2016, Volume 32, Number 1, Page(s) 015-021
Twist Expression in Pleomorphic Adenoma, Adenoid Cystic Carcinoma and Mucoepidermoid Carcinoma of Salivary Glands
Soheil PARDİS1, Raziyeh ZARE1, Zohreh JAAFARİ-ASHKAVANDİ1, Mohammad Javad ASHRAF2, Bijan KHADEMİ3
1Department of Oral and Maxillofacial Pathology, School of Dentistry, Shiraz University of Medical Sciences, SHIRAZ, IRAN
2Department of Pathology, Cancer Research Center, School of Medicine, Shiraz University of Medical Sciences, SHIRAZ, IRAN
3Department of Otolaryngology, Cancer Research Center,School of Medicine, Shiraz University of Medical Sciences, SHIRAZ, IRAN
Keywords: Twist transcription factor, Immunohistochemistry, Pleomorphic adenoma, Adenoid cystic carcinoma, Mucoepidermoid carcinoma
Twist is an important transcription factor that induces epithelial-mesenchymal transition and therefore plays an important role in tumor progression. There are a few studies on Twist expression in salivary gland carcinomas. The aim of this study was to evaluate and compare
the expression of Twist in the most common benign and malignant salivary gland tumors and to determine whether its expression was correlated with any tumor characteristics.
Material and Method: In this retrospective cross-sectional study, 45 cases including 11 cases of normal salivary gland, 12 pleomorphic adenomas, 12 adenoid cystic carcinomas and 10 mucoepidermoid carcinomas were enrolled. The mean and intensity of Twist expression were evaluated
immunohistochemically and were compared using statistical analysis.
Results: The expression of Twist was higher in malignant salivary gland tumors in comparison with normal glands and benign tumors (p= 0.03). It was also higher in pleomorphic adenomas in comparison with normal tissue. Adenoid cystic carcinomas and mucoepidermoid carcinomas showed no significant difference in Twist expression (p= 0.50). There was no correlation with the size, stage or grade of tumor.
Conclusion: The findings showed that Twist might play a role in the formation of salivary gland neoplasm and also may affect malignant transformation and tumoral invasion. The exact mechanism of this marker and the possibility of using it as a therapeutic target require further investigation.
Salivary gland tumors are one of the crucial neoplasms in
maxillofacial pathology, and constitute 3-6% of all head
and neck tumors1
. These tumors show a wide spectrum
of clinical and pathologic variants that lead to difficult
diagnosis and management. Pleomorphic adenoma (PA),
mucoepidermoid carcinoma (MEC) and adenoid cystic
carcinoma (AdCC) are the most common benign and
malignant tumors and have histopathologic similarities in
many cases. Although, hematoxylin and eosin-stained (H&
E) tissue sections are used routinely for diagnosis in many
cases, the definite diagnosis is sometimes difficult. These
tumors are managed differently and there is therefore a
need to identify of diagnostic, prognostic, or therapeutic
markers to explain their different invasiveness and
It has recently been recognized that epithelial-mesenchymal
transition (EMT) play a key role in tumor invasiveness and
progression and is a necessary step for metastasis3,4.
In this process, the epithelial cells lose their adhesion and
polarity and acquire mesenchymal properties and increased
motility5. EMT is necessary for several developmental
processes during embryogenesis6. Twist is an important
transcription factor that induces EMT by dysregulation of
N-cadherin and E-cadherin expression7.
In adults, this marker has been demonstrated in precursor
cells such as the placenta, heart, and skeletal muscles8.
Twist proteins (Twist 1 and 2) play an important role in
tumor progression by promoting cell invasion, metastasis,
tumor growth, and angiogenesis7. Moreover, Twist
regulates matrix metalloproteinase (MMP) expression and
Overexpression of Twist has been reported in several
malignant tumors including head and neck squamous
cell carcinoma, nasopharyngeal carcinoma, prostate,
bladder, and breast cancer3,4,10-12. In several types
of cancer, Twist was an independent prognostic factor
and a predictor of survival. The studies showed that Twist
may be an oncogene that induced tumorigenesis as well as
tumor progression in the malignant cells. Twist may be a
contributing factor in anticancer drug resistance and Twist
inactivation has increased chemodrug-induced apoptosis
and suppressed invasion ability in prostate cancer cells3. One study showed that Twist expression contributed
to invasiveness of salivary AdCC13. Twist may also be
associated with perineural invasion of this tumor14.
Although a few studies explained Twist overexpression in
AdCC, there is no data about MEC as a common malignant
salivary gland tumor. It seems that Twist can be a novel
target for enhancing the efficacy of cancer treatment;
therefore, this study was designed to evaluate and compare
the expression of Twist in the most common benign and
malignant salivary gland tumors and to scrutinize whether
its expression was correlated with any tumor characteristics
such as tumor stage and grade.
The data of this cross-sectional study were collected from
the archive of Oral Pathology department of Shiraz of
Dental School, Iran. The selected cases included 12 PA, 12
AdCC, 10 MEC and 11 normal salivary glands (NSG). All
cases had adequate tissue. Cases with uncertain diagnosis
The baseline data including the patients’ age and gender,
tumor site and size as well as the grade and stage of the
tumors were recorded according to the patients´ medical
documents. Tumor stages were assessed based on the
stages adapted by the American Joint Committee on
Cancer (AJCC) TNM stage15. Tumor grade in MEC was
classified as grade I, if it demonstrated a well demarcated
border, macrocystic spaces and a bland cyst lining; grade
II, if it demonstrated a more solid growth with only few
microcysts, and focal infiltration; and grade III, with no
cystic spaces and a highly infiltrative growth pattern, and
pronounced nuclear atypia. In ADCC, grade I is referred to
as a tubular growth pattern, grade II as a cribriform growth
pattern, and grade III as a solid growth pattern16.
Immunohistochemical examinations (IHC) were performed
on 4 μ-sections of formalin-fixed and paraffinembedded
specimens. Tissue sections were incubated in 60°c
for 30 minutes, and then the sections were deparaffinized in xylene and rehydrated by alcohol. Antigen retrieval
was performed by citrate solution in pH= 9 at 121°C for
20 minutes. To block the endogenous peroxidase activity,
sections were immersed in 3% hydrogen peroxide for 30
minutes. The sections were then incubated with Twist1
polyclonal antibody (1:100, mouse, Abcam Corporation,
ab49254, UK). After applying secondary antibody
associated with the Envision system, sections were washed
in PBS. 3.3´-Diaminobenzidine tetrahydrochloride was
used as chromogen and slides were counterstained with
Mayer’s hematoxylin. A section of breast cancer was
considered as the positive control. By omitting the primary
antibody, sections were used as the negative control.
The cells with nuclear and/or cytoplasmic staining were
considered as positive. The percentage of positive cells out
of 500 tumoral cells was assessed in 10 microscopic fields at
high magnification (x 400). Moreover, a similar evaluation
was separately performed in 500 stromal fibroblasts. The
intensity of staining in the parenchymal cells was marked
as negative, weak, moderate, and intense in comparison
with the control sample.
The statistical analysis was performed using the SPSS
15 software. Data were analyzed by Kruskal-Wallis and
Dunn’s tests at the 0.05 significance level.
In the present cross-sectional study, 45 cases including 11
NSG, 12 PA, 12 AdCC and 10 MEC were enrolled. Baseline
data of all groups are summarized in Table I
. Some cases of
AdCC did not have any stage (2 cases) and grade (3 cases)
All cases were positive for Twist marker. Positive cells
revealed brown nucleus and/or cytoplasmic staining.
In all NSGs, ductal epithelium and some myoepithelial
cells were cytoplasmic and/or nuclear positive for Twist
(Figure 1A). In 5 cases (45.5%), acinar cells showed focal
Click Here to Zoom
|Figure 1: Moderate cytoplasmic Twist expression in A) ducts of normal salivary gland tissue (Twist; x200), B) ductal and myoepithelial
cells of pleomorphic adenoma (Twist; x400).
PAs showed Twist expression in ductal and some
myoepithelial cells. The expression was 66.8% cytoplasmic,
16.6% nuclear and 16.6% both cytoplasmic and nuclear.
Epithelial nests were also positive for this marker (Figure
1B). Cytoplasmic expression was predominant in PA
stromal cells, which are between epithelial nests and sheets
and includes myoepithelial, fibroblast, endothelial and
inflammatory cells. In Table II, the mean± SD expression
of Twist in tumoral parenchyma and stroma is shown.
Click Here to Zoom
|Table II: Twist expression in benign and malignant salivary gland tumors
Twist expression was positive in almost all cases of MEC.
The only case that did not stain was a clear cell type MEC which was then excluded from statistical analysis.
Epidermoid, mucous and intermediate cells showed 80%
cytoplasmic, 10% nucleus and 10% both cytoplasmic and
nucleus staining (Figure 2A). Clear cells were negative
for Twist. Similar to the other tumors, stromal cells were
stained with this marker. Table II shows mean±SD of Twist
expression in the parenchymal cells and stroma.
Click Here to Zoom
|Figure 2: A) Weak cytoplasmic Twist expression in mucoepidermoid carcinoma (Twist; x400), B) Strong nuclear and cytoplasmic
expression in adenoid cytic carcinoma (Twist; x200).
AdCCs showed extensive nuclear expression in 67%
of cases. Cytoplasmic expression was found in 16.5%
and simultaneous cytoplasmic and nuclear staining was
also found in 16.5% of the cases. All ductal cells were
positive for Twist (Figure 2B). Stromal cells also showed
immunoreaction. The percentage of expression mean is
depicted in Table II.
PAs and MECs showed weak or moderate staining, whereas,
AdCCs revealed moderate or strong staining in most of
the cases; all NSGs showed moderate staining. Table III
illustrates the intensity of Twist expression in benign and
malignant salivary gland tumors as well as NSG tissues.
The Kruskal-Wallis test showed a significant difference
between the mean expression of Twist in the parenchymal
component of the tumoral groups (p=0.03). Dunn’s test
revealed that PAs were significantly different from both
AdCCs (p= 0.004) and MECs (p= 0.005) in Twist expression,
but AdCC and MEC did not show any statistical difference
in its immunoreaction (p= 0.50).
Using the Kruskal-Wallis test, the intensity of staining was
significantly different among the groups (p=0.018) and by
Dunn’s test, the intensity of Twist was higher in AdCCs
than those of MECs (p= 0.001) and PA (p= 0.003) but no
significant difference was seen between MEC (P= 0.4) and
Stromal cells in all tumors showed expression of Twist and
there was no statistical difference among groups in stromal
staining (Kruskal-Wallis test, p= 0.756).
The percentage and intensity of Twist expression in
tumoral parenchyma were not statistically different among
the groups as regards tumor size, stage, and grade (Kruskal-
Wallis and Dunn’s test, all p> 0.05)
EMT is one of the essential steps in metastasis of malignant
tumors and is regulated by several genetic pathways17
Twist is one of the important proteins that play a crucial
role in EMT by reduction in E-cadherin and upregulation
of mesenchymal markers18
. Twist includes two proteins:
Twist1 and Twist2, which have shown similar expression in
. Twist expression was previously shown in some
normal tissues such as prostate and breast11,19
. It was also
demonstrated that this protein showed higher expression
in malignant tumors compared to benign tumors such
as prostate, parathyroid and lung19-21
. In the salivary
gland tumors, there has been some investigation on Twist
expression in AdCCs and PA. These studies demonstrated
expression of Twist in PA and overexpression of Twist in
AdCC in relationship with perineural invasion and tumoral
; however, this protein was not studied in
MECs as a common malignant salivary gland tumor. In the
present study, we found Twist overexpression in the most
common benign and malignant SGTs (MECs and AdCCs)
and its possible role in tumor formation and malignant
transformation of SGs.
The expression of Twist in this study was both nuclear
and/or cytoplasmic. This pattern of expression was also
found in cancers of breast, oral cavity, and pancreas and in
The present study showed that Twist was expressed in all
NSGs in the ductal epithelium and some myoepithelial
cells, but not in acinar cells. Zhao et al. demonstrated the
expression of E-cadherin in normal salivary gland acinar
cells with no expression of N-cadherin26. Regarding the
mechanism of Twist in downregulation of E-cadherin, our
finding is indirectly in the same line with that study18. Lee
et al. stated that Twist had a role during the organogenesis
of the parathyroids27. As Twist has been shown to be a
crucial protein in gastrulation and organogenesis of several
organs20, its expression in NSGs might be related to its
role in the organogenesis of salivary glands.
The current study showed Twist expression in all benign
tumors (PAs), predominantly with a cytoplasmic pattern
and moderate intensity. Yuen et al. also explained that
cytoplasmic expression of Twist was related to neoplastic
transformation in prostate tissues19. It seems that
overexpression of Twist in our study was also related to
the neoplasm formation in salivary glands. In agreement
with our findings, Shen et al. reported overexpression of
Twist in 30% of PAs13. Overexpression of Twist has been
previously demonstrated in some benign and malignant tumors. Benign tumors of prostate, parathyroid, lungs and
precancerous lesions of the oral cavity have shown this
protein in their parenchymal cells19-21,28.
All malignant tumors (cases of MEC and AdCCs) also
revealed immunoreaction for Twist. MEC exhibited Twist
expression in squamous and mucous cells. Cytoplasmic
expression of Twist protein was dominant in this tumor.
Most cases of MEC in this study had low histopathologic
grade, and their cytoplasmic pattern of staining was similar
to the samples included in the study of Lee at al. They
reported the cytoplasmic expression of this marker in the
well-differentiated squamous cell carcinomas27.
In AdCCs, staining was predominantly nuclear with
moderate and strong intensity. AdCC is a high-grade tumor
with a high ability of metastasis29. It was demonstrated
that nuclear expression of Twist was related to tumor
metastasis19. Previous studies have demonstrated
higher expression of Twist in high grade AdCC and its
relationship with perineural invasion13,14. Another
study also showed nuclear immunoreaction with Twist in
high-grade squamous cell carcinoma12. According to
invasive behavior of AdCC, our results support previous
studies that have shown a relationship between nuclear
Twist expression and increasing tumor invasiveness10,19.
The findings showed overexpression of Twist in malignant
tumors in comparison with NSGs and benign tumors.
Previous studies have demonstrated a reduction in
E-cadherin expression in the majority of AdCC cases
in comparison with PA, and also overexpression of this
protein in benign salivary glands tumors26,30. According
to the explained mechanism of Twist in downregulation
of E-cadherin, these findings indirectly supported the
switching mechanism of Twist in salivary glands tumors18. Our findings might indicate the role of Twist
protein in malignant transformation of SGTs. Kwok et al.
demonstrated Twist overexpression in malignant prostate
cancer cells and concluded that Twist may be a novel
oncogene resulting in malignant transformation of the
cells3. That result was a noticeable finding in our study.
Several studies have demonstrated that Twist expression
might be an independent prognostic factor which is
useful for predicting patients’ survival in breast cancer
and melanoma31,32. AdCCs and high-grade MEC are
chemo-and radiotherapy-resistant malignant tumors33.
It was reported that inactivation of Twist increased the
drug-induced apoptosis and therefore, it could promote
the efficacy of anticancer treatments in cases of prostate
cancer3. Due to the overexpression of Twist in MEC and AdCCs, Twist might be a novel target of treatment that
needs further investigation.
In the present study, we observed no statistically significant
difference between Twist expression and tumor grade,
stage, or size. According to the different roles of Twist in
tumor progression, it is possible that the limited number of
our cases with complete clinical data have resulted in these
findings. Also, future studies are suggested to evaluate the
relationship between Twist overexpression with clinical
behavior of salivary gland tumors.
In conclusion, the overexpression of Twist in salivary
gland tumors in comparison with normal glands and also
in carcinomas in comparison with benign tumors may
indicate the role of this protein in the tumorigenesis of
salivary glands and also in the malignant transformation
and tumor invasion. In this regard, we suggest further
investigations to evaluate the exact mechanism of Twist
protein and its possible use as a therapeutic target.
The authors thank the Vice-Chancellery of Shiraz
University of Medical Sciences for supporting this research
(Grant #92-01-03-6530). The authors would like to thank
Dr Sh. Hamedani (DDS, MSc) for helping with the English
and editorial assistance in the manuscript and Dr M.
Vossoughi from the Dental Research Development Centre,
for the statistical analysis. This manuscript is related to
postgraduate thesis of R. Zare (Shiraz University of Medical
Science, Shiraz, Iran).
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