Early Oral Submucous Fibrosis Research Articles

Expression profile of diagnostic genes in oral submucous fibrosis

Oral Submucous Fibrosis (OSMF) is a chronic precancerous disorder of the oral mucosa caused by chewing of areca nut and its other variants. Chewing of areca nuts leads to dysregulated expression of specific genes, leading to various premalignant or malignant disorders. This study aimed to determine the differential expression of the diagnostic genes (MYH6, TNNT3, MYL1, and TPM2) in healthy controls and OSMF patients using saliva and tissue samples, determining the histopathological grade of the clinical samples. A total of 20 patients were included in the study and were divided into two groups: Group I consisted of 10 healthy patients (control group) and Group II consisted of 10 OSMF patients. Unstimulated whole saliva samples were collected from both groups, and the tissue samples were divided into two parts: one for RT-qPCR analysis and the other for histopathological assay. The expression profile of genes concerning OSMF saliva and tissue samples was significantly upregulated compared to the healthy control, and all the clinical samples of the study were categorized into histopathological grade 1. The findings of this study concluded that these genes can be referred to as diagnostic genes for OSMF in early and very early clinical samples, and saliva can be used as a promising diagnostic tool for early OSMF studies.

Evaluation of the Importance of Genetic Polymorphisms in Genes Expressing Cancer-Metabolizing Enzymes (Cyp1a1 and Gstm1) in Oral Submucous Fibrosis.

Genetic polymorphisms are common and contribute significantly to human illnesses. This study was carried out to evaluate the importance of genetic variations in the genes expressing cancer-metabolizing enzymes (CYP1A1 and GSTM1) in individuals experiencing oral submucous fibrosis (OSMF). Based on the clinical and histological characteristics of OSMF, 40 patients were chosen for the study; 10 of these patients had considerable polymorphism and malignant transformation; therefore, they were placed in a different group. After receiving written agreement, 30 normal subject patients were also picked for the study. For both normal and OSMF patients, tissue samples and 2 ml of peripheral venous blood were drawn from the arm vein and placed in a heparinized test tube. Electrophoresis on 0.8% agarose gel was used to verify genomic DNA. The GSTM1 polymorphism, CYP1A1 polymorphism was 10.41% and 15.27% in normal subjects. The GSTM1 polymorphsm, CYP1A1 polymorphism was 16.21% and 8.14% in early OSMF. The GSTM1 polymorphsm, CYP1A1 polymorphism was 20.31% and 28.56% in moderate OSMF. The GSTM1 polymorphsm, CYP1A1 polymorphism was 8.12% and 12.54% in moderate OSMF. Finally, GSTM1 polymorphism was 60.47% in OSMF+ CA, while CYP1A1 polymorphism was 40.21%. The GSTM1 gene polymorphism and CYP1A1 gene polymorphism were maximum in OSMF+ CA category. There is a significant role of genetic variations in the genes expressing cancer-metabolizing enzymes (CYP1A1) and GSTM1 in individuals experiencing OSMF.

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.

Role of programmed cell death 4 in myofibroblast differentiation in oral submucous fibrosis.

Background:Fibrosis is an uncontrolled healing process, led by persistent differentiation of fibroblast to alpha-smooth muscle actin (αSMA) positive activated fibroblast or myofibroblast. Oral submucous fibrosis (OSMF) is one such condition that is associated with areca nut use. Recently, Programmed Cell Death 4 (PDCD4), a pro-apoptotic marker, has been shown to modulate fibroblast differentiation in various organ fibrosis. The present study aimed to evaluate the role of PDCD4 in the regulation of fibroblast differentiation in OSMF.Materials and Methods:Paraffin-embedded tissue sections from 45 cases of the normal oral mucosa, early OSMF and advanced OSMF were examined for PDCD4 and αSMA expression by immunostaining. Co-expression of PDCD4 and αSMA in fibroblasts was examined using Spearman's correlation test.Results:The stromal fibroblasts showed minimal expression of αSMA in the normal mucosa and early OSMF, while advanced OSMF groups demonstrated a higher frequency of αSMA myofibroblasts. The PDCD4 expression was noted in the normal stromal fibroblasts. However, this expression appeared to progressively reduce with an increasing grade of OSMF. Thus, a negative correlation was noted between stromal PDCD4 and αSMA expression with progressive OSMF.Conclusion:This study demonstrated a putative role for PDCD4 in oral fibrosis consistent with its role in other tissues. The lack of PDCD4 expression with increasing myofibroblast expression in OSMF suggests that targeting its dysregulation may be an attractive translational therapeutic target.

COMPARE TO INTRALESIONAL THERAPY AND PHYTOCHEMICAL BASED SYSTEMIC DRUG THERAPY IN ORAL SUBMUCOUS FIBROSIS-CLINICOPHAMACOLOGICAL STUDY

Introduction: Oral sub-mucous fibrosis (OSMF) is characterized by abnormal collagen deposition. It is a precancerous disorder and transforms into a malignant tumor in 1.5–15% of all cases. Symptoms include submucous fibrosis, ulceration, xerostomia, a burning sensation restricted mouth opening and restricted tongue protrusion followed by oral mucosal blanching and uvla deviation. All of these greatly interfere with patient quality of life. Curcuma longa has been used for thousands of years as a remedy in the traditional Indian and folk medicine for the cure of a large variety of illnesses, such as inflammation, infectious diseases, and gastric, hepatic, and blood disorders. Curcumin is a major isolated polyphenol from the rhizome of turmeric (Curcuma longa). It has a wide range of pharmacological effects such as antioxidant, anti-inflammatory, antimicrobial, antitumor, and hepatoprotective activities. In our study curcumin used in OSMF as a systemic drug therapy and compare with intra-lesional therapy that’s widely used in OSMF Aims and objective: To check the efficacy of systemic drug therapy (curcumin longa) in OSMF, and compare with intra-lesional therapy (Dexamethasone with Hyaluronidase) in OSMF. Material and method 100 patients with clinically proven Oral Submucous Fibrosis were selected for the study and were randomly divided into 2 groups. The first group was treated with weekly Intralesional injection of 4 mg Dexamethasone & 1500 I.U Hyaluronidase and the second group by oral administration of two Curcumin tablets (Turmix 300 mg) per day for 4 months each. Improvement of burning sensation, interincisal distance, tongue protrusion and ulceration was evaluated on a weekly basis and compare to initial visit to end of treatment duration. Statistical analysis and Results: Burning sensation improved in both the groups from early to late stages. Complete Resolution of burning sensation was noted with Turmix early compare to intra-lesional therapy. The mean increase in inter-incisal distance or mouth opening was more compare to systemic drug therapy. The inter-incisal distance improved in both the groups gradually, but not more significant results at systemic drug therapy the end of both therapies. Tongue protrusion showed greater recovery at the end of last stmonth in group 1 when compared with group 2. Conclusion: Turmix is beneficial and effective in reducing burning sensation in early OSMF Patients so we can concluded on the basis of symptomatic relief systemic drug therapy with phyto-chemical based drug given better results compare to intralesional therapy.

Expression of p63 and Proliferating Cell Nuclear Antigen in Oral Submucous Fibrosis.

ABSTRACTAim:Oral squamous cell carcinoma (OSCC) may be preceded by potentially malignant disorders such as oral submucous fibrosis (OSMF). p63 can detect genetic changes in OSMF and it facilitates early detection of malignant transformation, whereas proliferating cell nuclear antigen (PCNA) is a marker of proliferation and may prove to be a useful objective indicator of the biological behavior of various tumors. The aim of this study was to investigate the expression and pattern of the intensity of p63 protein and PCNA in normal oral mucosa and OSMF using immunohistochemistry (IHC) and to correlate the expression of these biomarkers.Materials and Methods:A total of 15 archival paraffin-embedded blocks obtained from our department, which were histopathologically diagnosed early OSMF (n = 4), intermediate OSMF (n = 4), and advanced OSMF (n = 2) and normal mucosa (n = 5), were taken as the standard for comparison. p63 and PCNA positivity was analyzed using Kruskall-Wallis test followed by pairwise comparison using Mann-Whitney U test. The pattern of staining and intensity was compared using Chi-Square test for which Statistical Package for the Social Sciences (SPSS, v 22.0, IBM Corporation, Armonk, New York) was used.Results:All samples showed positive staining for p63 and PCNA. A statistically significant difference (P < 0.05) was seen between the frequency of occurrence of p63 and the PCNA pattern of expression among all the groups. The intensity of staining was mild to intense in the basal layer, as there was a progression toward the severity of the disease. Almost 75.4% correlation existed between p63 and PCNA, with high correlation and marked relationship.Conclusions:The OSMF is considered a potentially malignant disorder that has the potential to get transformed into OSCC. The malignant transformation is often associated with changes at the genetic level, and these are reflected by the altered expression of proteins. Our results showed that biomarkers such as p63 and PCNA are significant in predicting the malignant transformation in OSMF, so in future they may serve as a prognostic tool in the early detection of malignancies.

Long-term effectiveness of lycopene in the management of oral submucous fibrosis (OSMF): A 3-years follow-up study.

Long-term follow-ups after receiving lycopene therapy for management of oral submucous fibrosis (OSMF) are scarce. The study aimed to assess the long-term efficacy of lycopene for management of OSMF symptoms. In this prospective clinical study, 400 clinically diagnosed early OSMF patients were assessed for the efficacy of lycopene in alleviation of burning sensation (BS) and reduced mouth opening (MO) symptoms in comparison to placebo. After 1-year follow-up, group A (lycopene group) was divided equally into group A1 and group A2. group A1 patients were retreated with lycopene and the A2 group was followed without retreatment. After 2-year follow-up, the Group A2 patients were advised retreatment but not followed as most of the patient did not agree for follow-up. However, group A1 patients were continued to follow-up every 6months for a total threeyears. Statistical analysis was by independent sample t-test and P-value <.05 were considered as significant. A statistically significant difference (P < .05) in BS and MO was found between group A and B with lycopene showing better results. At one-year follow-up, a statistically significant recurrence in the symptoms was found (P < .05) in the lycopene group (group A). After the second intervention, there was a statistically significant difference in the improvement of symptoms between the group A1 and A2 at 6months and 1year (P < .05) with group A1 (retreatment) showing better results. Treatment with lycopene led to improvement in the symptoms of OSMF in the present study. The results highlight the importance of retreatment of lycopene for its long-term effect on alleviation the symptoms of OSMF.

Histochemical and Immunohistochemical Analysis of Collagen Fibers and Microvascular Density in Various Grades of Oral Submucous Fibrosis.

Background and Objective:This study was aimed to evaluate the collagen fibers qualitatively and its correlation with microvascular density in various grades of oral submucous fibrosis (OSMF). Material and Methods:The present study comprised of total 40 cases of oral submucous fibrosis. Picrosirius red staining was done on all the specimens’ sections. They were analyzed for the colour and orientation of collagen fibers. Morphometric measurements were done using image analysis on immunohistochemical stained sections for Factor VIII-related antigen and analyzed for microvascular density.Results:Picrosirius red polarizing microscopy results revealed that there was a shift in the colour of collagen fibers from greenish yellow to orange red and red colour as the severity of the oral submucous fibrosis increased. The collagen fibers showed mixed orientation in early oral submucous fibrosis and parallel orientation in advanced oral submucous fibrosis. There was a significant decrease in microvascular density from early to advanced oral submucous fibrosis.Conclusion:The change in the colours and orientation of collagen fibers in early and advanced oral submucous fibrosis could be attributed to the fibre thickness, type of collagen, alignment and packing, cross-linking of the fibers and the section thickness. However, in advanced cases the vascularity is reduced which may predispose to epithelial atrophy and subsequent malignant changes.

Interleukin 13 (IL-13) acts as a profibrotic cytokine for recruitment of myofibroblasts in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory condition due to areca nut (Areca catechu) chewing which induces fibrosis leading to the difficulty in mouth opening. Various profibrotic cytokines and chemokines have been found involved in fibrosis. The present study was aimed to find the role of interleukin 13 (IL-13) in recruiting the myofibroblasts and as a causative factor for fibrosis in OSF. Twenty-five sections of OSF tissue and ten sections of normal mucosa were stained with IL-13, TGF-α1 and β-SMA antibodies using immunohistochemical method. Immunostaining was done to assess staining intensity and percentage of positive cells. The results were analyzed by SPSS software. The expression of all three markers IL-13, TGF- β1 and α-SMA showed significant difference between control and OSF (p < 0.001), control and early OSF (p < 0.001) control and advanced OSF (p < 0.001). No statistical difference was found between early OSF and advanced OSF. Significant expression of IL-13 and its correlation with TGF-β and α-SMA suggests its role in pathogenesis of OSF.

Assessing the gustatory threshold and salivary flow rate in very early oral submucous fibrosis

Assessing the gustatory threshold and salivary flow rate in very early oral submucous fibrosis

Myofibroblasts could be recruited in a chemokine (C-C motif) ligand 2-dependent manner in pathogenesis of oral submucous fibrosis.

Monocyte chemoattractant protein-1 (CCL2) is a major profibrotic mediator with a proven role in fibrosis of different organs of the body. Recently, increased fibrosis in oral submucous fibrosis (OSMF) is linked with betel quid-related chronic irritation and myofibroblast. Hence, this study is designed to investigate expression of CCL2 in OSMF and its correlation with myofibroblasts. Paraffin-embedded specimens of 30 OSMF and 10 tissues of normal buccal mucosa were subjected to immunohistochemical analysis for CCL2 and alpha-smooth muscle actin (α-SMA) expression. CCL2 expression in basal cells (CCL2-B) and connective tissue (CCL2-CT), and α-SMA showed significantly increased expression in advanced OSMF as compared with early OSMF and controls. Significant differences were observed in the expression of CCL2-B between control vs. OSMF (P = 0.002), control vs. advanced OSMF (P = 0.005), and early vs. advanced OSMF (P = 0.0377). Similarly, differences in the CCL2-CT expression were statistically significant between control vs. OSMF (P = 0.00086), control vs. early OSMF (P = 0.02914), and control vs. advanced OSMF (P = 0.0006). For α-SMA expression, significant differences were observed between control vs. OSMF (P = 0.0003), control vs. early OSMF (P = 0.036), control vs. advanced OSMF (P = 0.00008), and early vs. advanced OSMF (P = 0.0009). In OSMF group, a significant correlation was observed between CCL2-B and CCL2-CT (P < 0.00001), CCL2-B and α-SMA (P < 0.00001), and CCL2-CT and α-SMA (P < 0.00001). CCL2 could be responsible for pathogenesis of OSMF by recruiting myofibroblasts.

Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis

Introduction: Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity characterized by generalized submucosal fibrosis with a multifactorial etiology. Myofibroblasts are a unique group of cells phenotypically intermediate between smooth muscle cells and fibroblast exhibiting contractile properties, expressing α-smooth muscle actin (α-SMA) and are considered primary producers of extracellular matrix after injury. Their accumulation has been established as a marker of progressive fibrosis in various organs. The aim of the present study is to evaluate and compare the myofibroblasts in various histological grades of OSMF. Materials and Method: Fifteen cases of OSMF, which were further categorized histologically into early (5 cases), moderately advanced (5 cases) and advanced (5 cases), were subjected to immunohistochemical evaluation using α-SMA antibody for detection of myofibroblasts. Fifteen benign mucosal proliferation specimens were also stained for comparison. Results: The number of myofibroblasts in OSMF was significantly increased when compared to that of benign mucosal proliferations (P < 0.05). Additionally, a statistically significant increase in the myofibroblasts population between early and advanced stages was observed (P < 0.05). Conclusions: The results of the present study showed that expression of myofibroblasts within the OSMF group showed a progressive increase from the early OSMF through moderate OSMF and the advanced OSMF group indicating that myofibroblasts could serve as effective prognostic marker for disease progression in oral submucous fibrosis.

Expression of transforming growth factor β and its correlation with lipodystrophy in oral submucous fibrosis: An immunohistochemical study

Objective: In our routine review of Oral Submucous Fibrosis (OSMF) biopsies, we observed decreased adipose tissue even though most are from buccal mucosa. Pathogenesis of OSMF has demonstrated the role of Transforming Growth Factor β (TGF β), in causing fibrosis. This study aims to correlate the role of TGF β with loss of adipose tissue in OSMF. Study Design: From our archives, 84 OSMF cases (24 early and 60 advanced OSMF) were screened for adipose tissue. Immunoexpression of TGF β in these cases were investigated. Results: Adipose tissue was seen in 67% of early OSMF and in 13% of advanced cases. Early cases showed more intense TGF β staining of epithelium, fibroblast, macrophages and inflammatory cells than the advanced cases. Conclusion: These findings suggest that TGF β plays a key role in causing lipodystrophy in OSMF and is secreted more during early course of the disease than in advanced stage. Key words:Adipose tissue, oral submucous fibrosis (OSMF), transforming growth factor β (TGF β), lipodystrophy.

Evaluation of PTEN Immunoexpression in Oral Submucous Fibrosis: Role in Pathogenesis and Malignant Transformation

Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity characterized by generalized submucosal fibrosis. Despite its precancerous nature, the molecular biology regarding its malignant potential has not been extensively studied. PTEN, a known tumor suppressor gene is mutated in a majority of human cancers and has also been implicated in several fibrotic disorders. The present study aims to evaluate the expression of PTEN in OSMF and oral squamous cell carcinoma (OSCC) and correlate it with the pathogenesis and malignant transformation of OSMF. 60 cases total of OSMF (30) and OSCC (30) were subjected to immunohistochemistry using PTEN antibody. Ten normal oral mucosa (NOM) specimens were also stained as controls. There was progressive loss of PTEN expression from normal mucosa to OSMF and OSCC (p≤0.001). Significant differences were observed for PTEN expression between NOM and OSMF, OSMF and OSCC as well as NOM and OSCC. Though a progressive loss of PTEN was noticed between early OSMF and advanced OSMF, the variation did not reach statistical significance (p≥0.001). Data suggest that there is a significant loss of PTEN expression in OSMF as compared to normal oral mucosa and that this trend increased from OSMF to OSCC. Thus, alteration of PTEN is likely an important molecular event in OSMF pathogenesis and oral carcinogenesis.

Mastication and Swallowing in Patients with Oral Submucous Fibrosis

Objective: Early stages of oral submucous fibrosis may lead to masticatory and swallowing impairments. This study was, conducted to evaluate the masticatory and swallowing functions in a group of oral submucous fibrosis patients.Patients and Methods: Masticatory and swallowing performance, maximum bite force, oral stereognosis, and surface electromyographic activity of the buccinator muscle were measured in 11 patients with early oral submucous fibrosis and controls matched for age and gender.Results: Statistical analysis revealed that there was no significant difference in maximum bite force between controls and patients with oral submucous fibrosis. However, the masticatory and swallowing performance, integrated surface electromyographic activity during maximum blowing in right buccinator, and oral stereognostic score in the oral submucous fibrosis group were significantly lower than in the control group.Conclusion: These findings suggest that patients with early oral submucous fibrosis may exhibit impaired masticatory and swallowing functions along with reduced oral stereognosis and buccinator muscle activity.