Biological Role of Synuclein-γ (SNCG) and Ki 67 in Oral Potentially Malignant Disorders and Squamous Cell Carcinomas of Buccal Mucosa: An Immunohistochemical Study

Biological Role of Synuclein-γ (SNCG) and Ki 67 in Oral Potentially Malignant Disorders and Squamous Cell Carcinomas of Buccal Mucosa: An Immunohistochemical Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biological Role of Synuclein-γ (SNCG) and Ki 67 in Oral Potentially Malignant Disorders and Squamous Cell Carcinomas of Buccal Mucosa: An Immunohistochemical Study Baishakhi Modak Postgraduate student, Monica Charlotte Solomon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7430987/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Oral squamous cell carcinoma (OSCC) is a subset of head and neck squamous cell carcinoma and constitutes about 94% of all oral malignant tumors. Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades, mainly due to delayed diagnosis, underdiagnosis or misdiagnosis. Hence, the data demonstrate the importance of early diagnosis and indirectly show that identifying diagnostic biomarkers is extremely urgent. Our study aims to determine Synuclein-γ and Ki 67 as potential biomarker for the diagnosis of OSCC. Identification of changes in this protein may be useful for promoting the early detection and early treatment of oral squamous cell carcinoma. Material and Methods The study was approved by the institutional ethics committee (IEC2: 67/2022). The material for the study comprised of archival formalin fixed paraffin embedded tumor blocks (FFPE) of oral potentially malignant disorders (n=40) and primary oral squamous cell carcinomas (n=40) cases. Sections were cut and immunohistochemically stained with Anti-SNCG Mouse monoclonal antibody [1H10D2] (STJ98409) and Ki-67 Mouse monoclonal antibody (prediluted/ready to use) [Clone: MIB1]. Statistical analysis was carried out using SPSS software and Chi square tests were used to determine the associations. A p value of < 0.05 was considered to be significant. Results In the study group for OPMDs, positive SNCG expression in the cytoplasm of the dysplastic cells was observed in 37/40 (92.5%) cases. Whereas positive Ki 67 expression in nucleus of the dysplastic cells was observed in 34/40 (85%) cases. In the study group for OSCC, positive SNCG expression in the cytoplasm of the tumor cells was observed in 38/40 (95%) cases. Whereas positive Ki 67 expression in nucleus of the tumor cells was observed in 39/40 (97.5%) cases. There was significant correlation between expression of SNCG with habits (p=0.005), lymphovascular invasion (p=0.037), margin clearance (p=0.039) and patient outcome (p=0.021). Also, there were significant correlation between the expression of Ki 67 with gender (p=0.004), histological grade of tumor (0.014) and stage of nodal status (p=0.026) Conclusion While Ki 67 as a standalone biomarker suffices in prognosticating both OSCC and dysplasia, there is a lack in significant correlations when it comes to certain parameters in which expression of SNCG was shown to be helpful in the present study. This complementary expression dynamic between Ki 67 and SNCG makes a strong case for using both these biomarkers, probably as a panel in the prognostication of dysplasia and OSCC. However further studies on a larger sample size and better patient follow –up data are needed for a better understanding of the molecular characteristic of the tumor cells which can be useful in developing effective treatment strategies. Synuclein-γ Ki 67 Oral squamous cell carcinoma prognosis cancer cell diagnosis cell division Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The most common malignant epithelial neoplasm affecting the oral cavity is Oral Squamous Cell Carcinoma (OSCC). Oral cancer makes up 2–4% of all cancers worldwide. However, the prevalence of oral cancer is slightly higher in certain regions like Southeast Asia and Indian subcontinents. In India, it is the leading malignancy in men and 3rd most common malignancy in women. The contributing factors for OSCC mainly consists of tobacco and tobacco related products, betel nut, and alcohol [1]. Oral Potentially Malignant Disorders (OPMDs) are the precursor lesions or conditions which can transform to oral cancer; leukoplakia being the most prevalent one. It has been observed that most of the oral squamous cell carcinomas develop from Potentially Malignant Disorders (PMDs). Therefore, early diagnosis and treatment of these disorders may aid in preventing their malignant transformation [2]. In case of leukoplakia, it has been seen that the people who fall under the age range of 25–40 are most affected. These lesions are also associated with the use of tobacco and tobacco related products [3]. The prevalence of these PMDs is also high in the Indian subcontinent due to the growing use of tobacco and tobacco-related products. The prevalence of leukoplakia in India varies from 0.2%-5.2% [4]. Beside this, the malignant transformation of these PMDs is also a concerning issue. The malignant transformation of leukoplakia, which ranges from 0.13%-10%, is alarmingly high in the Indian subcontinent because of widespread use of tobacco and associated items [5]. The various genetic and epigenetic changes due to exposure of carcinogens over a period could be the reason of PMD transforming to carcinomas or a normal epithelium converting to carcinoma [6]. Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades, mainly due to delayed diagnosis, under diagnosis or misdiagnosis [7]. Hence, the data demonstrate the importance of early diagnosis and indirectly show that identifying diagnostic biomarkers is extremely urgent. Biomarkers can be detected in serum, saliva, and tissue using convenient and rapid methods. Gradually, noninvasive methods are becoming the first choice for most biopsies, and it is apparent that saliva has an advantage in detecting OSCC. Currently, synuclein-γ (SNCG), found in both neurodegenerative diseases and cancer, is becoming the center of attraction for the researchers. γ-Synuclein belongs to the synuclein family of proteins, which consists of α-synuclein, β-synuclein, and γ-synuclein. The γ-synuclein shares 55.9% similarity with α-synuclein, with a similar N-terminal region, and a significantly different C-terminal region (amino acids 86–127) [8]. The role of -synuclein in the aetiology of human cancer was first discovered in 1998 [9]. Overexpression of SNCG is seen in various tumors, and predicts poor outcomes in breast, colon, and pancreatic cancer patients [10]. Several reports suggests that all OSCC cells show detectable SNCG expression at the mRNA and protein levels. Therefore, active secretion of SNCG by cancer cells and extracellular SNCG can promote malignant phenotypes of cancer cells [11]. There has been a fair amount of study done on the use of Ki-67 in head and neck cancer, demonstrating its use as a proliferative marker [12]. The Ki-67 is a 34 Kda Nuclear protein that is present during all active phases of the cell cycle (G1, S, G2, and M), but is absent in Go Phase [13]. Thus it represents the growth fraction of the tumor and serves as a reliable nuclear marker for cell proliferation in malignancies [14]. As, high proliferative activity is linked to poor prognosis, cell proliferation acts as a guide for predicting the prognosis of malignancies [15]. In this study, we analyzed the immunohistochemical expression of SNCG and Ki-67 in tissue samples from patients with OSCC, OPMDs and correlated the expression with various clinic-pathological features. Our study aims to determine the role of Synuclein-γ and Ki-67 as potential biomarkers for the diagnosis of OSCC. Identification of changes in this protein may be useful for promoting the early detection and early treatment of oral squamous cell carcinoma. Methodology The study was approved by the institutional ethics committee (IEC2: 67/2022). The material for the study comprised of archival formalin fixed paraffin embedded tumor blocks (FFPE) of oral epithelial dysplasias (OEDs) of buccal mucosa (n = 40) and squamous cell carcinomas of buccal mucosa (n = 40) cases. Since, FFPE blocks were used, consent of participation was not required for this study. The sections of normal buccal mucosa were the negative control and liver adenocarcinoma and tonsil were positive controls for SNCG and Ki 67 respectively. Patient selection Inclusion criteria Histopathologically diagnosed cases of epithelial dysplasia of the buccal mucosa. Histopathologically diagnosed cases of oral squamous cell carcinoma of the buccal mucosa. Exclusion criteria Tumors which are not oral squamous cell carcinoma. Tumors that have been treated. Cases with comorbidities and systemic diseases. The clinical and follow up details of all the patients were obtained from their medical records. All the patients were followed up for a period of 5 years. All biopsies specimens that were obtained prior to treatment were employed for this study. The follow-up data of the patients was available for all the patients, of which for dysplasias, 14 were transformed to oral squamous cell carcinoma, rest 26 were surviving free of disease. And for OSCC, 14 cases showed disease recurrence, 3 cases showed recurrence with metastasis and rest 23 were surviving free of disease. Immunohistochemistry Sections of 4 µm thick obtained from the FFPE tissue blocks were taken onto slides coated with 3-aminopropyl triethoxysilane (APES, Sigma – Aldrich Co. St. Louis, USA). Sections were then deparaffinised. Antigen retrieval were performed using trisodium citrate at pH 6 for SNCG and tris EDTA buffer at pH 9.0 for Ki 67. Endogenous peroxide was neutralized by treating the sections with pre-diluted 3% hydrogen peroxide. Following, these sections were incubated with Anti-SNCG Mouse monoclonal antibody [1H10D2] [Clementia Biotech] Ki-67 Mouse monoclonal antibody (prediluted/ready to use) [Clone: MIB1] [PathnSitu Biotechnologies] for 1 hour at room temperature. Then the sections were incubated with pre-diluted primary target binder (PolyExcel Target Binder, California PathnSitu, California, USA) at room temperature for 30 minutes for SNCG and 15 minutes for Ki 67 respectively. Slides were then incubated with secondary antibody (pre diluted PolyExcel Poly HRP, PathnSitu, California, USA), at room temperature for 30 minutes for both the antibodies. The peroxidase activity was developed with diaminobenzidine tetrahydrochloride (DAB PathnSitu, California, USA,). Finally, the sections were counter stained with Mayer’s haematoxylin, dehydrated, cleared and mounted with DPX (dibutyl phthalate xylene). Liver adenocarcinoma and tonsil were positive controls for SNCG and Ki 67 respectively. The negative control for the staining procedure was a tissue section of normal buccal mucosa for which the entire immunohistochemical procedure was carried out, except that the primary antibody was not used. Evaluation of SNCG and Ki 67 expression The tissue sections will be observed under light microscope (Olympus BX 41) attached with an Olympus DP20 microscope camera (Olympus Singapore Pte Ltd, Singapore), at 4x magnification gradually with the increase in magnification to 10x and 40x. Brownish-yellow stain in the cytoplasm of the cells will be considered positive for the expression of synuclein-γ. Brownish stain in the nucleus of the cells will be considered positive for the expression of Ki 67. The expression of the biomarkers was analyzed in a semi-quantitatively by two observers. Scoring system of SNCG The immunoreactivity of Synuclein-γ was cytoplasmic. The cytoplasm with brown-yellow stain, regardless of staining intensity, was regarded as positive. Five areas in the whole tumor sections were selected for each case. The percentages of positive tumor cells in all 5 fields were summed up along with the intensity of the expression SNCG. Score 0–1 = Negative Expression, Score 2–3 (Weak expression) = 25% cells were positive, Score 4–5 (Moderate Expression) = 26–50% cells were positive and Score 6–7 (Strong expression) = 50% of cells were positive. The cases were e further categorized as those with a low expression (Score 0–3) and those with a high expression (Score 4–7). Scoring system of Ki 67 The immunoreactivity of Ki-67 was seen in the nucleus. The nuclei with clear brown color, regardless of staining intensity, were regarded as positive. The positive cells of the tumor cells were evaluated in 5 representative fields at 40x magnification. Score 0 = Negative expression, Score 1 = Weak expression, Score 2 = Moderate Expression and Score 3 = Strong expression. The cases were e further categorized as those with a low expression (Score 0–1) and those with a high expression (Score 2–3). Statistical Analysis All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 16.0. Descriptive analysis was carried out for patient characteristics. Inter-observer variability was assessed using Cohen’s kappa coefficient and it was found to have good reproducibility (0.951). The association between clinicopathological parameters and the expression of SNCG and Ki 67 was assessed using the Chi-Square test. Survival analysis was carried out using Kaplan-Meier analysis. A p value of < 0.05 was considered to be statistically significant. Results The study group involved patients with OSCC and OEDs between the age of 25 to 85 years and 30 to 80years respectively. Among the 40 cases of OSCC and OEDs each, 82.5% were males and 17.5% were females. Only oral squamous cell carcinoma cases of the buccal mucosa were considered as the site for the study. Among the 40 patients of OSCC, 80%, patients had the habit of using tobacco either in the chewing form or smoking form. And out of the 40 patients of OEDs 62.5%, patients had the habit of using tobacco either in the chewing form or smoking form. In the study group for OEDs, positive SNCG expression in the cytoplasm of the dysplastic cells was observed in 92.5% cases, of which 45% cases showed a low expression of SNCG (Fig. 1A), and 55% cases showed high SNCG expression (Fig. 1B). Whereas positive Ki 67 expression in nucleus of the dysplastic cells was observed in 34/40 (85%) cases, of which 70% cases showed a low expression of Ki 67 (Fig. 2A), 30% cases showed high Ki 67 expression (Fig. 2B). In the study group for OSCC, positive SNCG expression in the cytoplasm of the tumor cells was observed in 95% cases, of which 42.5% cases showed a low expression of SNCG (Fig. 3A, 3B), and 57.5% cases showed high SNCG expression (Fig. 3C. 3D). Whereas positive Ki 67 expression in nucleus of the tumor cells was observed in 97.5% cases, of which 62.5% cases showed a low expression of Ki 67 (Fig. 4A, 4B), and 37.5% cases showed high Ki 67 expression (Fig. 4C, 4D). The association of the SNCG and Ki 67 expression with clinicopathological features of buccal epithelial dysplasias and buccal squamous cell carcinoma and patient outcome when assessed as individual markers is given in Table 1 and Table 2, respectively. Table 1 Association of the expression of Synuclein-γ and Ki-67 with clinic pathological features of Oral Leukoplakia Expression of SNCG Expression of Ki 67 Clinico-pathological parameters No. of cases Low (n = 18) High (n = 22) P value Low (n = 28) High (n = 12) p value Age Range (40–75 years) 40 18 22 0.529 28 12 0.180 Gender 0.900 0.414 Male 33 15 18 24 9 Female 7 3 4 4 3 Habits 0.204 0.025 Absent 8 2 6 3 5 Present 32 16 16 25 7 Histological grades 0.233 0.195 Mild 14 8 6 11 3 Moderate 14 7 7 11 3 Severe 12 3 9 6 6 Patient outcome 0.842 0.885 Disease free survival 26 12 14 18 8 Transformed into OSCC 14 6 8 10 4 Table 2 Association of the expression of Synuclein-γ and Ki-67 with clinic- pathological features of Oral squamous cell carcinoma Expression of SNCG Expression of Ki 67 Clinico-pathological parameters No. of cases Low (n = 17) High (n = 23) P value Low (n = 25) High (n = 15) p value Age Range (20–80 years) 40 17 23 0.373 25 15 0.345 Gender 0.983 0.004 Male 33 14 19 24 9 Female 7 3 4 1 6 Habits 0.055 0.411 Absent 11 2 9 8 3 Present 29 15 14 17 12 Histological grades 0.501 0.014 Well differentiated OSCC 16 8 8 6 10 Moderately differentiated OSCC 16 5 11 14 2 Poorly differentiated OSCC 8 4 4 5 3 Size of tumor 0.123 0.793 T2 16 5 11 11 5 T3 14 5 9 8 6 T4 10 7 3 6 4 Nodal status 0.516 0.026 N0 23 8 15 14 9 N1 6 3 3 1 5 N2 10 5 5 9 1 N3 1 1 0 1 0 Metastasis 0.384 0.433 M0 39 17 22 24 15 M1 1 0 1 1 0 Tumor staging 0.063 0.234 T2 13 4 9 7 6 T3 7 1 6 3 4 T4 20 12 8 15 5 Depth of invasion 0.342 0.202 10mm 4 2 2 2 2 Perineural invasion 0.185 0.285 Absent 28 10 18 16 12 Present 12 7 5 9 3 Lymphovascular invasion 0.037 1.000 Absent 24 7 17 15 9 Present 16 10 6 10 6 Margin clearance 0.039 0.864 No data found 18 6 12 11 7 Clear 15 10 5 9 6 Not clear 7 1 6 5 2 Patient outcome 0.169 0.037 Disease free survival 23 9 14 14 9 Recurrence 14 8 6 11 3 Recurrence with metastasis 3 0 3 0 3 Finally, survival analysis was done using the Kaplan-Meier method and log-rank test using the follow-up data of the patients. The patient outcomes are classified as good prognosis (disease free survival) and poor prognosis (cases of recurrence or recurrence with metastasis or death). The Kaplan Meier plots showed a significant difference in the clinical behavior of tumors based on the expression of SNCG (95% CI, p = 0.021, Fig. 5 ). Tumors that showed a high expression of SNCG had longer disease-free survival periods. However, there was no difference in the clinical behaviour of tumors based on the expression of Ki 67 (95% CI, p = 0.84) Discussion Numerous genes are dysregulated during the complicated, multigenic process of cancer initiation and progression. It is possible for individual cells to experience distinct sets of genetic alterations that lead to proliferation of tumour cells. The characteristic tumour cell phenotype that results from the acquisition of these sets of genetic alterations includes being able to promote and sustain angiogenesis and/or lymphangiogenesis, to produce autonomous cell growth factor production and signaling, to resist external growth inhibitory signals, to resist apoptosis, to have an infinite capacity for replication, and to invade and metastasize [16]. Situated on chromosome 10q23, the human γ-syn gene (SNCG) encodes for 127 amino acid proteins [17]. In OPMDs and OCSSs, metastasis and tumor progression has been attributed to overexpression of SNCG. In the present study, among 40 cases of OPMDs, 18 cases have low expression of SNCG, and 22 cases have high expression of SNCG. Out the cases of low expression, 11.1% cases have no habit history and 88.9% have habit history and among the patients of high expression, 27.3% cases have no habit history, and 72.7% cases have habit history. Also, among the cases of low expression 44.1% cases were mild dysplasia, 38.8% cases were moderate dysplasia, and 16.6% cases were of severe dysplasia. Out of the of high expression, 27.2% cases were mild dysplasia, 31.8% cases were of moderate dysplasia and 40.9% cases were of severe dysplasia. There was no significant association of SNCG expression with the habits of tobacco use (p = 0.204) and histological grades of dysplasia (p = 0.233) as well. Proliferation markers may be useful in enhancing the prognostic assessment of OSCC and premalignant lesions. In the current study, one such proliferative marker, Ki 67, was used. Among 40 cases of OPMDs, 28 cases have low expression of Ki 67, and 12 cases have high expression of Ki 67. Out of the cases of low expression, 10.8% cases have no habit history and 89.2% have habit history and among the patients of high expression, 41.7% cases have no habit history, and 58.3% cases have habit history. There was significant association between the expression of Ki 67 and habit of tobacco use (p = 0.025). Similar results were observed in the study by Veena Maheshwari et al. [18], which explains the fact that tobacco brings about structural changes in DNA, which leads to cancer. Also, out of the cases of low expression 39.3% cases were mild dysplasia, 39.3% cases were moderate dysplasia, and 21.4% cases were of severe dysplasia. Among the 12 cases of high expression, 35% cases were mild dysplasia, 25% cases were of moderate dysplasia and 50% cases were of severe dysplasia. There was no significant association of Ki 67 expression with histological grades of dysplasia (p = 0.145) as well. Being oral premalignant disorders, the progression of oral epithelial dysplasias, are measured in terms of their propensity for malignant transformation into OSCC. In this study, a detailed follow-up of patients with dysplasia was recorded in terms of whether they underwent malignant transformation or not. This data was correlated with the expression of SNCG and Ki 67. This correlation yielded the following results. For SNCG, out of cases of low expression, 33.3% have transformed into OSCC and among the cases of high expression, 36.4% cases have transformed into OSCC. And for Ki 67, out of the cases of low expression, 35.7% have transformed into OSCC and among the patients of high expression, 33.3% cases have transformed into OSCC. Over expression of SNCG in OSCC has been reported and in this present study a positive cytoplasmic expression was evident in 95% of the cases. There was significant association of SNCG expression with the habit of tobacco use (p = 0.055). Nicotine binding to nAChRs in cancer cells activates downstream mitogenic pathways, upregulates growth factor production, and stimulates intracellular signaling pathways in a tissue-specific way. Therefore, it is possible that the malignancy of nicotine-induced oral cancer is due to the elevation of SNCG expression, which may cause ECM breakdown and encourage malignant behaviors including invasion and migration [19]. γS interacts with the mitotic checkpoint protein BubR1 in breast cancer, causing BubR1 inactivation and mitotic checkpoint impairment [20]. Additionally, γS interferes with mitotic checkpoint control, conferring cellular resistance to anti-microtubule drugs; therefore, a γS-targeting peptide (ANK) has been shown to increase the sensitivity of antimicrotubule therapies to breast cancer cells [21]. Moreover, γS downmodulates JNK in all tumour types that it has been studied, increasing survival, and preventing apoptosis. The expression of γS is closely linked to the processes of invasion, metastatic spread, and EMT. According to reports, γS promotes the expression of metalloproteinases 2 and 9 in carcinomas of the breast, bladder, cervix, and liver in alongside retinoblastoma [22], and by controlling Rho GTPases, it enhances cell motility in ovarian and breast carcinomas [23]. In OSCC, the progression through several histological grades or stage of tumor may be attributed to these molecular changes. However, in the present study, out of the 17 cases of low expression, 47.1% cases were well differentiated OSCC, 29.4% cases were moderately differentiated OSCC, and 23.5% cases were of poorly differentiated OSCC and out of the 23 cases of high expression, 34.8% cases were well differentiated OSCC, 47.8% cases were of moderately differentiated OSCC, and 17.4% cases were of poorly differentiated OSCC. Also, among the 17 cases with low expression, 23.5% cases were of stage II, 5.9% case were of stage III, and 70.6% cases were of stage IV and out of the 23 cases with high expression, 39.1% cases were of stage II, 26.1% cases were of stage III, and 34.8% cases were of stage IV. But there was no significant association between the SNCG expression and histological grade (p = 0.501) and stage of tumor (p = 0.063). There was significant association of the SNCG expression with lymphovascular invasion (p = 0.037). According to Surgucheva et al.'s [22] hypothesis the strong induction of MMP9, tissue inhibitor of MMPs 1 and 2 (TIMP1, TIMP2) expression, and moderate increase in MMP2 activity and protein levels are potential mechanisms through which γ-syn overexpression contributes to tumour progression and malignancy. A study by Jia et al. [24], also showed that there was overexpression of SNCG which led to tumor growth and metastasis in nude mice with breast cancer cells. So, in cases of OSCC, this could be the possible reason of association of SNCG with lymphovascular invasion. Surgical excision as the primary treatment modality in cases of OSCC, especially early stage OSCCs is dictated by the presence of clear surgical margins with literature reporting the extent of normal mucosa beyond the excised tumor about 1-4mm [25] In our study, out of the 17 cases with low expression, 35.3% cases no data was found, 58.8% cases showed clear margins, and 5.9% case showed no clear margins. Out of the 23 cases with high expression, 52.2% cases no data was found, 21.7% cases showed clear margins, and 26.1% no clear margins. There was significant correlation between SNCG expression and margin clearance (p = 0.039). There is evidence indicating that γ-syn's atypical expression is initiated early in the formation of tumours and contributes to their metastasis and progression. A study by Ahmad et al [26], showed γ-syn may function as a marker of the malignant development of cancer, as evidenced by its stage-specific expression in pancreatic, ovarian, breast, and, more recently, hepatocellular carcinomas. Wu and colleagues (2006) [27] investigated this further and discovered a similar association between decreased disease-free survival and γ-syn expression. In our study also, there was significant association (p = 0.021) with the SNCG expression with survival rate. Chaperones are vital proteins that aid in the synthesis and maintenance of other important proteins for cell growth and survival in their appropriate form. Because of epigenetic control caused by the demethylation of CpG sites within the SNCG gene, SNCG is aberrantly produced in advanced malignant states and is not expressed in normal cells. This suggests that SNCG is a more tumor-oriented chaperone. The SNCG protein exhibits stage-specific patterns of very low expression in stage I and high expression in stages III to IV across a variety of cancer types. SNCG may encourage genomic instability, which could lead to carcinogenesis. In breast cancer cells, SNCG physically interacts with the mitotic checkpoint protein BubR1. The control of the mitotic checkpoint is overridden by SNCG expression in breast cancer cells, and this inhibitory effect of SNCG on the checkpoint can be overcome by forcing overexpression of BubR1 in SNCG-expressing cells. These findings imply that SNCG expression undermines the regulation of mitotic checkpoints by impeding BubR1's regular activity and therefore encouraging genetic instability [27]. Hence, SNCG expression is an early step in the malignant transformation of oral epithelium and could be used as a malignant index for OSCC and can be used for better prognostication. Many studies have indicated that aberrant cell proliferation appears to be a precursor and may be a predictor of carcinogenesis. It is believed that cancer loses the biological control over cell proliferation. For the purpose of analyzing cell proliferation, proliferating cell nuclear antigen and Ki-67 antigen are the most often implemented immunohistochemical markers. The current study shows significant correlation (p = 0.004) of Ki 67 with the gender of patients. This was not in accordance with the study done by Chandrakanta et al. [28]. Also, in the present study, among the of 25 cases of low expression, 32% cases have no habit history and 56% have habit history and out of the 15 patients of high expression, 20% cases have no habit history, and 80% cases have habit history. But there was no significant association between the Ki 67 expression and habit of tobacco use (p = 0.411). In the present study there was significant association (p = 0.014) of Ki 67 expression with histological grade of tumor. Among the 15 cases of high expression, 66.7% cases were of well differentiated OSCC, 13.3% cases were of moderately differentiated OSCC, and 20% cases were of poorly differentiated OSCC. These findings are similar to the findings of Birajdar SS et al. [29]. The periphery of tumour islands exhibited nuclear Ki-67 positivity in well-differentiated OSCC. This indicates that the tumour island's central cells lack Ki-67 expression because they are highly differentiated and capable of keratinization, while the peripheral layer contains less differentiated cells. Ki-67 expression was seen in both the periphery and portion of the central layer in moderately differentiated OSCC. In moderately differentiated OSCC, the cells were less differentiated than those in well-differentiated OSCC and had demonstrated a lower rate of proliferation. The nuclear Ki-67 positivity in poorly differentiated OSCC was observed in the periphery, while the majority of the tumour island's central region had diffuse Ki-67 staining. 31 But the proliferating rate of poorly differentiated OSCC was much lower than well differentiated OSCC, which is not in accordance with the previous studies. Also, in the present study, among the 25 cases with low expression, 25% cases were of stage II, 12% cases were of stage III, and 60% cases were of stage IV and out of the 15 cases with high expression, 40% cases were of stage II, 26.7% cases were of stage III, and 33.3% cases were of stage IV. However, there was no significant association between the Ki 67 expression and stage of the tumor (p = 0.234). Also, there is significant association (p = 0.026) of Ki 67 expression with the nodal status. A study by Jing Y et al., also has a similar result. Tumour aggression, as determined by tumour grade and stage, has been found to be correlated with tumour proliferative activity characterized by Ki-67. The correlation between Ki-67 and associated possible clinicopathological features, such as differentiation and lymph node metastasis, validates the biological behavior of OSCC with highly elevated Ki-67 [30]. In this study, a detailed follow-up of patients with oral squamous cell carcinoma was recorded in terms of whether they underwent recurrence or not. This data was again correlated with the expression of SNCG and Ki 67. This correlation yielded the following results. For SNCG, out of 17 cases of low expression, 47.05% have recurred and among the 23 patients of high expression, 26% cases have recurred as OSCC and/or associated with metastasis. And for Ki 67, out of 25 cases of low expression, 44% have recurred and among the 15 patients of high expression, 40% cases have recurred as OSCC and/or associated with metastasis within a span of time. There was significant association (p = 0.037) of Ki 67 expression with patient outcome. A higher risk of recurrence and a lower overall and disease-free survival have been associated with elevated Ki-67 expression. Risk stratification, treatment planning, and posttreatment surveillance may benefit from its analysis [31]. Conclusion Oral squamous cell carcinomas (OSCCs) are a growing problem in the world. The various existing treatments have not markedly improved the survival rate of patients with OSCC during the past three decades. Novel treatment strategies are required for which we have used two biomarkers’ markers SNCG and Ki 67. SNCG functions as a marker of the malignant development of cancer, as evidenced by its stage-specific expression while Ki67 proliferation index has been used in numerous studies to demonstrate its application as a prognostic indicator for cancer. In this study the expression SNCG and Ki 67 were evaluated in 40 cases of OPMDS and OSCC cases each. SNCG expression in OSCC was found to significantly correlate with tobacco habit, lymphovascular invasion, margin clearance and disease-free survival. The data points to the utility of SNCG as a prognostic marker for use in predicting progression of tumor and survival outcomes of patients with OSCC. Also, expression of SNCG can be used to identify the high-risk patients who may benefit from the various adjuvant therapies. In the present study, Ki 67 expression in OSCC is significantly correlated with the nodal status of tumor which shows tumor aggressiveness which is determined by tumor grade and stage. Also, the expression of Ki 67 has statistically significant correlation with histological grade, the expression scoring of those various histological grades are not showing a direct relation with the degree of proliferation especially when it comes to the more proliferative moderately differentiated OSCC, whose expression of Ki 67 was low in the majority of these cases. This is counter intuitive to the aspect of Ki 67 being the marker of proliferation. But in contrast, in cases of dysplasia, SNCG found no significant correlation in the association of their expression with parameters like that of age, gender, habits or histological grade. However, in case of Ki 67, significant correlation was found with respect to habits of tobacco use. While Ki 67 as a standalone biomarker suffices in prognosticating both OSCC and dysplasia, there is a lack in significant correlations when it comes to certain parameters in which expression of SNCG was shown to be helpful in the present study. This complementary expression dynamic between Ki 67 and SNCG makes a strong case for using both these biomarkers, probably as a panel in the prognostication of dysplasia and OSCC. However further studies on a larger sample size and better patient follow –up data are needed for a better understanding of the molecular characteristic of the tumor cells which can be useful in developing effective treatment strategies. Abbreviations SNCG: Synuclein-γ / γS / γ-Synuclein / γ-Syn OPMD: Oral Potentially Malignant Disorder OSCC: Oral Squamous Cell Carcinoma DPX: Dibutylpthalate Xylol APES: 3-aminopropyl triethoxy silane PBS: Phosphate-buffered saline TBS: Tris-buffered saline HIER: Heat induced epitope retrieval TNM: Tumor Node Metastasis AJCC: American Joint Committee WHO: World health Organization nAChRs: Nicotine acetylcholine receptors MMP: Matrix metalloproteinase ECM: Extra cellular matrix Declarations Funding: This study was not supported by any funding. Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval: For this type of study formal consent is not required. The study was approved by Institutional Ethics Committee (IEC2: 67/2022) and adhered to the Declaration of Helsinki. Consent to Participate: For this type of study informed consent is not required; as formalin fixed paraffin sections were used. The informed consent was waived by the Kasturba Medical College and Kasturba Hospital, Institutional Ethics Committee-2 (Student Research) which gave the waiver for retrospective study. Consent for publication: For this type of study consent for publication is not required. Availability of data and materials: NA Code Availability: NA Clinical trial number: Not applicable. Funding: Nil Author’s contribution: Dr. Baishakhi Modak: Conceptualisation, Data collection, Manuscript writing. Dr. Monica Charlotte Solomon: Conceptualisation, Review, Validation Acknowledgements: Nil Conflict of Interest: Nil References Markopoulos AK. Current aspects on oral squamous cell carcinoma. The open dentistry journal. 2012;6:126. George A, Sreenivasan BS, Sunil S, Varghese SS, Thomas J, Gopakumar D, Mani V. Potentially malignant disorders of oral cavity. Oral Maxillofac Pathol J. 2011 Jan 1;2(1):95-100. Shah S, Dave B, Shah R, Mehta TR, Dave R. Socioeconomic and cultural impact of tobacco in India. Journal of family medicine and primary care. 2018 Nov;7(6):1173. Saraswathi TR, Ranganathan K, Shanmugam S, Sowmya R, Narasimhan PD, Gunaseelan R. Prevalence of oral lesions in relation to habits: Cross-sectional study in South India. Indian Journal of Dental Research. 2006 Jul 1;17(3):121. Mohammed F, Fairozekhan AT. Oral Leukoplakia. 2022 Jul 18. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 28723042. Oral cancer prevention and the evolution of molecular – targeted drug development.JClinOncol.2005;23(2):346-56. Mascitti M, Orsini G, TOSCO V, Monterubbianesi R, Balercia A et al. An Overview on Current Noninvasive Diagnostic Devices in Oral Oncology. Front Physiol 2018; 9: 1510. Singh VK, Jia Z. Targeting synuclein-γ to counteract drug resistance in cancer. Expert Opin Ther Targets. 200. Lavedan C, Leroy E, Dehejia A, Buchholtz S, Dutra A, Nussbaum RL, et al. Identification, localization and characterization of the human γ-synuclein gene. Hum Genet. 1998;103(1):106–12. LIU C, SHI B, HAO C, WANG Q, LV Q et al. Urine gamma-synuclein as a biomarker for the diagnosis of bladder cancer. Oncotarget 2016; 7: 43432–43441. FENG Y, LI Q, CHEN J, YI P, XU X et al. Salivary protease spectrum biomarkers of oral cancer. Int J Oral Sci 2019; 11: 7. Daniel F.I, Fava M, Hoffmann R, Campos M, Yurgel L. Main Molecular Markers of Oral Squamous Cell Carcinoma. Applied Cancer Research 2010; 30(3): 279-88. Prayson RA.The utility of MIB-1/Ki-67 immunostaining in the evaluation of central nervous system neoplasms. Adv Anat Pathol 2005; 12: 144-148. Boas D.S, Takiya C.M, Sampaio T.L.C, Ribeiro L.C, Ramos E.A.G, Cabra M.G et al. Immunohistochemical detection of Ki-67 is not associated with tumor-infiltrating macrophages and cyclooxygenase-2 in oral squamous cell carcinoma; J Oral Pathol Med 2010; 39: 565–70. Pich A, Chiusa L &Navone R. Prognostic relevance of cell proliferation in head and neck tumors. Ann Oncol 2004; 15: 1319–29. Hanahan, D. Hallmarks of cancer: New dimensions. Cancer Discov 12, 31–46 (2022). Lavedan, C., Leroy, E., Torres, R., Dehejia, A., Dutra, A., Buchholtz, S., Nussbaum, R. L., and Polymeropoulos, M. H. (1998) Genomic organization and expression of the human beta-synuclein gene (SNCB). Genomics 54, 173–175. Veena Maheshwari et al., Prognostic and Predictive Impact of Ki-67 in Premalignant and Malignant Squamous Cell Lesions of Oral Cavity, International Journal of Head and Neck Surgery, May-August 2013;4(2):61-65 Hsu, CC., Su, YF., Tsai, KY. et al. Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy. Cancer Cell Int 20 , 300 (2020). Gupta, A.; Inaba, S.; Wong, O.K.; Fang, G.; Liu, J. Breast Cancer-Specific Gene 1 Interacts with the Mitotic Checkpoint Kinase BubR1. Oncogene 2003, 22, 7593–7599. Singh, V.K.; Zhou, Y.; Marsh, J.A.; Uversky, V.N.; Forman-Kay, J.D.; Liu, J.; Jia, Z. Synuclein-gamma targeting peptide inhibitor that enhances sensitivity of breast cancer cells to antimicrotubule drugs. Cancer Res. 2007, 67, 626–633. Surgucheva, I.G.; Sivak, J.M.; Fini, M.E.; Palazzo, R.E.; Surguchov, A.P. Effect of γ-Synuclein Overexpression on Matrix Metalloproteinases in Retinoblastoma Y79 Cells. Arch. Biochem. Biophys. 2003, 410, 167–176. Pan, Z.Z.; Bruening, W.; Godwin, A.K. Involvement of RHO GTPases and ERK in Synuclein-γ Enhanced Cancer Cell Motility. Int. J. Oncol. 2006, 29, 1201–1205. Jia, T., Liu, Y. E., Liu, J., and Shi, Y. E. (1999) Stimulation of breast cancer invasion and metastasis by synuclein gamma. Cancer Res. 59, 742–747. Brennan PA, Dylgjeri F, Coletta RD, Arakeri G, Goodson AM. Surgical tumour margins and their significance in oral squamous cell carcinoma. J Oral Pathol Med. 2022;51:311–314. Ahmad M, Attoub S, Singh MN, Martin FL, El-Agnaf OM. Gamma-synuclein and the progression of cancer. FASEB J. 2007 Nov;21(13):3419-30. Wu, K., Quan, Z., Weng, Z., Li, F., Zhang, Y., Yao, X., Chen, Y., Budman, D., Goldberg, I. D., and Shi, Y. E. (2006) Expression of neuronal protein synuclein gamma gene as a novel marker for breast cancer prognosis. Breast. Cancer Res. Treat. 101, 259–267. Chandrakanta, Nagayach P, Sonkar R, Bharti R, Kumar H. Ki-67 expression in human oral squamous cell carcinoma. Indian J Pathol Oncol 2021;8(4):473-477. Birajdar SS, Radhika M, Paremala K, Sudhakara M, Soumya M, Gadivan M. Expression of Ki-67 in normal oral epithelium, leukoplakic oral epithelium and oral squamous cell carcinoma. J Oral Maxillofac Pathol. 2014 May;18(2):169-76. Jing, Y., Zhou, Q., Zhu, H., Zhang, Y., Song, Y., Zhang, X., Huang, X., Yang, Y., Ni, Y., Hu, Q."Ki‑67 is an independent prognostic marker for the recurrence and relapse of oral squamous cell carcinoma". Oncology Letters 17.1 (2019): 974-980. Pekarek L, Garrido-Gil MJ, Sánchez-Cendra A, Cassinello J, Pekarek T, Fraile-Martinez O, García-Montero C, Lopez-Gonzalez L, Rios-Parra A, Álvarez-Mon M, Acero J, Diaz-Pedrero R, Ortega MA. Emerging histological and serological biomarkers in oral squamous cell carcinoma: Applications in diagnosis, prognosis evaluation and personalized therapeutics (Review). Oncol Rep. 2023 Dec;50(6):213. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7430987","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513533464,"identity":"57385f87-079d-4e2a-bcf0-767037c4b402","order_by":0,"name":"Baishakhi Modak Postgraduate student","email":"","orcid":"","institution":"Manipal College of Dental Sciences, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Baishakhi","middleName":"Modak Postgraduate","lastName":"student","suffix":""},{"id":513533465,"identity":"d7808106-d485-482e-97bc-888844072a61","order_by":1,"name":"Monica Charlotte Solomon","email":"data:image/png;base64,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","orcid":"","institution":"Manipal College of Dental Sciences, Manipal Academy of Higher Education","correspondingAuthor":true,"prefix":"","firstName":"Monica","middleName":"Charlotte","lastName":"Solomon","suffix":""}],"badges":[],"createdAt":"2025-08-22 05:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7430987/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7430987/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91204123,"identity":"65e42381-56b7-41c5-abb6-2aa133edfa62","added_by":"auto","created_at":"2025-09-12 16:14:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":682098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: Photomicrograph showing a Low expression of SNCG in Severe Dysplasia (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Photomicrograph showing a high expression of SNCG in Severe Dysplasia (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/3fd51def86993ccf7dc177f2.png"},{"id":91204126,"identity":"2de5ce55-98d5-49ff-a667-58902a59e1ac","added_by":"auto","created_at":"2025-09-12 16:14:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":749823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: Photomicrograph showing a Low expression of Ki-67 in Severe Dysplasia (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Photomicrograph showing a High expression of Ki-67 in Severe Dysplasia (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/b5858f6baac5ff358a566e6e.png"},{"id":91204735,"identity":"1091e88d-9227-4811-a1d3-f213e0271700","added_by":"auto","created_at":"2025-09-12 16:22:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":977884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA Photomicrograph showing a low expression of SNCG in Moderately differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB Photomicrograph showing a low expression of SNCG in Poorly differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC Photomicrograph showing a High expression of SNCG in Moderately differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D: Photomicrograph showing a High expression of SNCG in Poorly differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/b9f4755302a8954c932a2a06.png"},{"id":91204138,"identity":"b7863c5a-4a86-4fd7-bab2-593c101a5f92","added_by":"auto","created_at":"2025-09-12 16:14:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":914271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: Photomicrograph showing a low expression of Ki-67 in Moderately differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Photomicrograph showing a low expression of Ki-67 in Poorly differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC: Photomicrograph showing a high expression of Ki-67 in Moderately differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD: Photomicrograph showing a high expression of Ki-67 in Poorly differentiated Squamous cell carcinoma (IHC- 10x).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/5f790d7ebbeb8c50ea9b7ba0.png"},{"id":91205591,"identity":"c1cf55c5-1b4d-4586-964a-c7e183ba80f1","added_by":"auto","created_at":"2025-09-12 16:30:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation of SNCG expression with follow up status of the patients\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/0a1281a22b005ad24b0636fc.png"},{"id":101842080,"identity":"b4783db1-1c3e-4002-8020-34ae51591024","added_by":"auto","created_at":"2026-02-04 08:42:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4731693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7430987/v1/6b5ff333-7495-493f-93ec-0732e48165ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological Role of Synuclein-γ (SNCG) and Ki 67 in Oral Potentially Malignant Disorders and Squamous Cell Carcinomas of Buccal Mucosa: An Immunohistochemical Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe most common malignant epithelial neoplasm affecting the oral cavity is Oral Squamous Cell Carcinoma (OSCC). Oral cancer makes up 2\u0026ndash;4% of all cancers worldwide. However, the prevalence of oral cancer is slightly higher in certain regions like Southeast Asia and Indian subcontinents. In India, it is the leading malignancy in men and 3rd most common malignancy in women. The contributing factors for OSCC mainly consists of tobacco and tobacco related products, betel nut, and alcohol [1].\u003c/p\u003e\u003cp\u003eOral Potentially Malignant Disorders (OPMDs) are the precursor lesions or conditions which can transform to oral cancer; leukoplakia being the most prevalent one. It has been observed that most of the oral squamous cell carcinomas develop from Potentially Malignant Disorders (PMDs). Therefore, early diagnosis and treatment of these disorders may aid in preventing their malignant transformation [2].\u003c/p\u003e\u003cp\u003eIn case of leukoplakia, it has been seen that the people who fall under the age range of 25\u0026ndash;40 are most affected. These lesions are also associated with the use of tobacco and tobacco related products [3]. The prevalence of these PMDs is also high in the Indian subcontinent due to the growing use of tobacco and tobacco-related products. The prevalence of leukoplakia in India varies from 0.2%-5.2% [4]. Beside this, the malignant transformation of these PMDs is also a concerning issue. The malignant transformation of leukoplakia, which ranges from 0.13%-10%, is alarmingly high in the Indian subcontinent because of widespread use of tobacco and associated items [5]. The various genetic and epigenetic changes due to exposure of carcinogens over a period could be the reason of PMD transforming to carcinomas or a normal epithelium converting to carcinoma [6]. Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades, mainly due to delayed diagnosis, under diagnosis or misdiagnosis [7]. Hence, the data demonstrate the importance of early diagnosis and indirectly show that identifying diagnostic biomarkers is extremely urgent.\u003c/p\u003e\u003cp\u003eBiomarkers can be detected in serum, saliva, and tissue using convenient and rapid methods. Gradually, noninvasive methods are becoming the first choice for most biopsies, and it is apparent that saliva has an advantage in detecting OSCC. Currently, synuclein-γ (SNCG), found in both neurodegenerative diseases and cancer, is becoming the center of attraction for the researchers. γ-Synuclein belongs to the synuclein family of proteins, which consists of α-synuclein, β-synuclein, and γ-synuclein. The γ-synuclein shares 55.9% similarity with α-synuclein, with a similar N-terminal region, and a significantly different C-terminal region (amino acids 86\u0026ndash;127) [8]. The role of -synuclein in the aetiology of human cancer was first discovered in 1998 [9]. Overexpression of SNCG is seen in various tumors, and predicts poor outcomes in breast, colon, and pancreatic cancer patients [10]. Several reports suggests that all OSCC cells show detectable SNCG expression at the mRNA and protein levels. Therefore, active secretion of SNCG by cancer cells and extracellular SNCG can promote malignant phenotypes of cancer cells [11].\u003c/p\u003e\u003cp\u003eThere has been a fair amount of study done on the use of Ki-67 in head and neck cancer, demonstrating its use as a proliferative marker [12]. The Ki-67 is a 34 Kda Nuclear protein that is present during all active phases of the cell cycle (G1, S, G2, and M), but is absent in Go Phase [13]. Thus it represents the growth fraction of the tumor and serves as a reliable nuclear marker for cell proliferation in malignancies [14]. As, high proliferative activity is linked to poor prognosis, cell proliferation acts as a guide for predicting the prognosis of malignancies [15].\u003c/p\u003e\u003cp\u003eIn this study, we analyzed the immunohistochemical expression of SNCG and Ki-67 in tissue samples from patients with OSCC, OPMDs and correlated the expression with various clinic-pathological features. Our study aims to determine the role of Synuclein-γ and Ki-67 as potential biomarkers for the diagnosis of OSCC. Identification of changes in this protein may be useful for promoting the early detection and early treatment of oral squamous cell carcinoma.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003eThe study was approved by the institutional ethics committee (IEC2: 67/2022). The material for the study comprised of archival formalin fixed paraffin embedded tumor blocks (FFPE) of oral epithelial dysplasias (OEDs) of buccal mucosa (n\u0026thinsp;=\u0026thinsp;40) and squamous cell carcinomas of buccal mucosa (n\u0026thinsp;=\u0026thinsp;40) cases. Since, FFPE blocks were used, consent of participation was not required for this study. The sections of normal buccal mucosa were the negative control and liver adenocarcinoma and tonsil were positive controls for SNCG and Ki 67 respectively.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient selection\u003c/h2\u003e\u003cp\u003e\u003cem\u003eInclusion criteria\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eHistopathologically diagnosed cases of epithelial dysplasia of the buccal mucosa.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHistopathologically diagnosed cases of oral squamous cell carcinoma of the buccal mucosa.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eExclusion criteria\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTumors which are not oral squamous cell carcinoma.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTumors that have been treated.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCases with comorbidities and systemic diseases.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe clinical and follow up details of all the patients were obtained from their medical records. All the patients were followed up for a period of 5 years. All biopsies specimens that were obtained prior to treatment were employed for this study. The follow-up data of the patients was available for all the patients, of which for dysplasias, 14 were transformed to oral squamous cell carcinoma, rest 26 were surviving free of disease. And for OSCC, 14 cases showed disease recurrence, 3 cases showed recurrence with metastasis and rest 23 were surviving free of disease.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eSections of 4 \u0026micro;m thick obtained from the FFPE tissue blocks were taken onto slides coated with 3-aminopropyl triethoxysilane (APES, Sigma \u0026ndash; Aldrich Co. St. Louis, USA). Sections were then deparaffinised. Antigen retrieval were performed using trisodium citrate at pH 6 for SNCG and tris EDTA buffer at pH 9.0 for Ki 67. Endogenous peroxide was neutralized by treating the sections with pre-diluted 3% hydrogen peroxide. Following, these sections were incubated with Anti-SNCG Mouse monoclonal antibody [1H10D2] [Clementia Biotech] Ki-67 Mouse monoclonal antibody (prediluted/ready to use) [Clone: MIB1] [PathnSitu Biotechnologies] for 1 hour at room temperature. Then the sections were incubated with pre-diluted primary target binder (PolyExcel Target Binder, California PathnSitu, California, USA) at room temperature for 30 minutes for SNCG and 15 minutes for Ki 67 respectively. Slides were then incubated with secondary antibody (pre diluted PolyExcel Poly HRP, PathnSitu, California, USA), at room temperature for 30 minutes for both the antibodies. The peroxidase activity was developed with diaminobenzidine tetrahydrochloride (DAB PathnSitu, California, USA,). Finally, the sections were counter stained with Mayer\u0026rsquo;s haematoxylin, dehydrated, cleared and mounted with DPX (dibutyl phthalate xylene). Liver adenocarcinoma and tonsil were positive controls for SNCG and Ki 67 respectively. The negative control for the staining procedure was a tissue section of normal buccal mucosa for which the entire immunohistochemical procedure was carried out, except that the primary antibody was not used.\u003c/p\u003e\n\u003ch3\u003eEvaluation of SNCG and Ki 67 expression\u003c/h3\u003e\n\u003cp\u003eThe tissue sections will be observed under light microscope (Olympus BX 41) attached with an Olympus DP20 microscope camera (Olympus Singapore Pte Ltd, Singapore), at 4x magnification gradually with the increase in magnification to 10x and 40x. Brownish-yellow stain in the cytoplasm of the cells will be considered positive for the expression of synuclein-γ. Brownish stain in the nucleus of the cells will be considered positive for the expression of Ki 67. The expression of the biomarkers was analyzed in a semi-quantitatively by two observers.\u003c/p\u003e\n\u003ch3\u003eScoring system of SNCG\u003c/h3\u003e\n\u003cp\u003eThe immunoreactivity of Synuclein-γ was cytoplasmic. The cytoplasm with brown-yellow stain, regardless of staining intensity, was regarded as positive. Five areas in the whole tumor sections were selected for each case. The percentages of positive tumor cells in all 5 fields were summed up along with the intensity of the expression SNCG. Score 0\u0026ndash;1\u0026thinsp;=\u0026thinsp;Negative Expression, Score 2\u0026ndash;3 (Weak expression)\u0026thinsp;=\u0026thinsp;25% cells were positive, Score 4\u0026ndash;5 (Moderate Expression)\u0026thinsp;=\u0026thinsp;26\u0026ndash;50% cells were positive and Score 6\u0026ndash;7 (Strong expression)\u0026thinsp;=\u0026thinsp;50% of cells were positive. The cases were e further categorized as those with a low expression (Score 0\u0026ndash;3) and those with a high expression (Score 4\u0026ndash;7).\u003c/p\u003e\n\u003ch3\u003eScoring system of Ki 67\u003c/h3\u003e\n\u003cp\u003eThe immunoreactivity of Ki-67 was seen in the nucleus. The nuclei with clear brown color, regardless of staining intensity, were regarded as positive. The positive cells of the tumor cells were evaluated in 5 representative fields at 40x magnification. Score 0\u0026thinsp;=\u0026thinsp;Negative expression, Score 1\u0026thinsp;=\u0026thinsp;Weak expression, Score 2\u0026thinsp;=\u0026thinsp;Moderate Expression and Score 3\u0026thinsp;=\u0026thinsp;Strong expression. The cases were e further categorized as those with a low expression (Score 0\u0026ndash;1) and those with a high expression (Score 2\u0026ndash;3).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 16.0. Descriptive analysis was carried out for patient characteristics. Inter-observer variability was assessed using Cohen\u0026rsquo;s kappa coefficient and it was found to have good reproducibility (0.951). The association between clinicopathological parameters and the expression of SNCG and Ki 67 was assessed using the Chi-Square test. Survival analysis was carried out using Kaplan-Meier analysis. A p value of \u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe study group involved patients with OSCC and OEDs between the age of 25 to 85 years and 30 to 80years respectively. Among the 40 cases of OSCC and OEDs each, 82.5% were males and 17.5% were females. Only oral squamous cell carcinoma cases of the buccal mucosa were considered as the site for the study. Among the 40 patients of OSCC, 80%, patients had the habit of using tobacco either in the chewing form or smoking form. And out of the 40 patients of OEDs 62.5%, patients had the habit of using tobacco either in the chewing form or smoking form.\u003c/p\u003e\n\u003cp\u003eIn the study group for OEDs, positive SNCG expression in the cytoplasm of the dysplastic cells was observed in 92.5% cases, of which 45% cases showed a low expression of SNCG (Fig. 1A), and 55% cases showed high SNCG expression (Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhereas positive Ki 67 expression in nucleus of the dysplastic cells was observed in 34/40 (85%) cases, of which 70% cases showed a low expression of Ki 67 (Fig. 2A), 30% cases showed high Ki 67 expression (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eIn the study group for OSCC, positive SNCG expression in the cytoplasm of the tumor cells was observed in 95% cases, of which 42.5% cases showed a low expression of SNCG (Fig. 3A, 3B), and 57.5% cases showed high SNCG expression (Fig. 3C. 3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhereas positive Ki 67 expression in nucleus of the tumor cells was observed in 97.5% cases, of which 62.5% cases showed a low expression of Ki 67 (Fig. 4A, 4B), and 37.5% cases showed high Ki 67 expression (Fig. 4C, 4D).\u003c/p\u003e\n\u003cp\u003eThe association of the SNCG and Ki 67 expression with clinicopathological features of buccal epithelial dysplasias and buccal squamous cell carcinoma and patient outcome when assessed as individual markers is given in Table 1 and Table 2, respectively.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation of the expression of Synuclein-γ and Ki-67 with clinic pathological features of Oral Leukoplakia\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eExpression of SNCG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eExpression of Ki 67\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinico-pathological parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo. of cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh (n\u0026thinsp;=\u0026thinsp;22)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow (n\u0026thinsp;=\u0026thinsp;28)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ep value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRange (40\u0026ndash;75 years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.529\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.180\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.414\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHabits\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistological grades\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.195\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient outcome\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.842\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.885\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDisease free survival\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTransformed into OSCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation of the expression of Synuclein-γ and Ki-67 with clinic- pathological features of Oral squamous cell carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eExpression of SNCG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eExpression of Ki 67\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinico-pathological parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo. of cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow (n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow (n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ep value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRange (20\u0026ndash;80 years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.373\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.345\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHabits\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.411\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistological grades\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.501\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell differentiated OSCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerately differentiated OSCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePoorly differentiated OSCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSize of tumor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.793\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNodal status\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.026\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMetastasis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.384\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.433\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTumor staging\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.234\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDepth of invasion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.202\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;5mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-10mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePerineural invasion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.285\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLymphovascular invasion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMargin clearance\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.864\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo data found\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNot clear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient outcome\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDisease free survival\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence with metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFinally, survival analysis was done using the Kaplan-Meier method and log-rank test using the follow-up data of the patients. The patient outcomes are classified as good prognosis (disease free survival) and poor prognosis (cases of recurrence or recurrence with metastasis or death). The Kaplan Meier plots showed a significant difference in the clinical behavior of tumors based on the expression of SNCG (95% CI, p\u0026thinsp;=\u0026thinsp;0.021, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Tumors that showed a high expression of SNCG had longer disease-free survival periods. However, there was no difference in the clinical behaviour of tumors based on the expression of Ki 67 (95% CI, p\u0026thinsp;=\u0026thinsp;0.84)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNumerous genes are dysregulated during the complicated, multigenic process of cancer initiation and progression. It is possible for individual cells to experience distinct sets of genetic alterations that lead to proliferation of tumour cells. The characteristic tumour cell phenotype that results from the acquisition of these sets of genetic alterations includes being able to promote and sustain angiogenesis and/or lymphangiogenesis, to produce autonomous cell growth factor production and signaling, to resist external growth inhibitory signals, to resist apoptosis, to have an infinite capacity for replication, and to invade and metastasize [16].\u003c/p\u003e\u003cp\u003eSituated on chromosome 10q23, the human γ-syn gene (SNCG) encodes for 127 amino acid proteins [17]. In OPMDs and OCSSs, metastasis and tumor progression has been attributed to overexpression of SNCG. In the present study, among 40 cases of OPMDs, 18 cases have low expression of SNCG, and 22 cases have high expression of SNCG. Out the cases of low expression, 11.1% cases have no habit history and 88.9% have habit history and among the patients of high expression, 27.3% cases have no habit history, and 72.7% cases have habit history. Also, among the cases of low expression 44.1% cases were mild dysplasia, 38.8% cases were moderate dysplasia, and 16.6% cases were of severe dysplasia. Out of the of high expression, 27.2% cases were mild dysplasia, 31.8% cases were of moderate dysplasia and 40.9% cases were of severe dysplasia. There was no significant association of SNCG expression with the habits of tobacco use (p\u0026thinsp;=\u0026thinsp;0.204) and histological grades of dysplasia (p\u0026thinsp;=\u0026thinsp;0.233) as well.\u003c/p\u003e\u003cp\u003eProliferation markers may be useful in enhancing the prognostic assessment of OSCC and premalignant lesions. In the current study, one such proliferative marker, Ki 67, was used. Among 40 cases of OPMDs, 28 cases have low expression of Ki 67, and 12 cases have high expression of Ki 67. Out of the cases of low expression, 10.8% cases have no habit history and 89.2% have habit history and among the patients of high expression, 41.7% cases have no habit history, and 58.3% cases have habit history. There was significant association between the expression of Ki 67 and habit of tobacco use (p\u0026thinsp;=\u0026thinsp;0.025). Similar results were observed in the study by Veena Maheshwari et al. [18], which explains the fact that tobacco brings about structural changes in DNA, which leads to cancer. Also, out of the cases of low expression 39.3% cases were mild dysplasia, 39.3% cases were moderate dysplasia, and 21.4% cases were of severe dysplasia. Among the 12 cases of high expression, 35% cases were mild dysplasia, 25% cases were of moderate dysplasia and 50% cases were of severe dysplasia. There was no significant association of Ki 67 expression with histological grades of dysplasia (p\u0026thinsp;=\u0026thinsp;0.145) as well.\u003c/p\u003e\u003cp\u003eBeing oral premalignant disorders, the progression of oral epithelial dysplasias, are measured in terms of their propensity for malignant transformation into OSCC. In this study, a detailed follow-up of patients with dysplasia was recorded in terms of whether they underwent malignant transformation or not. This data was correlated with the expression of SNCG and Ki 67. This correlation yielded the following results. For SNCG, out of cases of low expression, 33.3% have transformed into OSCC and among the cases of high expression, 36.4% cases have transformed into OSCC. And for Ki 67, out of the cases of low expression, 35.7% have transformed into OSCC and among the patients of high expression, 33.3% cases have transformed into OSCC.\u003c/p\u003e\u003cp\u003eOver expression of SNCG in OSCC has been reported and in this present study a positive cytoplasmic expression was evident in 95% of the cases. There was significant association of SNCG expression with the habit of tobacco use (p\u0026thinsp;=\u0026thinsp;0.055). Nicotine binding to nAChRs in cancer cells activates downstream mitogenic pathways, upregulates growth factor production, and stimulates intracellular signaling pathways in a tissue-specific way. Therefore, it is possible that the malignancy of nicotine-induced oral cancer is due to the elevation of SNCG expression, which may cause ECM breakdown and encourage malignant behaviors including invasion and migration [19].\u003c/p\u003e\u003cp\u003eγS interacts with the mitotic checkpoint protein BubR1 in breast cancer, causing BubR1 inactivation and mitotic checkpoint impairment [20]. Additionally, γS interferes with mitotic checkpoint control, conferring cellular resistance to anti-microtubule drugs; therefore, a γS-targeting peptide (ANK) has been shown to increase the sensitivity of antimicrotubule therapies to breast cancer cells [21]. Moreover, γS downmodulates JNK in all tumour types that it has been studied, increasing survival, and preventing apoptosis. The expression of γS is closely linked to the processes of invasion, metastatic spread, and EMT. According to reports, γS promotes the expression of metalloproteinases 2 and 9 in carcinomas of the breast, bladder, cervix, and liver in alongside retinoblastoma [22], and by controlling Rho GTPases, it enhances cell motility in ovarian and breast carcinomas [23]. In OSCC, the progression through several histological grades or stage of tumor may be attributed to these molecular changes.\u003c/p\u003e\u003cp\u003eHowever, in the present study, out of the 17 cases of low expression, 47.1% cases were well differentiated OSCC, 29.4% cases were moderately differentiated OSCC, and 23.5% cases were of poorly differentiated OSCC and out of the 23 cases of high expression, 34.8% cases were well differentiated OSCC, 47.8% cases were of moderately differentiated OSCC, and 17.4% cases were of poorly differentiated OSCC. Also, among the 17 cases with low expression, 23.5% cases were of stage II, 5.9% case were of stage III, and 70.6% cases were of stage IV and out of the 23 cases with high expression, 39.1% cases were of stage II, 26.1% cases were of stage III, and 34.8% cases were of stage IV. But there was no significant association between the SNCG expression and histological grade (p\u0026thinsp;=\u0026thinsp;0.501) and stage of tumor (p\u0026thinsp;=\u0026thinsp;0.063).\u003c/p\u003e\u003cp\u003eThere was significant association of the SNCG expression with lymphovascular invasion (p\u0026thinsp;=\u0026thinsp;0.037). According to Surgucheva et al.'s [22] hypothesis the strong induction of MMP9, tissue inhibitor of MMPs 1 and 2 (TIMP1, TIMP2) expression, and moderate increase in MMP2 activity and protein levels are potential mechanisms through which γ-syn overexpression contributes to tumour progression and malignancy. A study by Jia et al. [24], also showed that there was overexpression of SNCG which led to tumor growth and metastasis in nude mice with breast cancer cells. So, in cases of OSCC, this could be the possible reason of association of SNCG with lymphovascular invasion.\u003c/p\u003e\u003cp\u003eSurgical excision as the primary treatment modality in cases of OSCC, especially early stage OSCCs is dictated by the presence of clear surgical margins with literature reporting the extent of normal mucosa beyond the excised tumor about 1-4mm [25] In our study, out of the 17 cases with low expression, 35.3% cases no data was found, 58.8% cases showed clear margins, and 5.9% case showed no clear margins. Out of the 23 cases with high expression, 52.2% cases no data was found, 21.7% cases showed clear margins, and 26.1% no clear margins. There was significant correlation between SNCG expression and margin clearance (p\u0026thinsp;=\u0026thinsp;0.039).\u003c/p\u003e\u003cp\u003eThere is evidence indicating that γ-syn's atypical expression is initiated early in the formation of tumours and contributes to their metastasis and progression. A study by Ahmad et al [26], showed γ-syn may function as a marker of the malignant development of cancer, as evidenced by its stage-specific expression in pancreatic, ovarian, breast, and, more recently, hepatocellular carcinomas. Wu and colleagues (2006) [27] investigated this further and discovered a similar association between decreased disease-free survival and γ-syn expression. In our study also, there was significant association (p\u0026thinsp;=\u0026thinsp;0.021) with the SNCG expression with survival rate. Chaperones are vital proteins that aid in the synthesis and maintenance of other important proteins for cell growth and survival in their appropriate form. Because of epigenetic control caused by the demethylation of CpG sites within the SNCG gene, SNCG is aberrantly produced in advanced malignant states and is not expressed in normal cells. This suggests that SNCG is a more tumor-oriented chaperone. The SNCG protein exhibits stage-specific patterns of very low expression in stage I and high expression in stages III to IV across a variety of cancer types. SNCG may encourage genomic instability, which could lead to carcinogenesis. In breast cancer cells, SNCG physically interacts with the mitotic checkpoint protein BubR1. The control of the mitotic checkpoint is overridden by SNCG expression in breast cancer cells, and this inhibitory effect of SNCG on the checkpoint can be overcome by forcing overexpression of BubR1 in SNCG-expressing cells. These findings imply that SNCG expression undermines the regulation of mitotic checkpoints by impeding BubR1's regular activity and therefore encouraging genetic instability [27]. Hence, SNCG expression is an early step in the malignant transformation of oral epithelium and could be used as a malignant index for OSCC and can be used for better prognostication.\u003c/p\u003e\u003cp\u003eMany studies have indicated that aberrant cell proliferation appears to be a precursor and may be a predictor of carcinogenesis. It is believed that cancer loses the biological control over cell proliferation. For the purpose of analyzing cell proliferation, proliferating cell nuclear antigen and Ki-67 antigen are the most often implemented immunohistochemical markers.\u003c/p\u003e\u003cp\u003eThe current study shows significant correlation (p\u0026thinsp;=\u0026thinsp;0.004) of Ki 67 with the gender of patients. This was not in accordance with the study done by Chandrakanta et al. [28]. Also, in the present study, among the of 25 cases of low expression, 32% cases have no habit history and 56% have habit history and out of the 15 patients of high expression, 20% cases have no habit history, and 80% cases have habit history. But there was no significant association between the Ki 67 expression and habit of tobacco use (p\u0026thinsp;=\u0026thinsp;0.411).\u003c/p\u003e\u003cp\u003eIn the present study there was significant association (p\u0026thinsp;=\u0026thinsp;0.014) of Ki 67 expression with histological grade of tumor. Among the 15 cases of high expression, 66.7% cases were of well differentiated OSCC, 13.3% cases were of moderately differentiated OSCC, and 20% cases were of poorly differentiated OSCC. These findings are similar to the findings of Birajdar SS et al. [29]. The periphery of tumour islands exhibited nuclear Ki-67 positivity in well-differentiated OSCC. This indicates that the tumour island's central cells lack Ki-67 expression because they are highly differentiated and capable of keratinization, while the peripheral layer contains less differentiated cells. Ki-67 expression was seen in both the periphery and portion of the central layer in moderately differentiated OSCC. In moderately differentiated OSCC, the cells were less differentiated than those in well-differentiated OSCC and had demonstrated a lower rate of proliferation. The nuclear Ki-67 positivity in poorly differentiated OSCC was observed in the periphery, while the majority of the tumour island's central region had diffuse Ki-67 staining.\u003csup\u003e31\u003c/sup\u003e But the proliferating rate of poorly differentiated OSCC was much lower than well differentiated OSCC, which is not in accordance with the previous studies. Also, in the present study, among the 25 cases with low expression, 25% cases were of stage II, 12% cases were of stage III, and 60% cases were of stage IV and out of the 15 cases with high expression, 40% cases were of stage II, 26.7% cases were of stage III, and 33.3% cases were of stage IV. However, there was no significant association between the Ki 67 expression and stage of the tumor (p\u0026thinsp;=\u0026thinsp;0.234).\u003c/p\u003e\u003cp\u003eAlso, there is significant association (p\u0026thinsp;=\u0026thinsp;0.026) of Ki 67 expression with the nodal status. A study by Jing Y et al., also has a similar result. Tumour aggression, as determined by tumour grade and stage, has been found to be correlated with tumour proliferative activity characterized by Ki-67. The correlation between Ki-67 and associated possible clinicopathological features, such as differentiation and lymph node metastasis, validates the biological behavior of OSCC with highly elevated Ki-67 [30].\u003c/p\u003e\u003cp\u003eIn this study, a detailed follow-up of patients with oral squamous cell carcinoma was recorded in terms of whether they underwent recurrence or not. This data was again correlated with the expression of SNCG and Ki 67. This correlation yielded the following results. For SNCG, out of 17 cases of low expression, 47.05% have recurred and among the 23 patients of high expression, 26% cases have recurred as OSCC and/or associated with metastasis. And for Ki 67, out of 25 cases of low expression, 44% have recurred and among the 15 patients of high expression, 40% cases have recurred as OSCC and/or associated with metastasis within a span of time. There was significant association (p\u0026thinsp;=\u0026thinsp;0.037) of Ki 67 expression with patient outcome. A higher risk of recurrence and a lower overall and disease-free survival have been associated with elevated Ki-67 expression. Risk stratification, treatment planning, and posttreatment surveillance may benefit from its analysis [31].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOral squamous cell carcinomas (OSCCs) are a growing problem in the world. The various existing treatments have not markedly improved the survival rate of patients with OSCC during the past three decades. Novel treatment strategies are required for which we have used two biomarkers\u0026rsquo; markers SNCG and Ki 67. SNCG functions as a marker of the malignant development of cancer, as evidenced by its stage-specific expression while Ki67 proliferation index has been used in numerous studies to demonstrate its application as a prognostic indicator for cancer.\u003c/p\u003e\u003cp\u003eIn this study the expression SNCG and Ki 67 were evaluated in 40 cases of OPMDS and OSCC cases each. SNCG expression in OSCC was found to significantly correlate with tobacco habit, lymphovascular invasion, margin clearance and disease-free survival. The data points to the utility of SNCG as a prognostic marker for use in predicting progression of tumor and survival outcomes of patients with OSCC. Also, expression of SNCG can be used to identify the high-risk patients who may benefit from the various adjuvant therapies.\u003c/p\u003e\u003cp\u003eIn the present study, Ki 67 expression in OSCC is significantly correlated with the nodal status of tumor which shows tumor aggressiveness which is determined by tumor grade and stage. Also, the expression of Ki 67 has statistically significant correlation with histological grade, the expression scoring of those various histological grades are not showing a direct relation with the degree of proliferation especially when it comes to the more proliferative moderately differentiated OSCC, whose expression of Ki 67 was low in the majority of these cases. This is counter intuitive to the aspect of Ki 67 being the marker of proliferation.\u003c/p\u003e\u003cp\u003eBut in contrast, in cases of dysplasia, SNCG found no significant correlation in the association of their expression with parameters like that of age, gender, habits or histological grade. However, in case of Ki 67, significant correlation was found with respect to habits of tobacco use. While Ki 67 as a standalone biomarker suffices in prognosticating both OSCC and dysplasia, there is a lack in significant correlations when it comes to certain parameters in which expression of SNCG was shown to be helpful in the present study. This complementary expression dynamic between Ki 67 and SNCG makes a strong case for using both these biomarkers, probably as a panel in the prognostication of dysplasia and OSCC. However further studies on a larger sample size and better patient follow \u0026ndash;up data are needed for a better understanding of the molecular characteristic of the tumor cells which can be useful in developing effective treatment strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003col\u003e\n \u003cli\u003eSNCG: Synuclein-\u0026gamma; / \u0026gamma;S / \u0026gamma;-Synuclein / \u0026gamma;-Syn\u003c/li\u003e\n \u003cli\u003eOPMD: Oral Potentially Malignant Disorder\u003c/li\u003e\n \u003cli\u003eOSCC: Oral Squamous Cell Carcinoma\u003c/li\u003e\n \u003cli\u003eDPX: Dibutylpthalate Xylol\u003c/li\u003e\n \u003cli\u003eAPES: 3-aminopropyl triethoxy silane\u003c/li\u003e\n \u003cli\u003ePBS: Phosphate-buffered saline\u003c/li\u003e\n \u003cli\u003eTBS: Tris-buffered saline\u003c/li\u003e\n \u003cli\u003eHIER: Heat\u0026nbsp;induced\u0026nbsp;epitope\u0026nbsp;retrieval\u003c/li\u003e\n \u003cli\u003eTNM: Tumor Node Metastasis\u003c/li\u003e\n \u003cli\u003eAJCC: American Joint Committee\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;WHO: World health Organization\u003c/li\u003e\n \u003cli\u003enAChRs: Nicotine acetylcholine receptors\u003c/li\u003e\n \u003cli\u003eMMP: Matrix metalloproteinase\u003c/li\u003e\n \u003cli\u003eECM: Extra cellular matrix\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003col\u003e\n \u003cli\u003eFunding: This study was not supported by any funding.\u003c/li\u003e\n \u003cli\u003eConflict of Interest: The authors declare that they have no conflict of interest.\u003c/li\u003e\n \u003cli\u003eEthical approval: For this type of study formal consent is not required. The study was approved by Institutional Ethics Committee (IEC2: 67/2022) and adhered to the Declaration of Helsinki.\u003c/li\u003e\n \u003cli\u003eConsent to Participate: For this type of study informed consent is not required; as formalin fixed paraffin sections were used. The informed consent was waived by the Kasturba Medical College and Kasturba Hospital, Institutional Ethics Committee-2 (Student Research) which gave the waiver for retrospective study.\u003c/li\u003e\n \u003cli\u003eConsent for publication: For this type of study consent for publication is not required.\u003c/li\u003e\n \u003cli\u003eAvailability of data and materials: NA\u003c/li\u003e\n \u003cli\u003eCode Availability: NA\u003c/li\u003e\n \u003cli\u003eClinical trial number: Not applicable.\u003c/li\u003e\n \u003cli\u003eFunding: Nil\u003c/li\u003e\n \u003cli\u003eAuthor\u0026rsquo;s contribution:\u0026nbsp;\u003col\u003e\n \u003cli\u003eDr. Baishakhi Modak: Conceptualisation, Data collection, Manuscript writing.\u003c/li\u003e\n \u003cli\u003eDr. Monica Charlotte Solomon: Conceptualisation, Review, Validation\u003c/li\u003e\n \u003c/ol\u003e\u003c/li\u003e\n \u003cli\u003eAcknowledgements: Nil\u003c/li\u003e\n \u003cli\u003eConflict of Interest: Nil\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMarkopoulos AK. 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Genomics 54, 173\u0026ndash;175.\u003c/li\u003e\n\u003cli\u003eVeena Maheshwari et al., Prognostic and Predictive Impact of Ki-67 in Premalignant and Malignant Squamous Cell Lesions of Oral Cavity, International Journal of Head and Neck Surgery, May-August 2013;4(2):61-65\u003c/li\u003e\n\u003cli\u003eHsu, CC., Su, YF., Tsai, KY. \u003cem\u003eet al.\u003c/em\u003e Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy. \u003cem\u003eCancer Cell Int\u003c/em\u003e\u003cstrong\u003e20\u003c/strong\u003e, 300 (2020).\u003c/li\u003e\n\u003cli\u003eGupta, A.; Inaba, S.; Wong, O.K.; Fang, G.; Liu, J. Breast Cancer-Specific Gene 1 Interacts with the Mitotic Checkpoint Kinase BubR1. Oncogene 2003, 22, 7593\u0026ndash;7599.\u003c/li\u003e\n\u003cli\u003eSingh, V.K.; Zhou, Y.; Marsh, J.A.; Uversky, V.N.; Forman-Kay, J.D.; Liu, J.; Jia, Z. Synuclein-gamma targeting peptide inhibitor that enhances sensitivity of breast cancer cells to antimicrotubule drugs. Cancer Res. 2007, 67, 626\u0026ndash;633.\u003c/li\u003e\n\u003cli\u003eSurgucheva, I.G.; Sivak, J.M.; Fini, M.E.; Palazzo, R.E.; Surguchov, A.P. Effect of \u0026gamma;-Synuclein Overexpression on Matrix Metalloproteinases in Retinoblastoma Y79 Cells. Arch. Biochem. Biophys. 2003, 410, 167\u0026ndash;176.\u003c/li\u003e\n\u003cli\u003ePan, Z.Z.; Bruening, W.; Godwin, A.K. Involvement of RHO GTPases and ERK in Synuclein-\u0026gamma; Enhanced Cancer Cell Motility. Int. J. Oncol. 2006, 29, 1201\u0026ndash;1205.\u003c/li\u003e\n\u003cli\u003eJia, T., Liu, Y. E., Liu, J., and Shi, Y. E. (1999) Stimulation of breast cancer invasion and metastasis by synuclein gamma. Cancer Res. 59, 742\u0026ndash;747.\u003c/li\u003e\n\u003cli\u003eBrennan PA, Dylgjeri F, Coletta RD, Arakeri G, Goodson AM. Surgical tumour margins and their significance in oral squamous cell carcinoma. J Oral Pathol Med. 2022;51:311\u0026ndash;314.\u003c/li\u003e\n\u003cli\u003eAhmad M, Attoub S, Singh MN, Martin FL, El-Agnaf OM. Gamma-synuclein and the progression of cancer. FASEB J. 2007 Nov;21(13):3419-30.\u003c/li\u003e\n\u003cli\u003eWu, K., Quan, Z., Weng, Z., Li, F., Zhang, Y., Yao, X., Chen, Y., Budman, D., Goldberg, I. D., and Shi, Y. E. (2006) Expression of neuronal protein synuclein gamma gene as a novel marker for breast cancer prognosis. Breast. Cancer Res. Treat. 101, 259\u0026ndash;267.\u003c/li\u003e\n\u003cli\u003eChandrakanta, Nagayach P, Sonkar R, Bharti R, Kumar H. Ki-67 expression in human oral squamous cell carcinoma. Indian J Pathol Oncol 2021;8(4):473-477.\u003c/li\u003e\n\u003cli\u003eBirajdar SS, Radhika M, Paremala K, Sudhakara M, Soumya M, Gadivan M. Expression of Ki-67 in normal oral epithelium, leukoplakic oral epithelium and oral squamous cell carcinoma. J Oral Maxillofac Pathol. 2014 May;18(2):169-76.\u003c/li\u003e\n\u003cli\u003eJing, Y., Zhou, Q., Zhu, H., Zhang, Y., Song, Y., Zhang, X., Huang, X., Yang, Y., Ni, Y., Hu, Q.\u0026quot;Ki‑67 is an independent prognostic marker for the recurrence and relapse of oral squamous cell carcinoma\u0026quot;. Oncology Letters 17.1 (2019): 974-980.\u003c/li\u003e\n\u003cli\u003ePekarek L, Garrido-Gil MJ, S\u0026aacute;nchez-Cendra A, Cassinello J, Pekarek T, Fraile-Martinez O, Garc\u0026iacute;a-Montero C, Lopez-Gonzalez L, Rios-Parra A, \u0026Aacute;lvarez-Mon M, Acero J, Diaz-Pedrero R, Ortega MA. Emerging histological and serological biomarkers in oral squamous cell carcinoma: Applications in diagnosis, prognosis evaluation and personalized therapeutics (Review). Oncol Rep. 2023 Dec;50(6):213.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Synuclein-γ, Ki 67, Oral squamous cell carcinoma, prognosis, cancer cell, diagnosis, cell division","lastPublishedDoi":"10.21203/rs.3.rs-7430987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7430987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOral squamous cell carcinoma (OSCC) is a subset of head and neck squamous cell carcinoma and constitutes about 94% of all oral malignant tumors. Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades, mainly due to delayed diagnosis, underdiagnosis or misdiagnosis. Hence, the data demonstrate the importance of early diagnosis and indirectly show that identifying diagnostic biomarkers is extremely urgent. Our study aims to determine Synuclein-γ and Ki 67 as potential biomarker for the diagnosis of OSCC. Identification of changes in this protein may be useful for promoting the early detection and early treatment of oral squamous cell carcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the institutional ethics committee (IEC2: 67/2022). The material for the study comprised of archival formalin fixed paraffin embedded tumor blocks (FFPE) of oral potentially malignant disorders (n=40) and primary oral squamous cell carcinomas (n=40) cases. Sections were cut and immunohistochemically stained with Anti-SNCG Mouse monoclonal antibody [1H10D2] (STJ98409) and Ki-67 Mouse monoclonal antibody (prediluted/ready to use) [Clone: MIB1]. Statistical analysis was carried out using SPSS software and Chi square tests were used to determine the associations. A p value of \u0026lt; 0.05 was considered to be significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the study group for OPMDs, positive SNCG expression in the cytoplasm of the dysplastic cells was observed in 37/40 (92.5%) cases. Whereas positive Ki 67 expression in nucleus of the dysplastic cells was observed in 34/40 (85%) cases. In the study group for OSCC, positive SNCG expression in the cytoplasm of the tumor cells was observed in 38/40 (95%) cases. Whereas positive Ki 67 expression in nucleus of the tumor cells was observed in 39/40 (97.5%) cases. There was significant correlation between expression of SNCG with habits (p=0.005), lymphovascular invasion (p=0.037), margin clearance (p=0.039) and patient outcome (p=0.021). Also, there were significant correlation between the expression of Ki 67 with gender (p=0.004), histological grade of tumor (0.014) and stage of nodal status (p=0.026)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile Ki 67 as a standalone biomarker suffices in prognosticating both OSCC and dysplasia, there is a lack in significant correlations when it comes to certain parameters in which expression of SNCG was shown to be helpful in the present study. This complementary expression dynamic between Ki 67 and SNCG makes a strong case for using both these biomarkers, probably as a panel in the prognostication of dysplasia and OSCC. However further studies on a larger sample size and better patient follow –up data are needed for a better understanding of the molecular characteristic of the tumor cells which can be useful in developing effective treatment strategies.\u003c/p\u003e","manuscriptTitle":"Biological Role of Synuclein-γ (SNCG) and Ki 67 in Oral Potentially Malignant Disorders and Squamous Cell Carcinomas of Buccal Mucosa: An Immunohistochemical Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 16:14:48","doi":"10.21203/rs.3.rs-7430987/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c112042a-b39f-4239-9ad8-a39d3f81cc30","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T08:41:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 16:14:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7430987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7430987","identity":"rs-7430987","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}