, and vimentin. In ABT and ACC, positive reactivity was demonstrated for all antibodies except actin and vimentin. These findings indicate that the basaloid cells of BCH, ABT, and ACC are derived from basal cells of the normal prostate gland and suggest a continuum among the three lesions. The presence of S-100 protein in ABT and ACC may be related to the lack of this antigen's specificity for myoepithelial cells. The absence of reactivity with the HHF35 MoAb supports our belief that the S-100 positivity does not necessarily indicate myoepithelial cell differentiation.     

Immunocytochemical identification of cell types in pleomorphic adenoma, with particular reference to myoepithelial cells

An immunocytochemical study was carried out on normal salivary gland tissue and ten salivary gland pleomorphic adenomas. Antibodies to myosin were used to stain myoepithelial cells. Duct cells were stained using an antibody to total keratin and a subpopulation of basal duct cells with an antibody to 45/46K keratins. Basement membranes were stained with anti-type IV collagen. The results demonstrated that myoepithelial cells are relatively rare in the majority of pleomorphic adenomas and that many of the cells which have been classically described as myoepithelial in routine histological preparations do not clearly show this type of differentiation. However, the tumors presented a spectrum of differentiation patterns from those that were mainly ductal to the rare tumour which was largely myoepithelial. It is further suggested that the 45/46K keratin antibody is capable of identifying a subpopulation of cells which could possibly be important in the histogenesis of this tumour.     

Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells.

The distribution of immunostaining in normal major salivary gland and in 12 pleomorphic adenomas was studied using monospecific monoclonal antibodies to a number of cytokeratins, including cytokeratin 14, to smooth muscle actin and vimentin. A number of these antibodies enabled a distinction to be made between structural components of the normal gland, and to relate this to the different structures of pleomorphic adenomas. In the normal gland, the luminal duct cells expressed cytokeratins 7, 8, 18 and 19. Three antibodies were of particular value for the characterization of normal myoepithelial and basal cells; while the antibody to smooth muscle actin and the cytokeratin antibody Ks8.12 mutually exclusively stained the myoepithelial (basket) cells and the basal duct (light) cells, respectively, the recently established monospecific antibodies to cytokeratin 14 showed specific immunostaining with both cell types. These three antibodies left luminal cells virtually unstained. Ck 13 was found occasionally in single luminal excretory duct cells. Antibodies to cytokeratins 1/2, 10 and 10/11 did not show any staining in the normal gland. In the pleomorphic adenomas, the staining pattern of the two-layered tubular formation resembled that of the normal gland ducts: tumour luminal cells showed the characteristic, although more irregular, expression of cytokeratins 7, 8, 18 and 19; the outer cells resembled normal ductal basal cells with their anti-cytokeratin 14/Ks8.12-epitope staining and in that they virtually lacked staining for smooth muscle actin. Trabecular formations and cells in myxoid areas were reactive with Ks8.12 and for cytokeratin 14, occasionally also for cytokeratins 7, 18 and 19. Epidermoid cell islets expressed mainly cytokeratin 14 and inconsistently the squamous epithelial cytokeratin 13 and the epidermal cytokeratin 10/11. Vimentin was found in cells of myxoid areas. The results support the postulate that some of the normal duct basal cells act as reserve cells and can give rise to tumour formation with a primitive myxoid or trabecular pattern and a more differentiated tubular or epidermoid configuration.     

The pleomorphic adenoma of salivary glands transplanted on athmymic mice

Summary 10 pleomorphic adenomas of the human parotid gland were transplanted on several groups of nude mice. For comparative reasons, 10 other pleomorphic adenomas, a neurinoma and a chordoma and transplants of squamous cell carcinomas and of normal salivary gland tissue were also analysed.In the primary tumours and in the transplants, the presence of keratin, carcinoembryonic antigen, tissue polypeptide antigen, lactoferrin, lysozyme, immunoglobulins, secretory component, amylase, fibronectin and of several lectin-receptors (PNA, WGA, HPA, Ulex europaeus) was sought.The immunohistological observations show that many of the features of a pleomorphic adenoma are constant under the conditions of transplantation. In the transplanted tumour, the same heterogeneity as in the primary tumours can be observed. Autoradiographic studies show little labelling with 3-H thymidine, which is in good accordance with the biological behaviour of the tumour.The distribution of fibronectin shows an interesting association with myoepithelial-like cells.Our results support the hypothesis that the histogenetic origin of the pleomorphic adenoma is a cell pool of the terminal ductal segment. A differentiation towards ductal cells (with production of secretory substances) and towards myoepithelial cells (associated with large amounts of basal membrane like substances) is observed.Supported by the Deutsche Forschungsgemeinschaft and by the Hamburger Stiftung zur Förderung der Krebsbekämpfung     

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

Summary We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and -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 -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--smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for -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.     

Inmumogold Localization of Actin and Cytokeratin Filaments in Myoepithelium

Myoepithelial cells of salivary gland are uniquely specialized cells; their function is unclear, but the considerable complement of muscle-specific actin suggests contractility is one function. By routine transmission electron microscopy myofilament visualization is variable. Some myoepithelial cells appear to have limited and only focal aggregates of myofilaments, while others seem to have readily appreciated myofilaments within a longitudinally oriented cytoplasmic zone at the basal portion of the cell. However, immunogold electron microscopy using the anti-muscle-specific actin antibody, HHF35, while indicating a basal distribution for the muscle-isoform of actin in a platelike fashion in certain myoepithelial cells, also reveals that others associated with both intercalated ducts and acini have a more generalized distribution of myofilaments throughout the cytoplasm. Actin was also noted within tonofilaments and double immunogold labeling using both the HHF35 and AE1/AE3 (anticytokeratins) antibodies confirmed the variable interrelationship of these two filaments. Within any one myoepithelial cell, actin and cytokeratins might colocalize in some areas of the cytoplasm containing filaments, but not in adjacent zones. These results suggest that intermediate filaments and myofilaments are complexly organized in myoepithelial cells, and that quantitative and qualitative differences exist in the expression and distribution of intermediate filaments and myofilaments. These cells are likely structurally, if not functionally, heterogeneous of Human Parotid Salivary Gland     

Inmumogold Localization of Actin and Cytokeratin Filaments in Myoepithelium

Myoepithelial cells of salivary gland are uniquely specialized cells; their function is unclear, but the considerable complement of muscle-specific actin suggests contractility is one function. By routine transmission electron microscopy myofilament visualization is variable. Some myoepithelial cells appear to have limited and only focal aggregates of myofilaments, while others seem to have readily appreciated myofilaments within a longitudinally oriented cytoplasmic zone at the basal portion of the cell. However, immunogold electron microscopy using the anti-muscle-specific actin antibody, HHF35, while indicating a basal distribution for the muscle-isoform of actin in a platelike fashion in certain myoepithelial cells, also reveals that others associated with both intercalated ducts and acini have a more generalized distribution of myofilaments throughout the cytoplasm. Actin was also noted within tonofilaments and double immunogold labeling using both the HHF35 and AE1/AE3 (anticytokeratins) antibodies confirmed the variable interrelationship of these two filaments. Within any one myoepithelial cell, actin and cytokeratins might colocalize in some areas of the cytoplasm containing filaments, but not in adjacent zones. These results suggest that intermediate filaments and myofilaments are complexly organized in myoepithelial cells, and that quantitative and qualitative differences exist in the expression and distribution of intermediate filaments and myofilaments. These cells are likely structurally, if not functionally, heterogeneous of Human Parotid Salivary Gland     

CD10 and HHF35 actin in the differential diagnosis between Collagenous spherulosis and adenoid-cystic carcinoma of the breast

Collagenous Spherulosis (CS) and Adenoid-Cystic Carcinoma (AdCC) of the breast consist of cribriform proliferations of epithelial and myoepithelial cells with an immunophenotypic overlap of some myoepithelial markers, such as p63 and smooth muscle actin (SMA). To our knowledge, CD10 and HHF35 actin have not been assessed in the differential diagnosis of these two breast lesions. We performed an immunohistochemical study on 6 cases of CS and 9 cases of AdCC. We found CD10, muscle-specific actin (HHF35), Estrogen and Progesterone receptors (ER and PR) to be strongly expressed in CS, but not in AdCC; C-kit was diffusely positive in AdCC and scanty in CS; SMA, p63 and Cytokeratine 5/6 (CK5/6) were positive in both. Our results also confirm that AdCC could be true basal-like neoplasia, probably arising from a basal stem line tending to divergent differentiation toward CK5/6/C-kit+, ER/PR-, epithelial basal-like cell type, and toward a myoepitelial-like cell type, with an incomplete SMA/p63+, CD10/HHF35- immunophenotype. By contrast, CS is a reactive, benign proliferation of two well-differentiated cell types: epithelial (ER/PR+, C-kit-) and myoepithelial cells with a complete immunophenotype including CD10/HHF35 positivity. Our study highlights the usefulness of CD10 and HHF35 in the differential diagnosis and helps to understand the histogenesis of the two lesions.     

Myoepithelial carcinoma (malignant myoepithelioma) of the parotid gland arising in a pleomorphic adenoma.

A myoepithelial carcinoma, a rare malignant salivary gland neoplasm, arose in a pleomorphic adenoma of the parotid gland. The initial tumour was a pleomorphic adenoma with epithelial and myoepithelial elements. Subsequently the tumour recurred twice and was characterised by invasion of the mandible. Histological examination of the second recurrence showed a malignant spindle cell neoplasm with an infiltrative growth pattern and a high mitotic rate. There was involvement of local lymph nodes. The immunophenotype was characteristic of myoepithelial differentiation: tumour cells stained positively with anticytokeratin antibodies, S-100 protein, alpha smooth muscle actin, and vimentin. Electron microscopy confirmed myoepithelial differentiation, with small foci of keratinocytic phenotype. Large numbers of tumour cell nuclei were reactive with the anti-p53 antibody, DO-7, in contrast to the two previous resections. Thus malignant transformation of a pleomorphic adenoma may involve myoepithelial as well as epithelial elements. Accumulation of p53 protein, perhaps through mutational events, may have played a role in this malignant transformation.