APMIS 97: 49-55. 1989

Myoeptihelial cells in salivary gland neoplasms

HANS GUSTAFSSON, FRANK BERGMAN. ISM0 VlRTANEN and LARS-ERIC THORNELL

The Departments of Otorhinolaryngology - Head & Neck Surgery, Pathology and Anatomy, the University of Umea. Umea. Sweden. and the Department of Pathology, Helsinki, Finland

Gustafsson, H.. Bergman, F.. Virtanen, 1. & Thornell, L.-E. Myoepithelial cells in salivary gland neoplasms. APMIS 97: 49-55, 1989.

Archival paratfin sections from normal salivary gland tissue and salivary gland neoplasms were stained by immunoperoxidase tehcnique with a well characterized cytokeratin antibody (PKKI). In normal parotid tissue, myoepithelial cells and peripheral cells of larger ducts were selectively stained. In pleomorhic adenomas, most cells were stained, the staining being somewhat stronger towards the duct lumina. In basal cell adenomas, only cells adjacent to the duct lumina were stained where a differentiation of cells into peripheral and ductal was seen. In adenolymphomas basal cells were stained, and in oncocytornas small elongated cells reacted with the PKKI antibody. Only a few duct cells in an acinic cell1 carcinoma were reactive and in mucoepidermoid carcinoma, peripheral epidermoid cells were strongly stained. In adenoid cystic carcinoma, mostly duct cells were stained whereas the peripheral ones remained unstained. Although the intermediate filament protein expression is very stable during tumourgenesis, the staining with the presently used monoclonal antibody in salivary gland neoplasms differed markedly from what could be expected according to current views on the participation of this cell type. This supports our view that cells in tumours should be characterized on the basis of theirstaining, i.e. state of differentiation and not on their presumed histogenesis.

Key words: Salivary gland neoplasms: immunohistochemistry; myoepithelial cells: cytokeratins.

Hans Gustafsson, Department of Otorhinolaryngology - Head & Neck Surgery, University of Umeh, S-901 85 Umea. Sweden.

Morphological studies of salivary gland neoplasms are almost always concerned with the participation of myoepithelial cells. Numerous techniques have been employed to visualize this cell type in normal and neoplastic salivary tissue. By light and electron microscopy, peripheral cells of ducts, spindle shaped cells, hyaline cells, clear cells, glycogen rich cells, mesenchymal like cells have been denoted “myoepithelial”. By immunohistochemistry cy- tokeratins (Caselifz ef al. 198 I), actin, myosin (Caselilz et al. 1981. Gustafsson et al. 1985a, Palmer 1986) and S- 100 (Hara et af. 1983), and by enzyme histochemistry, ATP-ase (Garret & Har- rison 1970) have been detected in myoepithelial cells of normal salivary tissue.

Received May 25. 1988. Accepted September 2, 1988.

Since intermediate filament protein expression is generally found to be very stable during tumour transformation, these proteins have been used extensively in histogenical studies. Recently, we found that a commonly used monoclonal anti- body against cytokeratins, (PKK I ) , selectively stained myoepithelial cells and basal cells of larger ducts in trypsin-digested parafin sections of nor- mal gland (Gus~ufison et al. I988a). This antibody detects CK 8, I8 and 19 on immunoblots and on frozen sections it stains all epithelial cells in sali- vary glands (Gustajisan el al. 1988b).

In the present study we have examined a num- ber of benign and malignant salivary neoplasms to determine whether the PKK I staining characteris- tics could be related to the common theories of myoepithelial cell participation in the tumourgen- esis of salivary neoplasms.

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TABLE 1 . Reuctivilv qf Differenl Neoplasms lo PKKl ufier Trvpsin Digestionjor One Hour Tumour type reactive cell type

pleomorphic adenoma oncocytoma small elongated oncocytic cells adenolymphoma basal cell adenoma acinic cell carcinoma mucoepidermoid carcinoma adenoidcystic carcinoma

cells in epithelial areas, a few cells in mesenchymal areas

basal cells (all cells in one tumour) central cells in ducts (all cells in monomorphic variants) negative (a few “intercalated duct” cells in one tumour) epidennoid areas, often positioned peripherally scattered cells more often towards duct structures

MATERIALS AND METHODS

Murerials From the files of the Department of Pathology at the

University of Umea, paraffin blocks from the major groups of salivary gland neoplasms were obtained. All sections were reexamined, and only cases where the diagnosis was confirmed were further processed.

Met / I 0d.s Paraffin sections were after deparafination, exposed

to . I % trypsin (Sigma Chemical Corp.) dissolved in . I % Calcium cloride at pH 7.8 for 1 hours.

Antibody PKKl used in the present study has been characterized earlier against simple epithelia (e.g. Ho1lhQfl.r el ul. 1983, 1984, Virianen el ul. 1985, 1986). In terms of the current numbering system for CKs (Moll et ul. 1982). PKKl reacts with CKs of Mr 40,000 (No. 19), Mr 45,000 (No. 18) and Mr 52,000 (No. 8).

This primary antibody PKK I ws used at a concentra- tion of 20-50 mg per litre at room temperature. After thorough washing, the sections were exposed to sec- ondary antibodies (rabbit anti-mouse immunoglobu- lins; Dakopatts, Glostrup, Denmark) and after further washing exposed to a peroxidase-antiperoxidase (PAP) complex (PAP - mouse - monoclonal; Dakopatts). This method was performed as previously decribed (e.g. Sternhcrgcr d ul. 1910, Bourne 1983). The sections were mounted in Mowiol 4-88 (Hoechst, Frankfurt, West Germany), and viewed in a Zeiss microscope. For control sections were prepared as above but omitting the PKKl antibody.

RESULTS

Normal Salivary Gland (5 cases) In sections digested with trypsin for one hour

and then stained with PKKl, a strong staining was seen in myoepithelial cells and basal cells of larger ducts (Fig. 1). In all sections, acinar cells and other duct cells displayed just a very faint or no staining at all.

Salivary Neoplasms BENIGN TUMOURS Pleomorphic adenoma. ( 5 cases). Most tumour cells were stained. The staining was often some- what stronger towards the lumina of the ducts than in the periphery of the ducts and in the mesenchy- ma1 areas (Fig. 2).

Monomorphic adnoma. Warthin ‘.r tumour. (3 cases). In two tumours, the apical epithelial cell layer was unstained whereas the basal cell layer was strongly stained. In one tumour, a moderate to strong staining was seen in all cells of the two-lay- ered epithelium (Fig. 3).

Oncocytoma. (3 cases). The typical larger sized oncocytic cells were unreactive. However. a strong reaction was seen in the small elongated tumour cells intermingled in the tumour mass (Fig. 4).

Basal cell adenoma. ( 2 cases). The epithelial

Fig 1. Normal parotid gland. Immunoperoxidase staining with PKKl after trypsin digestion. Strong staining is seen in myoepithelial cells and peripheral cells of larger ducts. x 160. Fig. 2. Pleomorphic adenoma. Immunoperoxidase staining with PKKl after trypsin digestion. Strong staining is seen in duct cells, and most other cells show a moderate to strong staining. x 160. Fig. 3. Adenolymphoma. Immunoperoxidase staining with PKK 1 after trypsin digestion. Basal cells are strongly stained, and the apical cylindrical cells are weakly stained. x 160. Fig 4. Oncocytoma. Immunoperoxidase staining with PKKl after trypsin digestion. Most cells are unstained, but a population of small elongated cells is intensely stained. x 160.

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GUSTAFSSON el al.

cells were moderately to strongly stained. In areas with a differentiation of ducts into luminal and peripheral layers, the central cells had a more intense staining (Fig. 5) .

MALIGNANT TUMOURS Acinic cell carcinoma ( 5 cases). The tumour cells were generally unstained. In one neoplasm some duct-like cells were, however, moderately stained (Fig. 6).

Mucoepidermoid carcinoma ( 5 cases). The epi- dermoid cells, most often located peripherally in cords, showed an intense staining whereas the glandular areas which are often more centrally located in tumour cords, were unreactive (Fig. 7).

Adenoid cystic carcinoma ( 5 cases). A clear separation between “central” cells extending to- wards real duct lumina and ‘‘peripheral’’ cells towards stroma or pseudocysts was seen in many areas of all tumours. In most cases the “central” cells were heavily stained and peripheral ones unstained, but the reverse reactivity pattern was also seen. Furthermore, sometimes the patterns varied within the same tumour. In some areas the wide majority of cells were unstained and only scattered cells stained irrespective of their position in the tumour (Fig. 8).

DISCUSSION

With the present technique, an intense staining of myoepithelial cells and basal cells of larger ducts was seen in normal parotid gland tissue. Since the CK-expression is very stable during cell transfor- mation (Moll et al. 1983) PKKl would thus be an ideal marker for “myoepithelial cells” in tumour tissue. However, the present findings of PKKl staining in salivary neoplasms violate current

opinions of the presence of myoepithelial cells in salivary neoplasms in a number of ways.

Most reports on mucoepidermoid carcinomas using both light and electron microscopy, deny the existence of myoepithelial cells (Mylius 1960; Kleinsasser 1967, Hamper1 1970, Hzjbner et al. 197 1, Batsakis 1985). Biengraher (1 978) and Dardick ef al. ( 1984) have, however, described myoepithelial cells in these tumours. This is in a wide contrast to our findings of a strong reactivity in the epidermoid parts of the tumours.

Similarly surprising were our findings in ade- noid cystic carcinoma. In current theories (Tan- dfer 197 1) these tumours are made up of myoep- ithelial cells towards the stroma, and pseudocysts and ductlike cells towards the real duct lumina. We found, however, immunoreactivity only in isolat- ed groups of cells and they were more often positioned towards the ducts than towards the stroma. The basal cell adenoma showed the same unexpected pattern, with an intense staining to- wards the ductal lumen. Also in the pleomorphic adenoma, this pattern of staining was sometimes seen. Current theories (Batsakis 1983, Batsakis et al. 1986, Erlandson et al. 1983, Dardick et al. 1982, 1983a. 1983b) consider the peripheral cells as myoepithelial and the cells in the stromal regions as modified myoepithelial cells.

In oncocytomas PKKl -reactive smaller elon- gated cells could be readily differentiated from the negative larger cells. This difference has also been seen in frozen sections with polyclonal anticytok- eratin antibodies (Gusfafsson ef al. 1985a) and mAb PKK2 (Gustqfison 1986), but not with mAbs PKKl or PKK3 on frozen sections (Gusfqfison 1986). The smaller cell type has been claimed to be myoepithelial on the basis of ultrastructural find- ings of fine filaments (Mylius 1960, Askew el al. 197 1 ). However, this has been questioned because of the lack of immunodetectable actin in this cell

Fig. 5. Basal cell adenoma. Immunoperoxidase staining with PKKl after trypsin digestion. In areas where a differentiation into central and peripheral cells is seen, the former cell type is strongly stained, and the latter is almost unstained. x 160. Fig. 6. Acinic cell carcinoma. Immunoperoxidase staining with PKKl after trypsin digestion. In one tumour some ductlike cells were moderately stained. x 160. Fig. 7. Mucoepidermoid carcinoma. Immunoperoxidase staining with PKKl after trypsin digestion. Epidermoid cells, mostly in the periphery, were strongly stained. x 160. Fig. 8. Adenoid cystic carcinoa. Immunoperoxidase staining with PKKl after trypsin digestion. In most areas, cells adjacent to duct structures were strongly stained wheres the peripheral cells were almost unstained. x 160.

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MYOEPITHELIAL CELLS IN SALIVARY GLAND NEOPLASMS

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type (Gustafsson et al. 1985a). Similarily in Warthin’s tumours, two types of

epithelial cells were found: Basal cells stained by PKKl and unstained apical cylindrical cells. In this respect, the tumour cysts have similarities with normal excretory ducts. The basal cells have ultra- structural similarities with myoepithelial cells (Hubner et al. 1971, Gustafsson et al. 1985). Furthermore, in the basal cells as in myoepithelial cells, ATPase can be demonstrated at the cell borders (Cutler et al. 1977). However, actin, which is typically found in myoepithelial cells, could not be detected in these basal cells (Gustafsson et al. 1985a).

In the acinic cell carcinomas the staining pattern was more or less as could be expected. Although Chaudhry et al. (1986) have found myoepithelial cell and ductal reserve cells in this type of tumour, most studies have only described ductal and acinar cells (Erlandsson & Tandler 1972, Gustafsson et al. 1985a, Batsakis 1979). The general lack of stain- ing in the neoplasms that we observed, would fit well with the findings of these latter studies, except for the staining of the ductal cells in one tumour.

The other type of cell besides the myoepithelial cell that was stained by PKKl in normal parotid gland, was a peripheral cell of larger ducts. Im- munological similarity between myoepithelial cells and this cell type has been found with other mAbs as vim 24 (Gustafsson et al. 1988a). Other mAbs, however, stain this cell type selectively (Palmer el al. 1985, 1987) and also some tumour cells in pleomorphic adenomas. The identity of these normal and neoplastic cells should, however, be interpretated carefully in view of the above discussion.

In conclusion, the present study showed that a monoclonal antibody can be used to distinguish different types of malignant salivary neoplasms: Acinic cell carcinomas were unstained, adenoid cystic carcinomas were mainly stained towards the duct lumina and in mucoepidermoid carcinomas staining was peripheral. Furthermore, the pattern of reactivity was similar in adenoid cystic car- cinomas, basal cell adenomas, and pleomorphic adenomas, showing a close relationship between these types of neoplasms.

The present study also illustrates the difficulties in using monoclonal antibodies to distinguish special types of cells in tumours. Although the cytokeratin expression is very stable during malig- nant cell transformation, and normal myoepithe-

lial cells show reactivity to the monoclonal anti- body PKK 1 in parafin sections, the staining with this monoclonal antibody in salivary gland neo- plasms differed markedly from what could be expected according to current views on the partic- ipation of this cell type. This supports our view that cells in tumours should be typed on their staining characteristics, and, thus, by their state of differentiation, and not on their presumed histoge- nesis.

~

Supported by grants from the Lion Fund (project 4891 87), Department of Oncology, University of UmeA. Umea. Sweden, the Swedish Medical Research Council (12X - 3934) and the UmeA University Faculty of Medicine.

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