Immunohistochemical evaluation of Ki-67 and Nucleostemin in a model of oral potentially malignant disorders to malignant lesions

Immunohistochemical evaluation of Ki-67 and Nucleostemin in a model of oral potentially malignant disorders to malignant lesions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunohistochemical evaluation of Ki-67 and Nucleostemin in a model of oral potentially malignant disorders to malignant lesions Shreyasi Polley, Sunitha Carnelio, Srikant Natarajan, Ravishankar Nagaraja This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7737960/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Proliferative biomarkers are one among the tumor markers that assist in tailored clinical management, thus could aid in improving the quality of life of patients with head and neck cancers. The present study aims to explore the clinical significance and relevance of Nucleostemin (NS) as a proliferative marker along with Ki-67, a known diagnostic proliferative marker in oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC), by evaluating the immune expression of these biomarkers. Methods A retrospective immunohistochemical (IHC) analysis of NS & Ki-67 was performed on a total of 90 primary cases, i.e., leucoplakia with dysplasia (LWD, n = 15), oral submucous fibrosis (OSMF, n = 15), OSCC (n = 60), correlated with clinicopathological features. Results An overexpression of these markers was observed in the upper strata of the epithelium of histologically diagnosed severe dysplasia, moderately advanced OSMF (among the OPMD) and similar expression was seen in moderately and poorly differentiated carcinoma. The clinicopathological features did exhibit a significant correlation in advanced stage of the disease. Normal oral mucosa, adjacent to the lesion showed NS expression only in the basal strata with few cells in the lower spinous layer. Among OPMD, dysplasia (n = 5) and advanced OSMF (n = 2) exhibiting increased expression, progressed to malignancy. Conclusions The presence of NS and Ki-67 in various strata of the diseased mucosa was able to identify a few cases in OPMD with a cut-off value of 31.9 for NS and 23.9 for Ki-67 progressing into OSCC. However, this method is challenging for routine diagnosis. Oral cancer oral potentially malignant disorders cancer stem cells oral squamous cell carcinoma leucoplakia dysplasia Figures Figure 1 Figure 2 BACKGROUND Oral squamous cell carcinoma (OSCC) is the most prevalent malignant epithelial neoplasm affecting the oral cavity. Two to four percent of all cancers worldwide are oral cancers (OC). However, South East Asia and the Indian subcontinent have a slightly higher rate of OC with a male preponderance. Alcohol consumption and tobacco usage in various forms are the major causative factors implicated among these populations [ 1 ]. Oral potentially malignant disorders (OPMD) are the precursor lesions or conditions that can transform into OC and the most seen are leukoplakia and oral submucous fibrosis (OSMF) [ 2 ]. Various genetic and epigenetic changes due to exposure to carcinogens over a period of time could be the reason for OPMD transforming to carcinomas or normal epithelium converting to carcinoma [ 3 ]. Cancer stem cells (CSC), a subset of cancer cells possess stem cell-like properties that could initiate self-renewal, differentiate into various cell lineages, progress and disseminate to various organs. Much research is focused on understanding the behavior of these cells. Due to the accumulation of genetic and epigenetic modifications over time, it has been speculated that these cells are derived from human embryonic or adult or normal stem cells and some of the CSC markers have been employed to understand the biological nature of these cells [ 4 ]. One such stem cell marker for the current study is nucleostemin (NS), a nucleolar GTP-binding protein that has been demonstrated to play a pivotal role in carcinogenesis of breast, prostate and gastric cancers [ 5 – 7 ]. Studies have proven NS to be a proliferative marker of various cancer cell lines, including NS knock-down studies [ 8 – 10 ]. However, studies of NS on OPMD are minimal. Ki-67 is a large nonhistone protein present in the cell nuclei during all the active phases of the cell cycle (G1, S, G2, and M phases) but is absent in the quiescent G0 phase and studies have correlated it with growth fracture, which is indicative of cell proliferation [ 11 ]. In the present study, immunohistochemical (IHC) appraisal of NS in OPMDs [leukoplakia with dysplasia (LWD) and OSMF] and OSCC has been evaluated. Also, an attempt has been made to correlate the expression of Ki-67 and NS with various clinicopathological parameters [ 12 , 13 ]. MATERIALS AND METHODS Materials Formalin fixed paraffin embedded (FFPE) tissues were retrieved between January 2018 to December 2020 after obtaining the Institutional Ethics Committee approval. The study included a total of 90 primary cases, i.e., 15 cases each of LWD [ 14 ], OSMF [ 15 ] and 60 cases of OSCC [ 16 ]. 10 cases of normal tissue obtained from the extraction site of impacted third molar were included as controls. Follow-up data was obtained from medical records. The slides were stained with hematoxylin and eosin for comparison. Immunohistochemical staining The IHC staining of NS and Ki-67 was carried out on the tissue sections of 4µm from the FFPE tissue blocks. 3-aminopropyl-tri-ethoxy-silane (APES) coated slides (Sigma-Aldrich Ltd, Massachusetts, USA) were used. The primary antibody, NS rabbit polyclonal antibody (catalog-PA5-83350, RRID: AB_2790506 Dako, Glostrup, Denmark), anti Ki-67 monoclonal antibody (MIB, Dako, Denmark, 1:200) was diluted with tris buffered saline (TBS). After de-paraffinization and endogenous peroxidase block in hydrogen peroxide, antigen retrieval was done in ethylenediamine tetra-acetic acid (EDTA) diluted in distilled water for 1 hour in heat-induced epitope retrieval at 110°C. The sections were incubated at 4°C overnight with the diluted rabbit polyclonal primary antibody for NS, while sections were incubated for 1 hour at 37◦C for Ki-67 monoclonal antibody. The secondary antibody kit containing the target binder or amplifier and HRP (EnVision + System-HRP Labelled Polymer, Dako) was applied as per manufacturer instructions. The marker was detected by using chromogen containing diaminobenzidine (DAB) as per manufacturer’s instructions. The sections were further counterstained with Mayer’s hematoxylin for better visualization and mounted with DPX (Sigma-Aldrich Ltd, Massachusetts, USA). Negative control sections were without the primary antibody. Immunostaining evaluation The nucleus was considered as positive reactivity when stained brown. The slides were evaluated with a light microscope (Olympus BX41) attached to an Olympus DP20 microscope camera (Olympus Singapore Pvt Ltd, Singapore) at 20X & 40X magnification. Two independent pathologists blinded for the study assessed and scored the immunostaining. To begin with, the number of positively stained cells in five random fields (400X objective) was counted. A labelling index (LI) was calculated by dividing the number of positive cells by the total number of cells per case and multiplying by 100. Both pathologists re-evaluated inconsistent scores until a consensus score was established. The interpretation was according to a modified version of the scoring criteria given by Cada Z et al [ 17 ]. Statistical analysis The statistical data analysis was obtained with SPSS version 26.0 (IBM, Chicago Inc). P values less than 0.05 were considered to indicate statistical significance. The chi-square test was used to analyze the statistical significance for NS and Ki-67. An independent T-test was used to analyze the mean LI between the two groups. To determine the optimal cut-off value, the accuracy, area under the receiver operating characteristic (AUC), sensitivity and specificity were computed. 𝑃 values less than 0.05 were considered to indicate statistical significance. RESULTS Of the 90 cases, 21 were females and 69 males. Among these 21 females, 2 had LWD, 4 OSMF and 15 OSCC whereas among 69 males, 13 had LWD, 11 OSMF and 45 OSCC. 10 normal oral mucosa (NOM) were controls. Most of our patients were found to have both smoking and smokeless tobacco usage [Table 1 ]. Table 1 NS, Ki-67 and clinicopathological variables Characteristics n Low High p-value S/NS Total 90 Age (Years) < 60 72 59 13 P < 0.0001 S ≥ 60 18 6 12 Gender Male 69 29 40 P < 0.021 S Female 21 3 18 Histology (differentiation ) Leukoplakia/Dysplasia 15 Mild 4 4 0 P < 0.061 NS Moderate 6 2 4 Severe 5 1 4 OSMF 15 Early 4 4 0 P < 0.119 NS Moderate 6 2 4 Advanced 5 2 3 OSCC 60 TNM staging I 8 5 3 P < 0.048 S II 14 7 7 III 27 5 22 IV 11 3 9 Histologic grade 60 Well differentiated 21 8 13 P < 0.192 NS Moderately differentiated 27 7 20 Poorly differentiated 12 1 11 Metastasis 60 Yes 8 31 P < 0.22 NS No 8 13 Vascular invasion 60 Positive 9 38 P < 0.029 S Negative 7 6 Perineural invasion 60 Positive 11 15 P < 0.212 NS Negative 5 29 NS: Nucleostemin; S: Significant; NS: Nonsignificant Among LWD (n = 15), 4 were mild, 6 moderate and 5 severe. In the OSMF group (n = 15), 4 were early, 6 moderately advanced and 5 advanced. Among the OSCC group (n = 60), 12 were poorly differentiated, 27 moderately differentiated and 21 well differentiated. There was a gradual increase in the expression of NS & Ki-67 in various strata ranging from basal to suprabasal in most of the cases of mild and moderate dysplasia. However, as the degree of dysplasia ranged to severe there was an increase in the expression of the markers in the parabasal layer [Figs. 1 & 2 ]. Similar features were also observed in various histological grading of OSMF, especially in advanced cases [Fig. 1 D, E] to the histological grades of OSCC [Figs. 1 & 2 ]. We also observed that cases of OSMF with atrophic epithelium exhibited expression of NS confined to the basal compartment. The nuclear LI of NS and Ki-67 in NOM, LWD, OSMF and OSCC was determined [Table 2 ]. The values of LI in NS and Ki-67 for NOM, LWD, OSMF and OSCC being 13.44 ± 2.07, 27.20 ± 5.54, 24.79 ± 5.41 and 39.13 ± 12.69 in the former, while in the latter (Ki-67) were 11.41 ± 2.58, 24.38 ± 5.40, 22.36 ± 5.16 and 30.61 ± 8.43. To categorize lesions as low and high-expression an AUC, sensitivity and specificity analysis was performed. A cut-off value distinguishing OPMDs (LWD and OSMF) from OSCC was 31.9 for NS and 23.9 for Ki-67 [Table 3 ]. Table 2 LI of NS, Ki-67 in NOM, LWD, OSMF and OSCC Cases NS Ki-67 P value NOM (Group 1) 10 13.44 ± 2.07 11.41 ± 2.58 < 0.001(S)* LWD (Group 2) 15 27.20 ± 5.54 24.38 ± 5.40 OSMF (Group 3) 15 24.79 ± 5.41 22.36 ± 5.16 OSCC (Group 4) 60 39.13 ± 12.69 30.61 ± 8.43 Group 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group 4, Group 2 & Group 3 vs Group 4 = S*, Mean ± Standard deviation, *S - Significant NOM-normal oral mucosa, LWD-leukoplakia with dysplasia, OSMF-oral submucous fibrosis, OSCC-oral squamous cell carcinoma, NS-nucleostemin; LI-labelling index Table 3 AUC analysis with cut-off values to determine low expression and high expression Group1 vs Group 2 (NOM vs LWD) NS 1.000 17.5 100 100 Ki-67 1.000 15.9 100 100 Group1 vs Group 3 (NOM vs OSMF) NS 0.941 17.96 93.3 100 Ki-67 0.937 16 93.3 100 Group1 vs Group 4 (NOM vs OSCC) NS 0.917 23.6 91.7 100 Ki-67 0.956 17.4 95.0 100 Group 2 & 3 vs Group 4 (LWD & OSMF vs OSCC) NS 0.908 31.9 90.0 87.8 Ki-67 0.807 23.9 90.0 56.1 AUC-area under the receiver operating characteristic, NOM-normal oral mucosa, LWD-leukoplakia with dysplasia, OSMF-oral submucous fibrosis, OSCC-oral squamous cell carcinoma, NS-nucleostemin Molecular markers and clinical course A follow-up of these patients showed that 5 of the 15 patients with LWD and 2 of OSMF in an advanced stage exhibited malignant transformation in 18 months. DISCUSSION Cancer stem cells (CSC) are subpopulations of cancer cells that contribute to the growth and heterogeneity of tumors. During cancer progression, CSC undergoes constant self-renewal and differentiation either via adaptation and interaction with the tumor microenvironment or responses from therapeutic pressures, ultimately contributing to the heterogeneous phenotype. Therefore, CSC markers provide a molecular target for various cancers and their therapeutic uses [ 18 ]. NS is present in the nucleolus and is a stem cell marker belonging to the YlqF/YawG GTPase subfamily. Human NS is located on chromosome 3p21.1. and plays an important role in embryogenesis, maintaining the genome integrity and repairing DNA damage caused by nuclear stress and ribosome biosynthesis [ 19 ]. Though widely expressed in proliferating cells, it quickly and almost completely disappears as the cells differentiate and thus maintain the homeostasis of the stem cell compartment and self-renewal.20 Apart from all these functions in normal physiology, the expression of NS has also been reported in various benign hyperplastic and malignant tumor tissues. These findings indicate that the NS gene may have regulated the growth of both stem cells and cancer cells [ 17 , 20 ]. Ki-67, a known diagnostic proliferative marker has been studied in many neoplasms viz lung, prostate, and OC [ 21 – 23 ]. NS is said to interact with molecules such as p53, MDM2 and other nucleolar proteins in the G1, G2 phases of the cell cycle and thus functions in cell proliferation and maintains homeostasis [ 24 , 25 ]. In the present study, a high expression of NS and Ki-67 was observed in neoplastic tissue compared to normal tissue, ranging from various grades of dysplasia and histopathological grades of OSMF to OSCC. However, the mean LI of Ki-67 was lower when compared to NS since Ki-67 cells passing the early events of mitogen-triggered transition from G0 to G1 lacked the Ki-67 nuclear antigen [ 23 ]. Also, we observed an increase in the expression in different epithelial strata as the degree of dysplasia increased which were compared with the hematoxylin and eosin-stained sections. Further the staining intensity of NS expression was less when compared to Ki-67, which requires further evaluation. Both Ki-67 and NS were expressed above the parabasal layer in OSMF (moderately advanced and advanced stages). These findings were concurrent with that of Yoshida et al., who opined that this protein is not affected by differentiation [ 26 ]. Similar findings were also observed in the human epidermis [ 27 ]. Recent studies have disproved its role in cell proliferation and suggested it could bring about transcriptome remodelling resulting in its involvement in various stages in the growth of cancer development [ 28 ]. Expression of NS also insinuates an early multipotent state, apart from the proliferative state. It has been further observed that the mutant NS missing the N-terminal basic region results in diffuse distribution of the protein in the nucleoplasm. Lack of GTP binding site results in the nucleolar aggregation and disruption of nucleolar structure, hence accumulation in the nucleoplasm contributing to over-expression requires further investigation [ 24 ]. A study by Crawford M et al., reveals the over-expression of NS beyond the normal basal epithelium that activates the STAT3 signalling pathway which leads to the early progression of the said disease into OSCC [ 29 ]. However, the NS marker was not expressed in 7 cases of OSCC, 2 of leukoplakia and 3 cases of OSMF. We speculate this could be due to the loss of antigenic receptors or mutation, which requires further investigation. Our study believes that NS is an indicator of prognosis. The observation of this marker in differentiated cells requires further exploration. CONCLUSIONS The expression of Ki-67 was visible in all while NS in most of the cases. The expression of these markers was observed both in the proliferative as well as in the differentiated strata of the epithelium with an increased degree of dysplasia. Patients with higher grades of dysplasia also had increased expression of these proliferative markers. Hence, we conclude NS is a prognostic predictive marker which requires further analysis with a larger sample size. Studies on NS apart from IHC to understand the functional state i.e., mutated or non-mutated (genomic characterization) is a matter of paramount importance and the association with other proteins like mini chromosome maintenance (MCM) family proteins, tumor suppressor markers along with other nucleolar proteins and stromal markers could establish a better understanding of the cancer biology and stemness of the cells involved along with other properties of the marker. Declarations Ethics approval and consent to participate: The study is approved by the Institutional Ethical committee at the Manipal Academy of Higher Education (Reference No: IEC729/2020). The study design was conducted in accordance with the Declaration of Helsinki. Informed consent to participate was obtained from all of the participants whose tissue were used in the study. Consent for publication: Not applicable. Availability of data and materials: The raw data supporting the conclusions of this article will be made available by the corresponding author upon reasonable request. Requests for data should be directed to [email protected] Competing interests: The authors declare no competing interests. Funding: This research received no external funding. Authors' contributions: S.P., S.N. data curation, formal analysis, investigation, methodology, validation, visualization, writing – original draft. R.N. statistics, analysis, writing – review and editing. S.C. conceptualization, data curation, investigation, methodology, project administration, resources, supervision, validation, writing – review and editing. Acknowledgements: None applicable. References Sarode G, Maniyar N, Sarode SC, Jafer M, Patil S, Awan KH. Epidemiologic aspects of oral cancer. Dis Mon. 2020;66:100988. Warnakulasuriya S. Oral potentially malignant disorders: A comprehensive review on clinical aspects and management. 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Nucleostemin upregulation and STAT3 activation as early events in oral epithelial dysplasia progression to squamous cell carcinoma. Neoplasia. 2012;23:1289–99. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers agreed at journal 29 Oct, 2025 Reviews received at journal 27 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 27 Oct, 2025 Editor invited by journal 14 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 14 Oct, 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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12:50:09","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89349,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7737960/v1/d0b2bb846676dac71da8478e.html"},{"id":95203350,"identity":"17cbe67c-7e9a-4a5b-96c3-92e3d48a59d9","added_by":"auto","created_at":"2025-11-05 12:50:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1027248,"visible":true,"origin":"","legend":"\u003cp\u003eNS expression in leukoplakia with dysplasia (LWD): (A) moderate dysplasia, (B) severe dysplasia, (C) mild dysplasia; oral submucous fibrosis (OSMF): (D) advanced, (E) moderately advanced; oral squamous cell carcinoma (OSCC): (F) well differentiated, (G) moderately differentiated, (H) poorly differentiated. IHC, 10X\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737960/v1/30f5f9e12346ea98a609d945.jpeg"},{"id":95228597,"identity":"511dbcc4-8c27-4e49-a4ba-f063ebc2560c","added_by":"auto","created_at":"2025-11-05 16:33:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":640253,"visible":true,"origin":"","legend":"\u003cp\u003eKi-67 expression in leukoplakia with dysplasia (LWD): (A) moderate dysplasia, (B) severe dysplasia, (C) mild dysplasia; oral squamous cell carcinoma (OSCC): (D) well differentiated, (E) moderately differentiated, (F) poorly differentiated. IHC,10X\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7737960/v1/bd57b813fe6de47483d2cf63.png"},{"id":95523457,"identity":"7e48e9a7-2f01-4bab-a887-aad1687bfdfa","added_by":"auto","created_at":"2025-11-10 09:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2471852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7737960/v1/1b22c169-81d1-4dda-af24-38a602a37182.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunohistochemical evaluation of Ki-67 and Nucleostemin in a model of oral potentially malignant disorders to malignant lesions","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eOral squamous cell carcinoma (OSCC) is the most prevalent malignant epithelial neoplasm affecting the oral cavity. Two to four percent of all cancers worldwide are oral cancers (OC). However, South East Asia and the Indian subcontinent have a slightly higher rate of OC with a male preponderance. Alcohol consumption and tobacco usage in various forms are the major causative factors implicated among these populations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOral potentially malignant disorders (OPMD) are the precursor lesions or conditions that can transform into OC and the most seen are leukoplakia and oral submucous fibrosis (OSMF) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Various genetic and epigenetic changes due to exposure to carcinogens over a period of time could be the reason for OPMD transforming to carcinomas or normal epithelium converting to carcinoma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCancer stem cells (CSC), a subset of cancer cells possess stem cell-like properties that could initiate self-renewal, differentiate into various cell lineages, progress and disseminate to various organs. Much research is focused on understanding the behavior of these cells. Due to the accumulation of genetic and epigenetic modifications over time, it has been speculated that these cells are derived from human embryonic or adult or normal stem cells and some of the CSC markers have been employed to understand the biological nature of these cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. One such stem cell marker for the current study is nucleostemin (NS), a nucleolar GTP-binding protein that has been demonstrated to play a pivotal role in carcinogenesis of breast, prostate and gastric cancers [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies have proven NS to be a proliferative marker of various cancer cell lines, including NS knock-down studies [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, studies of NS on OPMD are minimal.\u003c/p\u003e\u003cp\u003eKi-67 is a large nonhistone protein present in the cell nuclei during all the active phases of the cell cycle (G1, S, G2, and M phases) but is absent in the quiescent G0 phase and studies have correlated it with growth fracture, which is indicative of cell proliferation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the present study, immunohistochemical (IHC) appraisal of NS in OPMDs [leukoplakia with dysplasia (LWD) and OSMF] and OSCC has been evaluated. Also, an attempt has been made to correlate the expression of Ki-67 and NS with various clinicopathological parameters [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eFormalin fixed paraffin embedded (FFPE) tissues were retrieved between January 2018 to December 2020 after obtaining the Institutional Ethics Committee approval. The study included a total of 90 primary cases, i.e., 15 cases each of LWD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], OSMF [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and 60 cases of OSCC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. 10 cases of normal tissue obtained from the extraction site of impacted third molar were included as controls. Follow-up data was obtained from medical records. The slides were stained with hematoxylin and eosin for comparison.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eThe IHC staining of NS and Ki-67 was carried out on the tissue sections of 4\u0026micro;m from the FFPE tissue blocks. 3-aminopropyl-tri-ethoxy-silane (APES) coated slides (Sigma-Aldrich Ltd, Massachusetts, USA) were used. The primary antibody, NS rabbit polyclonal antibody (catalog-PA5-83350, RRID: AB_2790506 Dako, Glostrup, Denmark), anti Ki-67 monoclonal antibody (MIB, Dako, Denmark, 1:200) was diluted with tris buffered saline (TBS). After de-paraffinization and endogenous peroxidase block in hydrogen peroxide, antigen retrieval was done in ethylenediamine tetra-acetic acid (EDTA) diluted in distilled water for 1 hour in heat-induced epitope retrieval at 110\u0026deg;C. The sections were incubated at 4\u0026deg;C overnight with the diluted rabbit polyclonal primary antibody for NS, while sections were incubated for 1 hour at 37◦C for Ki-67 monoclonal antibody. The secondary antibody kit containing the target binder or amplifier and HRP (EnVision\u0026thinsp;+\u0026thinsp;System-HRP Labelled Polymer, Dako) was applied as per manufacturer instructions. The marker was detected by using chromogen containing diaminobenzidine (DAB) as per manufacturer\u0026rsquo;s instructions. The sections were further counterstained with Mayer\u0026rsquo;s hematoxylin for better visualization and mounted with DPX (Sigma-Aldrich Ltd, Massachusetts, USA). Negative control sections were without the primary antibody.\u003c/p\u003e\n\u003ch3\u003eImmunostaining evaluation\u003c/h3\u003e\n\u003cp\u003eThe nucleus was considered as positive reactivity when stained brown. The slides were evaluated with a light microscope (Olympus BX41) attached to an Olympus DP20 microscope camera (Olympus Singapore Pvt Ltd, Singapore) at 20X \u0026amp; 40X magnification. Two independent pathologists blinded for the study assessed and scored the immunostaining. To begin with, the number of positively stained cells in five random fields (400X objective) was counted. A labelling index (LI) was calculated by dividing the number of positive cells by the total number of cells per case and multiplying by 100. Both pathologists re-evaluated inconsistent scores until a consensus score was established. The interpretation was according to a modified version of the scoring criteria given by Cada Z et al [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003e The statistical data analysis was obtained with SPSS version 26.0 (IBM, Chicago Inc). \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered to indicate statistical significance. The chi-square test was used to analyze the statistical significance for NS and Ki-67. An independent T-test was used to analyze the mean LI between the two groups. To determine the optimal cut-off value, the accuracy, area under the receiver operating characteristic (AUC), sensitivity and specificity were computed. \u0026#119875; values less than 0.05 were considered to indicate statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOf the 90 cases, 21 were females and 69 males. Among these 21 females, 2 had LWD, 4 OSMF and 15 OSCC whereas among 69 males, 13 had LWD, 11 OSMF and 45 OSCC. 10 normal oral mucosa (NOM) were controls. Most of our patients were found to have both smoking and smokeless tobacco usage [Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\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\u003eNS, Ki-67 and clinicopathological variables\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCharacteristics\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLow\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eHigh\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eS/NS\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003e90\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (Years)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistology (differentiation\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLeukoplakia/Dysplasia\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.061\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c8\" namest=\"c7\" rowspan=\"3\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOSMF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEarly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.119\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c8\" namest=\"c7\" rowspan=\"3\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdvanced\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOSCC\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTNM staging\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c8\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"3\" nameend=\"c8\" namest=\"c7\" rowspan=\"4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistologic grade\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell differentiated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c8\" namest=\"c7\" rowspan=\"3\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerately differentiated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePoorly differentiated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMetastasis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVascular invasion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePerineural invasion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eNS: Nucleostemin; S: Significant; NS: Nonsignificant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAmong LWD (n\u0026thinsp;=\u0026thinsp;15), 4 were mild, 6 moderate and 5 severe. In the OSMF group (n\u0026thinsp;=\u0026thinsp;15), 4 were early, 6 moderately advanced and 5 advanced. Among the OSCC group (n\u0026thinsp;=\u0026thinsp;60), 12 were poorly differentiated, 27 moderately differentiated and 21 well differentiated.\u003c/p\u003e\u003cp\u003eThere was a gradual increase in the expression of NS \u0026amp; Ki-67 in various strata ranging from basal to suprabasal in most of the cases of mild and moderate dysplasia. However, as the degree of dysplasia ranged to severe there was an increase in the expression of the markers in the parabasal layer [Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. Similar features were also observed in various histological grading of OSMF, especially in advanced cases [Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E] to the histological grades of OSCC [Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. We also observed that cases of OSMF with atrophic epithelium exhibited expression of NS confined to the basal compartment. The nuclear LI of NS and Ki-67 in NOM, LWD, OSMF and OSCC was determined [Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. The values of LI in NS and Ki-67 for NOM, LWD, OSMF and OSCC being 13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07, 27.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.54, 24.79\u0026thinsp;\u0026plusmn;\u0026thinsp;5.41 and 39.13\u0026thinsp;\u0026plusmn;\u0026thinsp;12.69 in the former, while in the latter (Ki-67) were 11.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58, 24.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.40, 22.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16 and 30.61\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43. To categorize lesions as low and high-expression an AUC, sensitivity and specificity analysis was performed. A cut-off value distinguishing OPMDs (LWD and OSMF) from OSCC was 31.9 for NS and 23.9 for Ki-67 [Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\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\u003eLI of NS, Ki-67 in NOM, LWD, OSMF and OSCC\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eKi-67\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNOM (Group 1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001(S)*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLWD (Group 2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOSMF (Group 3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.79\u0026thinsp;\u0026plusmn;\u0026thinsp;5.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOSCC (Group 4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.13\u0026thinsp;\u0026plusmn;\u0026thinsp;12.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.61\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eGroup 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group 4, Group 2 \u0026amp; Group 3 vs Group 4\u0026thinsp;=\u0026thinsp;S*, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation, *S - Significant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNOM-normal oral mucosa, LWD-leukoplakia with dysplasia, OSMF-oral submucous fibrosis, OSCC-oral squamous cell carcinoma, NS-nucleostemin; LI-labelling index\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAUC analysis with cut-off values to determine low expression and high expression\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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=\"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\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup1 vs Group 2\u003c/p\u003e\u003cp\u003e(NOM vs LWD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKi-67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup1 vs Group 3\u003c/p\u003e\u003cp\u003e(NOM vs OSMF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.941\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e93.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKi-67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.937\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e93.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup1 vs Group 4\u003c/p\u003e\u003cp\u003e(NOM vs OSCC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKi-67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup 2 \u0026amp; 3 vs Group 4\u003c/p\u003e\u003cp\u003e(LWD \u0026amp; OSMF vs OSCC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e87.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKi-67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.807\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e56.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eAUC-area under the receiver operating characteristic, NOM-normal oral mucosa, LWD-leukoplakia with dysplasia, OSMF-oral submucous fibrosis, OSCC-oral squamous cell carcinoma, NS-nucleostemin\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular markers and clinical course\u003c/h2\u003e\u003cp\u003eA follow-up of these patients showed that 5 of the 15 patients with LWD and 2 of OSMF in an advanced stage exhibited malignant transformation in 18 months.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCancer stem cells (CSC) are subpopulations of cancer cells that contribute to the growth and heterogeneity of tumors. During cancer progression, CSC undergoes constant self-renewal and differentiation either via adaptation and interaction with the tumor microenvironment or responses from therapeutic pressures, ultimately contributing to the heterogeneous phenotype. Therefore, CSC markers provide a molecular target for various cancers and their therapeutic uses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. NS is present in the nucleolus and is a stem cell marker belonging to the YlqF/YawG GTPase subfamily. Human NS is located on chromosome 3p21.1. and plays an important role in embryogenesis, maintaining the genome integrity and repairing DNA damage caused by nuclear stress and ribosome biosynthesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Though widely expressed in proliferating cells, it quickly and almost completely disappears as the cells differentiate and thus maintain the homeostasis of the stem cell compartment and self-renewal.20 Apart from all these functions in normal physiology, the expression of NS has also been reported in various benign hyperplastic and malignant tumor tissues. These findings indicate that the NS gene may have regulated the growth of both stem cells and cancer cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eKi-67, a known diagnostic proliferative marker has been studied in many neoplasms viz lung, prostate, and OC [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. NS is said to interact with molecules such as p53, MDM2 and other nucleolar proteins in the G1, G2 phases of the cell cycle and thus functions in cell proliferation and maintains homeostasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the present study, a high expression of NS and Ki-67 was observed in neoplastic tissue compared to normal tissue, ranging from various grades of dysplasia and histopathological grades of OSMF to OSCC. However, the mean LI of Ki-67 was lower when compared to NS since Ki-67 cells passing the early events of mitogen-triggered transition from G0 to G1 lacked the Ki-67 nuclear antigen [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Also, we observed an increase in the expression in different epithelial strata as the degree of dysplasia increased which were compared with the hematoxylin and eosin-stained sections. Further the staining intensity of NS expression was less when compared to Ki-67, which requires further evaluation. Both Ki-67 and NS were expressed above the parabasal layer in OSMF (moderately advanced and advanced stages). These findings were concurrent with that of Yoshida et al., who opined that this protein is not affected by differentiation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similar findings were also observed in the human epidermis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recent studies have disproved its role in cell proliferation and suggested it could bring about transcriptome remodelling resulting in its involvement in various stages in the growth of cancer development [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Expression of NS also insinuates an early multipotent state, apart from the proliferative state. It has been further observed that the mutant NS missing the N-terminal basic region results in diffuse distribution of the protein in the nucleoplasm. Lack of GTP binding site results in the nucleolar aggregation and disruption of nucleolar structure, hence accumulation in the nucleoplasm contributing to over-expression requires further investigation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA study by Crawford M et al., reveals the over-expression of NS beyond the normal basal epithelium that activates the STAT3 signalling pathway which leads to the early progression of the said disease into OSCC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the NS marker was not expressed in 7 cases of OSCC, 2 of leukoplakia and 3 cases of OSMF. We speculate this could be due to the loss of antigenic receptors or mutation, which requires further investigation. Our study believes that NS is an indicator of prognosis. The observation of this marker in differentiated cells requires further exploration.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe expression of Ki-67 was visible in all while NS in most of the cases. The expression of these markers was observed both in the proliferative as well as in the differentiated strata of the epithelium with an increased degree of dysplasia. Patients with higher grades of dysplasia also had increased expression of these proliferative markers. Hence, we conclude NS is a prognostic predictive marker which requires further analysis with a larger sample size. Studies on NS apart from IHC to understand the functional state i.e., mutated or non-mutated (genomic characterization) is a matter of paramount importance and the association with other proteins like mini chromosome maintenance (MCM) family proteins, tumor suppressor markers along with other nucleolar proteins and stromal markers could establish a better understanding of the cancer biology and stemness of the cells involved along with other properties of the marker.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e The study is approved by the Institutional Ethical committee at the Manipal Academy of Higher Education (Reference No: IEC729/2020).\u0026nbsp;The study design was conducted in accordance with the Declaration of Helsinki. Informed consent to participate was obtained from all of the participants whose tissue were used in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The raw data supporting the conclusions of this article will be made available by the corresponding author upon reasonable request. Requests for data should be directed to [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eS.P., S.N. data curation, formal analysis, investigation, methodology, validation, visualization, writing \u0026ndash; original draft. R.N. statistics, analysis, writing \u0026ndash; review and editing. S.C. conceptualization, data curation, investigation, methodology, project administration, resources, supervision, validation, writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e None applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSarode G, Maniyar N, Sarode SC, Jafer M, Patil S, Awan KH. Epidemiologic aspects of oral cancer. Dis Mon. 2020;66:100988.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarnakulasuriya S. Oral potentially malignant disorders: A comprehensive review on clinical aspects and management. Oral Onco. 2020;102:104550.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbati S, Bramati C, Bondi S, Lissoni A, Trimarchi M. Oral cancer and Precancer: A narrative review on the relevance of early diagnosis. Int J Environ Res Public Health. 2020;17:9160.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkvortsova I. Cancer stem cells: What do we know about them? Cells. 2021;10:1528.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSami MM, Hachim MY, Hachim IY, Elbarkouky AH, L\u0026oacute;pez-Ozuna VM. Nucleostemin expression in breast cancer is a marker of more aggressive phenotype and unfavorable patients' outcome: A STROBE-compliant article. Med (Baltim). 2019;98:e14744.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu RL, Zhang ZH, Zhao WM, Wang M, Qi SY, Li J, et al. Expression of nucleostemin in prostate cancer and its effect on the proliferation of PC-3 cells. Chin Med J (Engl). 2008;121:299\u0026ndash;304.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYıldırım GD, Arık D, Yıldız B, Din\u0026ccedil;er M, Yaşar NF, Paşaoğlu \u0026Ouml;. High nucleostemin expression has a favorable prognostic effect on gastric carcinomas. Turk J Med Sci. 2018;48:484\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Wang L, Ji Q, Zhu H, Han S. Knockdown of Nucleostemin in an ovarian cancer SKOV-3 cell line and its effects on cell malignancy. Biochem Biophys Res Commun. 2017;487:262\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu SJ, Cai ZW, Liu YJ, Dong MY, Sun LQ, Hu GF, et al. Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies. World J Gastroenterol. 2004;10:1246\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmini S, Fathi F, Mobalegi J, Sofimajidpour H, Ghadimi T. The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines. Anat Cell Biol. 2014;47:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenon SS, Guruvayoorappan C, Sakthivel KM, Rasmi RR. Ki-67 protein as a tumor proliferation marker. Clin Chim Acta. 2019;491:39\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMuller S, Tilakaratne WM. Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: Tumors of the Oral Cavity and Mobile Tongue. Head Neck Pathol. 2022;16:54\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026ocirc;as DS, Takiya CM, Coelho-Sampaio TL, Mon\u0026ccedil;\u0026atilde;o-Ribeiro LC, Ramos EA, Cabral MG, et al. Immunohistochemical detection of Ki-67 is not associated with tumor-infiltrating macrophages and cyclooxygenase-2 in oral squamous cell carcinoma. J Oral Pathol Med. 2010;39:565\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHooghe B, Hulpiau P, van Roy F, De Bleser P. ConTra: a promoter alignment analysis tool for identification of transcription factor binding sites across species. Nucleic Acids Res. 2008;36:W128\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePassi D, Bhanot P, Kacker D, Chahal D, Atri M, Panwar Y. Oral submucous fibrosis: Newer proposed classification with critical updates in pathogenesis and management strategies. Natl J Maxillofac Surg. 2017;8:89\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBryne M, Koppang HS, Lilleng R, Kjaerheim A. Malignancy grading of the deep invasive margins of oral squamous cell carcinomas has high prognostic value. 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Mol Med Rep. 2015;11:1566\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJakobsen JN, S\u0026oslash;rensen JB. Clinical impact of Ki-67 labelling index in non-small cell lung cancer. Lung Cancer. 2012;79:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTodorov IT, Pepperkok R, Philipova RN, Kearsey SE, Ansorge W, Werner D. A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division. J Cell Sci. 1994;107:253\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsai RY, Meng L. Nucleostemin: a latecomer with new tricks. Int J Biochem Cell Biol. 2009;41:2122\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAndisheh-Tadbir A, Ranjbar MA, Shiri AA, Mardani M. Expression of nucleostemin in odontogenic cysts and tumors. Exp Mol Pathol. 2020;113:104376.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa H, Pederson T. Nucleostemin: a multiplex regulator of cell-cycle progression. Trends Cell Biol. 2008;18:575\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshida R, Fujimoto T, Kudoh S, Nagata M, Nakayama H, Shinohara M, et al. Nucleostemin affects the proliferation but not differentiation of oral squamous cell carcinoma cells. Cancer Sci. 2011;102:1418\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLacina L, Smetana K Jr, Dvor\u0026aacute;nkov\u0026aacute; B, Stork J, Plz\u0026aacute;kov\u0026aacute; Z, Gabius HJ. Immunocyto- and histochemical profiling of nucleostemin expression: marker of epidermal stem cells? J Dermatol Sci. 2006;44:73\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMrouj K, Andr\u0026eacute;s-S\u0026aacute;nchez N, Dubra G, Singh P, Sobecki M, Chahar D, et al. Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis. Proc Natl Acad Sci U S A. 2021;118:e2026507118.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrawford M, Liu X, Cheng YL, Tsai RY. Nucleostemin upregulation and STAT3 activation as early events in oral epithelial dysplasia progression to squamous cell carcinoma. Neoplasia. 2012;23:1289\u0026ndash;99.\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oral cancer, oral potentially malignant disorders, cancer stem cells, oral squamous cell carcinoma, leucoplakia, dysplasia","lastPublishedDoi":"10.21203/rs.3.rs-7737960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7737960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eProliferative biomarkers are one among the tumor markers that assist in tailored clinical management, thus could aid in improving the quality of life of patients with head and neck cancers. The present study aims to explore the clinical significance and relevance of Nucleostemin (NS) as a proliferative marker along with Ki-67, a known diagnostic proliferative marker in oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC), by evaluating the immune expression of these biomarkers.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA retrospective immunohistochemical (IHC) analysis of NS \u0026amp; Ki-67 was performed on a total of 90 primary cases, i.e., leucoplakia with dysplasia (LWD, n\u0026thinsp;=\u0026thinsp;15), oral submucous fibrosis (OSMF, n\u0026thinsp;=\u0026thinsp;15), OSCC (n\u0026thinsp;=\u0026thinsp;60), correlated with clinicopathological features.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAn overexpression of these markers was observed in the upper strata of the epithelium of histologically diagnosed severe dysplasia, moderately advanced OSMF (among the OPMD) and similar expression was seen in moderately and poorly differentiated carcinoma. The clinicopathological features did exhibit a significant correlation in advanced stage of the disease. Normal oral mucosa, adjacent to the lesion showed NS expression only in the basal strata with few cells in the lower spinous layer. Among OPMD, dysplasia (n\u0026thinsp;=\u0026thinsp;5) and advanced OSMF (n\u0026thinsp;=\u0026thinsp;2) exhibiting increased expression, progressed to malignancy.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe presence of NS and Ki-67 in various strata of the diseased mucosa was able to identify a few cases in OPMD with a cut-off value of 31.9 for NS and 23.9 for Ki-67 progressing into OSCC. However, this method is challenging for routine diagnosis.\u003c/p\u003e","manuscriptTitle":"Immunohistochemical evaluation of Ki-67 and Nucleostemin in a model of oral potentially malignant disorders to malignant lesions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 12:50:04","doi":"10.21203/rs.3.rs-7737960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-26T10:58:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T05:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24521295960507001931881041260111314536","date":"2026-03-21T04:02:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90126766633559927801366849439948256178","date":"2026-03-21T00:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71225520570773670954141499432844807247","date":"2025-10-29T17:33:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T07:47:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310083744970429572355166266661944250125","date":"2025-10-27T07:24:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T07:20:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T07:17:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-14T13:28:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T10:44:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-10-14T10:41:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"73706237-0bda-46ee-bd14-9b8964372f6b","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T12:50:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 12:50:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7737960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7737960","identity":"rs-7737960","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}