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High Tumor Grade in Salivary Gland Mucoepidermoid Carcinomas and Loss of Expression of Transforming Growth Factor ß Receptor Type II
David G. Dillard, MD;
Susan Muller, DMD, MS;
Cynthia Cohen, MD;
Dov Bloch, MD;
John M. Del Gaudio, MD;
Anthony A. Gal, MD
Arch Otolaryngol Head Neck Surg. 2001;127:683-686.
ABSTRACT
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Background Mucoepidermoid carcinoma (MEC) of salivary glands is a malignant, locally
aggressive neoplasm with metastatic potential. The clinical course is usually
dependent on histology; however, low-grade carcinomas can result in metastases
and tumor-related death. Transforming growth factor ß1 (TGF-ß1)
is a potent cytokine that affects growth inhibition of various cells and stimulates
extracellular matrix production and angiogenesis. Loss of TGF-ß receptor
type II (TGF-ß RII) expression has been related to resistance of TGF-ß1–mediated
growth control and tumor progression. In this study, we correlate MEC tumor
grade with expression of TGF-ß1 and TGF-ß RII.
Design Immunohistochemical staining was performed on 16 MEC specimens for activated
forms of TGF-ß1 and TGF-ß RII. The percentage of cells in which
staining yielded positive findings for activated TGF-ß1 and TGF-ß
RII was correlated with tumor grade.
Results Activated TGF-ß1 was detected in 16 specimens (100%) of MEC and
showed strong positive and diffuse staining. Predominately cytoplasmic staining
of TGF-ß1 was seen in salivary gland ducts, stroma, and endothelial cells.
There was an inverse correlation between tumor grade and loss of expression
of TGF-ß RII. All low-grade MEC tumors yielded positive staining results,
whereas only one case of intermediate-grade MEC had TGF-ß RII expression.
No high-grade MEC showed TGF-ß RII expression.
Conclusions Loss of expression of TGF-ß RII correlates with tumor grade. The
localization of activated TGF-ß1 within neoplastic epithelium, tumor-associated
stroma, and endothelium suggests that it might play a role in the stromal
proliferation and/or angiogenesis associated with MEC.
INTRODUCTION
SALIVARY GLAND tumors represent 6% of head and neck cancers and 0.3%
of all cancer in the United States. Mucoepidermoid carcinoma (MEC) is a malignant,
locally invasive neoplasm of the salivary glands. The incidence rate for MEC
has been reported at 0.44 per 100 000.1
It is the most common malignant neoplasm of the salivary glands, especially
in the parotid gland, but MEC also can occur in submandibular and minor salivary
glands.1, 2, 3, 4
Based on the histological finding, MEC of salivary glands is graded as low
(well differentiated), intermediate, or high (poorly differentiated) grade.
Although there is good correlation between tumor grade and clinical stage,
metastasis and tumor-related death have been noted with low-grade tumors,
particularly in the submandibular gland.2, 3
In the Armed Forces Institute of Pathology series of 227 cases of MEC, 17
patients (7%) with low-grade tumors had local metastases (n = 7) or died of
disease (n = 10).3
Transforming growth factor ß1 (TGF-ß1) is a polypeptide growth
factor associated with fibroblast proliferation, angiogenesis, tumor inhibition,
and apoptosis.5, 6, 7, 8, 9
Tumor inhibition and promotion by TGF-ß1 suggest an intricate relationship
between TGF-ß1 and its receptor complex. Transforming growth factor ß1
first binds to TGF-ß receptor type II (TGF-ß RII), which leads to
phosphorylation of receptor type I. Signals generated by this complex receptor-ligand
interaction are involved in cell growth regulation, chemotaxis, and extracellular
matrix production.5
It has been reported that loss of expression of TGF-ß RII appears
to render cells susceptible to DNA replicative errors.5, 10, 11, 12, 13
This decrease in TGF-ß RII expression or signaling intermediates may
be a mechanism by which cancer cells are not susceptible to TGF-ß1–mediated
inhibition of cell proliferation. Elevated levels of TGF-ß1 increase
desmoplasia, angiogenesis, and tumor progression.
The pathogenesis of MEC is unclear. Few studies have explored the role
of growth factors in MEC. Although lack of expression of TGF-ß RII is
associated with malignant progression in an immortalized salivary duct cell
line, to our knowledge no published reports have investigated the role of
TGF-ß1 or TGF-ß RII in MEC.13 To
investigate this potential role, 16 cases of MEC were evaluated by means of
an immunohistochemical analysis for expression of activated TGF-ß1 and
TGF-ß RII.
MATERIALS AND METHODS
Sixteen acceptable cases of MEC were identified between January 1, 1985,
and December 31, 1998, in the surgical pathology files at Emory University
Hospital, Atlanta, Ga. Two pathologists (S.M. and A.A.G.) reviewed hematoxylin-eosin–stained
slides to assess the diagnosis and grade. Grading of MEC was based on published,
accepted standards for MEC.3
TGF-ß1 DETECTION
Formalin-fixed, paraffin-embedded tissue blocks in sections of 5 µm
were processed for immunohistochemical analysis using an avidin-biotin complex
kit (LSAB 2; Dako Corporation, Carpinteria, Calif) and for steam antigen retrieval
using an autostainer (Dako Corporation). The primary antibody, a polyclonal
chicken antihuman antibody (R & D Systems, Minneapolis, Minn) specific
for the activated TGF-ß1, was used at a dilution of 1:40. (The primary
antibody was purified by means of affinity chromatography using TGF-ß1.
As determined by sandwich enzyme-linked immunosorbent assay [ELISA] with monoclonal
antibody to TGF-ß1, cross-reactivity was less than 5% with TGF-ß1,2
and less than 1% with TGF-ß2, TGF-ß3, TGF-ß4, or TGF-ß5.
There was no significant cross-reactivity with any other cytokine tested.)
The secondary-linking antibody, a rabbit antichicken antiserum (Chemicon International
Incorporated, Temecula, Calif) was used at a dilution of 1:80.
Positive controls consisted of tissue sections from human myometrial
blood vessels (the endothelium is known to yield staining results positive
for TGF-ß1, and human myometrium was chosen because of its high concentration
of blood vessels). For negative control sections, buffer replaced the primary
antibody.
Sections were deparaffinized and rehydrated, then steamed in citrate
buffer (pH, 6) for 20 minutes and cooled for 5 minutes before immunostaining.
All tissues were then exposed to 3% hydrogen peroxide for 5 minutes, primary
antibody for 25 minutes, biotinylated secondary-linking antibody for 25 minutes,
avidin-biotinylated enzyme complex for 25 minutes, diaminobenzidine as chromogen
for 5 minutes, and hematoxylin counterstain for 1 minute. These incubations
were performed at room temperature; between incubations, sections were washed
with buffer.
TGF-ß RII DETECTION
The immunohistochemical technique used for the detection of TGF-ß
RII was identical to that used for TGF-ß1. The primary antibody, a polyclonal
goat antihuman antibody (R & D Systems) specific for TGF-ß RII, was
used at a dilution of 1:40. The antibody was purified by means of affinity
chromatography using TGF-ß RII. This antibody was chosen for its ability
to neutralize the biological activity mediated by TGF-ß1. Based on direct
ELISA findings, there was no significant cross-reactivity with any other cytokine
tested. Specificity was greater than 99%. Positive controls consisted of tissue
sections of healthy esophageal mucosa, which has a high concentration of TGF-ß
RII.
Two pathologists (S.M. and C.C.) independently assessed each case. Immunostained
sections of TGF-ß1 and TGF-ß RII were reviewed by means of semiquantitative
analysis according to the percentage of positive cells (1 indicates <25%;
2, 25%-75%; and 3, >75%). Staining intensity was graded as weak, moderate,
and strong by comparing staining results with the positive controls. The pathologists
scoring specimens for TGF-ß1 and TGF-ß RII were unaware of histological
grade. For statistical evaluation, 1-tailed Fisher exact test was used to
correlate positive and negative results of staining for TGF-ß RII with
respect to histological grade. The statistical analysis compared low- and
intermediate vs high-grade MEC and low vs intermediate- and high-grade MEC.
RESULTS
CLINICAL AND PATHOLOGICAL CHARACTERISTICS
The Table 1 shows the clinical
and pathological characteristics of the 16 MEC specimens used in the study.
The mean age of the patients was 44 years (range, 18-88 years). There were
10 women and 6 men. Thirteen tumors were located in the parotid gland, 1 in
the submandibular gland, and 2 in minor salivary glands (base of tongue and
hard palate). Seven MEC specimens were high grade; 5, intermediate grade;
and 4, low grade.
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Clinical and Pathological Characteristics of Mucoepidermoid Carcinoma
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TGF-ß1 DETECTION
All 16 MEC specimens showed strong (2+ to 3+), mostly diffuse cytoplasmic
staining for activated TGF-ß1 (Figure
1 and Figure 2). In addition,
staining of stromal fibroblasts, endothelial cells, smooth muscle cells, and
epithelium was positive for TGF-ß1. Staining of adjacent non-neoplastic
salivary gland tissue also showed greater than 75% positive findings for TGF-ß1.
The pattern of staining was consistent throughout the tumor, both at the peripheral
and central portions.
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Figure 1. A, Low-grade mucoepidermoid carcinoma
(hematoxylin-eosin). B, Results of immunostaining positive for transforming
growth factor ß1 (TGF-ß1). C, Results of staining positive for TGF-ß
receptor type II (original magnification x50).
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Figure 2. A, High-grade mucoepidermoid carcinoma
(hematoxylin-eosin). B, Results of immunostaining positive for transforming
growth factor ß1 (TGF-ß1). C, Absence of TGF-ß receptor type
II immunostaining (original magnification x50).
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TGF-ß RII DETECTION
All low-grade tumors showed diffuse cytoplasmic (2+ to 3+) staining
for TGF-ß IIR (Figure 1). The
intensity of the staining pattern was moderate. In intermediate-grade MEC,
there was focal (<5%) cytoplasmic staining of tumor cells in 1 case with
weak staining intensity. No TGF-ß RII was identified in 7 high-grade
MEC specimens (Figure 2). Statistical
analysis demonstrated significant differences in staining between low- and
intermediate-grade MEC vs high-grade MEC (P = .03)
and between low-grade MEC vs intermediate- and high-grade MEC (P = .003). When present, surface epithelium, endothelial cells, nonneoplastic
salivary gland ducts, and stromal fibroblasts yielded staining results positive
for TGF ß RII.
COMMENT
Transforming growth factor ß1 is a potent multifunctional cytokine
that regulates growth and differentiation via a complex interaction with other
cytokines, growth factors, and mediators. Transforming growth factor ß1
inhibits the growth of epithelial, endothelial, and hematopoietic cells and
stimulates extracellular matrix protein production by mesenchymal cells. Three
distinct isoforms of the peptide are expressed in mammalian species, with
the most concentrated source of TGF-ß being the type I isoform.5 Transforming growth factor ß1 exerts control
on the cell cycle through its antiproliferative effect that inhibits key transitions
required for progression from the G1 to the S phase of the cell
cycle.5, 10, 11, 12, 13
The current findings show that TGF-ß1 is strongly expressed in stromal
and endothelial cells and in MEC as well as in normal salivary gland tissue.
Expression of TGF-ß1 was independent of tumor grade. This suggests other
mechanisms by which tumor cells modify their response to TGF-ß1.
Alterations in TGF receptors have been reported in breast, colon, and
head and neck cancers.14 In our series, it
is apparent that loss of differentiation in MEC correlates with progressive
loss of TGF-ß RII expression. The greatest reduction in TGF-ß RII
expression was in high-grade MEC, although intermediate-grade tumors showed
little to no expression. There is strong evidence to suggest that TGF-ß
RII plays a key role in tumor suppression mediated by TGF-ß1. In prostatic,
gastric, and breast cancer and in leukemia, TGF-ß RII alterations are
suggested to be important factors in altered tumor suppression and apoptosis.10, 11, 12, 13, 14, 15, 16, 17
Loss of TGF-ß RII expression correlates with poorer prognosis and more
aggressive local behavior in human prostate and thyroid cancer.18, 19
Our finding of lack of expression of TGF-ß RII in high-grade MEC
with concomitant high expression of TGF-ß1 seems to support the prevailing
opinion in the literature that alterations in TGF-ß1 and TGF-ß RII
might play a key role in tumorigenesis. The prognosis of MEC is closely related
to clinical and histological stage, age, sex, and location. Our data suggest
that a loss of expression of TGF-ß RII may define a transition from low-
to high-grade MEC. There are, however, reports of histologically low-grade
MEC with aggressive biological behaviors.2, 3
Therefore, TGF-ß RII expression is not by itself an independent assessment
of tumor grade.
CONCLUSIONS
Our study shows the frequent loss of expression of TGF-ß RII in
intermediate- and high-grade MEC, whereas TGF-ß1 expression was consistently
present in all histological grades of MEC. Although our study was limited
to a small number of cases, additional, larger cooperative studies among several
centers are needed to evaluate TGF-ß1 and TFG-ß RII expression as
potential prognostic factors and to elucidate their roles in the pathogenesis
of MEC.
AUTHOR INFORMATION
Accepted for publication January 17, 2001.
Funding provided in part by Emory University Educational Research Funds,
Atlanta, Ga.
From the Departments of Otolaryngology–Head and Neck Surgery
(Drs Dillard, Muller, and Del Gaudio) and Pathology and Laboratory Medicine
(Drs Muller, Cohen, and Gal), and the School of Medicine (Dr Bloch), Emory
University, Atlanta, Ga.
Corresponding author: Anthony A. Gal, MD, Department of Pathology
and Laboratory Medicine, 1364 Clifton Rd NE, Atlanta, GA 30322 (e-mail: agal{at}emory.edu).
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