Path. Res. Pract. 191, 1186-1191 (1995)

Immunohistochemical Pattern of Bcl-2- and PTHrP-positive Cells in Primary, in Recurrent and in Carcinoma in Pleomorphic Adenomas

Introduction

S. Sunardhi-Widyaputra and B. Van Damme Laboratory of Histo- & Cytochemistry, Department of Pathology 1/, Sint Raphael University Hospital, Catholic University of Leuven, Leuven, Belgium

SUMMARY

Forty-seven samples of paraffin-embedded formalin-fixed (and 25 related frozen) sections of 27 primary pleomorphic adenomas, 15 recurrent pleomorphic adenomas and 5 carcinomas in pleomorphic adenomas were studied to analyse their immuno-histologic patterns with respect to the ratio of the expression of 'normally' and 'aber-rantly' differentiated cell types.

In primary pleomorphic adenoma PTHrP-positive cells are seen in the inner layer of tubulo-ductal structures, in part of the cells in the mucoid, chondroid, or myx-ochondroid matrix, and in the squamous metaplastic areas. Bcl-2-positive cells are found in the outer layer of tubulo-ductal structures, in part of the cells in the mucoid, chondroid, or myxochondroid matrix, and around the squamous metaplastic areas. In one case of primary pleomorphic adenoma, which recurred later, the positivity for Bcl-2 is more intense and seen in the periphery of this tumour with a predominantly myxoid pattern. In recurrent pleomorphic adenomas, which also mostly showed a predominantly myxoid pattern, the positivity for Bcl-2 showed a pattern similar to the primary-to-recur tumour. PTHrP-positive cells are found less frequently than Bcl-2-positive cells. In carcinoma in pleomorphic adenoma, the benign part shows the features of primary pleomorphic adenoma with its Bcl-2 and PTHrP-positivity pat-terns. The malignant part strongly shows Bcl-2-positive cells in the periphery of the tumour.

We conclude that the maintained presence of Bcl-2 and PTHrP-positive cells in the tumours we studied shows the variable capacity of tumour cells to differentiate.

Seifert and co-workers21 proposed a subclassifica-tion of pleomorphic adenoma based on the differentia-tion of the epithelial cells and the quality and quantity of the stroma. Type 1 is the classic tumour type in which the stroma constitutes 30 to 50 percent of the tumour mass. Type 2 is a pleomorphic adenoma rich in stroma (80 percent), while type 3 is rich in cells (80 percent) but poor in stroma. Type 4 is also a pleo-

morphic adenoma rich in cells but poor in stroma as in type 3, but with the difference that the epithelial com-ponent is rather uniformly differentiated, resembling a monomorphic adenoma. This classification is mostly based on histomorphological and ultrastructural stud-ies. Immunohistochemical techniques provide new data for the classification and functional differentiation of salivary gland tumour pathology. Morphological tu-mour markers give information about the cellular dif-ferentiation, proliferation and functional status of

0344-0338/95/0191-1186$3.50/0 1995 by Gustav Fischer Verlag, Stuttgart

Immunohistochemical Pattern of Pleomorphic Adenoma 1187

Fig. 1. Primary pleomorphic adenoma. Sections stained for Bcl-2 (a) and PTHrP (b) immunoreactivity. The inner layer cells of tubulo-ductal structures are stained for PTHrP and the outer layer stained for Bcl-2, surrounded by the spindle cells that also stained for Bcl-2 (three-step immunoperoxidase method, lightly counter stained with Harris' haematoxylin; original magnification a and b x 100).

Fig. 2. Recurrent pleomorphic adenoma. Sections stained for Bcl-2 immunoreactivity in the tubulo-ductal structures found mainly in the periphery (three-step immunoperoxidase meth-od, lightly counterstained with Harris' haematoxylin; original magnification x 100).

tumours. In our previous studies on pleomorphic ade-noma23,24, we found different lines of differentiation: first, tumour cells that differentiate 'normally', show-ing positivity for PTHrP, a marker of 'normal' or inci-pient differentiation, and second, tumour cells that are positive for Bcl-2, suggesting an aberrant differentia-tion.

In this study we compare the patterns of cell differ-entiation in primary pleomorphic adenoma of the sali-vary gland with those of recurrent pleomorphic adenoma and carcinoma in pleomorphic adenoma.

Results

Samples were divided into four groups: primary pleomorphic adenoma, primary-to recur pleomorphic adenoma that is primary pleomorphic adenoma with its corresponding recurrence, recurrent pleomorphic adenoma, and carcinoma in pleomorphic adenoma. In 25 cases (23 primary and 2 recurrent), frozen sec-tions were also available. The qualitative results for PTHrP were similar in frozen sections and in paraffin sections, although the intensity and contrast in the par-affin sections were somewhat less. On the contrary, for Bcl-2 the paraffin sections gave superior results.

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Fig. 3. Carcinoma in pleomorphic adenoma. Sections stained for Bcl-2 (a) and PTHrP (b) immunoreactivity. Bcl-2 positive cells were present in the periphery of the malignant areas of the tumours. PTHrP positivity was present in some squamous metaplastic cells of the benign part (three-step immunoperox-idase method, lightly counterstained with Harris' haem at ox-ylin; original magnification a and b x 100).

Primary Pleomorphic Adenoma (Fig. 1 a-b) The histopathologic features of the primary pleo-

morphic adenomas were typical and contained various combinations of epithelial and mesenchymal-like tis-sues. In the primary tumours we found subtype 1 in 10 cases, subtype 2 in 13 cases, subtype 3 in 3 cases and subtype 4 in 1 case. The epithelial components in-cluded tubulo-ductal structures composed of a double layer of cells with an inner layer of luminal cells that stained for PTHrP and an outer layer that stained for Bcl-2 surrounded by spindle cells that also stained

for Bcl-2. The mesenchymal tissue consisted of 'myoep-ithelial-like cells' that formed a mucoid, chondroid, or myxochondroid matrix, and showed partly Bcl-2-posi-tivity and partly PTHrP positivity. The proportions of these components varied from tumour to tumour and between areas within any single neoplasm. On occa-sion, squamous foci were also seen, of which a part of the cells stained for PTHrP. In the capsule tissue some spindle cells positive for Bcl-2 were also found.

Primary-to-recur Pleomorphic Adenoma The primary adenoma showed a more striking myx-

oid pattern than the other primary tumours. Most of the myxoid cells stained strongly for Bcl-2. The tumour showed a restricted number of tubulo-ductal struc-tures, mainly found in the periphery, which revealed Bcl-2 and PTHrP reactivity similar to the other primary pleomorphic adenomas. The capsule adjacent to the myxoid matrix was thinner than that overlying more cellular areas. The recurrent tumour was also predomi-nantly myxoid, and showed the same features as the other recurrent adenomas (see below).

Recurrent Pleomorphic Adenoma (Fig. 2) Twelve of fifteen recurrent pleomorphic adenomas

showed a predominantly myxoid pattern (subtype 2). Subtype 1 was found in 1 case, subtype 3 in 2 cases, and no subtype 4 was found. Most tumour cells stained intensely for Bcl-2. This was predominantly found in the peripheral areas, a feature similar to the primary-to-recur pleomorphic adenoma. A few tubulo-ductal structures and tumour cells that stained for Bcl-2 were also found. PTHrP-positive cells were found in the in-ner layer of tubulo-ductal structures and some small ducts.

Immunohistochemically, the predominantly cellular pleomorphic adenomas (subtype 3) showed less PTHrP-positivity but more Bcl-2 positive cells. The in-ner layer cells of the tubulo-ductal structures and of some squamous metaplastic cells stained for PTHrP. Tumours with myxoid and/or chondroid predomi-nance (subtype 2) showed less PTHrP - but more Bcl-2-positive cells. Clusters of cells in the matrices also stained for Bcl-2. Pleomorphic adenomas with predom-inant tubular and/or trabecular structures (subtype 1) showed more PTHrP-positive cells.

Carcinoma in Pleomorphic Adenoma (Figs. 3 a-b) In carcinoma in pleomorphic adenoma only the

epithelial component is malignant. The types of carci-noma were undifferentiated carcinoma (4 cases), and mixed differentiation (1 case).

The benign part of the tumour showed the features of primary pleomorphic adenoma with Bcl-2- and PTHrP-positivity patterns. A few tumour cells in the squamous metaplastic areas stained for PTHrP. Strongly Bcl-2-positive cells were present in the benign areas, and in the periphery of the malignant areas of the

Immunohistochemical Pattern of Pleomorphic Adenoma . 1189

tumours. PTHrP was almost completely absent from the malignant part.

Discussion

The differentiation of a tissue is characterized by the expression of a specific repertoire of genes and thus by the appearance of their protein products. In an adult organism it is generally accepted that the majority of cells are relatively fixed along specific lines of differen-tiation. In tumours, at least some of the tumour cells retain the capacity to differentiate7, 15, 16, 19,20.

Our previous studies on PTHrP23 and Bcl-224, demonstrated two types of differentiation in pleo-morphic adenoma. Regardless of the subtype of the tu-mour, a consistent positivity for Bcl-2 and PTHrP is found. The PTHrP-positivity in tumour cells indicates incipient differentiation, considered to be normal. This is found in 'normally' differentiated cell types such as tubulo-ductal structures and squamous metaplastic cell formations. On the other hand, the Bcl-2 -positivity in-dicates a persistence in an undifferentiated phase and leads to the formation of aberrantly differentiated cell types such as spindle-shaped type cells and tumour cells in myxoid and chondroid matrices24.

In this study, pleomorphic adenoma with predomi-nant tubulo-ductal structures (mostly in primary tu-mours) presented more PTHrP-positive cells in the inner layers, even with a predominance of spindle cells PTHrP-positive cells could still be found. On the other hand, in the predominantly myxoid tumours (in pri-mary-to-recur and mostly in recurrent tumours) more Bcl-2-positive cells were found. This could be related with the more common recurrence in tumour of sub-type 221. From our case of primary-to-recur pleo-morphic adenoma, the primary tumour showed abundant Bcl-2-positive cells. Bcl-2-positive cells in predominant myxoid tumours are generally found in the peripheral areas, and in some parts of the tumour capsule. With this strategic location of cells therefore, the Bcl-2-positive cells could be responsible for the spilling, 'spreading' and growing of the tumour, and, due to their protection from apoptosis3, 10, 11, be re-sponsible for recurrence and possibly malignancy. In early reports, spillage of tumour cells and the method of surgery were thought to be responsible for the re-currence and possibility of malignancy. Since in adeno-ma and carcinoma of other organs, the maintenance of Bcl-2 expression is important in tumour development9, it may be useful to perform a careful search for Bcl-2-positive cells in the surrounding connective tissue. In pleomorphic adenoma, tumour cells usually infiltrate the capsule and small foci may become walled off from the main mass, without indicating malignancy.

The patterns of PTHrP- and Bcl-2-positive cells do not seem to correspond with the quantity of stroma in pleomorphic adenoma. The PTHrP-positive cells are more related to the quantity of 'normally' differen-tiated cell types than to the cellular density of the tu-

mours. The squamous cells are thought to represent the terminal differentiation of a cuboidal or columnar epithelium.

In this study and in others8, combined tubulo-ductal and squamous epithelial differentiation in carcinoma in pleomorphic adenoma was found. As the carcinoma-tous elements became less well differentiated, the epithelial tumoural structures in this tumour were in-creasingly disrupted, though some lesions continued to exhibit tubulo-ductal structures and squamous metaplasia. The squamous and the tubulo-ductal ma-lignant areas are reminiscent of 'normally' differen-tiated cell type in benign pleomorphic adenoma, while the more anaplastic areas may represent aber-rantly differentiated cells lines.

Stromal interactions are important in the mainte-nance of differentiated functions in epithelial cells, and the degradation of extracellular matrix with con-sequent loss of specific cell interactions may be impor-tant in the loss of functions by tumour cells. In one study, Azuma and co-workers 1 showed the presence of cuboidal and squamous cells in cultures of pleo-morphic adenoma. In these tumour cells no positivity for c-myc was found, but instead p53, a tumour sup-pressor gene, was present. The inverse relationship be-tween Bcl-2 immunoreactivity and p53 accumulation may be of interest in this respect. It has been suggested that p53 and Bcl-2 have opposite functions: that p53 is a death pathway gene22, 25 and that Bcl-2 is an antidote to programmed cell death 10. It is tempting to speculate that loss of function of the mutated p53 protein might confer on the tumour cells a double growth avantage, because the uncontrolled proliferation is combined with a reduced cell death rate. Interestingly, a signifi-cant inverse relationship between Bcl-2 expression and p53 accumulation has also been documented re-cently in breast cancer4 and in non-Hodgkin's lympho-mas18. Differentiation of neoplastic salivary gland cells into keratinizing squamous cells had been demon-strated in an in vitro system2.

Over-production of the Bcl-2 has been shown to in-crease the relative resistance of cells to killing by all chemotherapeutic drugs that have been tested to date13,14. The fact that primary and recurrent pleo-morphic adenomas retain Bcl-2 expression may, in part, explain why cancers originating from them are notoriously difficult to treat by conventional che-motherapeutic drugs12, since Bcl-2 expression confers a pleiotropic drug resistance in other cell types by in-hibiting the process of apoptosis5, 6, 13, 17.

We conclude that the maintained presence of Bcl-2-and PTHrP-positive cells in the tumours we studied is related to the variable capacity of tumour cells to dif-ferentiate.

Material and Methods

Forty-seven samples of paraffin-embedded formalin-fixed sections of 27 primary pleomorphic adenomas (23 parotids

1190 . S. Sunardhi-Widyaputra et al.

and 4 submandibular glands), and 15 recurrent pleomorphic adenoma (all in parotid glands) were studied. In one patient the primary and the recurrent tumour (of the parotid gland) were both available. The relapse occurred 8 years after the primary tumour. Five carcinomas in pleomorphic adenomas (4 parotids and 1 submandibular gland) were also studied. All cases (16 men and 31 women, ages ranging from 16 to 72 years; mean, 45.3 years) were obtained from the surgical files from the Department of Pathology, Sint Raphael Univer-sity Hospital-Catholic University of Leuven, Leuven, Bel-gium. Diagnoses are based on haematoxylin and eosin-stained sections. The formalin-fixed paraffin embedded and frozen sections were stained for PTHrP (diluted 1:200, a kind gift from Dr. Drucker, Ontario, Canada) and monoclonal antibody against Bcl-2 (diluted 1:5, Dakopatts a/s, Denmark).

Dewaxed paraffin sections were incubated in 0.3% hydro-gen peroxide in methanol for 10 minutes to block endogenous peroxidase activity. For removal of nonspecific background staining, sections were allowed to react with 2 % normal se-rum in PBS for 10 minutes. Primary antibody was applied overnight at 4 C. The slides were sequentially incubated with alkaline phosphatase conjugated goat anti-mouse immuno-globulin antibodies. The alkaline phosphatase reaction was demonstrated using a 5-bromo-4-chloro-3-indolyl phosphate (Sigma, Belgium).

Frozen sections (5 11m) were dried overnight and fixed in acetone for 10 minutes. A three-step unlabelled peroxidase-anti-peroxidase method was used in this study. PTHrP anti-bodies (diluted 1:400) were applied overnight at 4C, Bcl-2-antibodies (diluted 1:10) were applied for 30 minutes, fol-lowed by incubation with secondary, peroxidase-conjugated swine anti-rabbit Ig (diluted 1:50, Dakopatts, Denmark) and tertiary antibodies rabbit PAP complex. To reduce unwanted background staining, both secondary and tertiary antibodies were diluted in PBS, pH 7.2, containing 10% normal human AB-serum. To reduce endogenous peroxidase, 0.3% H 20 2 in methanol for 20 minutes were used prior to the incubation with primary antibodies. Each incubation with antibody was performed for 30 minutes at room temperature and was followed by a wash in three changes of PBS, pH 7.2. Sec-tions were then incubated for 15 minutes in 0.05 M acetate buffer (pH 4.9) containing 0.05% 3-amino-9-ethylcarbazole and 0.01 % H20 2 resulting in a red precipitate, and were lightly counterstained with Harris' haematoxylin.

A squamous cell carcinoma served as a positive control in immunostaining for PTHrP, and a B cell lymphoma for Bcl-2. Negative controls were prepared by replacing the primary antibody with non-immune serum. The staining results were compared with frozen sections of the same case, where avail-able.

Acknowledgements

We thank E. Van Dessel, K. Van Meerbeek and B. Smets for technical assistance, and M. Rooseleers for preparation of the micrographs. The author (SS-W) appreciates the financial support from the Belgian Administration for Developmental Co-operation.

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Received March 6, 1995 Accepted in revised form August 20, 1995

Key words: PTHrP - Bcl-2 - Pleomorphic adenoma - Carcinoma In pleomorphic adenoma - Recurrent pleomorphic adenoma - Immunohistologic pattern

S. Sunardhi-Widyaputra, Department of Pathology, Faculty of Medicine - RS Hasan Sadikin, University of Padjadjaran, Jalan Pasteur 38, Bandung, West Java, Indonesia, Phone 062-22-2501447