Comparative Study of key cellular polarity proteins’ expression in oral dysplasia and oral squamous cell carcinoma using a combined immunohistochemistry and transcriptomics- based approach

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This groundbreaking study explores how key polarity proteins (PAR3, SCRIBBLE, and DLG7) are expressed in different tissue types: oral epithelial dysplasia (OED), oral squamous cell carcinoma (OSCC), and normal oral mucosa (NOM). The research combines histopathology, immunohistochemistry, and whole-transcriptome sequencing for a comprehensive analysis. Materials and Methods: The study examined tissue samples from 50 tobacco users in West Bengal, India. These samples, preserved in formalin and embedded in paraffin, were stained with hematoxylin and eosin to help identify and classify various stages of OED and OSCC (both well- and moderately-differentiated). Further testing used immunohistochemistry to detect PAR3, SCRIBBLE, and DLG7 proteins. Additionally, RNA sequencing was performed on samples from 25 oral cancer patients who had both OED and OSCC lesions from an independent patient cohort, to analyze differences in gene expression related to the same proteins. Findings: Using IBM SPSS version 20.0, statistical tests (Chi-square) showed that PAR3, SCRIBBLE, and DLG7 were either significantly reduced or completely missing in both OED and OSCC cases. In contrast, these proteins were moderately to strongly expressed in normal oral tissues. Conclusion: The study found a clear and consistent reduction or loss of cell polarity proteins as tissue progresses from a precancerous to a cancerous state. The immunohistochemistry results matched the RNA sequencing data, suggesting that the breakdown of cell polarity is an early and crucial step in the development of oral cancer. These proteins could serve as valuable biomarkers for identifying when oral potentially malignant disorders (OPMDs) begin turning into cancer and may play a key role in cancer initiation, progression, and spread. Health sciences/Oncology/Cancer Health sciences/Medical research Cell polarity proteins Oral epithelial dysplasia Oral squamous cell carcinoma Immunohistochemistry Transcriptomic data analysis Epithelial-Mesenchymal Transition Loss of polarity of basal cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The cellular world is highly polarized and this polarized state is governed by a set of evolutionary conserved self organizing protein complexes with definite spatial pattern. This striking feature of a cell, termed as cell polarity, is a basic and essential aspect of how cells are organized. It refers to the uneven distribution of cellular components—like organelles, proteins, and lipids—within a cell, helping it maintain its structure and function ( 1 ). Depending on the type of cell, polarity can take different forms. For instance, epithelial cells show apico-basal polarity, while cells that move—like fibroblasts, immune cells, and cancer cells—exhibit front-rear polarity ( 2 , 3 ) Specific protein complexes help establish and maintain this polarity by anchoring themselves to certain parts of the cell. These complexes don’t mix—proteins like PAR and CRUMBS stay in the apical part of epithelial cells, while SCRIBBLE and DLG are found in the basolateral regions. Many of these proteins are linked to tight junctions and adherens junctions, which are critical in organizing and maintaining the cell’s structure (4). For example, CRUMBS and PAR complexes help define the top region of cells, while SCRIBBLE and DLG help manage the lower regions ( 5 ). When these polarity proteins are lost or disrupted, cells begin to lose their cohesion. This breakdown is often seen in precancerous tissues and is an early sign of a process called epithelial-mesenchymal transition (EMT), a key step in cancer development ( 6 ). EMT allows normally stationary epithelial cells to acquire mesenchymal characteristics, making them more mobile and invasive. This shift includes losing apico-basal polarity and forming a new front-rear polarity, driven by changes in gene expression. Sometimes, cells go through a “partial EMT,” where they still show some epithelial traits while gaining mesenchymal behavior ( 7 ).These polarity changes are often temporary—cells may reduce polarity marker expression while migrating, then partially restore it after settling into new tissue. Polarity proteins are deeply involved in EMT and are regulated by master genes like TWIST and SNAIL. ( 8 , 9 ).In oral epithelial dysplasia (OED), one key feature pathologists look for is a disruption in the polarity of the basal cell layer ( 10 ). This often shows up as nuclei shifting positions, which is similar to what happens, in the ameloblast cells, during tooth development.( Reversal of polarity) The same histomorphological changes can be observed in histopathology of tall columnar cell breast carcinoma, or papillary renal cells ( 11 ). A loss of the usual palisaded (aligned) arrangement of cells is also considered a sign of polarity change.But whether these visual histomorphologicalchanges actually reflect molecular-level polarity disruption is still not clear and lacks objectivity in evaluation of OED (pramanick). Similarly, the exact mechanisms behind the malignant transformation of oral potentially malignant disorders (OPMDs) remain poorly understood. With that in mind, our study aims to uncover new information about how oral squamous cell carcinoma (OSCC) develops, focusing on the role of polarity protein disruption during EMT. We examined the expression of three key polarity proteins—PAR3, SCRIBBLE, and DLG7—in normal oral mucosa (NOM), OED, and OSCC, using standard histological (H&E) and immunohistochemical techniques. To strengthen our findings, we also analyzed RNA-sequencing data from a separate group of 25 patients, all of whom had leukoplakia (graded as OED), OSCC, and histologically confirmed normal oral tissue. Materials and Methods Tissue Sample Collection Biopsy samples were collected from patients visiting the Department of Oral Pathology at Dr. R. Ahmed Dental College & Hospital in Kolkata. These included samples from normal oral mucosa (NOM), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Immunohistochemical (IHC) analysis was conducted at the Chittaranjan National Cancer Institute, also in Kolkata. A total of 50 samples were selected using a combination of purposive and incidental sampling methods. The study included patients clinically diagnosed with oral potentially malignant disorders (OPMDs), such as leukoplakia, or oral cancer. Patients with systemic illnesses, infections, or poorly differentiated OSCC were excluded. Normal tissue samples were either taken from areas near benign lesions (like fibroma or pyogenic granuloma) or from the healthy opposite side of the mouth in oral cancer patients. In some cases, non-cancerous salivary gland tissue was used as an internal control. Operational Definitions OSCC : Clinically seen as ulcerated growths with thickened, rolled edges. Histology confirmed that the cancer had spread into underlying connective tissue. Leukoplakia : Appeared as white patches that couldn’t be scraped off, often in tobacco users. Under the microscope, these tissues showed signs of dysplasia (either low or high grade) but no invasion into deeper tissues. Immunohistochemistry (IHC) After histopathological confirmation as NOM, OED, and OSCC, immunohistochemical study using specificantibodies against cell polarity proteins (PAR3, SCRIBBLE, DLG 7) following standardprotocol was done. Formalin fixed paraffin embedded tissue blocks were collected. 4- 5micron tissue sections were prepared using microtome. After preparing the sections on slides (slides were precoated with Poly-l-lysine) with proper labelling, the specimens were subjected to IHC staining. The sections were stained with rabbit polyclonal antibody specific to PAR 3 (Dilution 1:400, Novus biologicals, Catalogue # NBP-88861), SCRIBBLE (dilution 1:200, Novus biologicals, Catalogue # NBP2-15110) & DLG 7 (1:200, Novus biologicals, Catalogue #NBP1-87976) and incubated overnight (Ref. colour plate: Fig. 3 ). The secondary antibody kit used was Polymer HRP/DAB (ScyTek Laboratory, USA). The IHC procedure was performed manually. ( 12 ). IHC was used to examine the expression of three key polarity proteins: PAR3, SCRIBBLE, and DLG7. Tissue sections were treated with specific antibodies: PAR3 : 1:400 dilution (Novus Biologicals, Cat # NBP-88861) SCRIBBLE : 1:200 dilution (Novus Biologicals, Cat # NBP2-15110) DLG7 : 1:200 dilution (Novus Biologicals, Cat # NBP1-87976) A detection system using Polymer HRP/DAB (ScyTek Laboratories, USA) was used to visualize staining. Protein expression was scored from 0 to III (0 = none, I = weak, II = moderate, III = strong). ( 13 ). Data Analysis Descriptive statistics were illustrated using bar charts and pie diagrams. Differences in protein expression among NOM, OED, and OSCC groups were assessed using Chi-square tests. A p-value of less than 0.05 was considered statistically significant. All statistical analysis was performed with IBM SPSS version 20.0. RNA-Seq Analysis RNA sequencing data from a separate set of 25 patients (available under project ID PRJEB42969) were obtained from the ENA database. The data were processed using a suite of standard bioinformatics tools: (i) Quality check :FastQC( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) ,MultiQC ( 14 ), (ii) Read alignment : STAR ( 15 ), (iii) Post-alignment QC :Qualimap ( 16 ), (iv) Gene count :HTSeq-count ( 17 ), (v) Differential expression : DESeq2 ( 18 ). Significant changes in gene expression were defined as: (i) Upregulation : log2 fold change > 1 and adjusted p-value < 0.1, (ii) Downregulation : log2 fold change < -1 and adjusted p-value < 0.1 Results Clinicopathological Profile Of the 50 patients initially recruited, 10 dropped out of the study. OSCC was confirmed in 35 cases, and 30 of those were ultimately included based on tissue quality. All patients were residents of West Bengal and had a history of tobacco use, either through smoking, chewing, or both (Table 1 ). In a separate, independent cohort, RNAseq data was generated for 25 patients with concurrent leukoplakia and frank oral tumor (Table 2 ). Histopathological Findings of samples showing normal oral mucosa, oral epithelial dysplasia, oral squamous cell carcinoma All the samples of clinically normal oral mucosa, leukoplakia and malignant oral ulcer, taken through incisional biopsy are processed for histopathological evaluation and stained with hematoxyline and eosine. The sections show normal oral mucosa, different grades of oral epithelial dysplasia and oral squamous cell carcinoma (Fig. 1 ). IHC Findings for Polarity Proteins PAR3 : In healthy tissues (NOM), this protein was absent in the basal layer but weakly expressed in the superficial layer. In OED cases, PAR3 expression dropped significantly or was missing altogether (p = 0.045). Among OSCC samples, 23 showed no expression and only 7 had weak staining (p = 0.04). SCRIBBLE : Strong staining was seen in the basal and moderate staining in the superficial membrane in 80% of NOM samples. However, in OED, 70% of samples showed no expression (p = 0.03). Among OSCC cases, 13 had no staining, 7 were weak, and just 1 showed moderate staining. DLG7 : This protein followed a pattern similar to SCRIBBLE. In OED, 70% of samples were negative (p = 0.03). In OSCC, 15 samples had no expression, 14 were weak, and only one sample showed moderate expression (p = 0.024). An evaluation of all the three polarity proteins’ ( PAR3, SCRIBBLE AND DLG7 ) immunohistochemical expressions in NOM (Fig. 2 ) and also a comparative evaluation between NOM and OED (Fig. 3 ) and NOM and OSCC (Fig. 4 ) are done. Transcriptomic Analysis PARD3 and SCRIB were significantly downregulated in tumor tissues compared to normal (log2FC: -0.78 and − 0.69). (Fig. 5 ) DLGAP5 , which encodes DLG7, was significantly upregulated in tumor tissues (log2FC: 1.19). (Fig. 5 ) In leukoplakia vs. normal comparisons, PARD3 was significantly downregulated (log2FC: -0.61, p = 0.000185), while SCRIB showed a non-significant decrease and DLGAP5 a slight, non-significant increase. (Fig. 5 ) Overall, the trend across all sample types (normal → leukoplakia → cancer) showed a consistent drop in PARD3 and SCRIB , and an increase in DLGAP5 . (Fig. 5 ) Table 1 Clinicopathological characteristics of OSCC patients (n = 30) who were recruited for immunohistochemistry-based characterization of polarity-associated proteins. S.no Patient Id Age and sex Tobacco smoking with/ without alcohol Tobacco chewing with/without alcohol Both tobacco smoking & chewing with or without alcohol Tumour staging (Clinical) Histological subtype 1 RRB1 29 M - - YES T2N1Mx WDSCC 2 RRB2 38 M - - YES T3N2cMx WDSCC 3 RRB3 36 M - YES - T2N1Mx MDSCC 4 RRB4 32 M - YES - T4aN2cMx WDSCC 5 RRB5 32 M - YES - T3N1Mx WDSCC 6 RRB6 40 F - - - T3N2CMx WDSCC 7 RRB7 38 M - - YES T3N2AMx MDSCC 8 RRB8 30 M - YES - T2N1Mx WDSCC 9 RRB9 38 M YES - - T3N2CMx MDSCC 10 RRB10 30 M - YES - T3N2BMx WDSCC 11 RRB11 30 M - YES - T4AN2CMx WDSCC 12 RRB12 30 M - - YES T4AN3Mx WDSCC 13 RRB13 40 M - - YES T2N2AMx WDSCC 14 RRB14 40 M - YES - T1N0Mx WDSCC 15 RRB15 29 M YES - - T3N3Mx WDSCC 16 RRA1 64 F - YES - T4aN1Mx WDSCC 17 RRA2 78 F - YES - T3N2aMx WDSCC 18 RRA3 70 M - - - T4aN2cMx WDSCC 19 RRA4 47 F - YES - T3N2bMx MDSCC 20 RRA5 42 M - - - T4aN2bMx WDSCC 21 RRA6 60 F - YES - T4aN2cMx MDSCC 22 RRA7 68 M - - YES T3N1Mx WDSCC 23 RRA8 47 F - YES - T2N1Mx WDSCC 24 RRA9 75 F - - YES T3N3Mx MDSCC 25 RRA10 48 M YES - - T4aN2cMx MDSCC 26 RRA11 55 M - - YES T4aN2cMx WDSCC 27 RRA12 60 M - YES - T4aN3Mx WDSCC 28 RRA13 44 M YES - - T4aN3Mx MDSCC 29 RRA14 60 M - YES - T3N1Mx WDSCC 30 RRA15 70 F - YES - T4bN2cMx WDSCC Table 2 :Clinicopathological characteristics of 25 patients for whom RNAseq data was generated. Histopathological characterization terms are :EI = Early invesive, MD = Moderately differentiated, WD = Well differentiated tumour, MID = Mild, MTMD = Mild to moderate, MOD = Moderate, SD = Severe dysplasia Patient ID Age Sex Tobacco chewing in form of "Khaini" Tobacco chewing with betel leaf Tobacco chewing (others) Smoking (Bidi) Alcohol Histopathological grade (Tumour) Histopathological grade (leukoplakia) 2 53 M No Yes No Yes No MD MID 3 40 F No Yes No No No MD MID 4 32 M Yes No No Yes Yes 5 50 F No No Yes No No WD MID 6 60 M No No No Yes No WD MID 7 65 M No No No Yes Yes WD MOD 8 51 M No No No Yes No WD MOD 10 65 M Yes No No No Yes WD MOD 13 50 F Yes No Yes No No WD SD 17 53 M Yes No Yes Yes No WD MID 18 58 M No No Yes Yes No WD MID 19 40 O Yes No Yes No No WD SD 23 45 M No Yes No No No WD MID 27 50 M No Yes No Yes No EI MOD 28 55 M Yes Yes No Yes No WD SD 29 36 M No Yes No No No WD MTMD 34 65 M No No No No No WD MID 36 39 M Yes No Yes Yes No WD MID 37 55 M Yes No Yes Yes No WD MID 39 45 F No Yes No No No WD MOD 42 53 M No No No Yes Yes WD MTMD 43 70 M Yes No No No No WD MOD 44 55 F No Yes No No No WD MID 47 65 F No Yes No No No WD SD 52 53 M No Yes No No No MD MID Discussion To gain a clearer picture of how precancerous conditions turn into oral cancer—especially in terms of how cell polarity is perturbed—we examined the expression of three key polarity-regulating proteins: PAR3, SCRIBBLE, and DLG7 (which is encoded by the DLG5 gene). We looked at their behavior across Normal Oral Mucosa (NOM), Oral Epithelial Dysplasia (OED), and Oral Squamous Cell Carcinoma (OSCC), using both immunohistochemistry (IHC) and transcriptomic analysis.In healthy oral mucosa, PAR3 showed only faint staining, limited to the upper epithelial layers, and was absent in the basal cells. This aligns with its known role in biogenesis and functionof tight junctions, which are located near the apical surface of epithelial cells ( 19 ). Similar findings have been observed in the skin, reinforcing the idea that PAR3 is primarily active in superficial cell layers ( 20 ). On the other hand, SCRIBBLE and DLG7 were expressed in both the basal and superficial layers—likely because these proteins are associated with the basolateral parts of cells, which contain structures like desmosomes common in oral epithelium ( 21 , 22 ). The absence of PAR3 in basal cells—cells considered progenitors—raises important questions. It suggests there might be unique regulatory mechanisms at play, and further studies are needed to understand how this affects tissue balance and regeneration.Traditionally, pathologists assess cell polarity by looking at where the nucleus sits within a cell (Hankinson). But in the oral epithelium, this is tricky—basal cells are cube-shaped with nuclei in the center, making it hard to determine if polarity is disrupted. Even though loss of basal cell polarity is one of the histological criteria for dysplasia, It’s still unclear whether these visual histo-morphological changes actually reflect deeper molecular disruptions ( 23 ), Because reliable biomarkers are lacking, dysplasia remains the go-to indicator for predicting cancer risk in potentially malignant oral disorders (OPMDs).To address this issue, our study focused on analyzing polarity proteins as potential molecular markers. We found that expression of PAR3, SCRIBBLE, and DLG7 was significantly reduced in dysplastic tissues compared to normal tissue ( 6 )The most notable change was the loss of SCRIBBLE and DLG7 in the basal layer, similar to patterns observed in other polarity proteins like LGL2 in gastric dysplasia and cancer ( 24 ).Whether loss of cell polarity is an early driver of cancer or simply a byproduct of other cellular changes is still up for debate. However, our findings suggest that polarity loss could be an early event in oral cancer development, making it a potential early warning sign for histopathologists and clinicians.Transcriptomic analysis supported these observations: PAR3 and SCRIB were downregulated in dysplasia, while DLG5 was unexpectedly upregulated. This mismatch between mRNA levels and protein expression, particularly for DLG7, hints at complex regulatory processes that could include post-transcriptional or translational mechanisms.In OSCC, a process known as partial epithelial-to-mesenchymal transition (EMT) plays a key role in tumor invasion. During EMT, cells begin to adopt features of mesenchymal cells while retaining some epithelial traits ( 25 ).Our IHC data showed consistent loss of all three polarity proteins in OSCC, regardless of tumor grade or stage, echoing findings from Drosophila studies, where loss of Scribble proteins leads to unchecked cell growth. This supports the idea that these proteins help prevent EMT and tumor progression. Interestingly, even though IHC showed DLG7 was lost in OSCC, transcriptomic data showed DLG5 levels increased from dysplasia to carcinoma. This inconsistency further underscores the need to explore how DLG7 is regulated after transcription.A study by Tao Xu et al. (2020) in breast cancer found that high DLG7 mRNA levels were linked to poor outcomes and advanced disease—echoing our transcriptomic results. Still, like our findings, their study found no correlation between DLG7 expression and patient age or tumor stage ( 26 ).Perhaps most importantly, we observed a clear, progressive drop in PARD3 and SCRIB levels from normal tissue to dysplasia to carcinoma—both at the protein and gene levels. This supports a progressive model of oral cancer development, with polarity loss playing a central role. These findings suggest that polarity proteins could become useful diagnostic or prognostic tools for oral precancers or at least can help in objective histopathological assessment of OED. In summary, our study provides strong molecular evidence that cell polarity disruptions are involved early in oral cancer development and continue through progression to OSCC. These insights pave the way for larger studies, ideally with long-term patient follow-up, to explore the potential of these proteins as reliable biomarkers for malignancy risk in OPMDs. As polarity rewiring gains recognition as a hallmark of cancer, our findings add valuable knowledge to the evolving story of oral cancer biology. Declarations Funding: This study received no external funding. All the patients were included in the study after taking their Informed consents. All methods in this study were carried out in accordance with relevant Ethical guidelines and regulations. All experimental protocols were approved by the Institutional Ethics Committee (IEC) RADCH. Author Contribution Author contributions: SG- was responsible for conceiving the study and histo-pathological assessment. URC was responsible for patient recruitment and clinical assessment. SC and NKB were responsible for generation and analysis of transcriptomic data. SS and DR carried out the drafting. 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(2022). 10.3389/fcell.2022.885133 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Sep, 2025 Reviews received at journal 17 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers invited by journal 29 Jun, 2025 Editor assigned by journal 29 Jun, 2025 Editor invited by journal 25 Jun, 2025 Submission checks completed at journal 25 Jun, 2025 First submitted to journal 23 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-6956981","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":478346344,"identity":"aff1a218-6c3d-433c-bb2b-14826ae8b39c","order_by":0,"name":"Dr Sandip Ghose","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYJCCAwxsDAxs7M3Hf3xgADGI1cLHcyxBcgZICzNR9gC1yEnkKEjzgDiEtMi3nz14uKDsnjybRA6Dsc2vbfJ8zAyMHz7m4NZicCYv4fCMc8WGbTxvDyTn9t02bGNmYJacuQ2PFoYcg8O8bQmMbexAvbk9txmBWtiYefFoke9/A9Zi38aQY9hs2XPbnqAWhhsQWxLbOHKMmRl+3E4kqMXgBtAWnnMJyW08x9IYextuJ7cxMzbj9Yt8f47xZ56yBNv57c3HGH78uQ1iHPzwEZ/DUABjG5hsIFY9CPwhRfEoGAWjYBSMFAAAURJQhBBZ3KEAAAAASUVORK5CYII=","orcid":"","institution":"Dr. R. Ahmed Dental College and Hospital","correspondingAuthor":true,"prefix":"Dr","firstName":"Sandip","middleName":"","lastName":"Ghose","suffix":""},{"id":478346345,"identity":"97f4f3a9-2ccc-4a29-9083-1bb50cf66c74","order_by":1,"name":"Dr U Renuka Chaudry","email":"","orcid":"","institution":"Dr. R. Ahmed Dental College and Hospital","correspondingAuthor":false,"prefix":"Dr","firstName":"U","middleName":"Renuka","lastName":"Chaudry","suffix":""},{"id":478346346,"identity":"fa632786-756e-4a1c-81f2-a87f3a217313","order_by":2,"name":"Mr Shouvik Chakravarty","email":"","orcid":"","institution":"National Institute of Biomedical Genomics","correspondingAuthor":false,"prefix":"Mr","firstName":"Shouvik","middleName":"","lastName":"Chakravarty","suffix":""},{"id":478346347,"identity":"d8ed6dba-e9bb-4cae-80fd-9264e100a978","order_by":3,"name":"Dr Snehanjan Sarangi","email":"","orcid":"","institution":"Dr. R. Ahmed Dental College and Hospital","correspondingAuthor":false,"prefix":"Dr","firstName":"Snehanjan","middleName":"","lastName":"Sarangi","suffix":""},{"id":478346348,"identity":"5921fca9-7bf8-4669-9711-cef2bb0ec893","order_by":4,"name":"Dr Nidhan K Biswas","email":"","orcid":"","institution":"National Institute of Biomedical Genomics","correspondingAuthor":false,"prefix":"Dr","firstName":"Nidhan","middleName":"K","lastName":"Biswas","suffix":""},{"id":478346349,"identity":"c02e1978-395b-407f-a1ed-cbc904b2a4dc","order_by":5,"name":"Dr Rathindranath Baral","email":"","orcid":"","institution":"Chittaranjan National Cancer Institute","correspondingAuthor":false,"prefix":"Dr","firstName":"Rathindranath","middleName":"","lastName":"Baral","suffix":""},{"id":478346350,"identity":"654d4172-b71b-4d09-bdeb-6168d8c675a6","order_by":6,"name":"Dr Debarati Ray","email":"","orcid":"","institution":"Dr. R. Ahmed Dental College and Hospital","correspondingAuthor":false,"prefix":"Dr","firstName":"Debarati","middleName":"","lastName":"Ray","suffix":""}],"badges":[],"createdAt":"2025-06-23 12:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6956981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6956981/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-27447-2","type":"published","date":"2025-12-03T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85924979,"identity":"cd73fb59-775e-41e4-bf06-9d3c7e2a2499","added_by":"auto","created_at":"2025-07-03 08:30:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65107,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/32d975021c34604ddeead38c.jpg"},{"id":85923386,"identity":"ef84e5ba-5d57-430a-be59-ae13adf50256","added_by":"auto","created_at":"2025-07-03 08:22:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49152,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/e4faaf514eb5473c558b4df3.jpg"},{"id":85924981,"identity":"b55d434b-a430-46a2-864d-eb6a8549717e","added_by":"auto","created_at":"2025-07-03 08:30:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":65553,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/0dac033d476a0ddf7dd2ecce.jpg"},{"id":85924980,"identity":"39a2804b-253a-4e62-bb63-7935526174eb","added_by":"auto","created_at":"2025-07-03 08:30:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63817,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/7b4b7e5cc7ed47db8f00b88d.jpg"},{"id":85923393,"identity":"35088fc5-8f0a-415d-a5e9-b6754f05c929","added_by":"auto","created_at":"2025-07-03 08:22:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28725,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/f38638f0873846b3dada8759.jpg"},{"id":97724084,"identity":"7018916e-ca2f-4a40-8ff4-5f98048d5aa5","added_by":"auto","created_at":"2025-12-08 16:11:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1485125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6956981/v1/02186483-bcbf-4696-924f-8835af883ee1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Study of key cellular polarity proteins’ expression in oral dysplasia and oral squamous cell carcinoma using a combined immunohistochemistry and transcriptomics- based approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cellular world is highly polarized and this polarized state is governed by a set of evolutionary conserved self organizing protein complexes with definite spatial pattern. This striking feature of a cell, termed as cell polarity, is a basic and essential aspect of how cells are organized. It refers to the uneven distribution of cellular components\u0026mdash;like organelles, proteins, and lipids\u0026mdash;within a cell, helping it maintain its structure and function (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Depending on the type of cell, polarity can take different forms. For instance, epithelial cells show apico-basal polarity, while cells that move\u0026mdash;like fibroblasts, immune cells, and cancer cells\u0026mdash;exhibit front-rear polarity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Specific protein complexes help establish and maintain this polarity by anchoring themselves to certain parts of the cell. These complexes don\u0026rsquo;t mix\u0026mdash;proteins like PAR and CRUMBS stay in the apical part of epithelial cells, while SCRIBBLE and DLG are found in the basolateral regions. Many of these proteins are linked to tight junctions and adherens junctions, which are critical in organizing and maintaining the cell\u0026rsquo;s structure (4). For example, CRUMBS and PAR complexes help define the top region of cells, while SCRIBBLE and DLG help manage the lower regions (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen these polarity proteins are lost or disrupted, cells begin to lose their cohesion. This breakdown is often seen in precancerous tissues and is an early sign of a process called epithelial-mesenchymal transition (EMT), a key step in cancer development (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). EMT allows normally stationary epithelial cells to acquire mesenchymal characteristics, making them more mobile and invasive. This shift includes losing apico-basal polarity and forming a new front-rear polarity, driven by changes in gene expression. Sometimes, cells go through a \u0026ldquo;partial EMT,\u0026rdquo; where they still show some epithelial traits while gaining mesenchymal behavior (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).These polarity changes are often temporary\u0026mdash;cells may reduce polarity marker expression while migrating, then partially restore it after settling into new tissue. Polarity proteins are deeply involved in EMT and are regulated by master genes like TWIST and SNAIL. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).In oral epithelial dysplasia (OED), one key feature pathologists look for is a disruption in the polarity of the basal cell layer (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This often shows up as nuclei shifting positions, which is similar to what happens, in the ameloblast cells, during tooth development.( Reversal of polarity) The same histomorphological changes can be observed in histopathology of tall columnar cell breast carcinoma, or papillary renal cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). A loss of the usual palisaded (aligned) arrangement of cells is also considered a sign of polarity change.But whether these visual histomorphologicalchanges actually reflect molecular-level polarity disruption is still not clear and lacks objectivity in evaluation of OED (pramanick). Similarly, the exact mechanisms behind the malignant transformation of oral potentially malignant disorders (OPMDs) remain poorly understood.\u003c/p\u003e \u003cp\u003eWith that in mind, our study aims to uncover new information about how oral squamous cell carcinoma (OSCC) develops, focusing on the role of polarity protein disruption during EMT. We examined the expression of three key polarity proteins\u0026mdash;PAR3, SCRIBBLE, and DLG7\u0026mdash;in normal oral mucosa (NOM), OED, and OSCC, using standard histological (H\u0026amp;E) and immunohistochemical techniques. To strengthen our findings, we also analyzed RNA-sequencing data from a separate group of 25 patients, all of whom had leukoplakia (graded as OED), OSCC, and histologically confirmed normal oral tissue.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue Sample Collection\u003c/h2\u003e \u003cp\u003eBiopsy samples were collected from patients visiting the Department of Oral Pathology at Dr. R. Ahmed Dental College \u0026amp; Hospital in Kolkata. These included samples from normal oral mucosa (NOM), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Immunohistochemical (IHC) analysis was conducted at the Chittaranjan National Cancer Institute, also in Kolkata. A total of 50 samples were selected using a combination of purposive and incidental sampling methods.\u003c/p\u003e \u003cp\u003eThe study included patients clinically diagnosed with oral potentially malignant disorders (OPMDs), such as leukoplakia, or oral cancer. Patients with systemic illnesses, infections, or poorly differentiated OSCC were excluded. Normal tissue samples were either taken from areas near benign lesions (like fibroma or pyogenic granuloma) or from the healthy opposite side of the mouth in oral cancer patients. In some cases, non-cancerous salivary gland tissue was used as an internal control.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOperational Definitions\u003c/h3\u003e\n\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eOSCC\u003c/b\u003e: Clinically seen as ulcerated growths with thickened, rolled edges. Histology confirmed that the cancer had spread into underlying connective tissue.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLeukoplakia\u003c/b\u003e: Appeared as white patches that couldn\u0026rsquo;t be scraped off, often in tobacco users. Under the microscope, these tissues showed signs of dysplasia (either low or high grade) but no invasion into deeper tissues.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eAfter histopathological confirmation as NOM, OED, and OSCC, immunohistochemical study using specificantibodies against cell polarity proteins (PAR3, SCRIBBLE, DLG 7) following standardprotocol was done. Formalin fixed paraffin embedded tissue blocks were collected. 4-\u003c/p\u003e \u003cp\u003e5micron tissue sections were prepared using microtome. After preparing the sections on\u003c/p\u003e \u003cp\u003eslides (slides were precoated with Poly-l-lysine) with proper labelling, the specimens were\u003c/p\u003e \u003cp\u003esubjected to IHC staining. The sections were stained with rabbit polyclonal antibody specific\u003c/p\u003e \u003cp\u003eto PAR 3 (Dilution 1:400, Novus biologicals, Catalogue # NBP-88861), SCRIBBLE (dilution\u003c/p\u003e \u003cp\u003e1:200, Novus biologicals, Catalogue # NBP2-15110) \u0026amp; DLG 7 (1:200, Novus biologicals,\u003c/p\u003e \u003cp\u003eCatalogue #NBP1-87976) and incubated overnight (Ref. colour plate: Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The secondary\u003c/p\u003e \u003cp\u003eantibody kit used was Polymer HRP/DAB (ScyTek Laboratory, USA). The IHC procedure\u003c/p\u003e \u003cp\u003ewas performed manually. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIHC was used to examine the expression of three key polarity proteins: PAR3, SCRIBBLE, and DLG7. Tissue sections were treated with specific antibodies:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePAR3\u003c/b\u003e: 1:400 dilution (Novus Biologicals, Cat # NBP-88861)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSCRIBBLE\u003c/b\u003e: 1:200 dilution (Novus Biologicals, Cat # NBP2-15110)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDLG7\u003c/b\u003e: 1:200 dilution (Novus Biologicals, Cat # NBP1-87976)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eA detection system using Polymer HRP/DAB (ScyTek Laboratories, USA) was used to visualize staining. Protein expression was scored from 0 to III (0\u0026thinsp;=\u0026thinsp;none, I\u0026thinsp;=\u0026thinsp;weak, II\u0026thinsp;=\u0026thinsp;moderate, III\u0026thinsp;=\u0026thinsp;strong). (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eDescriptive statistics were illustrated using bar charts and pie diagrams. Differences in protein expression among NOM, OED, and OSCC groups were assessed using Chi-square tests. A p-value of less than 0.05 was considered statistically significant. All statistical analysis was performed with IBM SPSS version 20.0.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA-Seq Analysis\u003c/h3\u003e\n\u003cp\u003eRNA sequencing data from a separate set of 25 patients (available under project ID PRJEB42969) were obtained from the ENA database. The data were processed using a suite of standard bioinformatics tools: (i) \u003cb\u003eQuality check\u003c/b\u003e:FastQC(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) ,MultiQC (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), (ii) \u003cb\u003eRead alignment\u003c/b\u003e: STAR (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), (iii) \u003cb\u003ePost-alignment QC\u003c/b\u003e:Qualimap (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), (iv) \u003cb\u003eGene count\u003c/b\u003e:HTSeq-count (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), (v) \u003cb\u003eDifferential expression\u003c/b\u003e: DESeq2 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Significant changes in gene expression were defined as: (i) \u003cb\u003eUpregulation\u003c/b\u003e: log2 fold change\u0026thinsp;\u0026gt;\u0026thinsp;1 and adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1, (ii) \u003cb\u003eDownregulation\u003c/b\u003e: log2 fold change \u0026lt; -1 and adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological Profile\u003c/h2\u003e \u003cp\u003eOf the 50 patients initially recruited, 10 dropped out of the study. OSCC was confirmed in 35 cases, and 30 of those were ultimately included based on tissue quality. All patients were residents of West Bengal and had a history of tobacco use, either through smoking, chewing, or both (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In a separate, independent cohort, RNAseq data was generated for 25 patients with concurrent leukoplakia and frank oral tumor (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological Findings of samples showing normal oral mucosa, oral epithelial dysplasia, oral squamous cell carcinoma\u003c/h3\u003e\n\u003cp\u003eAll the samples of clinically normal oral mucosa, leukoplakia and malignant oral ulcer, taken through incisional biopsy are processed for histopathological evaluation and stained with hematoxyline and eosine. The sections show normal oral mucosa, different grades of oral epithelial dysplasia and oral squamous cell carcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIHC Findings for Polarity Proteins\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePAR3\u003c/b\u003e: In healthy tissues (NOM), this protein was absent in the basal layer but weakly expressed in the superficial layer. In OED cases, PAR3 expression dropped significantly or was missing altogether (p\u0026thinsp;=\u0026thinsp;0.045). Among OSCC samples, 23 showed no expression and only 7 had weak staining (p\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSCRIBBLE\u003c/b\u003e: Strong staining was seen in the basal and moderate staining in the superficial membrane in 80% of NOM samples. However, in OED, 70% of samples showed no expression (p\u0026thinsp;=\u0026thinsp;0.03). Among OSCC cases, 13 had no staining, 7 were weak, and just 1 showed moderate staining.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDLG7\u003c/b\u003e: This protein followed a pattern similar to SCRIBBLE. In OED, 70% of samples were negative (p\u0026thinsp;=\u0026thinsp;0.03). In OSCC, 15 samples had no expression, 14 were weak, and only one sample showed moderate expression (p\u0026thinsp;=\u0026thinsp;0.024).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eAn evaluation of all the three polarity proteins\u0026rsquo; (\u003cb\u003ePAR3, SCRIBBLE AND DLG7\u003c/b\u003e) immunohistochemical expressions in NOM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and also a comparative evaluation between NOM and OED (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and NOM and OSCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) are done.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic Analysis\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePARD3\u003c/b\u003e and \u003cb\u003eSCRIB\u003c/b\u003e were significantly downregulated in tumor tissues compared to normal (log2FC: -0.78 and \u0026minus;\u0026thinsp;0.69). (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDLGAP5\u003c/b\u003e, which encodes DLG7, was significantly upregulated in tumor tissues (log2FC: 1.19). (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eIn leukoplakia vs. normal comparisons, \u003cb\u003ePARD3\u003c/b\u003e was significantly downregulated (log2FC: -0.61, p\u0026thinsp;=\u0026thinsp;0.000185), while SCRIB showed a non-significant decrease and DLGAP5 a slight, non-significant increase. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eOverall, the trend across all sample types (normal \u0026rarr; leukoplakia \u0026rarr; cancer) showed a consistent drop in \u003cb\u003ePARD3\u003c/b\u003e and \u003cb\u003eSCRIB\u003c/b\u003e, and an increase in \u003cb\u003eDLGAP5\u003c/b\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\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\u003eClinicopathological characteristics of OSCC patients (n\u0026thinsp;=\u0026thinsp;30) who were recruited for immunohistochemistry-based characterization of polarity-associated proteins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS.no\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatient Id\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eAge and sex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTobacco smoking with/ without alcohol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTobacco chewing with/without alcohol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBoth tobacco smoking \u0026amp; chewing with or without\u003c/p\u003e \u003cp\u003ealcohol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTumour staging (Clinical)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHistological subtype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT2N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT2N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2CMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2AMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT2N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2CMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2BMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4AN2CMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4AN3Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT2N2AMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT1N0Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRB15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N3Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2aMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N2bMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2bMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT2N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N3Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN3Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4aN3Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e29\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N1Mx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRA15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT4bN2cMx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWDSCC\u003c/p\u003e \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\u003e:Clinicopathological characteristics of 25 patients for whom RNAseq data was generated. Histopathological characterization terms are :EI\u0026thinsp;=\u0026thinsp;Early invesive, MD\u0026thinsp;=\u0026thinsp;Moderately differentiated, WD\u0026thinsp;=\u0026thinsp;Well differentiated tumour, MID\u0026thinsp;=\u0026thinsp;Mild, MTMD\u0026thinsp;=\u0026thinsp;Mild to moderate, MOD\u0026thinsp;=\u0026thinsp;Moderate, SD\u0026thinsp;=\u0026thinsp;Severe dysplasia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTobacco chewing in form of \"Khaini\"\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTobacco chewing with betel leaf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTobacco chewing (others)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSmoking (Bidi)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAlcohol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHistopathological grade (Tumour)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHistopathological grade (leukoplakia)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMTMD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMTMD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMID\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo gain a clearer picture of how precancerous conditions turn into oral cancer\u0026mdash;especially in terms of how cell polarity is perturbed\u0026mdash;we examined the expression of three key polarity-regulating proteins: PAR3, SCRIBBLE, and DLG7 (which is encoded by the DLG5 gene). We looked at their behavior across Normal Oral Mucosa (NOM), Oral Epithelial Dysplasia (OED), and Oral Squamous Cell Carcinoma (OSCC), using both immunohistochemistry (IHC) and transcriptomic analysis.In healthy oral mucosa, PAR3 showed only faint staining, limited to the upper epithelial layers, and was absent in the basal cells. This aligns with its known role in biogenesis and functionof tight junctions, which are located near the apical surface of epithelial cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Similar findings have been observed in the skin, reinforcing the idea that PAR3 is primarily active in superficial cell layers (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). On the other hand, SCRIBBLE and DLG7 were expressed in both the basal and superficial layers\u0026mdash;likely because these proteins are associated with the basolateral parts of cells, which contain structures like desmosomes common in oral epithelium (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The absence of PAR3 in basal cells\u0026mdash;cells considered progenitors\u0026mdash;raises important questions. It suggests there might be unique regulatory mechanisms at play, and further studies are needed to understand how this affects tissue balance and regeneration.Traditionally, pathologists assess cell polarity by looking at where the nucleus sits within a cell (Hankinson). But in the oral epithelium, this is tricky\u0026mdash;basal cells are cube-shaped with nuclei in the center, making it hard to determine if polarity is disrupted. Even though loss of basal cell polarity is one of the histological criteria for dysplasia, It\u0026rsquo;s still unclear whether these visual histo-morphological changes actually reflect deeper molecular disruptions (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), Because reliable biomarkers are lacking, dysplasia remains the go-to indicator for predicting cancer risk in potentially malignant oral disorders (OPMDs).To address this issue, our study focused on analyzing polarity proteins as potential molecular markers. We found that expression of PAR3, SCRIBBLE, and DLG7 was significantly reduced in dysplastic tissues compared to normal tissue (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)The most notable change was the loss of SCRIBBLE and DLG7 in the basal layer, similar to patterns observed in other polarity proteins like LGL2 in gastric dysplasia and cancer (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).Whether loss of cell polarity is an early driver of cancer or simply a byproduct of other cellular changes is still up for debate. However, our findings suggest that polarity loss could be an early event in oral cancer development, making it a potential early warning sign for histopathologists and clinicians.Transcriptomic analysis supported these observations: PAR3 and SCRIB were downregulated in dysplasia, while DLG5 was unexpectedly upregulated. This mismatch between mRNA levels and protein expression, particularly for DLG7, hints at complex regulatory processes that could include post-transcriptional or translational mechanisms.In OSCC, a process known as partial epithelial-to-mesenchymal transition (EMT) plays a key role in tumor invasion. During EMT, cells begin to adopt features of mesenchymal cells while retaining some epithelial traits (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).Our IHC data showed consistent loss of all three polarity proteins in OSCC, regardless of tumor grade or stage, echoing findings from Drosophila studies, where loss of Scribble proteins leads to unchecked cell growth. This supports the idea that these proteins help prevent EMT and tumor progression.\u003c/p\u003e \u003cp\u003eInterestingly, even though IHC showed DLG7 was lost in OSCC, transcriptomic data showed DLG5 levels increased from dysplasia to carcinoma. This inconsistency further underscores the need to explore how DLG7 is regulated after transcription.A study by Tao Xu et al. (2020) in breast cancer found that high DLG7 mRNA levels were linked to poor outcomes and advanced disease\u0026mdash;echoing our transcriptomic results. Still, like our findings, their study found no correlation between DLG7 expression and patient age or tumor stage (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).Perhaps most importantly, we observed a clear, progressive drop in PARD3 and SCRIB levels from normal tissue to dysplasia to carcinoma\u0026mdash;both at the protein and gene levels. This supports a progressive model of oral cancer development, with polarity loss playing a central role. These findings suggest that polarity proteins could become useful diagnostic or prognostic tools for oral precancers or at least can help in objective histopathological assessment of OED. In summary, our study provides strong molecular evidence that cell polarity disruptions are involved early in oral cancer development and continue through progression to OSCC. These insights pave the way for larger studies, ideally with long-term patient follow-up, to explore the potential of these proteins as reliable biomarkers for malignancy risk in OPMDs. As polarity rewiring gains recognition as a hallmark of cancer, our findings add valuable knowledge to the evolving story of oral cancer biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study received no external funding.\u003c/p\u003e \u003cp\u003e All the patients were included in the study after taking their Informed consents. All methods in this study were carried out in accordance with relevant Ethical guidelines and regulations. All experimental protocols were approved by the Institutional Ethics Committee (IEC) RADCH.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions: SG- was responsible for conceiving the study and histo-pathological assessment. URC was responsible for patient recruitment and clinical assessment. SC and NKB were responsible for generation and analysis of transcriptomic data. SS and DR carried out the drafting. RNB was responsible for guiding and coordinating IHC investigations. SG, URC and SC wrote the article.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eTranscriptome sequence data used in this study is publicly available and has been downloaded from the European Nucleotide Archive accession - PRJEB42969\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJossin, Y. Molecular mechanisms of cell polarity in a range of model systems and in migrating neurons. \u003cem\u003eMol. 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Journal of Oral and Maxillofacial Pathology 24(1):p 189, Jan\u0026ndash;Apr 2020. | \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/jomfp.JOMFP_334_19\u003c/span\u003e\u003cspan address=\"10.4103/jomfp.JOMFP_334_19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. et al. The Key Role of RNA Modification in Breast Cancer. Front Cell Dev Biol. ;10:885133. Published 2022 Jun 1. (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcell.2022.885133\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2022.885133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cell polarity proteins, Oral epithelial dysplasia, Oral squamous cell carcinoma, Immunohistochemistry, Transcriptomic data analysis, Epithelial-Mesenchymal Transition, Loss of polarity of basal cells","lastPublishedDoi":"10.21203/rs.3.rs-6956981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6956981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective:\u003c/h2\u003e \u003cp\u003eThe process by which oral precancerous lesions turn malignant\u0026mdash;especially through Epithelial-Mesenchymal Transition (EMT)\u0026mdash;is closely tied to changes in certain cell polarity proteins. This groundbreaking study explores how key polarity proteins (PAR3, SCRIBBLE, and DLG7) are expressed in different tissue types: oral epithelial dysplasia (OED), oral squamous cell carcinoma (OSCC), and normal oral mucosa (NOM). The research combines histopathology, immunohistochemistry, and whole-transcriptome sequencing for a comprehensive analysis.\u003c/p\u003e\u003ch2\u003eMaterials and Methods:\u003c/h2\u003e \u003cp\u003eThe study examined tissue samples from 50 tobacco users in West Bengal, India. These samples, preserved in formalin and embedded in paraffin, were stained with hematoxylin and eosin to help identify and classify various stages of OED and OSCC (both well- and moderately-differentiated). Further testing used immunohistochemistry to detect PAR3, SCRIBBLE, and DLG7 proteins. Additionally, RNA sequencing was performed on samples from 25 oral cancer patients who had both OED and OSCC lesions from an independent patient cohort, to analyze differences in gene expression related to the same proteins.\u003c/p\u003e\u003ch2\u003eFindings:\u003c/h2\u003e \u003cp\u003eUsing IBM SPSS version 20.0, statistical tests (Chi-square) showed that PAR3, SCRIBBLE, and DLG7 were either significantly reduced or completely missing in both OED and OSCC cases. In contrast, these proteins were moderately to strongly expressed in normal oral tissues.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eThe study found a clear and consistent reduction or loss of cell polarity proteins as tissue progresses from a precancerous to a cancerous state. The immunohistochemistry results matched the RNA sequencing data, suggesting that the breakdown of cell polarity is an early and crucial step in the development of oral cancer. These proteins could serve as valuable biomarkers for identifying when oral potentially malignant disorders (OPMDs) begin turning into cancer and may play a key role in cancer initiation, progression, and spread.\u003c/p\u003e","manuscriptTitle":"Comparative Study of key cellular polarity proteins’ expression in oral dysplasia and oral squamous cell carcinoma using a combined immunohistochemistry and transcriptomics- based approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 08:22:34","doi":"10.21203/rs.3.rs-6956981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-18T15:03:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-17T08:10:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T05:22:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36222406110177287675437324437935284960","date":"2025-09-03T08:33:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233704636426351648957944371790534450493","date":"2025-09-01T08:29:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67960086420522152629237621385839119652","date":"2025-07-18T12:18:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-30T02:27:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-30T02:08:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-25T16:12:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-25T09:44:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-23T12:41:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c36b405f-1725-4e1a-b26a-be4a9ed9f901","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50784462,"name":"Health sciences/Oncology/Cancer"},{"id":50784463,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-12-08T16:06:47+00:00","versionOfRecord":{"articleIdentity":"rs-6956981","link":"https://doi.org/10.1038/s41598-025-27447-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-03 15:57:19","publishedOnDateReadable":"December 3rd, 2025"},"versionCreatedAt":"2025-07-03 08:22:34","video":"","vorDoi":"10.1038/s41598-025-27447-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-27447-2","workflowStages":[]},"version":"v1","identity":"rs-6956981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6956981","identity":"rs-6956981","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}