|Year : 2015 | Volume
| Issue : 4 | Page : 893-898
Evaluation of stromal myofibroblasts in oral leukoplakia, oral submucous fibrosis, and oral squamous cell carcinoma - an immunohistochemical study
Kanupriya Gupta1, Rashmi Metgud2, Jatin Gupta3
1 Department of Oral and Maxillofacial Pathology, Mithila Minority Dental College and Hospital, Darbhanga, Bihar, India
2 Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
3 Oral Medicine and Radiology, Mithila Minority Dental College and Hospital, Darbhanga, Bihar, India
|Date of Web Publication||15-Feb-2016|
Assistant Professor, Department of Oral and Maxillofacial Pathology, Mithila Minority Dental College and Hospital, Mansukhnagar, Ekmighat, Lehariyasarai, Darbhanga, Bihar - 846 004
Source of Support: None, Conflict of Interest: None
Background: Oral leukoplakia (OL) and oral submucous fibrosis (OSMF) are the main potentially malignant disorders and oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral mucosa. Myofibroblasts (MFs) secrete numerous growth factors and inflammatory mediators that stimulate epithelial cell proliferation and play an important role in tumoral invasion and use a combination of different factors in the course of neoplastic growth and development. Hence the present study was undertaken to evaluate and compare the distribution of MFs using alpha smooth muscle actin (α-SMA) in OL, OSMF, and various histopathological grades OSCC.
Materials and Methods: Sixty formalin-fixed paraffin-embedded tissue blocks consisting of histopathologically diagnosed cases of normal mucosa (n = 10), OL (n = 14) hyperkeratosis with various grades of dysplasia, OSMF (n = 11), and OSCC (n = 25) were subjected to immunohistochemistry using α-SMA antibody for detection of MFs.
Results: MFs were not detected in normal oral mucosa. On comparison of frequency of mean scores in OL, OSMF, and OSCC the values were 0.6 ± 0.2 (0-2), 1.2 ± 0.68 (1-2), and 2.6 ± 1.34 (0-4), respectively. The results were statistically significant (P < 0.001).
Conclusion: These findings are suggestive of role of MFs with the creation of a permissive environment for tumor invasion in OSCC. Hence the presence of MF is a prognostic marker and evaluation of the frequency in the stroma can be used as therapeutic targets.
Keywords: Immunohistochemistry, myofibroblasts, tumor microenvironment
|How to cite this article:|
Gupta K, Metgud R, Gupta J. Evaluation of stromal myofibroblasts in oral leukoplakia, oral submucous fibrosis, and oral squamous cell carcinoma - an immunohistochemical study. J Can Res Ther 2015;11:893-8
|How to cite this URL:|
Gupta K, Metgud R, Gupta J. Evaluation of stromal myofibroblasts in oral leukoplakia, oral submucous fibrosis, and oral squamous cell carcinoma - an immunohistochemical study. J Can Res Ther [serial online] 2015 [cited 2020 Aug 14];11:893-8. Available from: http://www.cancerjournal.net/text.asp?2015/11/4/893/147700
| > Introduction|| |
Recent years have witnessed a paradigm shift in which the microenvironment is increasingly recognized to make a significant contribution to tumor progression.  Traditionally, the stroma of different organs is described as a structural scaffold dominated by extracellular matrix (ECM) with sparsely embedded cells. The stroma ECM includes different types of collagens, elastin, fibronectin, hyaluronic acid, proteoglycans, and glycoproteins.  In addition to playing important roles during development, and in maintaining tissue architecture, ECM components can modify the activity of growth factors and cytokines.  It can act as a reservoir for growth factors that are deposited and can be rapidly released upon cellular request. , ECM molecules protect growth factors from degradation and provide biological latency. 
Potentially malignant disorders are those in which the risk of malignancy being present in a lesion or condition either at time of initial diagnosis or at a future dates. Among these are oral submucous fibrosis (OSMF) and oral leukoplakia (OL). 
Transformation of normal oral mucosa to squamous dysplasia and ultimately squamous cell carcinoma (SCC) represents a complicated process involving numerous etiologic factors.  Approximately 10-20% of oral dysplasias develop carcinomatous features and they eventually invade beyond the basement membrane.  There is an increasing amount of interest in the molecular and biological events that occur during the transition of dysplastic epithelium to SCC. , The presence of cancer is followed by some changes that happen in epithelium and the normal stroma and normal stroma becomes a reactive one. The formation of reactive stroma is associated with the secretion of cytokines such as transforming growth factor beta-1 from cancerous cells that promotes differentiation of fibroblasts/myofibroblasts (MFs) in to cancer associated fibroblasts (CAF) exhibiting different cell biological properties compared with normal fibroblasts. This is histologically represented by a desmoplastic stroma reaction. Along with increase in the number of blood vessels, increase in the inflammatory cells, decrease in the expression of epithelial markers (cadherins), and increased expression of mesenchymal markers such as vimentin. s in turn secrete cytokines and matrix metalloproteases, which in turn contribute to the destruction of extracellular matrix and cause tumor growth. ,
There has been always a question concerning whether the stromal cells distribution in carcinogenesis is random or their distribution is involved in invasive tumor behavior and prognosis. Few studies have been carried out for unlocking this mystery in oral squamous cell carcinoma (OSCC); ,,,,,,,,,, and on literature review we found only one study that has analyzed role of stroma in OSMF according to the severity of lesion by evaluating the MFs.  Thus, the present study aimed to evaluate and inter-compare the presence and distribution of alpha smooth muscle actin (α-SMA) positive MFs in OL, OSMF, and different histopathological grades of OSCC.
| > Materials and methods|| |
This study was approved by the local ethics committee. A total of 60 formalin-fixed paraffin-embedded tissue blocks consisting of histopathologically diagnosed cases of normal mucosa (n = 10), OL (n = 14), OSMF (n = 11), and OSCC (n = 25) were retrieved from the archives of Department Of Oral Pathology.
Ten samples of normal oral mucosa were used as a control obtained from surrounding mucosa of asymptomatic impacted tooth during disimpaction. Of the 14 patients with OL, which were evaluated, 12 were men and 2 were women, with mean age of 35.5 years. In OSMF samples, all patients were males with mean age 32.27 years. In OSCC samples, out of 25 patients, 17 were men and 8 were women, with mean age of 51.96 years [Table 1]. Diagnosis was confirmed by two oral pathologists using hematoxylin and eosin-stained sections. OL cases histopathologically showed hyperkeratosis with varying degrees of dysplasia. The OSCC cases were histologically graded as well-, moderately-, and poorly differentiated.  Inflammation was minimal in the normal tissues and all were devoid of pathologic conditions.
The immunohistochemical analysis for α-SMA was performed using supersensitive one-step polymer- Horseradish peroxidase technique (Biogenex life sciences, San Ramon, CA, USA). Paraffin-embedded tissue blocks were cut into 4-5 µm thickness and taken onto 2%, 3-aminopropylethoxysilane solution (APES) (Sigma Aldrich, St. Louis, MO, USA) adhesive-coated slides. The sections were then deparaffinized and rehydrated through xylene and descending grades of alcohol. Antigen retrieval was carried out using commercial microwave antigen retrieval system where the sections were placed in a container containing 10 mM sodium citrate buffer (pH 6.0) at 96°C for three cycles of 6 min each (EZ-Retriever System, Biogenex life sciences). After rinsing in phosphate buffered saline (PBS), the sections were treated with peroxidase block consisting of 3% H 2 O 2 in water for 15 min to block the endogenous peroxidase activity. This was followed by a power block for 20 min to block any nonspecific antigenic sites. The sections were then incubated for 1 h at room temperature with optimally prediluted antibody against α-SMA (Biogenex, USA: Clone 1A4). After washing with PBS, the sections were then incubated with one-step polymer-HRP reagent for 30 min. Visualization was performed using freshly prepared diamino benzadine tetrahydrochloride (DAB). The slides were counterstained with Harris hematoxylin, subsequent to which sections were dehydrated, cleared, and mounted with dibutyl pthalate xylene (DPX). For each batch of staining, positive and negative controls were run simultaneously with the study specimens. The endothelial staining of blood vessels known to be reactive to α-SMA was used as the internal positive control, while the primary antibodies were replaced by nonimmune mouse serum at the same dilutions for the negative controls.
Scoring of immunostaining results
The cytoplasm of those α-SMA-stained MFs in the OSCC of adjacent islands and tumor layers [Figure 1] a-c] and in leukoplakia [Figure 2] and OSMF [Figure 3] located under mucosa were counted in 100 cells at 40× magnification and the calculated average number was considered as the percentage of stained cells. The α-SMA-stained endothelial cells of blood vessel were not included in the calculation. Each section was counted twice and the counting was controlled by another pathologist afterward.
|Figure 1: (a) α-SMA staining in well differentiated SCC i) Low power, ii) High power, (b) α-SMA staining in moderately differentiated SCC i) Low power, ii) High power, (c) α-SMA staining in poorly differentiated SCC i) Low power, ii) High power|
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|Figure 2: α-SMA staining in oral leukoplakia (a) Low power, (b) High power|
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|Figure 3: α-SMA staining in oral submucous fibrosis (a) Low power, (b) High power|
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Kellerman's criteria  was modified and utilized to evaluate the slides for MFs.
Score 0: No MFs
Score 1: 1-20% MFs observed
Score 2: 21-40% MFs observed
Score 3: 41-60% MFs observed
Score 4: >60% MFs observed
Considering the distribution pattern of MFs, the arrangement of positive-stained cells was classified into three groups:
1 - Focal: If MFs had a focal arrangement or had no special arrangement in different areas of connective tissue and stroma
2 - Spindle: MFs arrange in one to three rows in a regular order in the periphery of the neoplastic islands or in the connective tissues with distinctive cell margins around MFs and malignant tissue
3 - Network: MFs with vesicular nucleus and abundant cytoplasm arranged in multiple rows with interwoven network of cytoplasmic extensions forming a network in the stroma of the connective tissue.  [Figure 4] a-c].
|Figure 4: (a) α-SMA staining showing focal pattern in OSCC (a) Low power (b) High power view, (b) α-SMA staining showing spindle pattern in OSCC (a) Low power (b) High power view, (c) α-SMA staining showing network pattern in OSCC (a) Low power (b) High power view|
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The results of the study on each lesion was recorded in Statistical packages for Social Science version 17.0; SPSS Inc., Chicago, IL and analyzed using Mann-Whitney and Kruskal-Wallis tests.
| > Results|| |
The clinical data of age, sex, and the location of the lesion are summarized in [Table 1] and [Table 2].
On comparison of scores in study groups, the scores of MFs in OSCC ranged from 1 to 4 with maximum number of cases (44%) showing grade 2 expression, 85% OL, and 100% of normal mucosa did not express MF when these scores were compared using Kruskal-Wallis test in the study group, the scores were statistically significant [Table 3].
For comparison of mean scores of frequency in study groups, Mann-Whitney test was used for analysis. The mean score of OSCC was 2.6 ± 1.34, which means most of the cases had score 3 or 2 (i.e. 20-60 MFs in the stroma). The mean score of OL was 0.6 ± 0.2, which means most of the cases had score 0 or 1 (i.e. 0-20 MFs in the stroma). The mean score of OSMF was 1.2 ± 0.68, which means most of the cases had score 1 or 2 (i.e. 20-40 MFs in the stroma). The result was statistically significant [Table 4].
|Table 4: Comparison of mean scores of frequency of Myofibroblasts in the study groups |
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Comparison of mean scores in different grades of OSCC was done by Kruskal-Wallis test. The mean score of Well differentiated squamous cell carcinoma was 2.00 ± 0.94, which means that most of WDSCC expressed 20-60 MFs in their stroma, the scores of MFs of WDSCC ranged from score 1 to score 3 with maximum cases in score 2. The mean score of moderately differentiated squamous cell carcioma was 2.5 ± 1.31 and it ranged from score 1 to score 4 with maximum cases expressing score 2, that is, 21-40 MFs. The mean score of Poorly differentiated squamous cell carcinoma was 3.4 ± 1.02 suggesting that most of PDSCC expressed 40-60 MFs in their stroma. When means were compared, the difference was not statistically significant [Table 5].
|Table 5: Comparison of mean scores of Myofibroblasts in histopathological grades of oral squamous cell carãnoma |
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On comparison of scores of expression MFs in all study groups using Mann-Whitney test relationship between cellular distribution and the lesion type (premalignant and malignant types) was statistically significant (P < 0.05) [Table 6].
|Table 6: Intergroup comparison for scores of expression of Myofibroblasts |
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| > Discussion|| |
The term MFs was introduced in the early 1970s to name fibroblasts that, under certain conditions as granulation tissue contraction, are capable of modulating toward a cell type structurally and functionally close to smooth muscle.  MF expression, as highlighted by α-SMA, is undetectable in normal oral mucosa and low resistant epithelial dysplasia but increases as the disease progresses from potentially malignant disorders as high grade epithelial dysplasia to OSCC. Thus, proliferation of MFs may be used as a stromal marker of oral premalignancy and malignancy. 
In the present study when α-SMA-stained slides of OSCC (n = 25) were evaluated for frequency of MF expression, we found that α-SMA positive MFs were expressed in all cases of OSCC. These results were in agreement with those of Vered et al.,  Chaudhary et al.,  Vered et al.,  and Moghadam et al. who have reported 98%, 97%, 100%, and 100% tumors, respectively, expressing MFs. Whereas Kellerman et al. (2008), Safora Seifi et al.,  and Kapse et al. have reported the presence of α-SMA in 60%, 67%, and 70% tumors only, respectively.
The frequency of MFs in OSCC ranged from 1% to 20% to more than 60% MFs (from score 1 to score 4) and the mean score being 2.6 ± 1.34, that is, most of the OSCC had more than 40% of MFs in the stroma. The findings are in agreement with those of Seifi et al. and Chaudhary et al.,  but Vered et al. reported much lesser frequency; this may be attributed to the difference of counting and use of double immunostaining. Vered et al. studied combined immunostained slides of OSCC with α-SMA and epithelial membrane antigen (EMA), to study epithelial-mesenchymal transition and prove that MFs originate from epithelial cells. They have considered only those cells that expressed for both EMA and α-SMA to be MF. Hence because of difference of methodology, the frequency of MFs reported by them has varied considerably.
In the present study, MFs were found surrounding the tumor islands and cords in the stroma, and often in the deep invasive front of the tumors. Some tumors expressed only a few MFs in delicate rows surrounding and abutting the tumor islands, while others showed abundance of MF in the stroma, which were organized in syncytium. These differences might reflect the biological behavior of the tumors. Vered et al., proposed that higher the number of MFs, more aggressive is the tumor with increased recurrence and poor survival rate. This can be attributed to the fact that the MFs are 'factories' of a range of cytokines and growth factors, such as matrixmetalloproteases (MMPs), vascular endothelial growth factors (VEGFs), fibroblast growth factor (FGF). Through production of cytokines, chemokines, and proteolytic enzymes MFs modulate the tumor stroma. They boost tumor angiogenesis, drive tumor invasion, and metastasis thereby helping tumor progression. Increased frequency of MFs in the stroma has been associated with poor prognosis because of increased recurrence and reduced survival. Hence the carcinomas with higher expression of MF might be more aggressive and have poor prognosis, and hence are recommended for aggressive treatment and close follow-up.
In different grades of carcinoma, a slight difference in the mean expression of MFs was observed. The mean scores of MF for WDSCC, MDSCC, and PDSCC were 2.00 ± 0.94, 2.5 ± 1.31, and 3.4 ± 1.02, respectively. But when the expression of MF was compared among various grades of OSCC, the differences were not statistically significant. Moghadam et al. and Kellermann et al. reported similar results. The expression of MF was slightly higher in PDSCC than other grades but larger sample size is necessary to illustrate the true difference and significance.
MFs are part of tumor milieu and they fulfill tumor needs in terms of angiogenesis, production of metalloproteinases for collagen breakdown, and further invasion and suppression of the host immune response. Vered et al. were able to demonstrate the presence of tumor cells undergoing epithelial mesenchymal transition in cases of human tongue carcinoma by using a double immunostaining technique using epithelial membrane antigen and α-SMA. This was further supported by using the triple immunostaining procedure, which showed that loss of expression of E-cadherin (found only in epithelial cells and is responsible for cell to cell junction) was a frequent event among the carcinoma cells. Furthermore, the high plasticity of the neoplastic phenotype can produce an inverse process of mesenchymal-to-epithelial transition (MET) whereby the carcinoma-derived stromal cells regain epithelial characteristics and establish new foci of tumor.
Transformation of normal oral mucosa to squamous dysplasia and ultimately to OSCC represent a complicated process involving numerous etiological factors. There is an increasing amount of interest in the molecular and biological events that occur during the transition of dysplastic epithelium to OSCC.
In the present study, when α-SMA stained slides of OL (n = 14) were evaluated for frequency of MF expression, we found that α-SMA positive MFs were expressed in two cases of OL. These results were in agreement with those of Seifi et al.,  Vered et al.,  Chaudhary et al.,  and Kapse et al.,  whereas Eliene-Magda de-Assis,  Moghadam et al.,  and Vered et al. have reported complete absence of MF.
The frequency of MFs in OL ranged from 0% no MFs to 21-40% MFs (from score 0 to score 2) and the mean score being 0.6 ± 0.2 (0-2), that is, most of the OL had absence of MFs in the stroma. The cases of OL, which showed higher degree of dysplasia, showed positivity. Statistical evaluation was not performed for the different grades of intraepithelial dysplasias due to the small number of cases.
In the present study when α-SMA-stained slides of OSMF (n = 12) were evaluated for frequency of MF expression, we found that α-SMA-positive MFs were expressed in all cases of OSMF. These results were in agreement with those of Angadi et al. who found the frequency of α-SMA positive cells to be 86%. This was the only study to our best knowledge in the English literature that has evaluated MFs in OSMF.
No study till now has simultaneously evaluated the frequency of MFs in OL, OSMF, and OSCC. As for OL, the rate of malignant transformation ranges from 0.13% to 2.2% per year. For OSMF, increased risk of undergoing carcinomatous changes ranges from 4% to 8%, this is known to be more than that of leukoplakia.
A major issue in this study is that there was no way of knowing whether the examined dysplasias would have transformed into SCCs or remained as dysplastic entities. Another problem was that we were unable to obtain samples of carcinoma in situ and superficially invasive SCC. This would have been helpful to draw probable conclusions as to where and when MFs are first observed.
Considering the lack of MFs in normal, dysplastic oral epithelium and their appearance in OSMF and OSCC, it seems that the genetically altered epithelium (carcinomatous epithelium) may have an inductive effect on the adjacent stroma to produce MFs. However, more sophisticated techniques are suggested to further clarify the exact mechanism by which these important cellular elements exert their effects on stromal and epithelial tissue compartments. In the event that our findings are confirmed by future investigations, therapeutic targeting of MFs, their byproducts or factors responsible for their transdifferentiation from fibroblasts may be beneficial to OSCC patients.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]