|Ahead of print publication
Epithelial–mesenchymal transition in serous and mucinous epithelial tumors of the ovary
Geet Bhuyan, Rashmi Arora, Charanjeet Ahluwalia, Preeti Sharma
Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
Department of Pathology, 4th Floor, College Building, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Context: The concept of epithelial–mesenchymal transition (EMT) in cancer origin, progression, and metastasis is of recent origin and not fully understood. So far, many cell culture studies have been done to investigate the role of EMT in epithelial ovarian cancer, but only a few human studies have been conducted.
Aims: The aim of the study is to study the expression of E-cadherin and vimentin in serous and mucinous tumors of the ovary and to compare their expression in benign and malignant serous and mucinous ovarian tumors.
Methods: This study was a prospective study done on 60 patients with a histological diagnosis of serous and mucinous ovarian malignancy. The study was conducted in the Department of Pathology and Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi. The expression of epithelial marker E-cadherin and mesenchymal marker vimentin was studied in each of the tumors.
Statistical Analysis: Unpaired t-test/Mann–Whitney test, Chi-Square test/Fisher's exact test, and Kolmogorov–Smirnov test were used.
Results: Of the total 60 cases included in the study, 30 benign and 30 malignant cases of serous and mucinous tumors were taken. Of the 30 benign cases, 22 cases (73.3%) were that of serous cystadenomas, whereas 8 (26.67%) cases were of mucinous cystadenomas. Among the malignant cases, 21 cases (70%) were serous surface epithelial ovarian carcinoma, whereas 9 (30%) were mucinous surface epithelial ovarian carcinoma. Subsequently, the malignant cases were graded according to their glandular differentiation. Immunohistochemistry was performed in each of the 60 cases
Conclusion: In the malignant cases with increasing grade of the tumor, a reduced expression of E-cadherin and an increased expression of vimentin were seen in the epithelial cells.
Keywords: Epithelial–mesenchymal transition, mucinous tumors, serous tumors
| > Introduction|| |
Ovarian cancer is the seventh most common cancer in women worldwide with approximately 200,000 cases diagnosed annually and 115,000 deaths occurring due to it. Among the ovarian tumors, the surface epithelial tumor of the ovary is the most common primary neoplasm of the ovary.
It has been suggested that the majority of surface tumors of the ovary arise not from the outer epithelium itself but rather from the portion of this epithelium that has invaginated to produce surface epithelial glands and cysts. The common histological types are serous, mucinous, endometrioid, and clear cell type.
Epithelial–mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal cell characteristics which are characterized by reduced cell adhesiveness and increased cell motility. It is seen that there is a transition of epithelial cells to mesenchymal cells, which promotes tumor progression and metastasis.
EMT is seen in various physiological processes. During embryogenesis, EMT facilitates the migration of cells through the extracellular environment and settlement in areas of new organ formation. Fibroblasts and immune cells during an inflammatory response release cytokines and other pro-inflammatory factors which result in stimulation of cells to undergo EMT resulting in fibrosis and organ damage.
| > Methods|| |
We studied 60 patients with a diagnosis of benign and malignant serous and mucinous surface epithelial ovarian malignancy. The tissue was obtained from patients who had undergone total/pan-hysterectomy or oophorectomy in the Department of Obstetrics and Gynaecology, Safdarjung Hospital, New Delhi, during a period of 2014–2015. None of the patients had received any preoperative treatment before their surgery. Clinicopathological data were obtained which included the age, presenting symptoms, any significant past history, and clinical examination.
Formalin-fixed paraffin-embedded tumor samples were prepared and these were categorized into two main groups – benign surface epithelial ovarian tumors (n = 30) and malignant surface epithelial ovarian tumors (n = 30). All histological slides were reviewed for the histological diagnosis and grading of the tumor. Tumor classification was done according to the WHO classification of ovarian tumors, 2013. Representative blocks were chosen for immunohistochemistry (IHC).
Two markers were selected to evaluate the expression of proteins in EMT. This included E-cadherin, an epithelial marker, and vimentin which is a known mesenchymal marker [Table 1]. Representative paraffin sections of the 60 benign and malignant tumor tissues were selected. Tissue sections of 4 μ were used on poly-L-lysine-coated slides for IHC analysis.
The sections on the slides were marked using a diamond pencil. The 3-μM sections were deparaffinized by putting on a hot plate and then by dipping them in xylene, hydrated with graded ethanol followed by water, placed in 3% hydrogen peroxide in methanol (hydrogen peroxide block) for 30 min. Antigen retrieval was done using 10-mM citrate buffer (pH 6.0) for 9–10 min. The slides along with the buffer were allowed to cool down. Three washes with Tris buffer were given followed by 5% milk block. Three washes with Tris buffer were given. The tissue was incubated with primary antibody overnight at 4° centigrade. Three washes with Tris buffer were given. Secondary antibody (biotinylated goat antipolyvalent antibody) was added for 20 min followed by Tris. Tertiary antibody (peroxidase-labeled streptavidin–peroxidase complex) was added for another 20 min followed by 3,3'-Diaminobenzidin (DAB). The slides were immersed in water as soon as crisp golden brown nuclear staining was seen. The slides were counterstained with hematoxylin. They were then dehydrated in graded alcohol solutions. They were then passed through xylene which acted as a clearing agent.
One thousand tumor cells were evaluated with a magnification of ×40. The tissue sections were evaluated semiquantitatively, assessing the intensity and localization including membrane, cytoplasm, or nuclear staining.
For E-cadherin and vimentin, a staining intensity scale of 0–3 was applied and the percentage of tumor cells expressing the marker was noted on a score of 0–3. Cells positive showed a membranous positivity for E-cadherin. Vimentin, on the other hand, showed a diffuse cytoplasmic positivity.
For E-cadherin, a score 0 was given when none of the cells showed positive staining, 1 when 1%–10% of the cells were positive, 2 when 11%–50% of the cells were positive, and 3 when >50% of the tumor cells were positive. A score of 1 was given for mild staining intensity, 2 for moderate staining, and 3 when the positivity was strong. For vimentin, when <5% of the cells were positive, a score of 0 was given and 1 when >5% of the cells were positive. Similarly, the staining intensity of the tumor cells was noted, and a score of 1 was given for mild, 2 for moderate, and 3 when then the staining was strong.
A combined score was given, summing the average percentage of tumor cells expressing the marker and the intensity with which the tumor cells expressed it. Both membranous and cytoplasmic staining of E-cadherin was noted. However, only membranous was taken into account for statistical analysis. The cutoff combined score was taken as 4. Score of μ4 was reported as strong expression and <4 was reported as reduced expression. Diffuse cytoplasmic positivity was noted for vimentin. The final expression of vimentin was reported as expressed and not expressed. The cutoff was taken as 1. A score of μ1 was taken as expressed and <1 was taken as not expressed.
All samples used in this study were collected from the Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi. Ethical principles related to patients' rights and privacy are fully respected by authors in accordance with the norms laid down by the Ethical Committee of Safdarjung Hospital, New Delhi.
Categorical variables were presented in number and percentage (%) and continuous variables were presented as mean ± standard deviation and median. Normality of data was tested by Kolmogorov–Smirnov test. If the normality was rejected, then nonparametric test was used.
Quantitative variables were compared using unpaired t-test/Mann–Whitney test (when the data sets were not normally distributed) between the two groups. Qualitative variables were correlated using Chi-square test/Fisher's exact test. Diagnostic test was used to find out the sensitivity, specificity, negative predictive value, and positive predictive value for predicting benign and malignant of E-cadherin expression and vimentin expression. P < 0.05 was considered statistically significant.
The data were entered in MS Excel spreadsheet and analysis was done using the Statistical Package for the Social Sciences (SPSS) version 21.0 (IBM Corp, Armonk, NY, USA).
| > Results|| |
Of the total 60 cases included in the study, the 30 cases were of benign surface epithelial ovarian tumors and 30 of malignant surface epithelial ovarian carcinomas. Of the 30 benign cases, 22 cases (73.3%) were that of serous cystadenomas, whereas 8 (26.67%) cases were of mucinous cystadenomas. Similarly, for the malignant cases, 21 cases (70%) were serous surface epithelial ovarian carcinoma, whereas 9 (30%) were mucinous surface epithelial ovarian carcinoma.
The overall age for all ovarian tumors ranged from 21 to 63 years with a mean age of diagnosis of 39 ± 11.24 years. The age range in the benign cases was 21–60 years with a median age of diagnosis of 34.63 ± 10.41 years. The age range in the malignant cases was 25–63 years with a median age of diagnosis of 43.37 ± 10.44 years [Table 2].
The malignant serous and mucinous tumors were divided into well-differentiated, moderately differentiated, and poorly differentiated tumor according to the glandular differentiation of the tumors. Of the 30 malignant cases, 19 cases (63.3%) were well-differentiated, 6 cases (20%) were moderately differentiated, and 5 cases (16.67%) were that of poorly differentiated carcinomas.
IHC was performed and the expression of EMT markers – E-cadherin and vimentin – was correlated with type and grade of tumor. E (epithelial) cadherin is a transmembrane cell adhesion molecule with a molecular weight of 120 kDa. It plays an important role in the intercellular adhesion of epithelial cells and the establishment of epithelial polarization, glandular differentiation, and stratification. E-cadherin expression was studied in all the cases. Out of the 30 benign cases, all (30/30) showed a strong expression of E-cadherin. On the other hand, of the 30 malignant ovarian tumors studied, 25 (83.33%) showed strong E-cadherin expression. About 5 (16.67%) of the malignant cases showed reduced E-cadherin expression. Thus, stronger expression of E-cadherin was found in benign tumors as compared to malignant tumors. This correlation was statistically significant with P < 0.05 [Table 3].
The E-cadherin expression was studied separately for the benign serous and mucinous tumors. All the cases (100%) showed strong E-cadherin expression in both categories, i.e., serous and mucinous benign tumors. However, of the nine malignant mucinous tumors, seven cases (77.78%) showed strong expression for E-cadherin, while two cases (22.2%) showed a reduction in E-cadherin expression. Eighteen (85.71%) malignant serous tumors strongly expressed E-cadherin, and the remaining 3 (14.28%) cases showed reduced expression.
The expression of E-cadherin was also correlated with the grade of the malignant tumors. Out of the 19 cases of well-differentiated malignant tumors, all (100.0%) cases showed a strong E-cadherin expression. Out of 6 cases of moderately differentiated tumors, 4 (66.67%) cases showed a strong expression for E-cadherin, while 2 (33.33%) cases showed a reduced E-cadherin expression. Among the poorly differentiated tumors, 2 (40%) cases showed strong E-cadherin, whereas 3 (60%) showed a reduction in the expression of E-cadherin [Figure 1]. Thus, the expression of E-cadherin reduced with an increase in the grade of the tumor. Thus, an association of E-cadherin expression and grade of the malignant tumors was found to be statistically significant with P = 0.003 [Table 4].
|Figure 1: (a) Tumors cells in well-differentiated cystadenocarcinoma arranged in well-formed papillae. (b) Membranous staining with E-cadherin in well-differentiated adenocarcinoma. (c) Moderately differentiated cystadenocarcinoma. (d) E-cadherin staining of reduced intensity in moderately differentiated adenocarcinoma. (e) Poorly differentiated serous cystadenocarcinoma. (f) E-cadherin staining is almost absentin the epithelial cells of poorly differentiated tumor|
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|Table 4: E-Cadherin expression in malignant tumours according to differentiation|
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Vimentin is 57-kDa intermediate filament protein which forms part of the cytoskeleton of vertebrate cells and is characteristically found in cells of mesenchymal origin. The expression of vimentin was studied in all the 60 cases. Of the 30 benign (both serous and mucinous) cases, none of the cases showed any staining for vimentin. On the other hand, of the 30 malignant ovarian tumors studied, 9 (30%) cases showed expression of vimentin. About 21 (70%) malignant cases showed no expression of vimentin. Thus, only the malignant cases showed expression for vimentin. This correlation was statistically significant with P = 0.002 [Table 5].
Out of the 9 malignant mucinous tumors, 3 cases (33.33%) showed expression for vimentin, while 6 (28.57%) out of the 21 malignant serous tumors showed expression for vimentin.
The expression of vimentin was studied in all of the malignant tumors according to the grade of the tumor. Out of the 19 cases of well-differentiated malignant tumors, only 1 (5.26%) case showed an expression for vimentin, while 18 (94.73%) showed no expression at all. About 3 (50%) moderately differentiated tumors showed expression for vimentin, while among the poorly differentiated tumors, all 5 (100%) cases showed an expression for vimentin [Figure 2]. Thus, vimentin expression was observed with increasing grade of malignant surface epithelial ovarian tumor. Hence, the association of vimentin expression and grade of the malignant tumors was found to be statistically significant with P = 0.0001 [Table 6].
|Figure 2: (a) Vimentin staining is absent in the epithelial cell of a well-differentiated adenocarcinoma. (b) Vimentin staining in few of the epithelial cells of moderately differentiated adenocarcinoma. (c) Vimentin staining of high intensity in poorly differentiated adenocarcinoma|
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|Table 6: Vimentin expression in malignant tumours according to differentiation|
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The correlation between E-cadherin and vimentin expression was studied. It was seen that all of the 5 cases which showed a reduced expression of E-cadherin showed an expression of vimentin. However, out of the total 55 cases which showed a strong expression of E-cadherin, only 4 cases showed vimentin expression [Table 7].
| > Discussion|| |
Metastasis is the cause of mortality in most of the malignant tumors including ovarian carcinoma. Chemotherapy and debulking surgery is the preferred modality of treatment for advanced cases which is as per the ESMO recommendations for the treatment of ovarian carcinomas. However, patients on chemotherapy present with a variety of toxic side effects. The hunt for the “metastasis signature” and the “metastatic oncogene” has pointed in the direction of EMT. The role of various genes involved in EMT and favoring a promigratory phenotype has been demonstrated in various epithelial cancers.
A host of markers are known to be upregulated/downregulated in the process of EMT. However, only a few studies have been done to study this phenomenon in ovarian tumors. This study has thrown light on that a combination of epithelial and mesenchymal markers provides greater accuracy for predicting metastatic potential of a tumor and chances of future recurrence and mortality.
E-cadherin is a transmembrane cell adhesion molecule. It plays a significant role in the cellular intercellular adhesions of epithelial cells and the establishment of epithelial polarization, glandular differentiation, and stratification. A reduction or loss of E-cadherin is recognized as an important primary event in metastasis and is often linked to poor prognosis.
In a study by Voutilainen et al., the tumor samples from surface epithelial ovarian tumors showed a reduction in the E-cadherin expression. It was further seen that the reduced cell surface expression of E-cadherin predicted poor disease-related survival or recurrence-free survival or both.
In our study, strong expression of E-cadherin was noted in all the 30 cases of benign surface epithelial ovarian tumors. On the other hand, out of the 30 malignant cases, 25 cases showed strong expression of E-cadherin. Most of these cases (63.3%) were well-differentiated carcinomas which showed strong E-cadherin expression. Among the moderately differentiated tumors which comprised 6 (20%) of the 30 malignant cases, 2 cases (40%) showed a reduced E-cadherin expression, while the other 4 cases showed a strong expression of E-cadherin. In the poorly differentiated tumors, 3 cases (60%) showed a reduced E-cadherin expression, while only 2 cases showed a strong E-cadherin expression. Thus, it was seen that as there was an increase in the grade of the tumor, there was a decrease in the expression of E-cadherin (P = 0.003).
In a previous study by Sundfeldt et al., it was seen that there was a decrease in the expression of E-cadherin in the malignant epithelial ovarian tumors in contrast to the benign cases which showed a strong expression. It was further noted that the poorly differentiated cases showed limited or no staining for E-cadherin. This is similar to our findings that we have noted in the present study.
Intermediate filaments are a family of proteins that form one of the tripartite components of the cytoskeleton. They are expressed in a highly regulated manner in terms of tissue and developmental stage specificity and have important functions in human health and disease. Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. Vimentin regulates cell migration in many cell types. In fibroblasts, vimentin filaments connect the nucleus to the plasma membrane and contribute to the formation of vimentin-associated matrix adhesions that are dynamically turned over in migrating cells. Vimentin can also be expressed in epithelial cells undergoing EMT both under physiological and pathological situations.
Vimentin expression has been linked to high metastatic potential of the tumor due to motile mesenchymal phenotype. Accordingly, in this study, none of the benign tumors demonstrated vimentin expression. Vimentin was expressed in 9 cases (30%) of the total 30 malignant tumors. Out of these 9 cases, 3 were moderately differentiated. Therefore, 3 (50%) out of the total 6 moderately differentiated cases showed a reduced vimentin expression. All the 5 (100%) cases of poorly differentiated cases showed an expression of vimentin. It was seen that vimentin expression was related to the grade of the tumor and an increase in expression of vimentin was seen as the grade increased (P = 0.0001). Thus, the expression of vimentin was confined to high-grade tumors and it implies that the novel expression of vimentin is a late event in the pathway of EMT.
Vuoriluoto et al. studied the role of vimentin in EMT in breast cancer. It was seen that vimentin regulated certain other genes such as tyrosine kinase Axl, integrin b4, and plasminogen activator, urokinase, during the process of EMT. These genes have earlier been linked with breast cancer invasion and the basal-like phenotype.,, The results showed that vimentin functions as a regulator of Axl and enhances cell migration by inducing Axl. Therefore, Axl acts as a downstream regulator of EMT. Thus, it was seen that advanced cases of breast cancer showed an upregulation of vimentin, thus facilitating the process of EMT.
In a previous study on cervical carcinoma by Hussain et al., vimentin expression was seen in 40% of the cervical squamous cell carcinomas studied and expression pattern was increased according to the grade of cervical cancer.
Zhai et al. studied the expression of vimentin in hepatocellular carcinoma (HCC) and its role in EMT, high expression of vimentin was significantly associated with high-grade HCC, and high VIM expression was significantly associated with a high serum α-fetoprotein levels (P = 0.016) suggesting a poorer prognosis. Further, the authors examined the co-expression of vimentin with E-cadherin and it was seen that a low E-cadherin expression was significantly associated with overexpression of vimentin (P = 0.001).
The above studies suggest an increase in the expression of vimentin in various malignant epithelial tumors. Due to its expression in tumors of higher grade, we can infer that the expression of vimentin may have a possible role in the later stages of the pathway of EMT. Although the literature regarding the expression of vimentin in surface epithelial ovarian carcinoma is scant, our results in the present study are similar and are in agreement to its expression as seen in other malignant epithelial carcinomas.
EMT is accompanied by altered expression of a wide variety of epithelial/mesenchymal markers. The loss or gain of these markers at a given point of time may not be complete. It is a dynamic process with a continuum of changes occurring in sequential fashion. The loss of E-cadherin and the novel expression of vimentin (cadherin switch) represent defining features of EMT.
The present study demonstrates altered immunoexpression of E-cadherin and vimentin in higher grade of surface epithelial ovarian carcinoma. Statistically significant association of E-cadherin and vimentin was noted with the grade of surface epithelial ovarian carcinoma. Since these markers form part of the spectrum of changes associated with EMT, the study establishes proof of concept of existence of this process in vivo.
Since the discovery of the role of EMT in cancer progression, numerous studies have been done related to the subject. Further studies on EMT can lead elucidation of those molecules which are central to regulation of this process. Comparing the expression of various markers of EMT in benign and various grades of malignant surface epithelial ovarian tumors can help in identifying a prognostic marker for tumors that are in high risk of progression. However, studies on larger cohorts are needed to establish their true biological impact in the course of the disease of various malignancies. This could lead us one step forward in the direction of targeted therapy in ovarian malignancy.
| > Conclusion|| |
Ovarian cancer is a leading cause of morbidity and mortality in women. Among the ovarian carcinomas, surface epithelial tumors form majority of the cases.
The concept of EMT states that the epithelial cells acquire mesenchymal phenotype to migrate and disseminate to distant places. This phenomenon is accompanied by change in differentiation markers. This acquisition of markers of mesenchymal phenotype might help in predicting the aggressive behavior of malignant tumors.
This study evaluated the expression of epithelial and mesenchymal markers, E-cadherin and vimentin, in surface epithelial ovarian tumors. The expression of these markers was also noted in different grades of the malignant tumors. Thus, this study evaluated the role of EMT by comparative evaluation of immunohistochemical expression of the above markers in benign and different grades of malignant surface epithelial ovarian tumors.
Downregulation of epithelial adhesion molecule E-cadherin is a hallmark of EMT. In the present study, strong E-cadherin expression was seen in the benign cases. Similar strong expression was seen in the well-differentiated malignant cases. However, a reduced expression was noted with the increasing grade of the tumor (P ≤ 0.05). Thus, the moderately differentiated and poorly differentiated malignant cases showed a reduced E-cadherin expression.
Vimentin expression is linked to high invasive ability of the tumor due to motile mesenchymal phenotype. Accordingly, in this study, none of the benign tumors demonstrated vimentin expression. Novel vimentin expression was confined to higher grades of the malignant cases (P ≤ 0.05).
Thus, we conclude that the benign surface epithelial ovarian tumors show a normal expression of E-cadherin and an absence of vimentin in the epithelial cells. However, in the malignant cases with increasing grade of the tumor, there is a reduction in expression of E-cadherin and the epithelial cells begin to express vimentin. Since these markers form part of the spectrum of changes associated with EMT, the study establishes proof of concept of the existence of this process in vivo.
We recommend further studies on a larger population size to verify the results obtained in our study for a stronger biological impact. Moreover, further research on other molecules central to the process of EMT needs to be carried out. This could lead us to one step forward in the direction of targeted therapy in the field of ovarian cancer.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]