Different Grades Of Oral Submucous Fibrosis Research Articles

Evaluation of extracellular matrix changes among oral submucous fibrosis and oral squamous cell carcinoma patients of Malwa region of Punjab using special histochemical stains: An insight into cancerous transformation.

To assess extracellular matrix changes among oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC) patients using special histochemical stains. Twenty biopsy specimens of OSMF and 30 biopsy specimens of OSCC were included in the present study. Among 20 OSMF specimens, 10 were of early OSMF and the remaining 10 were of advanced OSMF. Out of 30 OSCC specimens, 10 cases each were of well-differentiated OSCC, moderately differentiated OSCC and poorly differentiated OSCC. Three sections, each 4 μm thick, were obtained from all specimens. One section was stained with routine H&E staining, whereas the other section was stained with Masson's trichrome (MT) stain for collagen and Verhoeff-Van Gieson (VVG) for elastic fibres. Evaluation of all specimens was performed under the light microscope. The arrangement of collagen fibres and elastic fibres was compared between the OSMF group and OSCC group, in between different grades of OSMF and in between different grades of OSCC. The results were evaluated using SPSS software. Early OSMF cases were associated with fibrosis in the superficial lamina propria, whereas advanced OSMF had fibrosis involving deeper muscle fibres. In all early OSMF cases, elastic fibres were arranged in thin bundles, whereas in advanced OSMF cases, elastic fibres were in thick bundles. In well- and moderately differentiated OSCCs, the collagen fibres were arranged in thick bundles and in poorly-differentiated OSCCs, the collagen fibres appeared to be fragmented. The elastic fibres in well-differentiated OSCC and moderately-differentiated OSCC were thickly arranged, and poorly-differentiated OSCC showed thin fibres and 70% of cases showed the absence of elastic fibres. Changes observed in both collagen and elastic fibres in the extracellular matrix (ECM) can be taken as a study model to further understand the progression of OSMF to OSCC using histochemical stains.

Analysis of Expression of Myofibroblasts in Oral Submucous Fibrosis: An Immunohistochemistry Study

Background: Oral submucous fibrosis (OSMF) is a chronic disease that produces tissue fibrosis and is considered to be a potentially malignant disorder. The exact pathogenesis and malignant conversion mechanism of this disorder are still unknown. Myofibroblasts have been implicated as one of the possible pathological mechanisms responsible for the pathophysiology of OSMF. The present study was conducted to evaluate the expression of myofibroblasts (MF) in normal mucosa and different grades of OSMF. Materials & Methods: The sample consisted of a total of 80 specimens. The study group included specimens from clinically and histopathologically confirmed OSMF patients. The specimens were divided into four groups. Group 1 consisted of 19 specimens of grade III OSMF. Group II had 20 specimens of grade II OSMF, Group 3 with 21 specimens of grade I OSMF, and Group 4 constituted a control group of 20 normal epithelium specimens. Two sections each from all the four groups were obtained. While one section was stained with H and E, the other section was stained immunohistochemically using α-smooth muscle antibody. For analysis, the expression of myofibroblasts was categorized as strong, moderate, weak, or absent. All the results were recorded and subjected to statistical analysis. Results: In OSMF patients, irrespective of the grade, the expression of myofibroblast was strong in 28.33 percent of the patients, while it was moderate and weak in 30.00 percent and 40.00 percent of the patients, respectively. Expression of myofibroblast was noted to be significantly increased in grade III OSMF patients as compared to controls as well as grade I OSMF patients (p-value <0.05). Conclusion: Myofibroblasts expression is significantly raised in OSMF patients. The expression can also be correlated within different grades of OSMF where advanced stages show comparatively high expression of these smooth muscles like fibroblasts. Hence, we suggest that myofibroblasts could be assessed as markers for analyzing the progression of OSMF.

New Insights for Consummate Diagnosis and Management of Oral Submucous Fibrosis Using Reactive and Reparative Fibrotic Parameter Derived Algorithm.

Objective:Reproducibility of qualitative changes in histopathological diagnosis involving narrow variation is often challenging. This study aims to characterize the histological fibrotic events in detail so as to derive an in-depth multiparametric algorithm with individually quantified histological parameters for effective monitoring of the. disease process in oral submucous fibrosis and for potential therapeutic targets for early intervention.Methods:Formalin fixed paraffin embedded (FFPE) blocks of oral submucous fibrosis (OSMF), were taken and sections were stained with Hematoxylin & Eosin stain and Masson Trichrome stain. Photomicrographs were assessed for various morphometric parameters with Image J software version 1.8. Linear Regression was used to model the relationship using Inflammatory Cell Count, Extent of Inflammation collagen stained area, Epithelial thickness integrated density of collagen, MVPA, Area, Perimeter, were taken as variables.Result:Inflammatory cell count and the extent of inflammation also decreased with increasing grades of OSMF. Collagen proportionate area, integrated collagen density and epithelial thickness were compared among different grades of OSMF. Grade IV OSMF had greatest mean collagen proportionate area , highest integrated collagen density and lowest epithelial thickness when compared to other grades of OSMF. Linear regression model revealed smaller variation between Grade I to Grade II. Whereas Grade II to Grade IV exhibited larger variation suggestive of increased growth rate and all the coefficients were found to lie within 95% confidence limitsConclusion:Diagnostic algorithm with multiparametric regression model were derived and combinatorial therapeutic approaches have been suggested for more effective management of oral submucous fibrosis

Epithelial and Connective Tissue Changes in Oral Submucous Fibrosis ?? A Morphometric Analysis

Introduction: Oral Submucous Fibrosis (OSF) is a common oral health problem in the Indian subcontinent. It was first reported by Schwartz and defined by Pindborg. OSF is within a group of conditions that are classified under potentially malignant disorders of the oral cavity. Current evidence suggests that are coline in the areca nut is the key factor in initiating the disease process. Objective: The purpose of this study is to conduct the morphometric analysis of the epithelial and selected connective tissue changes related to capillaries and to determine mast cell density in different grades of OSF. Method: The study comprises 25 cases of OSF in varying grades (according to Pindborg and Sirsat classification) retrieved from the archives of department of oral pathology. The slides were prepared and stained in Hematoxylin and Eosin stain and 1% toluidine blue. All the slides are being studied under Trinocular research microscope Olympus BX-53 and further quantified in image capture software for morphometric analysis. Result: Morphometrical analysis of epithelial thickness and connective tissue changes highlightening the parameters of capillary density, luminal diameter and circumferences of the capillaries have shown that varying degree of alterations in different grades of OSF when the severity of the disease increases. This study also included the mast cell density analysis which have proven to undergo changes as the severity of the disease progress.

Immunohistochemical detection of 8-hydroxydeoxyguanosine: A biomarker of oxidative DNA damage in oral submucous fibrosis.

Background:Oral submucous fibrosis (OSMF) is one of the common potentially malignant disorders prevailing in India. The primary etiological factors include tobacco and arecanut, which contain numerous reactive oxygen species (ROS). ROS attack guanine bases in DNA and form 8-hydroxydeoxyguanosine (8-OHdG), which can be detected in patients who have diseases associated with oxidative stress. The oxidative DNA damage produced by oxidative stress may induce malignant transformation.Aim:The aim of the present study is to detect the expression of 8-OHdG in OSMF patients and compare the expression within different grades of OSMF and also normal buccal mucosa.Materials and Methods:A total of 30 samples were examined for the immunohistochemical expression of 8-OHdG. The control group included 10 formalin-fixed paraffin-embedded tissue blocks of the normal buccal mucosa. The study group includes 20 cases of formalin-fixed paraffin-embedded tissue blocks of OSMF (5 cases in each grade of very early, early, moderately advanced and advanced cases of OSMF). Three-micron thick tissue sections were made from each sample and stained with 8-OHdG antibody. The results were statistically analyzed using Kruskal–Wallis and Mann–Whitney U testResults:Statistically significant difference exists in the intensity of 8-OHdG expression between the study groups. The P-value obtained was <0.001, which was highly statistically significant.Conclusion:The present study is the first attempt to evaluate the expression of 8-OHdG in tissue samples of OSMF that revealed the role of free radicals and oxidative DNA damage in these patients. Further research with larger sample size, clinicopathologic correlation and long-term follow-up will shed more light on the pathogenesis of OSMF. It will also be useful for the development of new therapeutic strategies targeting treatment modalities for OSMF.

Role of angiogenesis in oral submucous fibrosis using vascular endothelial growth factor and CD34: An immunohistochemical study.

Oral submucous fibrosis (OSF) is an insidious, chronic, disabling disease, in which there is lack of perfusion due to reduced level of the vasculature and this is said to be responsible for the epithelial atrophy seen in OSF. The degree of vasculature of the affected mucosa and its effects on the epithelial thickness remains controversial till date. This study attempts to analyze the role of angiogenesis in OSF and its progression using vascular endothelial growth factor (VEGF) and CD34 markers. The study samples for the present study comprised of 10 cases each of early OSF, moderately advanced, advanced OSF, and 10 cases of normal oral mucosa were used as controls. All the cases were subjected to immunohistochemical staining with VEGF and CD34 markers. Among the different grades of OSF, we did not find any noticeable difference in VEGF expression although we found a upregulation in microvessel density (CD34) in early and moderately advanced OSF followed by a downregulation in advanced OSF. As the disease progresses, there is an increased production of the extracellular matrix component (collagen I and II and fibronectin) and results in fibrosis. Subsequently, it leads to the reduction in the level of corium vascularity and results in hypoxia which ultimately causes reduction and constriction of the vascular channels. This sequence of events alerts us to the relevance of early disease diagnosis and management in an irreversible pathology such as OSF.

Expression of cyclooxygenase 2 in oral submucous fibrosis: An immunohistochemical pilot study.

Introduction:Oral submucous fibrosis (OSF) is associated with inflammatory changes in at least some stages of the disease. Prostaglandin is one of the main inflammatory mediators and its production is controlled by various enzymes such as cyclooxygenase (COX). The genetic and pharmacological data strongly indicate that COX-2 should be investigated as a potential target for the prevention and treatment of OSF.Methodology:The study group comprised histologically confirmed specimens (n = 10 each) of early OSF, moderate OSF, advanced OSF and normal oral mucosa for comparison. Immunohistochemistry was performed with avidin–biotin technique and evaluated with scoring methods.Results:The difference in percentage of expression in normal tissue and OSF was statistically highly significant (P < 0.001). Positive COX-2 exhibited cytoplasmic staining. One-way analysis of variances test was performed to evaluate COX-2 expression in different grades of OSF. Cytoplasmic staining assessed in terms of intensity, percentage of expression and Q Score did not show any statistical difference (percentage of expression F = 0.029, P = 0.971; Q Score F = 0.154, P = 0.858).Conclusions:Our study indicates that COX-2 may be an important marker of disease progression and might be a reliable prognostic indicator.

Role of hypoxia in malignant transformation of oral submucous fibrosis

Context: In the Indian subcontinent and Southeast Asia, habit of chewing areca nut has been recognized as one of the most important risk factors leading to a ubiquitous oral potentially malignant disorder (OPMD), oral submucous fibrosis (OSMF).In OSMF, owing to fibrosis in the connective tissue, there is narrowing of blood vessels which further results in compromised blood supply to the local tissue milieu. This tissue hypoxia elicits the activation of the transcription factor Hypoxia-inducible factor 1-alpha (HIF-1 α). In OSMF, its expression shows significant correlation with degree of epithelial dysplasia. Aim: This study aims to evaluate the expression of HIF-1 α in OSMF. Settings and Design: Seventy-five histopathologically proven cases of OSMF were included in the study. The tissue sections were studied for histopathological evaluation and HIF-1α expression. Statistical Analysis Used: Descriptive and inferential statistics using one-way ANOVA and multiple comparison using Kruskal–Wallis test were used. Results: Comparison of HIF-1α expression in different grades of OSMF was done. There were 47 cases without dysplasia, whereas 16 and 12 were of low-risk and high-risk dysplasia, respectively. On comparison of HIF-1α expression in different grades of OSMF, it was found to increase significantly from normal oral mucosa (1.43 ± 0.89) to no dysplasia (3.97 ± 2.75) to low-risk epithelial dysplasia (4.93 ± 1.76) to high-risk epithelial dysplasia (5.66 ± 2.01). Conclusion: The altered expression of HIF-1α can signify the disturbed epithelial-mesenchymal interaction which indicates progression toward the malignant transformation of OSMF. Thus, HIF-1 α expression showed good correlation with increase in grades of epithelial dysplasia and thus can assist for grading/quantifying oral epithelial dysplasia in OSMF.

Evaluation of myofibroblasts in oral submucous fibrosis and oral squamous cell carcinoma: The pathogenesis and correlation.

Oral submucous fibrosis (OSMF) is a chronic debilitating disease of the oral cavity having premalignant potential and unclear pathogenesis. Recently, myofibroblast has been postulated to play an important role in its pathogenesis and in the process of carcinogenesis. The purpose of this study was to evaluate and compare the presence of myofibroblasts in normal mucosa, different grades of OSMF, and oral squamous cell carcinoma (OSCC). The present in vitro cross-sectional descriptive study sample consisted of three groups, including 40 OSCCs, 40 OSMF, and 10 sections of normal oral epithelium taken as control group. Alpha-smooth muscle actin was used to identify myofibroblasts using immunohistochemical technique. P < 0.05 was taken as statistically significant. The presence of myofibroblasts was significantly higher in OSMF cases when compared with normal epithelium specimens. The presence of myofibroblasts was significantly higher in OSCC compared to OSMF cases. A significant difference was not observed between the different grades of OSCC. These findings favor the possibility that OSMF actually represents an abnormal healing process in response irritation caused by areca nut. A significant increase in myofibroblasts in OSCC as compared to OSMF also highlights the possible role it may play in the malignant transformation of OSMF.

Caspase-3 expression in normal oral epithelium, oral submucous fibrosis and oral squamous cell carcinoma.

Context:The epithelium atrophy, as the oral submucous fibrosis (OSMF) progresses, is believed to be an after effect of stromal fibrosis, hyalinization, decrease in vascularity and cellularity and is considered as “ischemic atrophy.” Due to hypoxia, caspase-3 get activation and subsequent decrease in viable cell count can occur.Aims and Objectives:To determine caspase-3 expression in various grades of OSMF and oral squamous cell carcinoma (OSCC) to find out whether upregulation of apoptosis is responsible for the epithelial changes in OSMF.Subjects and Methods:The control tissue (15 samples from normal oral mucosa) and study group comprising 97 cases of OSMF of different grades and OSCC associated with OSMF were stained with caspase-3 antibody, and the percentage of positive cells was calculated using ImageJ software.Statistical Analysis:The results obtained were statistically analyzed using ANOVA and Tukey's honest significance difference test and Mann–Whitney U-test.Results:There was a nuclear expression of caspase-3 in basal and parabasal layers of normal epithelium. There was cytoplasmic expression of caspase-3 in OSMF without dysplasia, total absence of caspase-3 expression in dysplastic epithelium and in majority cases of OSCC. The caspase-3 percentage was increased in OSMF (0%–53%) when compared with OSCC (0%–8%). The statistical comparison of caspase-3 among normal, OSMF and OSCC patients revealed significant correlation (P < 0.00010). The comparison within different grades of OSMF and between dysplastic and nondysplastic epithelium OSMF also showed significance (P < 0.019).Conclusions:The decreased expression of caspase-3 in disease progression reflects its role in the malignant transformation.

Estimation of argyrophilic nucleolar organizer regions in different grades of oral submucous fibrosis.

Context:Assessment of potential for malignant transformation of oral submucous fibrosis (OSF) through clinical or light microscopic examination of hematoxylin and eosin stained tissue sections is not totally satisfactory. The search is for such a tissue marker that will differentiate those cases of OSF, which carry a higher risk for malignant transformation. During the past few years, numerous workers have validated the usefulness of enumerating argyrophilic nucleolar organizer regions (AgNORs) in predicting the malignant potential of lesions. The present study was carried out to validate the diagnostic potential of this marker.Objectives of the Study:Quantitative and qualitative assessment of AgNORs in different grades of OSF and to compare the count of AgNORs in different grades of OSF and normal mucosa.Materials and Methods:AgNORs were investigated in tissue specimens from 90 patients diagnosed with different histopathological grades of OSF. AgNORs were identified in tissue specimens stained with silver nitrate, using light microscope. AgNORs were counted as small, large and total count to analyze them both quantitatively and qualitatively.Statistical Analysis Used:Results were subjected to statistical analysis for obtaining significance value (P value) by unpaired Student's t-test.Results:The mean total count of AgNORs was 2.464 ± 0.101, 4.358 ± 0.108, 3.704 ± 0.106 and 3.279 ± 0.161 in normal mucosa, Grades I, II and III of OSF, respectively. A qualitative difference was observed in the presentation of AgNORs in different grades of OSF. Mean value of small nucleolar organizer regions (NORs) decreased while the mean of large NORs increased as the grade of OSF increased.Conclusions:The study findings suggest that AgNORs are increased in OSF and they can serve as a reliable tool adjunct to histopathological diagnosis. Their ease of demonstration and high specificity to cellular proliferation make them the best available histopathological marker in the arsenal of an oral pathologist.

Quantitative and qualitative analysis of collagen in oral submucous fibrosis

Background: Oral submucous fibrosis (OSMF) is considered as a potentially malignant disease, which is associated with betel quid and pan masala chewing. It is thought to be due to the defective collagen synthesis and degradation. The amount of collagen in the diseased varies with the functional stages and histopathological grades of the disease. Aims: The aim of the study was to assess the severity of the disease in oral submucous fibrosis, by correlating the functional staging with the histopathological grading and analyze the collagen distribution in different grades of OSMF using picrosirius red stain under polarizing microscope and also to assess the quantitative changes in collagen with respect to various histopathological grades of OSMF using spectrophotometry. Materials and Methods: The study conducted on 40 subjects, which included 30 subjects with different functional and histopathological grades of oral submucous fibrosis (OSMF), and 10 were in control group. Functional staging and histopathological grading was done based on the definite criteria. A Histopathological and quantitative analysis of collagen was conducted using picrosirius red stain, polarizing microscope and spectrophotometer, respectively. The results were analyzed using chi-square test, and the significance was tabulated. Results: The functional staging and the histopathological grading did not show statistical correlation, but as the histopathological grade of the disease increased; there was a shift in the polarizing color from yellow-green to orange-red. In the colorimetric estimation, the optical densities of the sample decreased as with increased grades of OSMF. Conclusion: In the present study, we observed that the histopathological grading of OSMF is not correlating with the functional staging of OSMF. Therefore, it mandates the proper correlation between the functional staging and the histopathological grading before the commencement of the treatment to render better outcome of the treatment.

Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucous fibrosis

Oral submucous fibrosis (OSMF) is a chronic, progressive, scarring disease that predominantly affects the people of south-east Asian origin. The present study is aimed at comparing and correlating the mast cell density (MCD) and micro vascular density (MVD) in normal mucosa and different grades of OSMF and to analyze their role in the disease progression. MCD was assessed immunohistochemically using anti mast cell tryptase and MVD was assessed using anti-factor VIII related von Willebrand factor. The one way comparison of MVD and MCD in normal mucosa and among different grades of OSMF showed a significant increase in MCD and MVD among OSMF cases. Correlation analysis using Pearson correlation coefficient, showed positive correlation between MCD and MVD i.e. as MCD increases there is an exponential increase in MVD. The increase in MVD and MCD reveals their role in the pathogenesis of OSMF, a lesion characterized by progressive fibrosis in early stages and failure of degradation or remodeling in the advanced stages.