Expression profile of miR-99a and miR-218 across oral submucous fibrosis and oral squamous cell carcinoma

Introduction: Oral leukoplakia, submucous fibrosis (OSMF), oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC) are the commonly occurring oral diseases, with characteristic clinical and histological features. These diseases at some stage are associated with chronic inflammation in adjacent connective tissue. Mast cells are the local residents of the connective tissue, and are said to be pro-inflammatory, immunoamplifying in action and producing mitogenic cytokines. These functions of mast cells may play a significant role in the pathogenesis of other oral diseases. Aims: This study was done to histologically evaluate the number of mast cells in tissue sections of oral leukoplakia, submucous fibrosis, lichen planus and squamous cell carcinoma. Materials and Methods: Five cases each of normal oral mucosa, oral leukoplakia, oral submucous fibrosis, lichen planus and squamous cell carcinoma were studied for mast cell number using 1% Toluidine blue. Results: Increase in mast cell number were seen in all the four above mentioned oral diseases, with the highest mast cell count obtained in oral lichen planus. The mast cell number/sq.mm in oral leukoplakia, submucous fibrosis, lichen planus, squamous cell carcinoma were; 59.50, 48.25, 59.75 and 56.75 respectively. Conclusion: As compared to normal oral mucosa, increase in the mast cell number was noted in all the four conditions. Mast cell hyperplasia in oral leukoplakia, OSMF, OLP, OSCC suggests their probable role in the pathogenesis of these diseases.

Introduction Oral submucous fibrosis (OSF) is a chronic and potentially malignant oral condition that poses a significant public health issue due to its insidious nature. Transforming growth factor-beta (TGF-β) is a key player in the pathogenesis of OSF and is responsible for fibrosis. This study aims to investigate the relationship between the expression of TGF-β1 and TGF-β3 in OSF and its malignant transformation by using immunohistochemistry. Materials and method The present study comprised of 120 formalin-fixed paraffin-embedded tissue samples, which included 20 normal oral mucosa (NOM), 80 OSF (20 each of stage 1- 4), and 20 oral squamous cell carcinoma (OSCC) (10 each of OSCC with and without OSF), and were stained for TGF-β1 and TGF-β3 by immunohistochemistry. Data were analyzed using R software version 4.1.2, GraphPad Prism 9.3.1 (GraphPad Software,San Diego, CA, USA) and Excel (Microsoft Corp., Redmond, WA). Results TGF-β1 immunoexpression was negative in NOM with no significant difference among OSF and OSCC (with or without OSF). TGF-β3 was significantly higher in OSCC (with or without OSF) than in OSF, and no significant difference was noted between OSF and NOM and between OSCC and NOM. A significant increase was seen in TGF-β3 compared to TGF-β1 in NOM, OSF (stage 1- 4), and OSCC with and without OSF. Conclusion TGF-β3 has higher immunoexpression levels than TGF-β1 in NOM, OSF, and OSCC. We speculate that quantitative or qualitative TGF- β3 may be inadequate to prevent or attenuate fibrosis in OSF patients. There is also a modicum of probability that TGF-β3 has a preventive rather than causative role in OSF pathogenesis. The role of TGF-β3 in OSF needs further clarification.

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

Context: It has been proposed that oral squamous cell carcinoma (OSCC) arising in preexisting oral submucous fibrosis (OSMF) constitutes a clinicopathologically distinct disease than the OSCC arising without OSMF. Expression of myofibroblasts is an early event in tumorogenesis, and its expression in malignant lesions has been suggested to represent an important participant in invasion. Aim: To evaluate the expression of myofibroblasts in OSCC arising in OSMF and OSCC without OSMF. Settings and Design: Thirty each histopathologically proven cases of OSMF, OSCC with OSMF, and OSCC without OSMF were included in the study. The tissue sections were studied for histopathological evaluation and alpha-smooth muscle actin (α-SMA) expression. Statistical Analysis Used: Descriptive and inferential statistics using Kruskal–Wallis test, Mann–Whitney test, and Chi-square test. Results: The expression of α-SMA was found to be high in OSCC with OSMF and OSCC without OSMF group as compared to OSMF group. However, the difference between α-SMA expression in OSCC with OSMF and OSCC without OSMF group was found statistically nonsignificant. Comparison of α-SMA score with histopathological grading and tumor, node, metastasis staging in OSCC with OSMF and OSCC without OSMF groups showed statistically nonsignificant difference, whereas comparison of α-SMA score with lymph node metastasis was statistically significant. Conclusion: The altered extracellular matrix in OSCC with OSMF could be responsible for modified epithelial–mesenchymal transition, which is further proven by reduced intensity and distinct pattern of α-SMA staining in OSCC with OSMF.

To investigate the clinical characteristics, pathological and biological behaviors of oral squamous cell carcinomas (OSCC) originated from oral submucous fibrosis (OSF). A total of 528 patients with OSCC treated at Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University from 2002 to 2010 were divided into two groups, OSCC originated from OSF and OSCC not originated from OSF. The clinical data regarding age, gender, location, pathological type, metastasis and recurrence rate of these patients were analyzed retrospectively. The mean age of the group of OSCC originated from OSF (45.8 years) was younger than that of the group of OSCC not originated from OSF (55.9 years). The male female ratio of the group of OSCC originated from OSF (32.3:1) was higher than that of the group of OSCC not originated from OSF (2.3:1). The metastasis and recurrence rate of the group of OSCC originated from OSF (13.5%, 39.1%) was higher than that of the group of OSCC not originated from OSF (7.6%, 27.8%.). OSCC originated from OSF occurs at younger age and more in male, and is clinically more invasive and metastatic.

TRPV4-mediated mechanotransduction of matrix stiffness in the pathogenesis, progression, and malignant transformation of oral submucous fibrosis.

The aim of our study was to determine the impact of Th17/Treg imbalance on the progression and malignant transformation of oral submucosal fibrosis (OSF). To assess Th17 and Treg expression, overall 52 peripheral blood samples from OSF, oral squamous cell carcinoma (OSCC) patients, and healthy donors were analyzed by flow cytometry. Thirty normal oral mucosa, 72 OSF, and 90 OSCC samples were analyzed by immunohistochemistry. In peripheral blood samples, in OSCC with OSF, Th17 and Treg expression were significantly higher than those in OSF and OSCC without OSF as confirmed by immunohistochemistry. During OSF progression, Th17 and Th17/Treg ratio showed an increasing trend, while Treg expression showed a decreasing trend. Treg expression was significantly higher in OSCC with OSF than in OSF and OSCC without OSF, whereas the Th17/Treg ratio was significantly lower in OSCC with OSF. Treg expression was significantly correlated with smoking and clinical stage. Th17/Treg ratio was significantly associated with tumor size, lymph node metastasis, and clinical stage. A low Th17/Treg ratio was significantly associated with poor prognosis. Th17/Treg ratio is a potential diagnostic indicator for OSF occurrence and malignant transformation and was an independent prognostic factor for OSCC.

Differential expression of programmed death-1 and its ligand, programmed death ligand-1 in oral squamous cell carcinoma with and without oral submucous fibrosis

Background: Oral submucous fibrosis (OSF) is the most common chronic, progressive and irreversible potentially malignant disorder with high probability of malignant transformation (MT). From a clinical as well as the histological point of view, establishing and understanding the molecular nature of malignant transformation mechanism in OSF is almost important. The majority of genetic alteration caused by exogenous and endogenous mutagens is restored by the cell’s ability by DNA Damage Response (DDR). DDR mechanism dysfunction is one of the leading causes of MT. In OSF, this investigation remains scare.
 Objectives: To determine the DDR molecules expression (γΗ2ΑΧ, 53BP1, pChk2 and p53) in subjects with habit of arecanut and tobacco without OSF, OSF and oral squamous cell carcinoma (OSCC) with OSF and compare and quantify the expression among them.
 Methodology: Material and Methods: 90 subjects with 30 individuals in one of the three groups would be included in the given study. Group A: Subjects with habit of arecanut and tobacco without OSF. Group B: Patients with OSF. Group C: Patients having OSCC with OSF. DDR molecule (γΗ2ΑΧ, 53BP1, pChk2 and p53) expression will bequantified by RT-PCR. The expression levels will be analyzed using SPSS software version 17 using one-way ANOVA, followed by post hoc comparisons using Tukey’s HSD and Categorial data will be analysed using the chi-squared test.
 Expected Results: The OSF lesion prone for development of OSCC, DDR markers (γΗ2ΑΧ, 53BP1, pChk2 and p53) will accumulate before the development of p53 mutation resulting in OSCC
 Conclusion: Thus, the present study assess and quantify DDR-related molecules (γΗ2ΑΧ, 53BP1, pChk2 and p53)in OSF patients suggesting the potential benefit in the prevention of OSCC due to early therapeutic exploitation of DDR.

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.

Background:Oral squamous cell carcinoma (OSCC) is a leading cause of cancer-related deaths, with India accounting for approximately one-fourth of global cases. OSCC often develops from oral potentially malignant disorders like leucoplakia, erythroplakia, and oral submucous fibrosis (OSF), each carrying varying risks for malignant transformation. Fibulin-4, a glycoprotein involved in extracellular matrix (ECM) remodelling, has been implicated in several cancers, but its role in oral tumours remains unexplored.Methodology:Paraffin-embedded sections of oral epithelial dysplasia (OED) (n = 24), OSF (n = 23), OSCC (n = 23), and normal mucosa (n = 23) were analysed for immunohistochemical expression of Fibulin-4. Staining intensity and percentage of positive cells were evaluated with a semi-quantitative scoring system. Statistical analyses included Kruskal–Wallis, Chi-square, Mann–Whitney tests, and Kappa statistics to assess interobserver and intraobserver variability.Results:Significant differences in Fibulin-4 expression were noted across groups. OSCC and OED displayed high proportions of strong Fibulin-4 expression (76–100%), while OSF showed distinct staining patterns mainly in the middle and upper tissue layers. Higher grades of staining intensity were observed in leucoplakia (82.6% were grade 2), OSMF (83.3% were grade 2), and OSCC groups (65.2% were grade 2) than the control group (78% were grade 1). The interobserver reliability and interobserver reproducibility of Fibulin-4 expression evaluated by kappa statistic was excellent( kappa ≥ 0.80). Fibulin-4 expression was high in OSCC, OED, OSF compared to normal oral mucosa and was statistically significant (p<0.001).Conclusion:Fibulin-4 expression varies significantly across normal mucosa, OED, OSF, and OSCC, suggesting its potential role in oral carcinogenesis and ECM dynamics. Elevated Fibulin-4 expression in OSCC and OED highlights its potential as a biomarker for malignant transformation. Further research is needed to clarify its role and explore its potential as a therapeutic target in oral cancer.

ObjectivesWith the rising number of cases of oral submucous fibrosis (OSMF) undergoing malignant transformation into oral squamous cell carcinoma (OSCC), it is needed to detect the changes at the earliest. Several molecular markers have been used for this purpose, and insulin-like growth factor receptor 1 (IGF-1R) is one of the markers in vogue for several human malignancies. Thus, we compared the expression of IGF-1R mRNA in normal oral mucosa (NOM), OSMF, and OSCC. Materials and methodsThirty biopsy tissue samples for each of the three groups, NOM, OSMF, and OSCC, were taken and analyzed histopathologically and then subjected to RT-PCR for evaluation of the expression of IGF-1R mRNA in each of them. ResultsThere was an upregulation of IGF-1R mRNA in OSMF tissue samples as compared to NOM, but a downregulation as compared to OSCC. There was a 3.24-fold upregulation of IGF-1R mRNA expression in OSMF as compared to NOM and a 1.31-fold downregulation as compared to OSCC. Likewise, there was a 4.2-fold upregulation of IGF-1R mRNA expression in OSCC as compared to NOM and 1.31-fold upregulation as compared to OSMF. Moreover, the expression was more in OSMF patients with interincisal opening (IIO) < 27 mm as compared to those with IIO >27 mm and also was found to increase with the grade of OSCC. ConclusionThe study reflected a potential role of IGF-1R in the pathogenesis of OSMF and OSCC, thus implying its possible exploitation as a diagnostic and prognostic marker and also as a therapeutic target in future.

Interleukin-1 beta in oral submucous fibrosis, verrucous hyperplasia and squamous cell carcinoma tissues.

Introduction: Cyclooxygenase 2 (COX-2) expression in oral potentially malignant disorders (OPMDs) such as oral submucous fibrosis (OSMF) and oral squamous cell carcinomas (OSCCs) has revealed inconclusive reports. Studies on loss of type IV collagen expression in oral epithelial dysplasias (OEDs) and OSCCs were subjective and lacked systemic approach. To evaluate the immunohistochemical (IHC) expression of COX-2 and type IV collagen in OED, OSMF, and OSCC. Materials and methods: IHC expression of COX-2 and type IV collagen on paraffin - embedded tissue section of 10 cases each in normal oral mucosa, mild OED, moderate OED, and severe OED, OSMF, and OSCC were evaluated using mean H score and discontinuity Score (DS) designed grades for every group, respectively. Mean H score of COX-2 was compared within and between the groups using analysis of variance (ANOVA), and DS designed specifically for type IV collagen expression was compared using Kruskal–Wallis ANOVA. Pairwise comparison between the groups were performed using Tukey multiple posthoc procedure and Mann–Whitney U test for COX-2 and type IV collagen, respectively. Results: Mean H scores of COX-2 expression increased significantly (P = 0.0001) as disease progressed from mild OED to severe OED. But COX-2 in OSCC was less than that observed in mild OED (P = 0.0001). Expression of COX-2 in OSMF was more than that observed in moderate OED. Type IV collagen expression decreased as disease progressed from OED to malignancy (P = 0.0001). OSMF indicated a variation in grades of loss of type IV collagen expression. Conclusion: Expression of COX-2 in OED, OSMF, and OSCC and DS for type IV collagen expression in our study could be effectively applied to assess the malignant potential of OPMDs. However, further studies need to be implemented on a larger sample size to conclude the above findings.

BackgroundPodoplanin is a type I transmembrane sialomucin-like glycoprotein that is specifically expressed in lymphatic endothelial cells. Studies have shown that assessment of podoplanin expression in the epithelial cells can be used to predict the malignant transformation of potentially malignant disorders and the metastatic tendency of primary head and neck squamous cell carcinoma. The aim of our study was to compare the expression of podoplanin in oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma with that in normal buccal mucosa by immunohistochemical methods.Material and MethodsImmunohistochemical expression of podoplanin was analyzed in 20 cases each of oral leukoplakia, oral submucous fibrosis, oral squamous cell carcinoma and normal buccal mucosa, with monoclonal antibody D2-40. The expression of podoplanin was graded from grade 0-4.ResultsThere was a statistically significant upregulation of the grades of podoplanin expression in oral squamous cell carcinoma(100%), oral submucous fibrosis (90%) and oral leukoplakia (65%) when compared to that in normal mucosa(35%). Podoplanin expression increased with decrease in grades of differentiation in oral squamous cell carcinoma . Podoplanin expression in the samples of oral submucous fibrosis was higher than that in oral leukoplakia.ConclusionsEvaluation of podoplanin expression in the epithelial cells of oral dysplastic lesions may provide valuable information to predict their risk of malignant transformation. Key words:Immunohistochemistry, Oral leukoplakia, Oral submucous fibrosis, Podoplanin, Squamous cell carcinoma.