Assessment of collagen birefringence in different grades of oral squamous cell carcinoma using Picrosirius red – polarizing microscopy and comparison with Picrosirius red – Fast green stain

Background:Solid tumors such as oral squamous cell carcinoma (OSCC) are composed of malignant epithelial cells and the stroma in which these cells are dispersed. As the tumor progresses, the extracellular matrix undergoes dramatic morphological and architectural changes. Special stains make analysis easy and less erroneous by highlighting the area of interest and can be used to study these changes.Aim:The aim of the study was to analyze morphological changes in collagen fibers in various histological grades of OSCC using Masson's trichrome (MT) and Picrosirius red (PSR).Study Design:The study comprised 74 tissue samples, divided into two groups: Group I consisted of 63 cases of histologically proven OSCC (39 cases of well-differentiated squamous cell carcinoma [WDSCC], 17 moderately differentiated squamous cell carcinoma [MDSCC] and 7 poorly differentiated squamous cell carcinoma [PDSCC]) and Group II consisted of 11 cases of normal mucosa as controls.Materials and Methods:Sections were stained with hematoxylin and eosin, MT and PSR and observed under light and polarizing microscope, respectively.Statistical Analysis:ANOVA, Tukey's honestly significant difference post hoc multiple comparison test, Chi-square test and paired t-test were used for the statistical analysis.Results:As the grade of OSCC progressed, collagen fibers became thin, loosely packed and haphazard. The mean area fraction also decreased. They exhibited orange–red hue and strong birefringence in WDSCC, yellowish-orange hue and strong birefringence in MDSCC and greenish-yellow hue and weak birefringence in PDSCC.Conclusion:Initially, there is a reorganization of the collagen fibers in an attempt to prevent the invasion of tumor cells, but as cancer progresses, the stromal change enhances movement of the tumor cells within it, leading to metastasis.

Introduction: Oral submucous fibrosis (OSMF) is a potentially malignant disease with a prevalence of 7-13%. It is characterised by the fibroelastic changes due to excessive deposition of collagen which results in dense fibrous bands and epithelial changes. About 90-95% of oral cancers are Oral Squamous Cell Carcinoma (OSCC). Stroma produced by the invading neoplastic cells are rich in collagen fibres. These collagen fibers have been the main focus of study to understand the pathogenesis of these lesions. Hence, these fibres have been evaluated under polarised microscopy following staining with Picrosirius red stain. Aim: To evaluate changes in birefringence, thickness and orientation of collagen fibres in different histopathological stages of OSMF and OSCC. Materials and Methods: This observational study involved sections of clinically and histopathologically confirmed cases of OSCC and OSMF from Department of Oral Pathology and Microbiology, Rajarajeswari Dental College and Hospital, Bangalore. The study was conducted for a duration of one year from April 2017 to March 2018. The study included total 60 cases among which 20 for each-normal mucosa, OSCC and OSMF. Tissue sections were stained with Picrosirius red stain and collagen fibres were analysed for colour, thickness and orientation under polarised microscope. Chi-square test was used for statistical analysis. Significance was set at p-value <0.05. Results: Comparison of birefringence of collagen fibres between OSMF, OSCC and normal mucosa was not statistically significant (p-value=0.37). Orientation of collagen fibres between OSMF, OSCC and normal mucosa was statistically significant with (p-value=0.02). Conclusion: This study showed a change in colour from yellow orange to orange red in advanced OSMF cases which indicated progression of disease and tightly packing of collagen fibres, suggestive of presence of thick fibres in the extracellular matrix. In OSCC, the colour change from yellow orange to orange red and haphazardly arranged collagen fibres was indicative of transformation of preneoplastic to carcinoma stage.

Objective: Determination of the type of stromal collagen fibers in the Oral Squamous Cell Carcinoma.
 Materials and Methods: Biopsies were taken from 71 patients of oral squamous cell carcinoma. Slides were stained with Picrosirius red (PSR) stain. All the samples were evaluated for the type of collagen fibers by examining under light microscope using polarizing lens.
 Results: Type III collagen was the predominant type of collagen seen in the poorly differentiated oral squamous cell carcinoma cases, whereas type I collagen fibers were sparse. The percentage of type I collagen was highest in well differentiated oral squamous cell carcinoma cases and type III collagen fibers were infrequent. While the percentage of type III collagen fibers was more than type I collagen fibers in the moderately differentiated oral squamous cell carcinoma cases. In the present study, this variation in types of collagen fibers in the three histological grades of oral squamous cell carcinoma was found to be statistically significant (p= 0.08).
 Conclusion: In the light of the results of the present study it is concluded the collagen profiling can be effectively used as a prognostic marker in oral squamous cell carcinoma cases. Abundance of type III collagen fibers in tumor stroma seems to be a poor prognostic indicator.

Polo-like kinase 1 (PLK1) is a critical molecule in the proliferation of several human cancers. Overexpression of PLK1 has been correlated with cancer cell proliferation and lower overall survival rates. Although PLK1 has been studied in various tumors, information regarding its expression in oral cancer and precancer is limited. Aims: This study is aimed at evaluating the expression of PLK1 in a potentially malignant and malignant disorder of the oral cavity, namely, oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively, using the immunohistochemistry technique. It also intended to evaluate the association of the various histological grades of OSCC with the intensity of PLK1 expression. Thirty OSMF, thirty OSCC tissues, and thirty control tissues were obtained, and the expression of PLK1 was detected by immunohistochemistry using rabbit antihuman PLK1 polyclonal antibodies (Abcam Ab47867). The association between staining intensity and histological grade of OSCC was evaluated. Using SPSS 20 version, a test for proportions, nonparametric Chi-square/correlation analysis was used to compare differences in proportions of categorical variables of interest between groups. Results: PLK1 was positively expressed in 27 (90%) OSCC tissues. OSMF showed no detectable staining in 27 (90%) tissues and positive staining in 3 (10%) tissues. PLK1 showed no staining (0%) in normal tissues. Statistically significant associations were not found between staining intensity and histological grade of OSCC. PLK1 could be a promising progression marker for OSCC. Therapeutically, targeting PLK1 may be a new approach to fight oral cancer.

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.

HPV infection in relation to OSCC histological grading and TNM stage. Evaluation by traditional statistics and fuzzy logic model

Oral squamous cell carcinoma (OSCC) is an important malignancy throughout the world; early detection is an important criterion for achieving high cure rate. Out of the many reported markers for OSCC, this study validated the efficacy of tumor necrosis factor-α (TNF- α) in differentially diagnosing premalignant oral lesions and OSCC. Also, the study aimed to correlate the levels of salivary and serum TNF- α with clinicopathologic factors. A prospective experimental laboratory study was designed. Serum and salivary samples from 100 subjects in each group of healthy control, premalignant disease (PMD) and OSCC were collected for the study following appropriate exclusion and inclusion criteria. Serum and salivary level of TNF-α was analysed by enzyme linked immunosorbent assay. The data obtained were subjected to appropriate statistical analysis. Increased level of both serum and salivary TNF- α was observed in OSCC subjects compared to healthy control and PMD group. Receiver operator characteristic curve analysis and area under curve values showed high specificity and sensitivity for salivary TNF-α in differentiating OSCC from PMD and healthy controls. There was significant increase in TNF- α level in moderately and poorly differentiated lesion compared to well differentiated lesion and in stage IV of clinical stage. A positive correlation was observed only with histological grading of OSCC and TNF- α. Salivary TNF-α is proved to be superior for detecting OSCC. Increase in TNF-α with histological grading and clinical staging suggests a role in prognosis.

Background:Oral squamous cell carcinoma (OSCC) is the most common type of head-and-neck cancer. It is a complex and relentless malignancy prone to local invasion and dissemination. An insight into the molecular alterations associated with metastasis will provide critical insights into the fundamental mechanisms underlying its progression and further contribute to improvements in the clinical management of H and N cancer patients. Hence, identifying specific biomarkers would pave the way for early detection and prognosis of OSCC. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a membrane-bound glycoprotein found on the surface of tumor cells. It plays a central role in the promotion of tumor invasion, progression and metastasis as it upregulates matrix metalloproteinases secreted from adjacent fibroblasts. There is a paucity of studies on the expression of EMMPRIN in OSCC.Objectives:The aim is to assess the immunohistochemical expression of EMMPRIN in OSCC and to compare it with the clinicopathological parameters and histological grades of OSCC.Materials and Methods:Thirty histopathologically diagnosed cases of OSCC were included in the study. The slides were immunohistochemically analyzed for EMMPRIN expression and correlated with the clinicopathological parameters and histological grades of OSCC.Results:EMMPRIN expression was noted in all 30 cases of OSCC. Strong EMMPRIN expression was noted in the advanced clinical stages of OSCC. Higher histological grades of OSCC exhibited strong EMMPRIN expression.Conclusion:EMMPRIN overexpression indicates that this protein could be used as an important biological prognostic marker to identify high-risk OSCC patients.

Objective:Objectives of this study were to compare expression of Programmed Death-Ligand 1(PD-L1) protein in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorder (OPMD) cases; and to compare the PD-L1 protein expression in histological grades of OSCC and also in OPMD’s with Dysplasia and without Dysplasia. Materials & Methods:In this study, 25 cases of Oral squamous cell carcinoms, 25 cases of Oral Potentially Malignant Disorders and 10 cases of non-neoplastic oral mucosa (control) cases were included. FFPE blocks of OSCC and OPMD cases were contributed by Department of Pathology, Histopathology Division,Pakistan Institute of Medical Sciences, Islamabad. Immunohistochemical staining of cases with PD-L1 monoclonal antibody (1:100; Dako) was carried out at Histopathology division , PMC Labs, Peshawar Medical College,Peshawar, Riphah International University, Islamabad . Epithelial cells (membranous and cytoplasmic) positivity was observed for PD-L1 Antibody. Data was analyzed in SPSS version20. For qualitative variables frequencies and percentages were calculated whereas for quantitative variables means and standard deviations were recorded. The Chi-square test was applied to evaluate the significant difference in categorical variables . p-value of ≤0.05 was taken as significant.Results:PD-L1 expression in OSCC cases turned out to be 48% (n=12/25) as compared to 8% of OPMD cases (n=2/25) with a significant p value of 0.002 and all non-neoplastic oral mucosa cases were negative. PD-L1 expression in high grade OSCC cases was quite high (61% n=11/18) as compared to low grade OSCC (14% n=1/7) cases with a significant p value of 0.035. Conclusion:A statistically significant increased PD-L1 expression was noted in OSCC as compared to OPMD. Expression of PD-L1 was more intense in high grade OSCC cases. The relation of PD-L1 expression to age ,gender or location of OSCC and OPMD cases , and presence of dysplasia in OPMD cases was statistically not significant.

Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked β-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFRtyr1173 , and anti-phosphorylated-Aktser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 were significantly higher in OSCC than normal tissues (P<.001 and P<.01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 (P<.01 and P<.001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC.

Oral squamous cell carcinoma (OSCC) accounts for over 90% of all oral malignancies and has a 50–60% five-year survival rate, despite advances in treatment. Ki67, a nuclear protein involved in cellular proliferation, has been studied as a prognostic marker in various malignancies, including OSCC. However, its link with different histological grades of OSCC is uncertain. The study aimed to evaluate Ki-67 expression in well-differentiated (WDOSCC), moderately differentiated (MDOSCC), and poorly differentiated (PDOSCC) tumors to ascertain its correlation with tumor differentiation and aggressiveness. Forty OSCC cases were classified using Broder’s histological grading system. Ki67 immunohistochemical staining was performed, and three groups were categorized based on Ki67 expression: low (1–25%), moderate (26–50%), and high ( > 50%) proliferation. Statistical analysis assessed the significance of Ki67 expression across OSCC grades. Among 40 cases, 35% were WDOSCC, 45% MDOSCC, and 20% PDOSCC. Low Ki67 proliferation was seen in 64% of WDOSCC, moderate in 61% of MDOSCC, and high in 75% of PDOSCC. There was a significant (p < 0.001) association between OSCC grading and Ki67 expression. Ki67 expression correlates with OSCC histological grades, increasing with tumor proliferation. These findings support Ki67 as a prognostic marker, warranting further large-scale validation studies

Introduction: Chemerin is a multifunctional chemoattractant cytokine, encoded by RARRES2 gene. Its role in oral carcinogenesis is largely unexplored. Study was undertaken to evaluate the role of salivary chemerin as a potential biomarker in the early diagnosis of oral cancer and to elucidate the clinicopathologic and prognostic significance of chemerin in different grades of Oral Squamous Cell Carcinoma (OSCC) and oral leukoplakia. Materials and Methods: The study comprised 96 subjects, divided into 3 groups, each consisting of 32 subjects. These groups were clinically and histopathologically diagnosed with OSCC, oral leukoplakia, and healthy controls, respectively. The whole unstimulated saliva (1.5 mL) was collected from the subjects using passive drool technique and immediately centrifuged at 3000 rpm for 10 minutes. The salivary chemerin level was estimated using the competitive sandwich enzyme-linked immunosorbent assay method. The statistical analysis was performed using R software EZR version 1.32. Independent t-tests, analysis of variance tests, and Scheffe Multiple Comparisons were carried out to compare quantitative parameters. A receiver operating characteristic (ROC) curve was plotted to assess diagnostic accuracy of salivary chemerin. Results: Significantly higher levels of salivary chemerin were observed in OSCC and oral leukoplakia patients compared to healthy controls (P &lt; 0.05). The study further established an increase in salivary chemerin concentration in different histological grades of OSCC and oral leukoplakia. ROC curve analysis suggested that salivary chemerin is a highly sensitive and specific indicator for early screening and detection of oral leukoplakia and OSCC. Conclusion: Salivary chemerin can be considered as a reliable and specific indicator for the early screening and detection of oral leukoplakia and OSCC and a highly sensitive biomarker which may aid in detection of malignant transformation of oral leukoplakia.

Background:Many studies have been carried out to study the role of extracellular matrix proteins, growth factors and matrix metalloproteinases on tumor invasion. However, literature related to the analysis of connective tissue fibers in varying grades of oral squamous cell carcinoma (OSCC) is very limited.Aim:To analyze the changes in collagen and elastic fibers in varying grades of (OSCC).Settings and Design:This retrospective study was carried out using a light and polarizing microscope.Materials and Methods:Three sections each were cut from fifty samples of varying grades of OSCC and ten samples of control followed by staining with H and E, Picrosirius-Red and Verhoeff–Van Gieson. Qualitative and quantitative analysis of collagen and elastic fibers were accomplished using set criteria.Statistical Analysis:Data were entered into the Statistical Package for Social Sciences (SPSS) version 13.5 for analysis.Results:A change in colors of collagen fibers was seen on progressing from well to poorly differentiated OSCC. Thin collagen fibers predominantly exhibited greenish yellow, but the thick fibers exhibited a variety of colors. As the grade of OSCC progressed, collagen fibers were loosely packed haphazardly arranged. Statistically insignificant results were obtained for quantitative analysis of collagen and qualitative analysis of elastic fibers.Conclusion:The collagen fibers undergo a change in color, orientation and packing in the stroma of varying grades of OSCC. The uniqueness of this study lies in the exploration of elastic fibers in OSCC which has not been done so far.

Verrucous carcinoma (VC) should be considered a distinct clinicopathologic entity different from the more common oral squamous cell carcinoma (OSCC) because of its unique biological behavior. Best way to understand the behavior of these carcinomas is to study them by means of molecular methods, especially in tumor progression tests and Bcl-X is an important antiapoptotic member of the Bcl-2 family and is one of the newest and most useful markers to determine the aggressiveness of many carcinomas. The relationship between this Bcl-X protein and carcinomatous behavior toward it is not studied extensively, which we attempted to evaluate using immunohistochemical analysis in selected carcinomas of the head and neck region. We studied Bcl-X protein expression in sections of thirty OSCC and ten VC samples and correlated this with tumor differentiation. There was a significant difference in cytoplasmic staining of Bcl-X expression with statistical analysis (P < 0.005) for VC and OSCC when compared as a group. No significance was seen among the different histological grades of OSCC and when compared with VC individually. The significant result between OSCC and VC suggests that their biologic course is comparable and can be helpful in differentiating them with each other for establishment of a better treatment protocol.

Introduction: Tumour Associated Tissue Eosinophilia (TATE) is an important phenomenon occurring in the tumour microenvironment and has a pivotal role. TATE is defined as “eosinophilic stromal infiltration of a tumour not associated with tumour necrosis or ulceration”. The exact role of TATE in malignancies is yet unclear but studies have shown that TATE usually has a favourable outcome in head and neck Squamous Cell Carcinoma (SCC). Therefore, more studies are needed to substantiate this data. Aim: To compare TATE between normal epithelium and Oral Squamous Cell Carcinoma (OSCC) and compare TATE between the histological grades of OSCC. Materials and Methods: A retrospective study was conducted at Dr. BR Ambedkar Medical College, Bangalore, Karnataka, India, from June 2019-January 2021 which included 50 cases, 10 from normal mucosa and 40 histopathologically diagnosed cases of OSCC. 1% congo red solution was used to stain 4 μm thick sections. The sections were examined at a high magnification (40X) and 10 microscopic fields were examined in succession for eosinophils. The average number of eosinophils observed were compared using univariate analysis, which included the Analysis of Variance (ANOVA) test and the Chi- square test and a p-value of 0.001 was considered statistically significant. Results: The mean value of tissue eosinophils increased in OSCC compared to normal mucosa, according to present study findings. When comparing different grades of carcinoma, statistical analysis revealed that well differentiated squamous cell carcinoma had a greater TATE than other grades, which was statistically significant (p-value &lt;0.001). Conclusion: The higher eosinophil count in well differentiated OSCC compared to the other grades could be associated with a better clinical outcome for the patient. In OSCC, TATE can be utilised as a predictor of a good prognosis.