The prognostic values of the expression of Vimentin, TP53, and Podoplanin in patients with cervical cancer
Lin et al. Cancer Cell Int
The prognostic values of the expression of Vimentin, TP53, and Podoplanin in patients with cervical cancer
Jiaying Lin 2
Jiaqi Lu 0 1
Xiaohong Xue 0 1
0 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University , 419 Fangxie Road, Shanghai 200011 , China
1 Department of Gynecology , Obstetrics and Gynecology
2 Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine , 639 Zhizaoju Road, Shanghai 200011 , China
Purpose: Epithelial-mesenchymal transition (EMT), TP53, and Podoplanin have been implicated in the tumorigenesis and metastasis of human cancers. Nevertheless, the clinical significance of these markers in cancer patients is still not clear. In this study, we sought to determine the prognostic values of Vimentin, TP53, and Podoplanin in patients with cervical cancer. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were performed to determine the messenger RNA and protein expression levels of Vimentin, TP53, and Podoplanin, respectively, in cervical squamous cell carcinoma and adjacent normal cervical tissues. Additionally, the expression levels of Podoplanin were also measured in 130 cervical cancer patients (FIGO stages Ib1-IIa2) using immunohistochemistry (IHC) staining. Results: The mRNA expression levels of Vimentin, TP53, and Podoplanin were considerably elevated in cervical cancer tissues, compared with those in the adjacent normal cervical tissues. Additionally, the protein expression levels of Vimentin were closely correlated with the age of onset (P = 0.007), lymph node metastasis (P = 0.007), lymphatic invasion (P = 0.024), disease recurrence (P < 0.001), and the clinical prognosis of patients with cervical cancer (P < 0.001). Our multivariate analysis also suggests that Vimentin is an independent marker for survival in cervical cancer patients. Furthermore, the expression levels of Vimentin are negatively correlated with the proliferation marker Ki67 expression. Conclusions: Our data show that Vimentin can serve as an independent prognostic marker for cervical cancer patients with primary surgery.
Vimentin; EMT; TP53; Podoplanin; Prognostic marker; Cervical cancer
With approximately 470,000 new cases and 233,000
deaths each year, cervical cancer is one of the most
common cancers found in women globally [
]. 85% of these
cases and deaths occurred in developing countries,
including China [
]. The malignant transformation of
human cells is characterised by their capacity to invade
neighbouring tissues and metastasize to distant organs.
Cancer invasion and metastasis are associated with a
physiological process, epithelial–mesenchymal transition
(EMT), by which epithelial cells lose their cell polarity
and cell–cell adhesion, and gain a mesenchymal
]. EMT plays a considerable role in normal
embryonic development as well as wound healing. It allows
epithelial cells to undergo dramatic morphological and
biochemical changes, which results in the loss of cell–cell
and cell–extracellular matrix connections and enables
the cells to migrate to other organs in the body
throughout the physiological process of tissue maintenance and
]. Notably, EMT plays a fundamental role in
the disease progression including the acquisition of the
invasive and metastatic potentials for not only head and
neck squamous cancer cells but also breast and hepatic
cancer cells [
]. The loss of epithelial phenotype and
gaining of mesenchymal properties enable cancer cells to
spread to distant organs of the body at a much faster pace
]. Vimentin is a key protein that composes the
interstitial cell skeleton. Its expression level is upregulated
during EMT and closely correlated with cancer invasion
and metastasis [
]. Additionally, Vimentin has also
been involved in cell cycle regulation and adhesion ,
which further validates its role in the development and
progression of human cancers.
Our previous studies have shown that cervical cancer
stem-like cells, which are extremely resistant to
chemoradiotherapy, play a crucial role in contributing to the
mechanism of metastasis in human cervical cancer. We
demonstrated that cervical cancer stem-like cells exhibit
typical EMT features, including upregulated expression
of EMT-related genes such as Vimentin, Twist 1, Twist 2,
Snail 1 and Snail 2 and the potential to migrate through
the basal membrane [
]. Intriguingly, Vimentin has also
been reported to form a complex with TP53 in the
cytoplasm and then suppress the translocation of TP53 into
the nucleus, thereby inhibiting the function of TP53 in
promoting apoptosis [
]. These findings suggest that
TP53 also plays an important role in the EMT pathway.
TP53 is a tumour suppressor nuclear protein that
exerts its anti-cancer function by inhibiting cell cycle
]. Compared with wild-type TP53,
mutant TP53 exhibits a longer half-life and is localized in
the nucleus, allowing us to readily examine its expression
using immunohistochemistry (IHC). Consistently,
previous reports have suggested a strong correlation between
TP53 mutations and immunohistochemical detection of
TP53 nuclear reactivity in human cancer tissues. TP53
mutations are undoubtedly involved in the development
and progression of human cancers. However, the
clinical prognostic values of TP53 mutations in cervical
cancer remains controversial [
]. It has been suggested that
TP53 overexpression is correlated with an unfavourable
prognosis in cervical cancer patients [
Nevertheless, other groups have also reported that TP53
mutations do not display important prognostic values in the
management of cervical cancer patients [
Invasion and migration of cancer cells into the nearby
tissues as well as their ingress into the microvasculature
of the lymphatic system are crucial steps for the
lymphatic dissemination of malignant tumours [
Podoplanin, a small membrane mucin-like type I glycoprotein,
is well expressed in lymphatic endothelial cells, but not in
blood vessel endothelium. Thus, it has been widely used
as a specific marker for lymphatic endothelial cells and
lymphangiogenesis in various species [
forms a complex with ezrin/moesin of the Ezrin, Radixin
and Moesin protein family through the interaction of its
cytoplasmic domain with the cytoskeleton protein, actin.
This interaction enables Podoplanin to activate GTPase
RhoA and its related RhoA-linked kinase, thereby
promoting EMT [
]. However, Wicki et al. have also
reported that Podoplanin can activate the rearrangement
of the cytoskeleton protein, actin, thereby promoting
tumour cell migration in the absence of the involvement
of EMT [
In this study, we aimed to investigate the
clinicopathologic properties of the expression of Vimentin, TP53 and
Podoplanin in human cervical cancer tissues. Our
findings will reveal the prognostic values of these protein
markers in cervical cancer patients and may provide
important guidelines for the management of the patients
in the future.
Materials and methods
130 cervical cancer tissues collected in the Obstetrics
and Gynecology Hospital of Fudan University
(Shanghai, China) between November 2007 and December
2012 were analysed to determine the clinical staging and
clinicopathological characteristics of the cervical
cancers. All protocols were carried out in accordance with
the altered International Federation of Gynecology and
Obstetrics staging system (FIGO) regarding cervical
cancer published in 2009. All patients enrolled in this study
were diagnosed with only gynaecological tumour(s).
Additionally, they did not received preoperative
radiotherapy, chemotherapy, or hormonal therapy. Following
the surgery, 93 patients underwent adjuvant radiotherapy
and (or) chemotherapy. All participants of this study
provided their written informed consent regarding the use of
the clinical materials for the research we described here.
Additionally, this study was approved by the Research
Ethics Committee of Fudan University, Shanghai, China.
In accordance with the ethical and legal standards, all
of the samples were rendered anonymous by removing
all identifiers. The patients’ characteristics related to the
samples have been illustrated in Table 1. The follow-up
time in respect of the initial cervical cancer group fell
between the ranges from 18 to 89 months, whereas the
median follow-up time was 53.2 months.
Quantitative real‑time‑polymerase chain reaction (qRT‑PCR)
Total RNA was purified from cervical cancer tissues as
well as from surrounding normal cervical tissues using the
Trizol reagent (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s protocol. The concentration of the
purified RNA was measured with a NanoDrop ND-1000
spectrophotometer (Thermo Scientific, Wilmington, DE,
USA). Following the first-strand cDNA synthesis with
the Reverse Transcription Kit (Invitrogen), qRT-PCR was
performed using the SYBR real-time PCR Kit (Invitrogen)
according to the manufacturer’s protocol. The primers
used in the PCR amplification were listed in Table 1.
Formalin-fixed, paraffin-embedded tissues were
sectioned to a thickness of 4 μm, mounted onto glass
slides, deparaffinized with xylene and then rehydrated
through the graded ethanol series (100, 95 and 70%) to
deionized H2O. The expression of Vimentin, TP53, and
Podoplanin was determined with IHC staining. The
antibodies used in the IHC staining included mouse
monoclonal antibodies against Vimentin (dilution 1: 200; Dako,
Denmark), TP53 rabbit monoclonal antibody (1:100
dilution, Carpinteria, CA), and mouse monoclonal
antibody against Podoplanin (1:100 dilution, Dako). Briefly,
retrieval of antigens was done through steam heat for
20 min in a 0.01 M trisodium citrate buffer (pH 6.0). The
slides were then immersed in the ChemMate
peroxidaseblocking solution (Dako) for 10 min at room temperature
to block endogenous peroxidase activity. The samples
were subjected to immunostaining with primary
antibodies for 2 h, followed by incubation with HRP-labeled
anti-mouse or anti-rabbit secondary antibodies.
Visualization of immunoreactive proteins was carried out using
3,3-diaminobenzidine (Sigma-Aldrich) as chromogen
and nuclei were counterstained with Mayer’s
hematoxylin. Following dehydration through an ethanol series (70,
90 and 100%), the slides were mounted and evaluated
by two pathologists (S.L. and C.W.) independently as
described below. To assess the quality control of our IHC
staining protocol, normal cervical tissue staining and an
isotype control (Dako) were used as positive and negative
Two pathologists (S.L. and C.W.) who were unaware
of the clinical data as well as other
immunohistochemical findings carried out semiquantitative evaluation
of the slides. When there were differences in scoring, a
consensus was reached through discussion between the
two pathologists. The expression levels and subcellular
localization of Vimentin, TP53, and Podoplanin were
determined using the positive and negative controls as
a reference. At least 1000 cells in five randomly chosen
areas of the tumour tissues were analysed in each section
at a ×400 magnification to obtain the labelling indices
(percentage of positive cells).
Vimentin expression was scored as positive when
cytoplasmic or nuclear staining of the cells was observed in
greater than 10% of the tumor tissues [
]. Tumor tissues
with a positive immunohistological staining of at least
50% of tumor cells were defined to have positive TP53
]. Podoplanin expression was
considered positive when there was moderate or strong
immunoreaction in more than 10% of the cells .
Statistical analysis was carried out using SPSS version
19.0 software (SPSS Inc., Chicago, Ill., USA. Disease-free
survival was defined as the interval falling between the
date of surgery and the date of tumor recurrence or the
date of the most recent follow-up with no proof of tumor
recurrence. At the time of the previous visit for regular
follow-ups, a censor was performed on overall survival
time. Chi squared test or Fisher’s exact test was
performed to compare the difference between groups. Cox’s
proportional hazards model was built to calculate the risk
ratios and their corresponding 95% confidence intervals
(CIs). The associations of the gene expression with
disease-free and overall survival was evaluated based on the
Univariate and multivariate Cox’s proportional hazards
models. Survival rates were estimated using the Kaplan–
Meier method and log-rank test. The results were
considered statistically significant, when P values are less than
In order to evaluate the expression of Vimentin, TP53,
and Podoplanin, 130 samples from cervical cancer
patients were used in this study. The clinicopathological
characteristics of the patients are presented in Table 1.
The patients have a median age at 48 years old, ranging
from 28 to 70 years. According to the 2009 FIGO criteria,
the patients were diagnosed with cervical cancers of
different clinical stages: stage Ib1 (57.7%), stage Ib2 (10.7%),
stage IIa1 (23.1%), and stage IIa2 (8.5%). Additionally, the
patients had cervical cancers of different degrees of
differentiation: well differentiated (11 cases), moderately
differentiated (97 cases), and poorly differentiated (22
cases). A total of 22 patients had lymph node metastases,
while the other 108 patients did not.
The mRNA expression of Vimentin, TP53, and Podoplanin in cervical cancer
The mRNA expression levels of Vimentin, TP53, and
Podoplanin were examined using qRT-PCR in cervical
cancer tissues and surrounding normal cervical tissues
from fifteen randomly selected cases of cervical cancer
patients. Our results demonstrated that cervical cancer
tissues exhibit considerably higher mRNA expression levels
of Vimentin, TP53, and Podoplanin than the normal
tissues (P < 0.05; Fig. 1).
The expression of Vimentin, TP53 and Podoplanin in cervical cancers
In order to examine the expression of Vimentin, TP53
and Podoplanin in cervical cancer, IHC analysis was
performed as described in the “Materials and methods”.
Representative images defined as positive staining of the
three proteins were shown in Fig. 2. Cells with positive
Vimentin expression display yellow or brown granules
in the cytoplasm that are close to the membrane. The
presence of yellow or brown granules in the nucleus and
cytoplasm of cancer cells is indicative of positive TP53
expression. On the other hand, Podoplanin is expressed
primarily in the cytoplasm as well as on the plasma
membrane of the tumour cells (Fig. 2).
The association of the expression of Vimentin,
TP53 and Podoplanin with the clinicopathological characteristics of cervical cancer
We next investigated whether the expression of
Vimentin, TP53, and Podoplanin is correlated with several
clinicopathological features in cervical cancer. Our data
showed that Vimentin expression is considerably
associated with the onset age (P = 0.007), lymph node
metastasis (P = 0.007), lymphatic invasion (P = 0.024), disease
recurrence (P < 0.001), and the clinical prognosis of
patients (P < 0.001) (Table 2).
Similarly, a significant association was observed
between the expression of TP53, and tumour size
(P = 0.037), vascular space involvement (P < 0.0001),
disease recurrence (P = 0.006) and the clinical prognosis of
cervical cancer patients (P = 0.001) (Table 2).
Moreover, Positive staining of Podoplanin was also
significantly correlated with onset age (P < 0.0001), lymph
node metastasis (P = 0.028), vascular space
involvement (P < 0.0001), lymphatic invasion (P < 0.0001), deep
stromal invasion (P = 0.007), positive parametrium
(P = 0.029), disease recurrence (P = 0.006), and the
clinical prognosis of patients (P = 0.004) (Table 2).
The association of the expression of Vimentin, TP53 and Podoplanin with the proliferation of cervical cancer
In order to investigate whether there is a link between
the expression of these three protein biomarkers and
the proliferation of cervical cancer, we next examined
the correlation of the expression of Vimentin, TP53 or
Podoplanin with Ki67, a cellular marker for proliferation
(Table 2). We found that Vimentin expression is closely
correlated with Ki67 expression in cervical cancer tissues
(P = 0.037). Nevertheless, there is no significant
association between the other two protein markers and Ki67
(P > 0.05).
Expression of Vimentin, TP53 and Podoplanin as prognostic factors in patients with cervical cancer
The cumulative OS and DFS rate of the 130 patients
with cervical cancer were 92.3 and 88.5%, respectively.
To evaluate the prognostic value of Vimentin, TP53
and Podoplanin in cervical cancer, we then examined
the correlation between the expression of Vimentin,
TP53 and Podoplanin and patients’ survival using the
Kaplan–Meier estimate and log-rank test. Our data
showed that patients with positive expression of
Vimentin exhibit shorter OS as compared with those with
negative expression (77.1% vs. 97.9%, P < 0.001). Similarly,
Vimentin-positive patients display significantly shorter
DFS (65.7%), compared with Vimentin-negative patients
(96.8%) (P < 0.001) (Fig. 3a, b).
Additionally, we also explored the impact of TP53
expression on the OS and DFS of cervical cancer patients.
The cumulative OS rate for the cervical cancer patients
with positive TP53 expression (76.9%) is significantly
lower than that for patients with negative TP53 expression
(96.2%, P < 0.001). Similarly, cervical cancer patients with
positive TP53 expression also display smaller cumulative
DFS rate (73.1%), compared with those with negative TP53
expression patients (92.3%, P = 0.006) (Fig. 4a, b).
Our data also show that Podoplanin expression is
negatively correlated with the OS (P = 0.004) and DFS
(P = 0.006) of cervical cancer patients (Fig. 5a, b). The
OS (85.2%) and DFS (80.3%) rates in the cervical cancer
patients with positive Podoplanin expression are smaller
than those for the patients without Podoplanin
expression (OS 98.6%, DFS 95.7%).
We next employed Cox proportional hazards model
to examine the clinicopathologic features of the
expression of Vimentin, TP53 and Podoplanin in cervical
cancer patients (Table 3). The results from our univariate
0 7 6
0 9 4
.0 .2 .2
0 0 0
a n N (n 65 31 65 31 06 9 51 18 62 7 60 9 49 17 3 5 64
= eN (n= 4 7 9 0 7 9 4 5 6 8 41
n 3 1 2 4 2 6 0 6 1 5 3 6 6 1
1 e =
0 a 9
sq P e 7 7
ih tse lau .7 .3 .6 .8 .7
7 3 0
C t v 0 0 0 0 0
t ve )5 m
en iit 3 ng .7± sou
iVm sPo (n= ,en 23 12 21 14 2 33 1 34 8 27 12 23 27 8 49 aum
)0 itang .503 seonq
3 a )
l 1 m n p
eud taTo (n= ircon 88 42 isaon 69 61 ium 01 210 irag 3 127 ten 23 98 15 115 l-uow 210 01 .533 ,SaCA
in v rt l
le2b trcaae s sauuom 5 5 rtseop se o iitsvep se o iitssveu se o iilanng se o rrcceeun se o lttsaau ilveA aedD i()67% (aenm± caaeond
a h ic q ≤ > e Y N o Y N o Y N a Y N e Y N it C
T C t S D P P V R V K A
l m ic
a ra rg
and multivariate analysis suggest that age and Vimentin
expression exhibit a considerable impact on the OS and
DFS of cervical cancer patients (Table 3). These data
indicate that age and Vimentin expression are independent
prognostic factors for cervical cancer patients.
Though both the incidence and mortality rate of
cervical cancer have considerably diminished globally in
the past five decades, cervical cancer remains a major
cause of cancer death in women [
]. The survival
of patients with primary cervical cancer is generally
determined by several factors, including lymph node
metastasis, parametrial invasion, tumor size,
lymphovascular involvement, and histologic grade [
Following surgical resection of tumors, patients with one
or more of the clinicopathological features mentioned
above need to undergo additional therapy.
Nevertheless, traditional pathological markers don’t offer reliable
prognostic values to guide optimal treatment strategies.
In this study, we showed that the expression of
Vimentin, TP53 and Podoplanin is correlated with the survival
of cervical cancer patients, indicating that the expression
of these proteins may serve as valuable biomarkers to aid
in the diagnosis and effective treatment of cervical cancer
Hazard ratios with 95% confidence intervals (95% CI) were estimated and adjusted by indicated clinical parameters
In this study, we employed immunohistochemistry to
investigate the expression of Vimentin, TP53, and
Podoplanin in cervical cancer. Our findings may help to reveal
the role of these proteins in cervical cancer
carcinogenesis and their prognostic significance in the management
of cervical cancer patients. Vimentin, which functions as
a key intermediate filament protein in mesenchymal cells,
is involved in EMT and plays a critical role in the growth,
invasion and metastasis of human cancer cells,
including cervical cancer [
3, 29, 30
]. Nevertheless, it is still
unknown whether or not the expression of Vimentin is
correlated with the clinicopathologic features of cervical
cancers. Interestingly, we found out that Vimentin
protein expression is strongly associated with the onset age,
lymph node metastasis, lymphatic invasion, Ki67
staining, recurrence, and survival in cervical cancer patients.
Furthermore, a multivariate analysis also reveals that
Vimentin expression is an independent prognostic
factor for the OS and DFS of cervical cancer patients. These
results indicate that Vimentin plays an important role
in the malignant phenotype in cervical cancer patients.
Importantly, our data suggest that positive Vimentin
expression may serve as a biomarker to predict a poor
prognosis in cervical cancer patients and provides
important insights into the design of novel therapeutic
strategies to treat cervical cancer patients.
Consistent with earlier reports [
3, 31, 32
], our data also
showed that 20% of the cases of cervical cancer exhibit
high levels of TP53 expression. Over-expression of TP53,
resulting from accumulation of defected TP53 protein
due to gene alterations, is commonly found in cervical
cancer. Nonetheless, its prognostic value in cervical
cancer remains controversial [
]. Here, we found that
upregulation of TP53 expression is correlated with the size
of tumor and vascular space involvement as well as poor
DFS and OS. However, when other co-variables were
included in the Cox regression model, over-expression of
TP53 is not an independent prognosis factor. Hence, in
agreement with the findings by Åvall-Lundqvist et al. our
data suggest that TP53 expression is not an independent
predictive factor for patients with cervical cancer [
Podoplanin, selectively expressed in lymphatic
endothelium, has been used to detect lymphatic
invasion in several malignant neoplasms, including cervical
]. Podoplanin, a transmembrane
glycoprotein, is up-regulated in a variety of human cancer
cells, especially those derived from squamous stratified
epithelia (SCCs). Its expression in tumor cells is linked
to increased cell migration and invasiveness . It has
been reported that CD44, the major hyaluronan (HA)
receptor and one of the cancer stem cell (CSC) markers,
is a novel partner for Podoplanin. Expression of the CD44
standard isoform (CD44s) is coordinately up-regulated
together with that of Podoplanin during progression to
highly aggressive SCCs in a mouse skin model of
carcinogenesis, and during the process of EMT. It has been
shown that aggressive squamous CSCs are enriched at
the invasive front with the extracellular matrix composed
of hyaluronic acids and Podoplanin [
previous reports indicate that presence of
lymphovascular invasion serves as a considerable risk factor for tumor
recurrence in cervical squamous cell carcinoma [
Consistent with these reports [
3, 36, 37
], our data suggest that
Podoplanin expression in the tumor cells displays strong
association with the onset age, the existence of lymphatic
invasion, lymph node metastasis, vascular space
involvement, deep stromal invasion, positive parametrium,
higher risk of tumor recurrence and shorter survival. Our
findings also confirms that Podoplanin plays an
important role in cell migration and in the lymphatic spread of
cervical cancer cells to regional lymph nodes.
Ki-67 has been widely used as a proliferation marker to
measure the growth fraction of human cancer cells [
High Ki67 expression has been suggested as a poor
prognostic indicator for Ewing’s sarcomas [
] or breast
]. Although Ki-67 expression has not been found
to be associated with patient’s general survival in studies
conducted by us and other groups [
], we found that
Vimentin expression was significantly associated with a
decreased proliferation rate of cervical cancer as
measured by the Ki-67 labeling index. The inversed correlation
between Vimentin and Ki-67 seems to be paradoxical.
However, it has been reported that cancer stem cells with
increased CD44 expression tend to form the negative
feedback machinery in terms of oxidative stress-induced
Wnt/beta-catenin signal transduction [
negative feedback regulation exerted by upregulated CD44/
Vimentin expression may be partially responsible for the
inversed expression pattern between CD44/Vimentin
and Ki-67/c-Myc [
It is worth noting that there are some limitations
regarding our research findings we presented here. First,
the sample size in our study is relatively small. Thus, it is
necessary to perform further studies with larger sample
sizes to validate our findings. Second, a population
selection bias may also exist, since it is a retrospective study.
Lastly, the patients recruited in our study all had
resectable tumors. Hence, it is not clear whether or not we can
extend our findings to the patients with advanced
nonresectable cervical cancer.
As of now, clinical TNM stage doesn’t serve well as a
practical indicator for the prognosis of patients with
cervical cancer. Patients with the same clinical stage may
display completely different clinical courses. Here, we
demonstrated that Vimentin expression can act as an
independent predictive factor for patients with cervical
cancer, providing important guidelines for the
management of cervical cancer patients.
JYL and XX conceived and designed the experiments. JYL, JQL and XX
performed the experiments. JYL analyzed the data. XX contributed reagents/
materials/analysis tools. JYL contributed to the writing of the manuscript. All
authors read and approved the final manuscript.
Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, China.
3 Department of Pathology, Obstetrics and Gynecology Hospital, Fudan
University, 419 Fangxie Road, Shanghai 200011, China.
This research was supported in part by Grants 81371517 from the National
Science Foundation of China.
The authors declares that they have no competing interests.
Availability of data and materials
The data and materials of this study are included in this published article.
Consent for publication
This manuscript is approved by all authors for publication in Cancer Cell
Ethics approval and consent to participate
The ethics committee at the Fudan University approved this study, and all the
patients provided written informed consent for the use of clinical specimens
for this study.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Parkin DM , Bray F , Ferlay J , Pisani P. Estimating the world cancer burden: globocan 2000 . Int J Cancer . 2001 ; 94 ( 2 ): 153 - 6 .
2. Ferlay J , Shin HR , Bray F , Forman D , Mathers C , Parkin DM . Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 . Int J Cancer . 2010 ; 127 ( 12 ): 2893 - 917 .
3. Brabletz T , Hlubek F , Spaderna S , Schmalhofer O , Hiendlmeyer E , Jung A . Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and betacatenin . Cells Tissues Organs . 2004 ; 179 ( 1-2 ): 56 - 65 .
4. Chen C , Zimmermann M , Tinhofer I , Kaufmann AM , Albers AE . Epithelialto-mesenchymal transition and cancer stem (-like) cells in head and neck squamous cell carcinoma . Cancer Lett . 2013 ; 338 ( 1 ): 47 - 56 .
5. Zeisberg M , Neilson EG . Biomarkers for epithelial-mesenchymal transitions . J Clin Investig . 2009 ; 119 ( 6 ): 1429 - 37 .
6. Yoshida GJ . Emerging role of epithelial-mesenchymal transition in hepatic cancer . J Exp Clin Cancer Res . 2016 ; 35 ( 1 ): 141 .
7. Kokkinos MI , Wafai R , Wong MK , Newgreen DF , Thompson EW , Waltham M. Vimentin and epithelial-mesenchymal transition in human breast cancer-observations in vitro and in vivo . Cells Tissues Organs . 2007 ; 185 ( 1-3 ): 191 - 203 .
8. Polyak K , Weinberg RA . Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits . Nat Rev Cancer . 2009 ; 9 ( 4 ): 265 - 73 .
9. Chikaishi Y , Uramoto H , Tanaka F . The EMT status in the primary tumor does not predict postoperative recurrence or disease-free survival in lung adenocarcinoma . Anticancer Res . 2011 ; 31 ( 12 ): 4451 - 6 .
10. Perlson E , Michaelevski I , Kowalsman N , Ben-Yaakov K , Shaked M , Seger R , Eisenstein M , Fainzilber M. Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase . J Mol Biol . 2006 ; 364 ( 5 ): 938 - 44 .
11. Thiery JP , Sleeman JP . Complex networks orchestrate epithelial-mesenchymal transitions . Nat Rev Mol Cell Biol . 2006 ; 7 ( 2 ): 131 - 42 .
12. Lin J , Liu X , Ding D . Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri . Int J Clin Exp Pathol . 2015 ; 8 ( 1 ): 847 .
13. Chang L , Goldman RD . Intermediate filaments mediate cytoskeletal crosstalk . Nat Rev Mol Cell Biol . 2004 ; 5 ( 8 ): 601 - 13 .
14. Meek DW . Tumour suppression by p53: a role for the DNA damage response ? Nat Rev Cancer . 2009 ; 9 ( 10 ): 714 - 23 .
15. Bremer GL , Tieboschb AT , van der Putten HW , de Haan J , Arends J-W. p53 tumor suppressor gene protein expression in cervical cancer: relationship to prognosis . Eur J Obstet Gynecol Reprod Biol . 1995 ; 63 ( 1 ): 55 - 9 .
16. Chen H-Y , Hsu C-T, Lin W-C , Tsai H-D, Chang W-C. Prognostic value of p53 expression in stage IB1 cervical carcinoma . Gynecol Obstet Invest . 1999 ; 49 ( 4 ): 266 - 71 .
17. Brenna S , Zeferino L , Pinto G , Souza R , Andrade L , Vassalo J , Martinez E , Syrjänen K. P53 expression as a predictor of recurrence in cervical squamous cell carcinoma . Int J Gynecol Cancer . 2002 ; 12 ( 3 ): 299 - 303 .
18. Khunamornpong S , Siriaunkgul S , Manusirivithaya S , Settakorn J , Srisomboon J , Ponjaroen J , Thorner PS . Prognostic value of p53 expression in early stage cervical carcinoma treated by surgery . Asian Pac J Cancer Prev . 2008 ; 9 : 48 - 52 .
19. Bremer GL , Tieboschb AT , van der Putten HW , de Haan J , Arends J-W. p53 tumor suppressor gene protein expression in cervical cancer: relationship to prognosis . Eur J Obstet Gynecol Reprod Biol . 1995 ; 1 ( 63 ): 55 - 9 .
20. Chaffer CL , Weinberg RA. A perspective on cancer cell metastasis . Science . 2011 ; 331 ( 6024 ): 1559 - 64 .
21. Ordóñez NG . Podoplanin: a novel diagnostic immunohistochemical marker . Adv Anat Pathol . 2006 ; 13 ( 2 ): 83 - 8 .
22. Martín-Villar E , Megías D , Castel S , Yurrita MM , Vilaró S , Quintanilla M. Podoplanin binds ERM proteins to activate RhoA and promote epithelialmesenchymal transition . J Cell Sci . 2006 ; 119 ( 21 ): 4541 - 53 .
23. Wicki A , Lehembre F , Wick N , Hantusch B , Kerjaschki D , Christofori G . Tumor invasion in the absence of epithelial-mesenchymal transition: Podoplanin-mediated remodeling of the actin cytoskeleton . Cancer Cell . 2006 ; 9 ( 4 ): 261 - 72 .
24. Sousa B , Paredes J , Milanezi F , Lopes N , Martins D , Dufloth R , Vieira D , Albergaria A , Veronese L , Carneiro V. P-cadherin, vimentin and CK14 for identification of basal-like phenotype in breast carcinomas: an immunohistochemical study . Histol Histopathol . 2010 ; 25 ( 8 ): 963 - 74 .
25. Allegra CJ , Paik S , Colangelo LH , Parr AL , Kirsch I , Kim G , Klein P , Johnston PG , Wolmark N , Wieand HS . Prognostic value of thymidylate synthase, Ki-67, and p53 in patients with Dukes' B and C colon cancer: a National Cancer Institute-National surgical adjuvant breast and bowel project collaborative study . J Clin Oncol . 2003 ; 21 ( 2 ): 241 - 50 .
26. Vinicius DL , Scapulatempo C , Perpetuo NM , Mohamed F , de Carvalho TS , de Oliveira ATT , Segalla JGM , Carvalho AL . Prognostic and risk factors in patients with locally advanced cutaneous squamous cell carcinoma of the trunk and extremities . J Skin Cancer . 2011 ; 2011 : 420796 .
27. Chung H , Jang M , Jung K , Won Y , Shin H , Kim J. LEE HP : Cervical cancer incidence and survival in Korea: 1993 - 2002 . Int J Gynecol Cancer . 2006 ; 16 ( 5 ): 1833 - 8 .
28. Chen C -A, Cheng W-F, Wei L-H , Su Y-N , Hsieh C-Y. Radical hysterectomy alone or combined with neoadjuvant chemotherapy in the treatment of early stage bulky cervical carcinoma . J Formos Med Assoc . 2002 ; 101 ( 3 ): 195 - 202 .
29. Luo W , Fang W , Li S , Yao K. Aberrant expression of nuclear vimentin and related epithelial-mesenchymal transition markers in nasopharyngeal carcinoma . Int J Cancer . 2012 ; 131 ( 8 ): 1863 - 73 .
30. Gilles C. POLETTE M , Piette J , DELVIGNE AC , Thompson EW , FOIDART JM , BIREMBAUT P: Vimentin expression in cervical carcinomas: association with invasive and migratory potential . J Pathol . 1996 ; 180 ( 2 ): 175 - 80 .
31. Helland A , Karlsen F , Due E , Holm R , Kristensen G. Mutations in the TP53 gene and protein expression of p53, MDM 2 and p21/WAF-1 in primary cervical carcinomas with no or low human papillomavirus load . Br J Cancer . 1998 ; 78 ( 1 ): 69 .
32. Denk C , Butz K , Schneider A , Dürst M , Hoppe-Seyler F . p53 mutations are rare events in recurrent cervical cancer . J Mol Med . 2001 ; 79 ( 5-6 ): 283 - 8 .
33. Oh M-J , Choi J-H , Lee YH , Lee JK , Hur JY , Park YK , Lee KW , Chough SY , Saw H-S. Mutant p53 protein in the serum of patients with cervical carcinoma: correlation with the level of serum epidermal growth factor receptor and prognostic significance . Cancer Lett . 2004 ; 203 ( 1 ): 107 - 12 .
34. Kahn HJ , Marks A. A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors . Lab Invest . 2002 ; 82 ( 9 ): 1255 - 7 .
35. Dumoff KL , Chu C , Xu X , Pasha T , Zhang PJ , Acs G . Low D2-40 immunoreactivity correlates with lymphatic invasion and nodal metastasis in early-stage squamous cell carcinoma of the uterine cervix . Mod Pathol . 2005 ; 18 ( 1 ): 97 - 104 .
36. Martin-Villar E , Fernandez-Munoz B , Parsons M , Yurrita MM , Megias D , Perez-Gomez E , Jones GE , Quintanilla M. Podoplanin associates with CD44 to promote directional cell migration . Mol Biol Cell . 2010 ; 21 ( 24 ): 4387 - 99 .
37. Yoshida GJ . The heterogeneity of cancer stem-like cells at the invasive front . Cancer Cell Int . 2017 ; 17 : 23 .
38. Schlüter C , Duchrow M , Wohlenberg C , Becker M , Key G , Flad H-D , Gerdes J. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins . J Cell Biol . 1993 ; 123 ( 3 ): 513 - 22 .
39. López-Guerrero JA , Machado I , Scotlandi K , Noguera R , Pellín A , Navarro S , Serra M , Calabuig-Fariñas S , Picci P , Llombart-Bosch A . Clinicopathological significance of cell cycle regulation markers in a large series of genetically confirmed Ewing's sarcoma family of tumors . Int J Cancer . 2011 ; 128 ( 5 ): 1139 - 50 .
40. Yerushalmi R , Woods R , Ravdin PM , Hayes MM , Gelmon KA. Ki67 in breast cancer: prognostic and predictive potential . Lancet Oncol . 2010 ; 11 ( 2 ): 174 - 83 .
41. Davidson B , Goldberg I , Lerner-Geva L , Gotlieb WH , Ben-Baruch G , Novikov I , Kopolovic J . Expression of topoisomerase II and Ki-67 in cervical carcinoma-clinicopathological study using immunohistochemistry . Apmis . 2000 ; 108 ( 3 ): 209 - 15 .
42. Yoshida GJ , Saya H . Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation . Biochem Biophys Res Commun . 2014 ; 443 ( 2 ): 622 - 7 .