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
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 immunohistologic patterns with respect to the ratio of the expression of 'normally' and 'aberrantly' 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 myxochondroid 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 Bcl2-positive cells. In carcinoma in pleomorphic adenoma, the benign part shows the
features of primary pleomorphic adenoma with its Bcl-2 and PTHrP-positivity patterns. 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.

Introduction
Seifert and co-workers21 proposed a subclassification of pleomorphic adenoma based on the differentiation 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 pleo0344-0338/95/0191-1186$3.50/0

morphic adenoma rich in cells but poor in stroma as in

type 3, but with the difference that the epithelial component is rather uniformly differentiated, resembling a
monomorphic adenoma. This classification is mostly
based on histomorphological and ultrastructural studies. Immunohistochemical techniques provide new
data for the classification and functional differentiation
of salivary gland tumour pathology. Morphological tumour markers give information about the cellular differentiation, proliferation and functional status of
1995 by Gustav Fischer Verlag, Stuttgart

Immunohistochemical Pattern of Pleomorphic Adenoma 1187

Fig. 2. Recurrent pleomorphic adenoma. Sections stained for

Bcl-2 immunoreactivity in the tubulo-ductal structures found
mainly in the periphery (three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin; original
magnification x 100).

tumours. In our previous studies on pleomorphic adenoma 23 ,24, we found different lines of differentiation:
first, tumour cells that differentiate 'normally', showing positivity for PTHrP, a marker of 'normal' or incipient differentiation, and second, tumour cells that are
positive for Bcl-2, suggesting an aberrant differentiation.
In this study we compare the patterns of cell differentiation in primary pleomorphic adenoma of the salivary gland with those of recurrent pleomorphic
adenoma and carcinoma in pleomorphic adenoma.
Results

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).

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 sections were also available. The qualitative results for
PTHrP were similar in frozen sections and in paraffin
sections, although the intensity and contrast in the paraffin sections were somewhat less. On the contrary, for
Bcl-2 the paraffin sections gave superior results.

1188

S. Sunardhi-Widyaputra et al.

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for Bcl-2. The mesenchymal tissue consisted of 'myoepithelial-like cells' that formed a mucoid, chondroid, or
myxochondroid matrix, and showed partly Bcl-2-positivity and partly PTHrP positivity. The proportions of
these components varied from tumour to tumour and
between areas within any single neoplasm. On occasion, 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 myxoid 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 structures, 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 predominantly myxoid, and showed the same features as the
other recurrent adenomas (see below).
Recurrent Pleomorphic Adenoma (Fig. 2)

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 immunoperoxidase method, lightly counterstained with Harris' haem at oxylin; original magnification a and b x 100).

Primary Pleomorphic Adenoma (Fig. 1 a-b)

The histopathologic features of the primary pleomorphic adenomas were typical and contained various
combinations of epithelial and mesenchymal-like tissues. 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 included 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

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 primaryto-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 inner 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 inner layer cells of the tubulo-ductal structures and of
some squamous metaplastic cells stained for PTHrP.
Tumours with myxoid and/or chondroid predominance (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 predominant 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 carcinoma 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 differentiation. In tumours, at least some of the tumour cells
retain the capacity to differentiate 7, 15, 16, 19,20.
Our previous studies on PTHrP23 and Bcl-224,
demonstrated two types of differentiation in pleomorphic adenoma. Regardless of the subtype of the tumour, 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 indicates 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 matrices 24 .
In this study, pleomorphic adenoma with predominant tubulo-ductal structures (mostly in primary tumours) 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 primary-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 subtype 221. From our case of primary-to-recur pleomorphic 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 apoptosis 3, 10, 11, be responsible for recurrence and possibly malignancy. In
early reports, spillage of tumour cells and the method
of surgery were thought to be responsible for the recurrence and possibility of malignancy. Since in adenoma and carcinoma of other organs, the maintenance of
Bcl-2 expression is important in tumour development 9,
it may be useful to perform a careful search for Bcl-2positive 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' differentiated 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 others 8, combined tubulo-ductal
and squamous epithelial differentiation in carcinoma in
pleomorphic adenoma was found. As the carcinomatous elements became less well differentiated, the
epithelial tumoural structures in this tumour were increasingly disrupted, though some lesions continued
to exhibit tubulo-ductal structures and squamous
metaplasia. The squamous and the tubulo-ductal malignant areas are reminiscent of 'normally' differentiated cell type in benign pleomorphic adenoma,
while the more anaplastic areas may represent aberrantly differentiated cells lines.
Stromal interactions are important in the maintenance of differentiated functions in epithelial cells,
and the degradation of extracellular matrix with consequent loss of specific cell interactions may be important 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 pleomorphic adenoma. In these tumour cells no positivity
for c-myc was found, but instead p53, a tumour suppressor gene, was present. The inverse relationship between 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 gene 22 , 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 significant inverse relationship between Bcl-2 expression
and p53 accumulation has also been documented recently in breast cancer4 and in non-Hodgkin's lymphomas 18 . Differentiation of neoplastic salivary gland cells
into keratinizing squamous cells had been demonstrated in an in vitro system2.
Over-production of the Bcl-2 has been shown to increase the relative resistance of cells to killing by all
chemotherapeutic drugs that have been tested to
date 13 ,14. The fact that primary and recurrent pleomorphic adenomas retain Bcl-2 expression may, in
part, explain why cancers originating from them are
notoriously difficult to treat by conventional chemotherapeutic drugs 12 , since Bcl-2 expression confers
a pleiotropic drug resistance in other cell types by inhibiting the process of apoptosis5, 6, 13, 17.
We conclude that the maintained presence of Bcl-2and PTHrP-positive cells in the tumours we studied is
related to the variable capacity of tumour cells to differentiate.
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 University Hospital-Catholic University of Leuven, Leuven, Belgium. Diagnoses are based on haematoxylin and eosinstained 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% hydrogen 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 serum 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 immunoglobulin 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 peroxidaseanti-peroxidase method was used in this study. PTHrP antibodies (diluted 1:400) were applied overnight at 4C, Bcl2-antibodies (diluted 1:10) were applied for 30 minutes, followed 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. Sections 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 % H2 0 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 Bcell 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 available.

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

pleomorphic adenoma - Immunohistologic pattern

In

pleomorphic adenoma - Recurrent

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