Ultrastructural and immunohistochemical aspects of carcinoma in mixed tumors

Am J Otolaryn~;ol 7:218-230, 198fi

Ultrastructural and Immunohistochemical Aspects of Carcinoma in Mixed Tumors HANS GUSTAFSSON, M.D., BENGTC~LsQQ, M.D., PH.D., UNO KI6RELL,D.D.S., PH.D., AND LARS-ERICTHORNELL,M.D., PH.D. Four cases of malignant mixed tumor (carcinoma in pleomorphic adenomas) were studied for ultrastructural appearance and for the presence of cytokeratins and vimentin. Ultrastructurally, both squamous and glandular epithelial differentiation were found not only in the same tumor but also within the same cell. One tumor showed mainly mesenchymal differentiation with fibroblast-like cells. The intermediate filament expression of benign mixed tumors (i.e., both cytokeratin and vimentin content) were found in two of the three malignant tumors investigated. In the third tumor, only cytokeratins were found. Thus, the filament content of mixed tumors may change when the tumor becomes malignant. This change does not always parallel a change in morphology. Although one tumor was clearly epithelial ultrastructurally and another mostly mesenchymal, both did contain both cytokeratins and vimentin.

The term mixed tumor was coined in 1874 by Minssen, 1 but later, as the epithelial origin of these neoplasms was emphasized, they were more commonly designated as pleomorphic adenomas. Different classifications into benign, semi-malignant, and malignant types have previously been applied. 2,3 Later, a differentiation into a benign and a malignant variety, the latter often arising from a benign mixed tumor, has generally been acceptedA s The benign mixed tumor, or pleomorphic adenoma, is microscopically characterized by its pleomorphic or "mixed" appearance of clearly recognizable epithelial tissue intermingled with tissue of mucoid, myxoid, or chondroid appearance. 6 Because the tumors show great variation in morphology, it is understandable that their histogenesis still is a matter of controversy. A large number of theories have been proposed Received July 19, 1985, from the Department of Otolaryngology-Head and Neck Surgery, the Department of Histology and Cell Biology,and the Department of Anatomy, University of Ume~i.Ume~. Sweden. Accepted for publication December4, 1985. This study was supported by grants from the Lion Research Fund (329-84), the Department of Oncology, the M~ngberg Fund of University of Ume~, and the Swedish Medical ResearchCouncil (12X-3934). Address reprint requests to Dr. Gustafsson:Departmentof Otorhinolaryngologyand Head and Neck Surgery, University of Ume~,S-901 85 Ume~,Sweden.

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during the years, 3 but present theories favor a purely epithelial progenitor, either myoepithelial cells 7 or undifferentiated pluripatential d u c t reserve cellsA 9 The malignant mixed tumor has been d e f i n e d by Foote and Frazell 4 as a tumor having t h e structural qualities f o u n d in o r d i n a r y m i x e d tumors, and also "areas of different structural makeup that by experience have been shown t o be associated with the ability to metastasize." The appearance of such tumors varies considerably, but the epithelial character of the malignant areas of the tumors, including the metastases, has been pointed out by several authors, and the term "carcinoma in pleomorphic a d e homo" has been proposed. 1°-12 In light microscopic studies of the m a l i g n a n t parts and metastatic tissue of these t u m o r s , mostly adenocarcinomatous differentiation6,1215 but also squamous differentiation has been reported. 6'12'14 However, other forms of s a l i v a r y gland epithelial malignancies have been reported. Moberger and Eneroth, 1° for e x a m p l e , found adenoidcystic carcinomas and m u c o e p i dermoid carcinomas, and Tortoledo et al. 16 described ductal carcinomas and terminal d u c t carcinomas. Still other designations that h a v e been applied to these tumors are myoepithelial carcinomas, 15'16 anaplastic carcinomas, lo,16 a n d

GUSTAFSSON ET AL.

"true" malignant mixed tumors,~Sn 7 the latter displaying both epithelial and mesenchymal parts in the metastases. However, although Boles et al. 12 could define six different cell types in the malignant parts, no cell type was found consistently more associated with metastasis. The present study investigated whether the malignant part of a carcinoma in mixed tumors expresses both glandular and squamous features within the same tumor, and whether the malignant cells display any mesenchymal differentiation. Furthermore, immunohistochemical identification of intermediate filaments has proved to be a reliable method for tissue typing and as a histogenical marker. This method was used, in addition to electron microscopy, to identify the tumors' cells of origin by their filament patterns. MATERIALS AND METHODS

Four cases of malignant mixed tumors were studied. Specimens for immunohistochemistry (cases 2 - 4 ) and electron microscopy (cases 1-4) were obtained at surgery. Specimens from three normal parotid glands and two benign pleomorphic adenomas were included for comparative immunohistochemical analysis.

Immunohistochemistry Small pieces of tissue were rapidly frozen in liquid isopentane, precooled in liquid nitrogen. Guinea pig and rabbit antibodies against the keratins were prepared according to KjSrell and Ostberg. 18 Monoclonal antibodies against vim e n t i n were p u r c h a s e d from Sanbio Life Products, Nistelrode, Holland. Indirect immunefluorescence m i c r o s c o p y of 5-1xm-thick, acetone-fixed or unfixed cryostat sections was performed as earlier d e s c r i b e d , is In addition, tetramethyl-rhodamine isothiocyanate (TRITC)conjugated secondary antibodies were used in this study.

Electron Microscopy Minute specimens from four cases of malignant mixed tumors were taken at operation and immediately fixed in 2.5 or 4 per cent glutaraldehyde in phosphate buffer overnight, rinsed in buffer, postfixed in osmium tetroxide for 1 hour, dehydrated in graded ethanol solutions followed by xylene, and embedded in epoxy resin. Thin sections were cut on an Ultratome, collected on copper grids, stained with uranyl a c e -

tate and lead citrate, and examined in an electron microscope.

Clinical Data Case 1. A woman born in 1937 had surgery for a pleomorphic adenama in the right parotid gland in 1971. A local recurrence was extirpated in 1973. Microscopic evaluation did not reveal any signs of malignancy, b u t the tumor was considered locally malignant, and radiotherapy was given. Later, new recurrences appeared, and a total facial paralysis was diagnosed. Despite extensive surgical intervention, the patient expired of her disease because of massive local rec u r r e n c e in 1983. Case 2. A 70-year-old w o m a n h a d been treated during adolescence for scraphula by rad i o t h e r a p y to the neck. She developed a left parotid tumor and underwent superficial parotidectomy. Fine needle aspiration had shown med i u m - g r a d e c a r c i n o m a . She h a d m u l t i p l e recurrences in the scar tissue, and although extended operations were performed, new local and cervical node recurrences had appeared. Case 3. A man born i n 1906 had surgery in 1938 for a tumor of the right parotid gland. He developed a slowly growing recurrence, and in 1981 the tumor was 5 x 5 cm, but the patient refused operation. During the spring of 1984, the tumor started growing rapidly, and the patient developed a facial palsy. Fine needle aspiration indicated a poorly differentiated adenocarcihomo. After preoperative radiation of 65 Gy, a wide resection of the parotid region was performed. Case 4. An 85-year-old woman was observed for a lump in the right parotid from which a fine needle aspiration biopsy had indicated pleomorphic adenoma. After 2 years, the tumor started growing faster, and repeated aspiration yielded cytoplasmic-rich cells with large nuclei and marked atypia. On suspicion of malignant mixed tumor, the woman underwent parotidectomy. RESULTS

Normal Parotid Gland Immunofluorescenee microscopy. The two a n t i - c y t o k e r a t i n p r e p a r a t i o n s gave s i m i l a r staining of all tissue examined. Therefore, they will not be discussed individually. For cytokeratins a strong fluorescence was found in all duct and myoepithelial tells. The duct cells were

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:e ~ Figure 1. Normal parotid gland (a, b, and c} and pieomorphic adonoma (d, e, and/~. Illustrated are hematoxylin-eosin {a, and dl and immunofluorescent staining with antibodies to cytokeratin (b and e) and vimentin (c and f). In normal glandular tissue, anti-cytokeratins are found in all epithelial cells (b). Acinar cell staining is indicated [arrowheads), whereas vimantin is found in stromal cells and vessels. In pleomorphic adenoma, most cells are positive for both eytokeratin (e) and vimentin (f) ( × 140 [a and d]; x 300 [b, c, e, a n d f]}.

mainly stained at the apical and basal cell surfaces, w h i l e the c y t o p l a s m of myoepithelial cells was h o m o g e n e o u s l y reactive. Acinar ceils disp l a y e d o n l y faint s t a i n i n g at t h e apical cell borders a n d along the intercellular canaliculi. With anti-vimentin, stromal and vascular tissue was stained, but all myoepithelial and other epit h e l i a l c e i l s w e r e u n s t a i n e d . A f e w small, r o u n d e d v i m e n t i n - p o s i t i v e cells w e r e sometimes f o u n d within the striated ducts, but they were regarded as l y m p h o c y t e s (Fig. 1, b and c}. American Journal

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Benign Pleomorphic Adenomas

Immunofluorescence microscopy. M o s t tumor cells displayed a very distinct staining for

both cytokeratin and vimentin. A f e w cells, mainly at the duct lumen, were vimentin-negative but cytokeratin-positive, while the opposite pattern was never found (Fig. 1, e and f).

Case 1 General morphology. All specimens showed a similar picture with a reticulum of epithelial cords with sites of moderate cellular and nuclear polymorphism in a fibrous stroma. Often, regressive changes were observed. A f e w mitotic cells were seen. Electron microscopy. No c l e a r l y d e f i n e d acinar formations were seen, but small intercellular canaliculi with cellular interdigitations

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Figure 2. Case 1. Above, tumor cells displaying tonofilament bundles and desmosomes as well as atypical secretory granules with multiple dense cores. (× 10,000). Right, light micrograph from primary recurrence showing areas of densely packed epithelial cells, ducts, or cysts and myxoid regions (van Cieson's stain, x 140],

a n d microvilli w e r e often e n c o u n t e r e d . Desmos o m e s were a b u n d a n t . In a large n u m b e r of cells, t h e r o u g h e n d o p l a s m i c r e t i c u l u m (RER) cisternae w e r e s w o l l e n and a p p e a r e d as secretory granules lined with a few ribosomes. These " g r a n u l e s " often c o n t a i n e d a finely granular material a n d focal densities a p p e a r i n g either as a central dense core or m u l t i p l e m i n o r densities (Figs. 2 and 3). S o m e l i p i d droplets and lyso-

s o m e s c o u l d also be f o u n d , T y p i c a l t o n o f i l a m e n t s w e r e often f o u n d as d e n s e b u n d l e s of parallel fibers (Fig, 3). No a r e a s w i t h m y o f i l a m e n t s were seen.

Case 2 G e n e r a l morphology. T h e t u m o r c o n s i s t e d of cords of epithelial aggregations, f i b r o u s a n d al-

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Figure 3. Case1. High magnification view of secretory granules and tonofilament bundles (x 3 5,000).

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most hyalinic tissue, and cartilage. The epithelial parts showed moderate to strong polymorphism and many cells in mitosis. A squamous differentiation was sometimes suspected. Areas of calcification and hemorrhage were seen, as well as signs of infiltration. In specimens obtained later, mesenchyme-like spindle cells predominated (Fig. 4a). lmmunofluorescence microscopy. Staining for both the cytokeratin and vimentin antibodies (Fig. 4, b and c) was found within the cytoplasm of these cells. Electron microscopy. In a collagen-rich stroma, pleomorphic dark cells, often in close apposition, were seen. The large nuclei often contained two or more nucleoli and sometimes exhibited protrusions or segmentations. No acinar or tubular aggregations were found. Microvillous protrusions could sometimes be seen directed toward the collagen-rich stroma, A well-developed RER, sometimes with dilated tubules containing a finely granular material, was found. A few secretory granules with a homogeneous material inside were observed. No desmosomes were found, although the cells were in

close apposition (Fig. 5). F i l a m e n t s , probably of the intermediate filament type, w e r e a b u n d a n t (Fig. 6). In contrast to case 1, no b u n d l e s of tonofilaments appeared, but areas o f parallel filaments giving large parts of the c y t o p l a s m a dark "hyaline" appearance with s c a t t e r e d mitochondria were seen. In the specimens o b t a i n e d later, the cells appeared more m e s e n c h y m a l and were mostly spindle-shaped with s p a r s e c y t o p l a s m (Fig. 7). Sometimes they could h a r d l y he distinguished from fibroblasts. Case 3

General morphology. A t y p i c a l p l e o m o r p h i c adenoma with signs of regression w a s seen, and at the periphery, a low differentiated tumor w i t h squamous cell differentiation c o u l d be recognized. No clear glandular differentiation w a s observed, b u t P A S - p o s i t i v e m a t e r i a l w a s s e e n in some cells, even after a m y l a s e d i g e s t i o n (Fig. 4d). Immunofluorescence microscopy. S p e c imens with large ceils invading a small n e r v e bunch were studied. Tumor cells w e r e cytoker-

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Figure 4. Hematoxylin-eosin (a, d, and g) and immunofluorescent staining with antibodies to cytakeratin (b, e, and h) and vimentin (c, f, and i) in case 2 (a, b, and c), case 3 (d, e, and f}, and case 4 (g, h, and i). In case 2, virtually all cells were heavily stained with both anti-cytokeratins (b) and anti-vimentin (c). [n case 3 (d, e, and f), malignant cells invading a facial nerve branch (N) are shown. The malignant ceils are cytokeratin-positive (e) but vimentin-negative (f}, whereas stromal ceils show the reversed reactivity. Schwann cells of the nerve are weakly vimentin-positive. In case 4, the basal tumor cells are both cytokeratin-positive (h) and vimentin-positive (i), whereas the apical luminal cells only express cytokeratins. The hyaline stroma is vimentin-positive but cytokeratin-negative (x 140 ([a, d, and g]; × 300 [b, c, e, f, h, and i]).

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The tumor produced a submandibular metastasis six months later. In a fine needle aspirate, the cytology was that of a high-grade trabecular adenocarcinoma. Case 4

General morphology. Epithelial cells with varying degrees of atypia were growing in solid trabecular sheets of follicles in a collagen-rich stroma. At some sites, spinocellular differentiation was identified. No chondroid tissue was observed. In some areas, suspected invasion into the collagen capsule was seen [Fig. 4g). Immunofluorescence microscopy. Tumor cords or cysts of tumor cells in hyaline stroma w e r e s t u d i e d . Basal t u m o r cell layers were clearly positive for both vimentin and cytokeratin, while the apical or central parts of cords were m a i n l y cytokeratin-positive. The stroma w a s d i s t i n c t l y c y t o k e r a t i n - n e g a t i v e b u t vimentin-positive (Fig. 4, h and i). Electron microscopy. All tumor ceils were clearly epithelial and organized in solid sheets or duct-like structures s u r r o u n d e d by a basal Figure 5. Case 2. High magnification of two tumor calls in close apposition. No d e s m o s o m e s can be seen at the cell borders { x 13,500).

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atin-positive b u t v i m e n t i n - n e g a t i v e . Stromal cells were v i m e n t i n - p o s i t i v e b u t cytokeratinnegative (Fig. 4, e and f). Electron microscopy. In the s p e c i m e n s examined, the epithelial ceils were f o u n d in cords or ducts. The cells were in close apposition with a large n u m b e r of desmosomes a n d intercellular interdigitations present (Fig, 8). Most cells exhibited a large n u m b e r of swollen mitochondria and dilated e n d o p l a s m i c reticulum. Vacuoles were seen in most cells. At least some of them r e s e m b l e d secretory granules b e c a u s e of their size and content (Fig. 9). Another cell population was better preserved with less mitocondrial swelling. The sparse RER w a s n o t d i l a t e d . N o v a c u o l e s or s e c r e t o r y granules were seen. More cytoplasmic filaments were f o u n d in this cell t y p e (Fig. 8). Otherwise, the general structure of the cells was similar to those p r e v i o u s l y mentioned. Typical, small duct lumina with a large n u m b e r of microvillous projections were observed (Fig. 9). In the periphery of the ceils and in the microvilli, large amounts of filaments were seen. Remnants of the basal lamina were observed at the basal cell surface.

Figure 6. Case 2. Fibroblast-like tumor celt in mitosis. Filaments and endoplasmic retieulum are seen in large amounts

( x 6,000).

Figure 7. Case 2. The gradual change from round cells with sparse cytoplasm [right) to elongated cells with large amounts of endoplasmic reticulum (middle}. The cells to the left represent fibroblasts (x 5,500}.

Figure 8. Case 3, Agranular ceil with two nuclei, Villous protrusions into intercellular spaces show s:imilarities to acini ( x 9,ooo).

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ysis of tumors is always complicated by the fact that only minute specimens can be studied at a time, sampling error can be minimized by examination of several s p e c i m e n s from the same tumor. In the present study, the ultrastructural morphology in general paralleled the light microscopic observation, which seems to exclude an artifactual sampling. Nevertheless, certain characteristics of the tumor obviously are excluded from ultrastructural observations. Ultrastructural differentiation toward glandular, squamous, and mesenchymal cells was observed in the malignant parts. Squamous differentiation evidenced by tonofilament bundles a t t a c h e d to d e s m o s o m e s w a s d e t e c t e d frequently in cases 1 and 4. Vestiges of acinar and/or duct differentiation could be identified in three tumors (cases 1, 3, and 4). Accumulation of secretory-like material was also obvious in certain cells, although all granules might not be secretory, s i n c e they might represent a defective protein synthesis of the cells resulting in accumulation of abnormal material within the RER. Furthermore, in case 1, a differentiation toward squamous as well as Figure 9, Case 3. Ductal lumina with microvilli packed with filaments. In the periphery, dilated endaplasmic reticulum, secretory granules, and swollen mitoehondria are shown (x 14,500],

lamina. In some areas, typical spinocellular differentiation was found with cells containing large amounts of tonofibrils and numerous desmosomes (Figs. 10 and 11). In other areas, ducts lined with two or three cell layers were observed. The basal cells were smaller and contained larger amounts of fine filaments. No focal densities or tonofibrillar arrangements were observed. The apical cells similarly contained filaments, most abundant in the apical c y t o p l a s m under the microvilli protruding into the duct lumen (Fig. 12). Ceils were connected by desmosomes, and cellular interdigitations were common. A few cells contained light secretory granules (Fig. 13), while others harbored dark, round, or polyglonal granules with a concentric lamellar structure. DISCUSSION

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The specimens of the four cases studied, all judged to be carcinoma in mixed tumors by routine histopathology, showed obvious variations in ultrastructural morphology within and between the tumors. Although ultrastructural anal-

Figure 10, Case 4. Squamous different[ation of cells in cords with desmosomes and dense bundles of tonofilament

( x 7,00o).

GUSTAFSSONET AL.

Figure 11. Case 4. Filaments are found both in a perinuclear position and as long bundles or strands (x 15,500).

glandular cells appeared within the same cell. These cells exhibited both secretory granules and typical tonofilaments within the cytoplasm. These latter structures, however, do not appear to be pathognomonic for squamous cells since tonofilaments have been described in glandular cells and even in oncocytomas. 19 With regard to the mesenchymal features, they were most pronounced in case 2. Apart from an exceedingly large nucleus, the cells strongly resembled fibroblasts, with spindle shape and a l a r g e a m o u n t of e n d o p l a s m i c r e t i c u l u m , whereas epithelial features such as basal lamina, desmosomes, tonofilament bundles, and secretory granules could not be observed. Interestingly, the histologic picture varied somewhat in this case. Tumor specimens were obtained on several occasions as the tumor locally recurred despite extensive surgery. The mesenchymal features were most pronounced in the latest obtained s p e c i m e n s . This indicates a gradual change of tumor type with time. However, although the tumor had lost most of its epithelial characteristics, distant metastases have not yet appeared. In cases 2, 3, and 4, some neoplastic cells were packed with actin microfilaments or

intermediate filaments. These cells resembled the hyaline cell originally described by LomaxSmith and Azzopardi 2° and primarily considered as myoepithelial cells pathognomonic for pleomorphic adenoma. However, Warner and Seo 21 found similar cells in other tumors from tissues not containing the my0epithelial cells. Filament diameters of approximately 10 nm were not consistent with actin but with different types of intermediate filaments. Recently, much interest has been focused on intermediate filaments for tissue and tumor typing. Intermediate f i l a m e n t s - - a l l ranging from 7 to 11 n m - - c a n be divided into five main classes of proteins all specific for a certain tissue type, viz, cytokeratin is found in epithelial tissues, v i m e n t i n in m e s e n c h y m a l tissues, desmin in striated and some smooth muscle tissue, and neurofilament proteins and glial acidic fibrillary protein in neural and glial tissue, respectively. In general, tumor cells only contain the filament pattern of the cells of origin.22. 23 Previously, as well as in the present study, mixed tumor cells have been found to contain both vimentin and cytokeratins, regardless of

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of origin may take place as seen in case 3. The malignant part did not express vimentin as did the benign pleomorphic adenoma. However, a qualitative change in tumor filament content does not necessarily parallel an alteration of morphology. In case 2, the tumor cells lost their ultrastructural epithelial features such as desmosomes and basal lamina, b u t they contained cytokeratins. Similarly, in case 4, all malignant cells were clearly epithelial in morphology and still expressed both vimentin and cytokeratins. Except for mixed tumors (benign or malignant) and adenoid cystic carcinoma, all other salivary gland neoplasms have been found to be cytokeratin-positive but vimentin-negative (squamous cell carcinomas, 24 rnucoepidermoid carcinoma, 24 Warthin's tumor, ~9,24 and oncocytomas. 19) Consequently, intermediate filament protein histochemistry can be useful in the differential diagnosis of these tumors. The only cell in normal parotid tissue that has been described as containing both vimentin and cytokeratins is a triangular, basal duct cell. 24 However, in the present study, no such ceils could be identified. The present findings did not indicate any particular cell as a progenitor. Figure 12. Case 4, Mucus-producing cell with secretary granules and filaments. Glycogen particles are heavily stained ( x 5,500).

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mucoid, chondroid, or epithelial location. 24-26 This intermediate filament pattern is virtually unique among benign tumors. Recently, Caselitz et el. 27 and Gustafsson et al. 28 also reported a dual filament content of both vimentin and keratin in tumor cells of adenoid cystic carcinoma. Furthermore, renal adenocarcinomas 29,a° and Wilms' tumors al exhibit a similar filament pattern. In a few embryonal tumors such as teratomas, a2 e n d o d e r m a l sinus t u m o r s , a2 chordomas, aa and synovial sarcomas, a4,a5 vimentinpositive and cytokeratin-positive tumor cells have been found. In our study, two of the three tumors exhibited cells containing vimentin as well as cytokeratins. Of these two tumors, one displayed mainly mesenchymal features, whereas the other exhibited typical epithelial cords surrounded by a thick basement membrane, and the ceils showed squamous cell as well as glandular differentiation. The third tumor, which was vimentin-negative, contained mainly large epithelial cells resembling clear cells or cells of glandular type. Thus, in carcinoma in mixed tumors, a change in the filament pattern from that of the neoplasm

Figure 13. Case 4. High-magnification view of the filaments in the apical cytoplasm ( × 22,000).

GUSTAFSSON ET AL. H o w e v e r , as most other t u m o r cells containing b o t h v i m e n t i n a n d cytokeratins originate from e m b r y o n a l u n d i f f e r e n t i a t e d cells, an origin in p l u r i p o t e n t i a l u n d i f f e r e n t i a t e d cells seems plausible. T h e existence of these cells is still to be proved. If the classification of c a r c i n o m a s expleomorp h i c a d e n o m a s of T o r t e l e d o et al. a6 is applied to t h e p r e s e n t t u m o r s , t h e y p r o b a b l y will be reg a r d e d as a n a p l a s t i c or ductal varieties. However, t h e terms ductal c a r c i n o m a and terminal d u c t c a r c i n o m a m u s t be c o n s i d e r e d merely morp h o l o g i c and not h i s t o g e n i c since studies of cell c u l t u r e s f r o m m i x e d t u m o r s h a v e indicated that t h e cell p o p u l a t i o n in t h e s e t u m o r s is m o n o c l o n a l , aa F u r t h e r m o r e , a l t h o u g h light and elect r o n m i c r o s c o p i c i n v e s t i g a t i o n only reveals epit h e l i a l c h a r a c t e r i s t i c s of a m a l i g n a n t m i x e d t u m o r , it still m a y retain the i m m u n o h i s t o c h e m i c a l l y m o s t constant feature of m e s e n c h y m a l cells and t u m o r s - - v i m e n t i n . Ill c o n c l u s i o n , in the m a l i g n a n t mixed t u m o r s w e h a v e studied, signs of a d e n o c a r c i n o m a t e u s , as w e l l as s p i n o c e l l u l a r differentiation, were f o u n d u l t r a s t r u c t u r a l l y , s o m e t i m e s even w i t h i n t h e s a m e cell, A d i f f e r e n t i a t i o n t o w a r d vim e n t i n - c a n t a i n i n g m e s e n c h y m a l ceils was evid e n t in o n e tumor. F u r t h e r m o r e , our s t u d y indic a t e s t h a t t h e f i l a m e n t p a t t e r n of t u m o r cells m a y be c h a n g e d d u r i n g malignant transformat i o n . T w o m a l i g n a n t tumors expressed both cyt o k e r a t i n s a n d v i m e n t i n as benign p l e o m o r p h i c a d e n o m a s , w h i l e o n e t u m o r o n l y c o n t a i n e d cyt o k e r a t i n s , the n o r m a l f i l a m e n t pattern of carcin o m a s f o u n d elsewhere. U l t r a s t r u c t u r e a n d f i l a m e n t patterns do not a l w a y s c o r r e s p o n d . Thus, the presence of both c y t o k e r a t i n s a n d v i m e n t i n , a p h e n o m e n o n seen o n l y in renal a d e n o c a r c i n o m a s and a few emb r y o n a l t u m o r s besides in salivary gland tumors, c a n still be r e t a i n e d w h e n a m i x e d t u m o r und e r g o e s m a l i g n a n t transformation, even if there is n o u l t r a s t r u c t u r a l e v i d e n c e of m e s e n c h y m a l d i f f e r e n t i a t i o n . This fact m a y be used in the diff e r e n t i a l diagnosis from other a d e n o c a r c i n o m a s , s q u a m o u s cell c a r c i n o m a s , a n d m u c o e p i d e r m o l d c a r c i n o m a s of salivary gland tumors and c a r c i n o m a s of other origin.

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