NANOG is a specific biomarker predicting malignant transformation of proliferative verrucous leukoplakia

NANOG is a specific biomarker predicting malignant transformation of proliferative verrucous leukoplakia | 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 NANOG is a specific biomarker predicting malignant transformation of proliferative verrucous leukoplakia Yanning Zhang, Xinning Zhang, Yanting Chi, Jiayin Song, Long Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7191114/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Proliferative verrucous leukoplakia (PVL) is a special type of oral leukoplakia with no unified diagnosis criteria. Objective: To evaluate the diagnostic values of NANOG for PVL and explore the associations between NANOG expression and the malignant transformation of PVL. Methods: A total of 112 patients were collected, including 79 cases of PVL (48 cases developed into oral squamous cell carcinoma) and 33 patients diagnosed as conventional oral leukoplakia (COL). The expression of NANOG was detected by immunohistochemical staining. Results: NANOG showed higher expression in 34 cases(70.83%) PVL with malignant transformation, significantly higher than that in PVL without malignant transformation and COL ( P <0.05), and the NANOG expression was correlated with non-homogeneous clinical manifestation and oral epithelial dysplasia ( P <0.001). Receiver operating characteristic (ROC) analysis revealed that it would be used to identify PVL from COL, AUC = 0.674, with sensitivity and specificity of 0.800 and 0.879. Kaplan-Meier plotter data revealed that patients with high NANOG expression had shorter malignant transformation-free survival than those with low expression ( P <0.001). Multivariate COX analysis further confirmed that high NANOG expression was an independent risk factor for malignant transformation of PVL. ROC analysis also revealed that it would be used to predict the malignant transformation of PVL, AUC = 0.806, with sensitivity and specificity of 0.900 and 0.903. The above resultswere also validated in external case cohort. Conclusions: NANOG would be a promising diagnostic and prognostic biomarker for PVL. proliferative verrucous leukoplakia canceration early diagnosis prognosis NANOG Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Proliferative verrucous leukoplakia (PVL), an independent entity different from leukoplakia, is defined as “Progressive, persistent, and irreversible disorder characterized by the presence of multiple leukoplakias that frequently become warty” by WHO 2022. 1 It is characterized by a progressive clinical course, typically demonstrating slow and persistent progression over an extended period before suddenly transitioning into rapid growth or malignant transformation. It exhibits the highest proportion of malignant transformation compared to other oral potentially malignant disorders (OPMDs), with a recurrence rate of 67.2% and a malignant transformation rate ranging from 26.7–72.4%. 2–5 However, due to the lack of unified criteria and specific biomarkers for early diagnosis and prognostic assessment, the current diagnosis usually needs to be combined with the medical history, which would be delayed, subjective and heterogeneous. On the other hand, PVL might be even misdiagnosed as other diseases, given its clinical manifestations resembling conventional oral leukoplakia (COL) or oral lichen planus. 6 , 7 Therefore, early diagnosis remains extremely challenging. A reliable predictive biomarker would facilitate the early diagnosis and accurate treatment of PVL. However, due to the low incidence and limited cases of PVL, most of the current studies are observational or case reports, lacking in-depth research and understanding of PVL. In this study, we included a relatively large number of cases cohort for nested case-control study, and combined the biomarker NANOG for analysis. NANOG is a transcription regulator involved in inner cell mass and embryonic stem cells proliferation, renewal and pluripotency. It plays a pivotal role in various aspects of tumori-genesis, metastasis, and chemo-sensitivity, 8 – 11 and has been proved to be potential biomarker to predict the prognosis of various human cancers. 12 NANOG showed strong expression in laryngeal dysplasia and association with laryngeal cancer risk superior to the histological classification. 13 Nevertheless, the role of NANOG in PVL and its possible implication in malignant transformation remain to be undetermined. In this study, we detected the NANOG expression in PVL progression, and combined it with clinical characteristics to analyze its association with malignant transformation prognosis. Materials and Methods Patients Data A total of 112 patients collected from Peking University School and Hospital of Stomatology (from 2013 to 2020) were enrolled in this study. Among them, 79 cases of PVL including 48 cases progressed to oral squamous cell carcinoma (OSCC), and 33 cases of COL as control. The studies involving human participants were reviewed and approved by The Biomedical Ethics Committee of the Department of Peking University (No. PKUSSIRB-201948111). Another cohort of 31 cases from Xiangya Stomalogical Hospital were included in the external validation cohort. There were 18 cases of COL and 15 cases of PVL. Eight cases of PVL underwent malignant transformation. Diagnostic criteria for PVL : 14 Major criteria: (1) Leukoplakia lesion involved in more than two different oral subsites; (2) The existence of a verrucous area; (3) Lesion spreading or engrossing during the disease development; (4) There has been a recurrence in a previously treated area; (5) Histopathological test: oral epithelial hyperkeratosis to verrucous hyperplasia, verrucous carcinoma, or squamous cell carcinoma, whether in situ or infiltrating. Minor criteria: (1) Oral leukoplakia lesion that occupies at least 3 cm when adding all the affected areas; (2) Female patient; (3) Non-smoker regardless of gender; (4) More than 5 years evolution. Diagnostic criteria: Three major criteria (histopathological test among them) or two major criteria (histopathological test among them) + two minor criteria. COL included criteria: (1) Histopathologic diagnosis of oral leukoplakia by two pathologists independently; (2) It cannot be diagnosed with other diseases or PVL as mentioned above. Exclusion criteria for PVL and COL: (1) The first diagnosis is OSCC; (2) With tumors of other origins or other serious diseases; (3) Insufficient follow up data. The clinical and histopathological characteristics were collected as following: sex, age, tobacco and alcohol habits, local irritants, primary site, clinical presentation, malignant transformation-free survival (MTFS) and oral epithelial dysplasia (OED). Local irritants, such as faulty restorations, residual roots, sharp tooth cusps, silver amalgam, were included in the possible influencing factors accessed in this analysis. The primary lesion sites included oral subsites: dorsum and ventrum of the tongue, floor of the mouth, gingiva, buccal mucosa, lip, soft palate and hard palate. Furthermore, based on the number of subsites involved, the lesion sites were categorized into those affecting more than two subsites and those affecting two or fewer subsites. The involvement of the ventral tongue and floor of the mouth as primary sites was also considered as potential risk factors and prognostic factors. Clinical presentation was considered homogeneous when predominantly white, flat, thin or wrinkled lesions; and non-homogeneous in the presence of mixed white-and-red, erosion, nodular, granular, or verrucous lesions. MTFS time was measured from the time of initial biopsy to the time of transformation to OSCC confirmed by pathology or the end of follow-up. Immunohistochemical staining All the samples were fixed with 10% formalin and embedded in paraffin. A series of 2.5µm thick sections of tissue were obtained for immunohistochemical staining. Following deparaffination and rehydration, heat slides with 10 mM citrate buffer (pH 6.0) for 10 minutes. Endogenous peroxidase activity was quenched with 10% H 2 O 2 for 15 min, which was followed by blocking the non-specific protein binding by using 5% normal goat serum 15 min at room temperature. The tissues were then incubated with the rabbit monoclonal anti-NANOG (#4903, D73G4, cell signaling technology, USA, 1:100), overnight at 4°C, the secondary goat anti-rabbit IgG antibodies (PV6000 Kit, ZSGB-BIO, China) for 60 min at 37°C, using the diaminobenzidine tetrahydrochloride (DAB) as the chromogen, haematoxylin was used as a counterstain. The immunohistochemical staining results were assessed semi-quantitatively; the staining was scored and graded by two pathologists as follows: low-expression (0: negative, 1+: weak positivity), high-expression (2+: moderate positivity, 3+: strong positivity). All readings were performed independently and without any prior knowledge of the clinical or histopathological characteristics of the cases. Statistical Analysis Statistical analyses were performed using SPSS software (version 22.0, IBM, United States) and R 4.4.0. For the univariate analysis, relationships between categorical characteristics were evaluated using the Chi-squared test and Fisher’s exact test, and between continuous variables using the T-test or Mann-Whitney U test, as appropriate. Kaplan-Meier was utilized to assess the prognostic significance of NANOG in PVL by analyzing the association between NANOG level and MTFS. Survival difference was evaluated using Log-rank test. Univariate and multivariate COX analyses were employed to evaluate the independent prognostic significance of NANOG expression level and other clinical parameters on MTFS in PVL patients. Receiver operator characteristic (ROC) curves were used to reflect the sensitivity and specificity of NANOG in diagnostic and prognostic of PVL. Results Patients As shown in Table 1 , a total of 112 patients were included in this study. The ratio of males to females was 1:2, and the average age was 57.21 years. The mean follow-up time was 77.78 months. The mean time for the malignant transformation of PVL was 47.5 months. Table 1 Clinico-demographic-pathologic characteristics of 112 cases of PVL cohort Characteristics PVL (N = 79) COL (N = 33) P .overall Age 57.27 ± 10.42 59.18 ± 14.66 0.499 Sex: 0.113 Female 57 (72.15%) 18 (54.55%) Male 22 (27.85%) 15 (45.45%) NANOG Expression: 0.001 L-NANOG 42 (53.16%) 29 (87.88%) H-NANOG 37 (46.84%) 4 (12.12%) Time 78.77 ± 47.17 120.82 ± 19.34 <0.001 Lesion sites: <0.001 2 51 (64.56%) 6 (18.18%) Clinical presentation: 0.16 homogeneous 42 (53.16%) 23 (69.70%) non-homogeneous 37 (46.84%) 10 (30.30%) Local irritants: 0.491 No 43 (54.43%) 21 (63.64%) Yes 36 (45.57%) 12 (36.36%) Smoker: 0.074 No 71 (89.87%) 25 (75.76%) Yes 8 (10.13%) 8 (24.24%) Alcohol use: 0.346 No 71 (89.87%) 27 (81.82%) Yes 8 (10.13%) 6 (18.18%) OED: 1 Non-OED 33 (41.77%) 14 (42.42%) OED 46 (58.23%) 19 (57.58%) VT or FOM involved: 0.007 No 47 (59.49%) 29 (87.88%) Yes 32 (40.51%) 4 (12.12%) PVL, proliferative verrucous leukoplakia; COL, conventional oral leukoplakia; OED, oral epithelial dysplasia; Non-OED, without oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved. PVL histopathological features were complex, ranging from hyperkeratosis to severely dysplastic, or OSCC. As shown in Fig. 1 , it would be manifested as corrugated (undulating) ortho(para)hyperkeratotic with verrucous or papillary architecture, or notably thickened epithelium with marked hyperorthokeratosis, even atrophic epithelium, but cytologic atypia was rare. The histopathology of OSCC derived from PVL (OSCC-PVL) more likely to be conventional OSCC ( Fig. 1 , E ) , sometimes manifested as a verrucous pattern ( Fig. 1 . F) , with keratin plunges down into the epithelial crypts. The characteristics of structural disorder were obvious, but the cytologic pleomorphism was rare. Immunohistochemical staining The initial biology samples of all patients were used for immunohistochemistry, in addition, biopsy samples of OSCC when first confirmed during the PVL progression (33 cases) were included. NANOG is mainly expressed in nuclear and cytoplasmic ( Fig. 2 ) . NANOG showed high expression in PVL with malignant transformation (70.83%), significantly higher than that in PVL without malignant transformation (9.68%) and COL group (12.12%). However, in the PVL progression, there was no difference in expression between the samples of initial biopsy and after malignant transformation ( P > 0.05) (Table 2 ) . Table 2 Immunohistochemical staining of NANOG Group NANOG expression Total High Low PVL-MT*# 48 34 (70.83%) 14 (29.17%) PVL-NMT 31 3 (9.68%) 28 (90.32%) OSCC-PVL 33 28 (84.85%) 5 (15.15%) COL 33 4 (12.12%) 29(87.88%) * compare with PVL-NMT: P < 0.05 #compare with COL: P 0.05 PVL-MT vs SCC-PVL: P > 0.05 PVL, proliferative verrucous leukoplakia; COL, conventional oral leukoplakia; PVL-MT, PVL with malignant transformation PVL-NMT, PVL without malignant transformation OSCC-PVL, OSCC occurs during the evolution of PVL NANOG protein showed negative expression in COL (A, ×200) and low expression in PVL-NMT (B, ×200), while strong expression in early stage of PVL-MT, even in the lesion present as hyperkeratosis (C, ×200), verrucous hyperplasia (D, ×200) or leukoplakia with dysplasia (E, ×200). NANOG showed strong expression in OSCC-PVL (F, ×100, red arrow), both conventional OSCC (F, ×200) and verrucous carcinoma (H, ×50), while negative expression in normal epithelium adjacent to cancer (F, ×100, black arrow) . The association between NANOG and clinicopathological characteristic was then evaluated in PVL patients, including sex, age, primary site, local irritants, tobacco and alcohol habits, clinical presentation, MTFS, and OED. NANOG high expression was associated with clinical presentation and OED, which showed high expression level in sample with non-homogenous presentation (62.16%) and histological OED (78.38%) ( Table 3 ). Table 3 Association between NANOG and clinicopathological Characteristic in PVL L-NANOG (N = 42) H-NANOG (N = 37) P .overall Malignant transformation <0.001 No 28 (66.67%) 3 (8.11%) Yes 14 (33.33%) 34 (91.89%) Age 57.79 (8.66) 56.68 (12.22) 0.647 Sex: 0.158 Female 27 (64.29%) 30 (81.08%) Male 15 (35.71%) 7 (18.92%) Time (Month) 103.31 (40.64) 50.92 (37.94) <0.001 Lesion sites: 0.11 2 31 (73.81%) 20 (54.05%) Clinical presentation: 0.019 homogeneous 28 (66.67%) 14 (37.84%) non-homogeneous 14 (33.33%) 23 (62.16%) Local irritants: 0.458 No 25 (59.52%) 18 (48.65%) Yes 17 (40.48%) 19 (51.35%) Smoker: 1 No 38 (90.48%) 33 (89.19%) Yes 4 (9.52%) 4 (10.81%) Alcohol use: 0.717 No 37 (88.10%) 34 (91.89%) Yes 5 (11.90%) 3 (8.11%) OED: 0.001 Non-OED 25 (59.52%) 8 (21.62%) OED 17 (40.48%) 29 (78.38%) VT or FOM involved: 0.487 No 27 (64.29%) 20 (54.05%) Yes 15 (35.71%) 17 (45.95%) PVL, proliferative verrucous leukoplakia; OED, oral epithelial dysplasia; Non-OED, without oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved. COX proportional hazards regression analysis COX proportional hazards regression analysis was used to analyze NANOG expression and clinicopathological characteristics related with malignant transformation of PVL, Multivariate COX analysis demonstrated that NANOG high expression was an independent prognostic factor for PVL patients, revealing that high expression of NANOG may affect the malignant characteristics of PVL and poor prognosis ( P < 0.05; Table 4 ). Table 4 Univariate and Multivariate COX proportional hazard model Univariate Multivariate Variable HR 95% CI P _value HR 95% CI P _value Age 1.026 0.995–1.057 0.100 Sex 2.049 0.695–6.041 0.194 NANOG Expression 3.379 1.565–7.296 0.002 2.992 1.516–5.906 0.002 Lesion sites 0.558 0.293–1.064 0.077 Clinical presentation 2.129 1.066–4.251 0.032 1.775 0.959–3.286 0.068 Local irritants 0.595 0.320–1.106 0.101 Smoker 0.572 0.123–2.658 0.476 Alcohol use 1.189 0.243–5.819 0.831 OED 2.906 1.240–6.809 0.014 2.876 1.317–6.281 0.008 VT or FOM involved 1.314 0.692–2.495 0.404 HR: Hazard Ratio; CI, confidence interval; OED, oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved. ROC analysis To determine the sensitivity and specificity of NANOG in distinguishing PVL patients from COL controls at early diagnosis, ROC curves were generated, AUC value of the model reached 0.674 (0.594–0.753) ( P < 0.001), the sensitivity and specificity were 0.800 and 0.879 respectively ( Fig. 3 . A) . To determine the sensitivity and specificity of NANOG in malignant transformation predicting for PVL, ROC curves were generated, AUC value of the model reached 0.806 (0.721–0.890) ( P < 0.001), the sensitivity and specificity were 0.900 and 0.903 respectively ( Fig. 3 . B) . Compared with OED ( Fig. 3 . C) , NANOG showed better sensitivity and specificity in identify and prognostic prediction of PVL. Survival Analysis Kaplan-Meier survival analysis was used to explore the effect of NANOG expression (high vs. low) on the malignant transformation of PVL patients. As shown in Fig. 4 , high expression of NANOG was associated with lower MTFS ( P < 0.001). Accordingly, overexpression of NANOG was closely associated to malignant transformation of patients. This was consistent with the results of the COX analysis. Malignant transformation-free survival with PVL. High expression of NANOG was associated with malignant transformation of PVL patients ( P < 0.001). NANOG Expression in the Validation Cohort NANOG expression in the validation cohort as shown in Table 5 and Figure S1. ROC analysis showed that the sensitivity and specificity of NANOG in distinguishing PVL patients from COL were 0.800 and 0.333, respectively. And the sensitivity and specificity in malignant transformation predicting for PVL were 0.800 and 0.869 ( Fig. 5 ). Table 5 NANOG expression in the validation cohort Group NANOG expression Total High Low PVL-MT*# 8 7 (87.50%) 1 (12.50%) PVL-NMT 7 1 (14.29%) 6 (85.71%) OSCC-PVL 8 6 (75.00%) 2 (25.00%) COL 18 4 (22.22%) 14(77.78%) * compare with PVL-NMT: P < 0.05 #compare with COL: P 0.05 PVL-MT vs SCC-PVL: P > 0.05 PVL, proliferative verrucous leukoplakia; COL, conventional oral leukoplakia; PVL-MT, PVL with malignant transformation PVL-NMT, PVL without malignant transformation OSCC-PVL, OSCC occurs during the evolution of PVL Discussion PVL presents the highest malignant conversion rate in OMPD. However, due to its complex clinical course, clinical and morphological manifestations variants, lacking specificity. The initial manifestation of PVL may be a flat white area or lichenoid appearance, with or without dysplasia, 15 similar to conventional oral leukoplakia, oral lichen planus or other oral white lesions. 7 , 16 Thus, these cases could be erroneously treated as other diseases, subsequently malignant transformation over an extended period. Therefore, there is an urgent need for an effective biomarker to facilitate the early diagnosis and accurate treatment of PVL. Oral epithelial dysplasia (OED) is considered a risk for OSCC, and used to assess the malignant transformation potential in OPMDs. However, research indicated that one-third of PVL cases did not exhibit OED, yet malignant transformation still occurred in 3.7% of these cases. 17 In this study, 11 cases of PVL without OED (22.92%) progressed to OSCC. Therefore, additional objective and reliable markers are needed to predict the risk of malignant transformation beyond current clinical and histopathological characteristics. which will undoubtedly help the clinicians to choose the most adequate therapeutic option. NANOG, as one of the main transcription factors participating in stemness, 18 It plays an important role in maintaining the stemness and self-renewal of tumor cells, 19 , 20 NANOG shows high expression in various cancers, participated in signaling pathways (JAK/STAT, Wnt/β-catenin, Notch and EGFR), induced stemness, self-renewal, metastasis, invasion, epithelial-mesenchymal transition and chemoresistance of cancer cells.[21–24] High NANOG expression was also associated with poor prognosis and might serve as an unfavorable prognostic biomarker. 12 , 25 – 27 Strong NANOG expression was found in laryngeal dysplasia, associated with laryngeal cancer risk superior to the histological classification, and no expression in normal epithelia. 13 NANOG was uniquely showed as a better prognosis in pharyngeal tumors. 28 Similar results were found in oral studies, showing that NANOG exhibited no expression in normal mucosa, very weak expression in low-grade dysplasia, and strong expression in high-grade dysplasia and OSCC, NANOG expression emerged as an early predictor of oral cancer risk in patients with OPMD, and was more frequent in early stages of OSCC tumorigenesis rather than in advanced neoplastic disease. 29 , 30 Unlike in laryngeal cancer, high NANOG expression in OSCC were associated with a worse overall survival. 31 In this study, NANOG was significantly higher in the early stage of PVL compared to COL, ROC analysis showed that, the sensitivity and specificity were 0.800 and 0.879, indicating that NANOG would be used to identified PVL from COL. In addition, NANOG showed higher expression in PVL with malignancy transformation than that without malignant transformation. However, it is worth noting that NANOG showed high expression in the early stage before the malignant transformation, but there was no difference before and after carcinogenesis of PVL, suggesting the NANOG high expression was associated with malignant transformation, but not associated with disease progress. Studies suggested that positive NANOG expression in OSCC was significantly associated with tobacco and alcohol consumption. 30 In this study, NANOG expression associated with non-homogeneous clinical presentation and OED ( P < 0.05). Multivariate COX analysis indicated that NANOG was an independent prognostic factor for PVL malignant transformation. ROC analysis showed that NANOG could be used to predict malignant transformation of PVL with sensitivity and specificity of 0.900 and 0.903, respectively. Kaplan-Meier analysis also demonstrated that the high expression of NANOG is associated with the increased malignant transformation. Thus, NANOG showed higher accuracy, sensitivity and specificity than that of OED in predicting the malignant transformation of PVL. Therefore, NANOG would be a specific predictor of early identification and prediction of malignant transformation in PVL patients. These results were confirmed in the validation cohort, especially the NANOG expression demonstrated superiority in sensitivity and specificity in predicting the malignant transformation of PVL. In the validation cohort, NANOG demonstrated relatively poor specificity in distinguishing PVL from COL, which might be related to the small sample size. Furthermore, NANOG was believed to be a factor in enhancing patient resistance to immune checkpoint inhibitors.[32] As shown in this study, NANOG high expression in PVL with malignant transformation, even in the early stages premalignancy, may contribute to the resistance of PVL patients to most therapies. 33 Therapeutically, since NANOG is highly expressed in PVL with malignant transformation but not in normal tissues, it could serve as an ideal target for disease treatment, minimizing off-target effects and enhancing specificity in gene-targeted therapy. Research suggested that targeting NANOG could improve chemosensitivity and inhibit tumorigenesis. 34 – 36 Consequently, the targeted therapy for NANOG might present a therapeutic potential in PVL and warranted further exploration. In summary, NANOG could be used as an independent risk factor for malignant transformation of PVL, and it had a good predictive value in early diagnosis and predicting malignant transformation of PVL superior to OED. It might be an ideal target in future gene-targeted therapy. Declarations Conflicts of Interest and Source of Funding: This work was supported by the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038). Acknowledgements Author Contributions Binbin Li performed study concept and design; Yanning Zhang & Xinning Zhang performed development of methodology and writing, review and revision of the paper; Yanting Chi provided acquisition, analysis and interpretation of data, and statistical analysis; Jiayin Song & Long Li contributed to data collection and slide evaluation. All authors contributed to review and revision of the manuscript. Ethics approval and con sent to participate This study was conducted according to the guidelines recommended in the Declaration of Helsinki and approved by The Biomedical Ethics Committee of the Department of Peking University (No. PKUSSIRB-201948111). Written informed consent was obtained from all individual participants included in the study. Consent for publication Not applicable. Data Availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request, without undue reservation. Declarations of interest None. Funding This work was supported by the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038). Declaration of Generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used ChatGPT in order to improve readability and make the language expression more in line with the native English expression habits. 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Lichenoid morphology could be an early feature of oral proliferative verrucous leukoplakia. J Oral Pathol Med . 2021; 50(2):229-235. https://doi.org/10.1111/jop.13129. Alabdulaaly L, Villa A, Chen T, et al. Characterization of initial/early histologic features of proliferative leukoplakia and correlation with malignant transformation: a multicenter study. Mod Pathol . 2022; 35(8):1034-1044. https://doi.org/10.1038/s41379-022-01021-x. Okamoto K, Fujita H, Okada Y, et al. Single-molecule tracking of Nanog and Oct4 in living mouse embryonic stem cells uncovers a feedback mechanism of pluripotency maintenance. Embo J . 2023; 42(18):e112305. https://doi.org/10.15252/embj.2022112305. Thiagarajan PS, Sinyuk M, Turaga SM, et al. Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase. Nat Commun . 2018; 9(1):578. https://doi.org/10.1038/s41467-018-02938-1. Semenza GL. Mechanisms of Breast Cancer Stem Cell Specification and Self-Renewal Mediated by Hypoxia-Inducible Factor 1. Stem Cells Transl Med . 2023; 12(12):783-790. https://doi.org/10.1093/stcltm/szad061. Lo UG, Chen YA, Cen J, et al. The driver role of JAK-STAT signalling in cancer stemness capabilities leading to new therapeutic strategies for therapy- and castration-resistant prostate cancer. Clin Transl Med . 2022; 12(8):e978. https://doi.org/10.1002/ctm2.978. Liu L, Zhu H, Liao Y, et al. Inhibition of Wnt/beta-catenin pathway reverses multi-drug resistance and EMT in Oct4(+)/Nanog(+) NSCLC cells. Biomed Pharmacother . 2020; 127:110225. https://doi.org/10.1016/j.biopha.2020.110225. Yang J, Huang Y, Song M, et al. SPC25 promotes proliferation and stemness of hepatocellular carcinoma cells via the DNA-PK/AKT/Notch1 signaling pathway. Int J Biol Sci . 2022; 18(14):5241-5259. https://doi.org/10.7150/ijbs.71694. Oh SJ, Lim JY, Son MK, et al. TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway. Nat Commun . 2023; 14(1):2691. https://doi.org/10.1038/s41467-023-38318-7. Zadvornyi TV, Lukianova NY, Borikun TV, Vitruk YV, Stakhovsky EO, Chekhun VF. NANOG as prognostic factor of prostate cancer course. Exp Oncol . 2020; 42(2):94-100. https://doi.org/10.32471/exp-oncology.2312-8852.vol-42-no-2.14673. Huang G, Zhang J, Wang X, Chen Y, Liu D, Guo S. Clinicopathological and prognostic significance of Nanog expression in non-small cell lung cancer: a meta-analysis. Onco Targets Ther . 2019; 12:3609-3617. https://doi.org/10.2147/OTT.S202081. Liang C, Zhang K, Ge H, Li W, Li G, Wu J. Prognostic and clinicopathological value of Nanog in hepatocellular carcinoma: A meta-analysis. Clin Chim Acta . 2018; 477:24-31. https://doi.org/10.1016/j.cca.2017.11.037. Pedregal-Mallo D, Hermida-Prado F, Granda-Diaz R, et al. Prognostic Significance of the Pluripotency Factors NANOG, SOX2, and OCT4 in Head and Neck Squamous Cell Carcinomas. Cancers (Basel) . 2020; 12(7). https://doi.org/10.3390/cancers12071794. Grubelnik G, Bostjancic E, Anicin A, Dovsak T, Zidar N. MicroRNAs and Long Non-Coding RNAs as Regulators of NANOG Expression in the Development of Oral Squamous Cell Carcinoma. Front Oncol . 2020; 10:579053. https://doi.org/10.3389/fonc.2020.579053. de Vicente JC, Rodriguez-Santamarta T, Rodrigo JP, et al. The Emerging Role of NANOG as an Early Cancer Risk Biomarker in Patients with Oral Potentially Malignant Disorders. J Clin Med . 2019; 8(9). https://doi.org/10.3390/jcm8091376. Rodrigues M, Xavier F, Andrade NP, et al. Prognostic implications of CD44, NANOG, OCT4, and BMI1 expression in tongue squamous cell carcinoma. Head Neck . 2018; 40(8):1759-1773. https://doi.org/10.1002/hed.25158. Oh SJ, Lee HJ, Song KH, et al. Targeting the NANOG/HDAC1 axis reverses resistance to PD-1 blockade by reinvigorating the antitumor immunity cycle. J Clin Invest . 2022; 132(6). https://doi.org/10.1172/JCI147908. Capella DL, Goncalves JM, Abrantes A, Grando LJ, Daniel FI. Proliferative verrucous leukoplakia: diagnosis, management and current advances. Braz J Otorhinolaryngol . 2017; 83(5):585-593. https://doi.org/10.1016/j.bjorl.2016.12.005. Liu X, Ye Y, Zhu L, et al. Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation. Nat Commun . 2023; 14(1):238. https://doi.org/10.1038/s41467-023-35856-y. Vasefifar P, Najafi S, Motafakkerazad R, et al. Targeting Nanog expression increased Cisplatin chemosensitivity and inhibited cell migration in Gastric cancer cells. Exp Cell Res . 2023; 429(2):113681. https://doi.org/10.1016/j.yexcr.2023.113681. Huang CE, Yu CC, Hu FW, Chou MY, Tsai LL. Enhanced chemosensitivity by targeting Nanog in head and neck squamous cell carcinomas. Int J Mol Sci . 2014; 15(9):14935-14948. https://doi.org/10.3390/ijms150914935. Supplementary Figure Supplementary Figure S1 is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7191114","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495482749,"identity":"300e7f55-8eed-48dd-9b3d-351144309a61","order_by":0,"name":"Yanning Zhang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanning","middleName":"","lastName":"Zhang","suffix":""},{"id":495482750,"identity":"7491f348-6620-457b-84c9-20481a7b5e17","order_by":1,"name":"Xinning Zhang","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology \u0026 National Center for Stomatology \u0026 National Clinical Research Center for Oral Diseases \u0026 National Engineering Research Center of Oral Biomaterials and Digital Medical Devices","correspondingAuthor":false,"prefix":"","firstName":"Xinning","middleName":"","lastName":"Zhang","suffix":""},{"id":495482751,"identity":"0ff3f6ee-ac5c-4191-970b-f81b4c0752ac","order_by":2,"name":"Yanting Chi","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology \u0026 National Center for Stomatology \u0026 National Clinical Research Center for Oral Diseases \u0026 National Engineering Research Center of Oral Biomaterials and Digital Medical Devices","correspondingAuthor":false,"prefix":"","firstName":"Yanting","middleName":"","lastName":"Chi","suffix":""},{"id":495482752,"identity":"d28ef751-7480-434f-9326-5e05ce6d2e03","order_by":3,"name":"Jiayin Song","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology \u0026 National Center for Stomatology \u0026 National Clinical Research Center for Oral Diseases \u0026 National Engineering Research Center of Oral Biomaterials and Digital Medical Devices","correspondingAuthor":false,"prefix":"","firstName":"Jiayin","middleName":"","lastName":"Song","suffix":""},{"id":495482753,"identity":"abdc024f-d16c-41d0-aeb4-fd1e32599bf3","order_by":4,"name":"Long Li","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Li","suffix":""},{"id":495482754,"identity":"2d4249d5-5f1d-4839-966a-7de8d2b9391c","order_by":5,"name":"Binbin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACCQbmhsMMBgwJbAzMx4A8EEggpIURpoUtjXgtzBBlPGZQMQJaJNsTGw8XFNzJ45Pu+fbAMucwAz97jgHDzx24tUjzPGw4PMPgWTGbzNntBpLbDjNI9rwxYOw9g1uLnERiw2Eeg8OJbRK52yRAWgxu5BgwM7YRpSXnGViLPSEt0kha2CC2SBDQItkD9svhYjaJNHOgX9J5JM48KzjYi0eLxPHkw58L/hzOk5+R/Oyx5DZrOf725I0PfuLRghILzMCo5AExDuDTgKKF8QN+paNgFIyCUTBCAQCs/lHkKkt48wAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University School and Hospital of Stomatology \u0026 National Center for Stomatology \u0026 National Clinical Research Center for Oral Diseases \u0026 National Engineering Research Center of Oral Biomaterials and Digital Medical Devices","correspondingAuthor":true,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-23 01:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7191114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7191114/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88644532,"identity":"f6d6465d-6684-4184-bc6f-86a3d651d200","added_by":"auto","created_at":"2025-08-08 16:20:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":658815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehistopathological features of proliferative verrucous leukoplakia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Corrugated (undulating) epithelium with hyperkeratosis showing moderate epithelial dysplasia, prominent inflammatory infiltrate presented at the interface (buccal, HE, \u003cstrong\u003e×200\u003c/strong\u003e); B. The epithelium was notably thickened, marked hyperorthokeratosis as a verrucous pattern, prominent granular layer (dorsum of the tongue, HE, \u003cstrong\u003e×200\u003c/strong\u003e); C. Hyper-orthokeratosis of buccal mucosal epithelium, the rete pegs were notably thickened and elongated; (buccal, HE, \u003cstrong\u003e×200\u003c/strong\u003e); D. Atrophic epithelium, hyperparakeratosis at the surface without a prominent granular cell layer (ventrum of the tongue, HE, \u003cstrong\u003e×200\u003c/strong\u003e); E. OSCC evolved from PVL (HE, \u003cstrong\u003e×200\u003c/strong\u003e); F. Verrucous carcinoma evolved from PVL (HE, \u003cstrong\u003e×50\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/e65dc7c804bf6508beaec1a3.png"},{"id":88644531,"identity":"88331585-343d-4f46-a688-8958378933d2","added_by":"auto","created_at":"2025-08-08 16:20:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1041219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining for NANOG (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNANOG protein showed negative expression in COL (A, ×200) and low expression in PVL-NMT (B, ×200), while strong expression in early stage of PVL-MT, even in the lesion present as hyperkeratosis (C, ×200), verrucous hyperplasia (D, ×200) or leukoplakia with dysplasia (E, ×200). NANOG showed strong expression in OSCC-PVL (F, ×100, red arrow), both conventional OSCC (F, ×200) and verrucous carcinoma (H, ×50), while negative expression in normal epithelium adjacent to cancer (F, ×100, black arrow) .\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/cd99b51033fff4d12d92a615.png"},{"id":88642820,"identity":"b5b325a1-c2d2-492c-9ce4-656dd69a8d45","added_by":"auto","created_at":"2025-08-08 16:12:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. ROC curve for NANOG in identifying PVL from COL; B. ROC curve for NANOG in predicting malignant transformation of PVL; C. ROC curve for OED in predicting malignant transformation of PVL.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/9343f839390d901733e6b7bc.png"},{"id":88642824,"identity":"df831440-f21f-4232-bb39-b1f1cdebb97f","added_by":"auto","created_at":"2025-08-08 16:12:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":187252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan–Meier analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMalignant transformation-free survival with PVL. High expression of NANOG was associated with malignant transformation of PVL patients (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/d5e4e332b1cf7798c4f57fcb.png"},{"id":88642817,"identity":"5aaa4d1f-6262-4f9a-a697-cb1971cc4688","added_by":"auto","created_at":"2025-08-08 16:12:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC curves in the External Validation Cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. ROC curve for NANOG in identifying PVL from COL; B. ROC curve for NANOG in predicting malignant transformation of PVL.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/58b53a918200347ae94ef74f.png"},{"id":91696575,"identity":"2cdbe346-9748-4cb4-8d87-cb1176f2317f","added_by":"auto","created_at":"2025-09-19 09:39:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3117634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7191114/v1/db543d3e-aa50-49eb-ab00-bca5448a9741.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNANOG is a specific biomarker predicting malignant transformation of proliferative verrucous leukoplakia\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProliferative verrucous leukoplakia (PVL), an independent entity different from leukoplakia, is defined as \u0026ldquo;Progressive, persistent, and irreversible disorder characterized by the presence of multiple leukoplakias that frequently become warty\u0026rdquo; by WHO 2022.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e It is characterized by a progressive clinical course, typically demonstrating slow and persistent progression over an extended period before suddenly transitioning into rapid growth or malignant transformation. It exhibits the highest proportion of malignant transformation compared to other oral potentially malignant disorders (OPMDs), with a recurrence rate of 67.2% and a malignant transformation rate ranging from 26.7\u0026ndash;72.4%.\u003csup\u003e2\u0026ndash;5\u003c/sup\u003e However, due to the lack of unified criteria and specific biomarkers for early diagnosis and prognostic assessment, the current diagnosis usually needs to be combined with the medical history, which would be delayed, subjective and heterogeneous. On the other hand, PVL might be even misdiagnosed as other diseases, given its clinical manifestations resembling conventional oral leukoplakia (COL) or oral lichen planus.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Therefore, early diagnosis remains extremely challenging.\u003c/p\u003e\u003cp\u003eA reliable predictive biomarker would facilitate the early diagnosis and accurate treatment of PVL. However, due to the low incidence and limited cases of PVL, most of the current studies are observational or case reports, lacking in-depth research and understanding of PVL. In this study, we included a relatively large number of cases cohort for nested case-control study, and combined the biomarker NANOG for analysis.\u003c/p\u003e\u003cp\u003eNANOG is a transcription regulator involved in inner cell mass and embryonic stem cells proliferation, renewal and pluripotency. It plays a pivotal role in various aspects of tumori-genesis, metastasis, and chemo-sensitivity,\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and has been proved to be potential biomarker to predict the prognosis of various human cancers.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e NANOG showed strong expression in laryngeal dysplasia and association with laryngeal cancer risk superior to the histological classification.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Nevertheless, the role of NANOG in PVL and its possible implication in malignant transformation remain to be undetermined. In this study, we detected the NANOG expression in PVL progression, and combined it with clinical characteristics to analyze its association with malignant transformation prognosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePatients Data\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 112 patients collected from Peking University School and Hospital of Stomatology (from 2013 to 2020) were enrolled in this study. Among them, 79 cases of PVL including 48 cases progressed to oral squamous cell carcinoma (OSCC), and 33 cases of COL as control. The studies involving human participants were reviewed and approved by The Biomedical Ethics Committee of the Department of Peking University (No. PKUSSIRB-201948111).\u003c/p\u003e\u003cp\u003eAnother cohort of 31 cases from Xiangya Stomalogical Hospital were included in the external validation cohort. There were 18 cases of COL and 15 cases of PVL. Eight cases of PVL underwent malignant transformation.\u003c/p\u003e\u003cp\u003eDiagnostic criteria for PVL :\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Major criteria: (1) Leukoplakia lesion involved in more than two different oral subsites; (2) The existence of a verrucous area; (3) Lesion spreading or engrossing during the disease development; (4) There has been a recurrence in a previously treated area; (5) Histopathological test: oral epithelial hyperkeratosis to verrucous hyperplasia, verrucous carcinoma, or squamous cell carcinoma, whether in situ or infiltrating. Minor criteria: (1) Oral leukoplakia lesion that occupies at least 3 cm when adding all the affected areas; (2) Female patient; (3) Non-smoker regardless of gender; (4) More than 5 years evolution. Diagnostic criteria: Three major criteria (histopathological test among them) or two major criteria (histopathological test among them)\u0026thinsp;+\u0026thinsp;two minor criteria.\u003c/p\u003e\u003cp\u003eCOL included criteria: (1) Histopathologic diagnosis of oral leukoplakia by two pathologists independently; (2) It cannot be diagnosed with other diseases or PVL as mentioned above.\u003c/p\u003e\u003cp\u003eExclusion criteria for PVL and COL: (1) The first diagnosis is OSCC; (2) With tumors of other origins or other serious diseases; (3) Insufficient follow up data.\u003c/p\u003e\u003cp\u003eThe clinical and histopathological characteristics were collected as following: sex, age, tobacco and alcohol habits, local irritants, primary site, clinical presentation, malignant transformation-free survival (MTFS) and oral epithelial dysplasia (OED). Local irritants, such as faulty restorations, residual roots, sharp tooth cusps, silver amalgam, were included in the possible influencing factors accessed in this analysis. The primary lesion sites included oral subsites: dorsum and ventrum of the tongue, floor of the mouth, gingiva, buccal mucosa, lip, soft palate and hard palate. Furthermore, based on the number of subsites involved, the lesion sites were categorized into those affecting more than two subsites and those affecting two or fewer subsites. The involvement of the ventral tongue and floor of the mouth as primary sites was also considered as potential risk factors and prognostic factors. Clinical presentation was considered homogeneous when predominantly white, flat, thin or wrinkled lesions; and non-homogeneous in the presence of mixed white-and-red, erosion, nodular, granular, or verrucous lesions. MTFS time was measured from the time of initial biopsy to the time of transformation to OSCC confirmed by pathology or the end of follow-up.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemical staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll the samples were fixed with 10% formalin and embedded in paraffin. A series of 2.5\u0026micro;m thick sections of tissue were obtained for immunohistochemical staining. Following deparaffination and rehydration, heat slides with 10 mM citrate buffer (pH 6.0) for 10 minutes. Endogenous peroxidase activity was quenched with 10% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 min, which was followed by blocking the non-specific protein binding by using 5% normal goat serum 15 min at room temperature. The tissues were then incubated with the rabbit monoclonal anti-NANOG (#4903, D73G4, cell signaling technology, USA, 1:100), overnight at 4\u0026deg;C, the secondary goat anti-rabbit IgG antibodies (PV6000 Kit, ZSGB-BIO, China) for 60 min at 37\u0026deg;C, using the diaminobenzidine tetrahydrochloride (DAB) as the chromogen, haematoxylin was used as a counterstain.\u003c/p\u003e\u003cp\u003eThe immunohistochemical staining results were assessed semi-quantitatively; the staining was scored and graded by two pathologists as follows: low-expression (0: negative, 1+: weak positivity), high-expression (2+: moderate positivity, 3+: strong positivity). All readings were performed independently and without any prior knowledge of the clinical or histopathological characteristics of the cases.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using SPSS software (version 22.0, IBM, United States) and R 4.4.0. For the univariate analysis, relationships between categorical characteristics were evaluated using the Chi-squared test and Fisher\u0026rsquo;s exact test, and between continuous variables using the T-test or Mann-Whitney U test, as appropriate. Kaplan-Meier was utilized to assess the prognostic significance of NANOG in PVL by analyzing the association between NANOG level and MTFS. Survival difference was evaluated using Log-rank test. Univariate and multivariate COX analyses were employed to evaluate the independent prognostic significance of NANOG expression level and other clinical parameters on MTFS in PVL patients. Receiver operator characteristic (ROC) curves were used to reflect the sensitivity and specificity of NANOG in diagnostic and prognostic of PVL.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, a total of 112 patients were included in this study. The ratio of males to females was 1:2, and the average age was 57.21 years. The mean follow-up time was 77.78 months. The mean time for the malignant transformation of PVL was 47.5 months.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinico-demographic-pathologic characteristics of 112 cases of PVL cohort\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePVL (N\u0026thinsp;=\u0026thinsp;79)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCOL (N\u0026thinsp;=\u0026thinsp;33)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e.overall\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.27\u0026thinsp;\u0026plusmn;\u0026thinsp;10.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.18\u0026thinsp;\u0026plusmn;\u0026thinsp;14.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.499\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57 (72.15%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (54.55%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (27.85%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (45.45%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNANOG Expression:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-NANOG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42 (53.16%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29 (87.88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-NANOG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37 (46.84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (12.12%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.77\u0026thinsp;\u0026plusmn;\u0026thinsp;47.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120.82\u0026thinsp;\u0026plusmn;\u0026thinsp;19.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLesion sites:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;=2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28 (35.44%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (81.82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51 (64.56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (18.18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClinical presentation:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42 (53.16%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (69.70%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-homogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37 (46.84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (30.30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocal irritants:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.491\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43 (54.43%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (63.64%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36 (45.57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (36.36%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmoker:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71 (89.87%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 (75.76%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (10.13%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (24.24%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlcohol use:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71 (89.87%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (81.82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (10.13%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (18.18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOED:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-OED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33 (41.77%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (42.42%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46 (58.23%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 (57.58%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVT or FOM involved:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47 (59.49%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29 (87.88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32 (40.51%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (12.12%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL, proliferative verrucous leukoplakia; COL, conventional oral leukoplakia; OED, oral epithelial dysplasia; Non-OED, without oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePVL histopathological features were complex, ranging from hyperkeratosis to severely dysplastic, or OSCC. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, it would be manifested as corrugated (undulating) ortho(para)hyperkeratotic with verrucous or papillary architecture, or notably thickened epithelium with marked hyperorthokeratosis, even atrophic epithelium, but cytologic atypia was rare. The histopathology of OSCC derived from PVL (OSCC-PVL) more likely to be conventional OSCC \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e(\u003c/span\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, E\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, sometimes manifested as a verrucous pattern \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e(\u003c/span\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eF)\u003c/span\u003e, with keratin plunges down into the epithelial crypts. The characteristics of structural disorder were obvious, but the cytologic pleomorphism was rare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial biology samples of all patients were used for immunohistochemistry, in addition, biopsy samples of OSCC when first confirmed during the PVL progression (33 cases) were included. NANOG is mainly expressed in nuclear and cytoplasmic \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e(\u003c/span\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e)\u003c/span\u003e. NANOG showed high expression in PVL with malignant transformation (70.83%), significantly higher than that in PVL without malignant transformation (9.68%) and COL group (12.12%). However, in the PVL progression, there was no difference in expression between the samples of initial biopsy and after malignant transformation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e)\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eImmunohistochemical staining of NANOG\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eNANOG expression\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePVL-MT*#\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34 (70.83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14 (29.17%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePVL-NMT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3 (9.68%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28 (90.32%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOSCC-PVL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28 (84.85%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5 (15.15%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4 (12.12%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29(87.88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e* compare with PVL-NMT: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e#compare with COL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-NMT vs COL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-MT vs SCC-PVL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL, proliferative verrucous leukoplakia;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eCOL, conventional oral leukoplakia;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-MT, PVL with malignant transformation\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-NMT, PVL without malignant transformation\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eOSCC-PVL, OSCC occurs during the evolution of PVL\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNANOG protein showed negative expression in COL (A, \u0026times;200) and low expression in PVL-NMT (B, \u0026times;200), while strong expression in early stage of PVL-MT, even in the lesion present as hyperkeratosis (C, \u0026times;200), verrucous hyperplasia (D, \u0026times;200) or leukoplakia with dysplasia (E, \u0026times;200). NANOG showed strong expression in OSCC-PVL (F, \u0026times;100, red arrow), both conventional OSCC (F, \u0026times;200) and verrucous carcinoma (H, \u0026times;50), while negative expression in normal epithelium adjacent to cancer (F, \u0026times;100, black arrow) .\u003c/p\u003e\n\u003cp\u003eThe association between NANOG and clinicopathological characteristic was then evaluated in PVL patients, including sex, age, primary site, local irritants, tobacco and alcohol habits, clinical presentation, MTFS, and OED. NANOG high expression was associated with clinical presentation and OED, which showed high expression level in sample with non-homogenous presentation (62.16%) and histological OED (78.38%) \u003cstrong\u003e(\u003c/strong\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAssociation between NANOG and clinicopathological Characteristic in PVL\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eL-NANOG (N\u0026thinsp;=\u0026thinsp;42)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH-NANOG (N\u0026thinsp;=\u0026thinsp;37)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e.overall\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMalignant transformation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28 (66.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3 (8.11%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14 (33.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34 (91.89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.79 (8.66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.68 (12.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.647\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.158\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27 (64.29%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30 (81.08%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15 (35.71%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7 (18.92%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTime (Month)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103.31 (40.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.92 (37.94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLesion sites:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;=2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11 (26.19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17 (45.95%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31 (73.81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20 (54.05%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClinical presentation:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28 (66.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14 (37.84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-homogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14 (33.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23 (62.16%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocal irritants:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25 (59.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18 (48.65%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17 (40.48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19 (51.35%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmoker:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38 (90.48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33 (89.19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4 (9.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4 (10.81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlcohol use:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.717\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37 (88.10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34 (91.89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5 (11.90%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3 (8.11%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOED:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-OED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25 (59.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8 (21.62%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17 (40.48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29 (78.38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVT or FOM involved:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27 (64.29%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20 (54.05%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15 (35.71%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17 (45.95%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL, proliferative verrucous leukoplakia; OED, oral epithelial dysplasia; Non-OED, without oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eCOX proportional hazards regression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCOX proportional hazards regression analysis was used to analyze NANOG expression and clinicopathological characteristics related with malignant transformation of PVL, Multivariate COX analysis demonstrated that NANOG high expression was an independent prognostic factor for PVL patients, revealing that high expression of NANOG may affect the malignant characteristics of PVL and poor prognosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eUnivariate and Multivariate COX proportional hazard model\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eUnivariate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eMultivariate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e_value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e_value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.995\u0026ndash;1.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.695\u0026ndash;6.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNANOG Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.565\u0026ndash;7.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.516\u0026ndash;5.906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLesion sites\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.558\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.293\u0026ndash;1.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical presentation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.066\u0026ndash;4.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.959\u0026ndash;3.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocal irritants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.320\u0026ndash;1.106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmoker\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.123\u0026ndash;2.658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlcohol use\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.243\u0026ndash;5.819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOED\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.240\u0026ndash;6.809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.317\u0026ndash;6.281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVT or FOM involved\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.692\u0026ndash;2.495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eHR: Hazard Ratio; CI, confidence interval; OED, oral epithelial dysplasia; VT or FOM involved, ventral tongue or floor of mouth involved.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eROC analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the sensitivity and specificity of NANOG in distinguishing PVL patients from COL controls at early diagnosis, ROC curves were generated, AUC value of the model reached 0.674 (0.594\u0026ndash;0.753) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the sensitivity and specificity were 0.800 and 0.879 respectively \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cstrong\u003eA)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo determine the sensitivity and specificity of NANOG in malignant transformation predicting for PVL, ROC curves were generated, AUC value of the model reached 0.806 (0.721\u0026ndash;0.890) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the sensitivity and specificity were 0.900 and 0.903 respectively \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cstrong\u003eB)\u003c/strong\u003e. Compared with OED \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cstrong\u003eC)\u003c/strong\u003e, NANOG showed better sensitivity and specificity in identify and prognostic prediction of PVL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKaplan-Meier survival analysis was used to explore the effect of NANOG expression (high vs. low) on the malignant transformation of PVL patients. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, high expression of NANOG was associated with lower MTFS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Accordingly, overexpression of NANOG was closely associated to malignant transformation of patients. This was consistent with the results of the COX analysis.\u003c/p\u003e\n\u003cp\u003eMalignant transformation-free survival with PVL. High expression of NANOG was associated with malignant transformation of PVL patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNANOG Expression in the Validation Cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNANOG expression in the validation cohort as shown in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cstrong\u003eand Figure S1.\u003c/strong\u003e ROC analysis showed that the sensitivity and specificity of NANOG in distinguishing PVL patients from COL were 0.800 and 0.333, respectively. And the sensitivity and specificity in malignant transformation predicting for PVL were 0.800 and 0.869 \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNANOG expression in the validation cohort\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eNANOG expression\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePVL-MT*#\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7 (87.50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1 (12.50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePVL-NMT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1 (14.29%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6 (85.71%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOSCC-PVL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6 (75.00%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2 (25.00%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4 (22.22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14(77.78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e* compare with PVL-NMT: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e#compare with COL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-NMT vs COL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-MT vs SCC-PVL: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL, proliferative verrucous leukoplakia;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eCOL, conventional oral leukoplakia;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-MT, PVL with malignant transformation\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003ePVL-NMT, PVL without malignant transformation\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eOSCC-PVL, OSCC occurs during the evolution of PVL\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePVL presents the highest malignant conversion rate in OMPD. However, due to its complex clinical course, clinical and morphological manifestations variants, lacking specificity. The initial manifestation of PVL may be a flat white area or lichenoid appearance, with or without dysplasia,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e similar to conventional oral leukoplakia, oral lichen planus or other oral white lesions.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Thus, these cases could be erroneously treated as other diseases, subsequently malignant transformation over an extended period. Therefore, there is an urgent need for an effective biomarker to facilitate the early diagnosis and accurate treatment of PVL.\u003c/p\u003e\u003cp\u003eOral epithelial dysplasia (OED) is considered a risk for OSCC, and used to assess the malignant transformation potential in OPMDs. However, research indicated that one-third of PVL cases did not exhibit OED, yet malignant transformation still occurred in 3.7% of these cases.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e In this study, 11 cases of PVL without OED (22.92%) progressed to OSCC. Therefore, additional objective and reliable markers are needed to predict the risk of malignant transformation beyond current clinical and histopathological characteristics. which will undoubtedly help the clinicians to choose the most adequate therapeutic option.\u003c/p\u003e\u003cp\u003eNANOG, as one of the main transcription factors participating in stemness,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e It plays an important role in maintaining the stemness and self-renewal of tumor cells,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e NANOG shows high expression in various cancers, participated in signaling pathways (JAK/STAT, Wnt/β-catenin, Notch and EGFR), induced stemness, self-renewal, metastasis, invasion, epithelial-mesenchymal transition and chemoresistance of cancer cells.[21\u0026ndash;24] High NANOG expression was also associated with poor prognosis and might serve as an unfavorable prognostic biomarker.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eStrong NANOG expression was found in laryngeal dysplasia, associated with laryngeal cancer risk superior to the histological classification, and no expression in normal epithelia.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e NANOG was uniquely showed as a better prognosis in pharyngeal tumors.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Similar results were found in oral studies, showing that NANOG exhibited no expression in normal mucosa, very weak expression in low-grade dysplasia, and strong expression in high-grade dysplasia and OSCC, NANOG expression emerged as an early predictor of oral cancer risk in patients with OPMD, and was more frequent in early stages of OSCC tumorigenesis rather than in advanced neoplastic disease.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Unlike in laryngeal cancer, high NANOG expression in OSCC were associated with a worse overall survival.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, NANOG was significantly higher in the early stage of PVL compared to COL, ROC analysis showed that, the sensitivity and specificity were 0.800 and 0.879, indicating that NANOG would be used to identified PVL from COL. In addition, NANOG showed higher expression in PVL with malignancy transformation than that without malignant transformation. However, it is worth noting that NANOG showed high expression in the early stage before the malignant transformation, but there was no difference before and after carcinogenesis of PVL, suggesting the NANOG high expression was associated with malignant transformation, but not associated with disease progress.\u003c/p\u003e\u003cp\u003eStudies suggested that positive NANOG expression in OSCC was significantly associated with tobacco and alcohol consumption.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In this study, NANOG expression associated with non-homogeneous clinical presentation and OED (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multivariate COX analysis indicated that NANOG was an independent prognostic factor for PVL malignant transformation. ROC analysis showed that NANOG could be used to predict malignant transformation of PVL with sensitivity and specificity of 0.900 and 0.903, respectively. Kaplan-Meier analysis also demonstrated that the high expression of NANOG is associated with the increased malignant transformation. Thus, NANOG showed higher accuracy, sensitivity and specificity than that of OED in predicting the malignant transformation of PVL. Therefore, NANOG would be a specific predictor of early identification and prediction of malignant transformation in PVL patients. These results were confirmed in the validation cohort, especially the NANOG expression demonstrated superiority in sensitivity and specificity in predicting the malignant transformation of PVL. In the validation cohort, NANOG demonstrated relatively poor specificity in distinguishing PVL from COL, which might be related to the small sample size.\u003c/p\u003e\u003cp\u003eFurthermore, NANOG was believed to be a factor in enhancing patient resistance to immune checkpoint inhibitors.[32] As shown in this study, NANOG high expression in PVL with malignant transformation, even in the early stages premalignancy, may contribute to the resistance of PVL patients to most therapies.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Therapeutically, since NANOG is highly expressed in PVL with malignant transformation but not in normal tissues, it could serve as an ideal target for disease treatment, minimizing off-target effects and enhancing specificity in gene-targeted therapy. Research suggested that targeting NANOG could improve chemosensitivity and inhibit tumorigenesis.\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Consequently, the targeted therapy for NANOG might present a therapeutic potential in PVL and warranted further exploration.\u003c/p\u003e\u003cp\u003eIn summary, NANOG could be used as an independent risk factor for malignant transformation of PVL, and it had a good predictive value in early diagnosis and predicting malignant transformation of PVL superior to OED. It might be an ideal target in future gene-targeted therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest and Source of Funding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBinbin Li performed study concept and design; Yanning Zhang \u0026amp; Xinning Zhang performed development of methodology and writing, review and revision of the paper; Yanting Chi provided acquisition, analysis and interpretation of data, and statistical analysis; Jiayin Song \u0026amp; Long Li contributed to data collection and slide evaluation. All authors contributed to review and revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and con\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003esent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted according to the guidelines recommended in the Declaration of Helsinki and\u0026nbsp;approved by The Biomedical Ethics Committee of the Department of Peking University (No. PKUSSIRB-201948111). Written informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeclarations of interest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used ChatGPT in order to improve readability and make the language expression more in line with the native English expression habits. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWarnakulasuriya S, Kujan O, Aguirre-Urizar JM, et al. Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer. \u003cem\u003eOral Dis\u003c/em\u003e. 2021; 27(8):1862-1880. https://doi.org/10.1111/odi.13704.\u003c/li\u003e\n \u003cli\u003eProano-Haro A, Bagan L, Bagan JV. Recurrences following treatment of proliferative verrucous leukoplakia: A systematic review and meta-analysis. \u003cem\u003eJ Oral Pathol Med\u003c/em\u003e. 2021; 50(8):820-828. https://doi.org/10.1111/jop.13178.\u003c/li\u003e\n \u003cli\u003eIocca O, Sollecito TP, Alawi F, et al. 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Single-molecule tracking of Nanog and Oct4 in living mouse embryonic stem cells uncovers a feedback mechanism of pluripotency maintenance. \u003cem\u003eEmbo J\u003c/em\u003e. 2023; 42(18):e112305. https://doi.org/10.15252/embj.2022112305.\u003c/li\u003e\n \u003cli\u003eThiagarajan PS, Sinyuk M, Turaga SM, et al. Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase. \u003cem\u003eNat Commun\u003c/em\u003e. 2018; 9(1):578. https://doi.org/10.1038/s41467-018-02938-1.\u003c/li\u003e\n \u003cli\u003eSemenza GL. Mechanisms of Breast Cancer Stem Cell Specification and Self-Renewal Mediated by Hypoxia-Inducible Factor 1. \u003cem\u003eStem Cells Transl Med\u003c/em\u003e. 2023; 12(12):783-790. https://doi.org/10.1093/stcltm/szad061.\u003c/li\u003e\n \u003cli\u003eLo UG, Chen YA, Cen J, et al. The driver role of JAK-STAT signalling in cancer stemness capabilities leading to new therapeutic strategies for therapy- and castration-resistant prostate cancer. \u003cem\u003eClin Transl Med\u003c/em\u003e. 2022; 12(8):e978. https://doi.org/10.1002/ctm2.978.\u003c/li\u003e\n \u003cli\u003eLiu L, Zhu H, Liao Y, et al. Inhibition of Wnt/beta-catenin pathway reverses multi-drug resistance and EMT in Oct4(+)/Nanog(+) NSCLC cells. \u003cem\u003eBiomed Pharmacother\u003c/em\u003e. 2020; 127:110225. https://doi.org/10.1016/j.biopha.2020.110225.\u003c/li\u003e\n \u003cli\u003eYang J, Huang Y, Song M, et al. SPC25 promotes proliferation and stemness of hepatocellular carcinoma cells via the DNA-PK/AKT/Notch1 signaling pathway. \u003cem\u003eInt J Biol Sci\u003c/em\u003e. 2022; 18(14):5241-5259. https://doi.org/10.7150/ijbs.71694.\u003c/li\u003e\n \u003cli\u003eOh SJ, Lim JY, Son MK, et al. 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Proliferative verrucous leukoplakia: diagnosis, management and current advances. \u003cem\u003eBraz J Otorhinolaryngol\u003c/em\u003e. 2017; 83(5):585-593. https://doi.org/10.1016/j.bjorl.2016.12.005.\u003c/li\u003e\n \u003cli\u003eLiu X, Ye Y, Zhu L, et al. Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation. \u003cem\u003eNat Commun\u003c/em\u003e. 2023; 14(1):238. https://doi.org/10.1038/s41467-023-35856-y.\u003c/li\u003e\n \u003cli\u003eVasefifar P, Najafi S, Motafakkerazad R, et al. Targeting Nanog expression increased Cisplatin chemosensitivity and inhibited cell migration in Gastric cancer cells. \u003cem\u003eExp Cell Res\u003c/em\u003e. 2023; 429(2):113681. https://doi.org/10.1016/j.yexcr.2023.113681.\u003c/li\u003e\n \u003cli\u003eHuang CE, Yu CC, Hu FW, Chou MY, Tsai LL. Enhanced chemosensitivity by targeting Nanog in head and neck squamous cell carcinomas. \u003cem\u003eInt J Mol Sci\u003c/em\u003e. 2014; 15(9):14935-14948. https://doi.org/10.3390/ijms150914935.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figure","content":"\u003cp\u003eSupplementary Figure S1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"proliferative verrucous leukoplakia, canceration, early diagnosis, prognosis, NANOG ","lastPublishedDoi":"10.21203/rs.3.rs-7191114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7191114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eProliferative verrucous leukoplakia (PVL) is a special type of oral leukoplakia with no unified diagnosis criteria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To evaluate the diagnostic values of NANOG for PVL and explore the associations between NANOG expression and the malignant transformation of PVL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA total of 112 patients were collected, including 79 cases of PVL (48 cases developed into oral squamous cell carcinoma) and 33 patients diagnosed as conventional oral leukoplakia (COL). The expression of NANOG was detected by immunohistochemical staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e NANOG showed higher expression in 34 cases(70.83%) PVL with malignant transformation, significantly higher than that in PVL without malignant transformation and COL (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), and the NANOG expression was correlated with non-homogeneous clinical manifestation and oral epithelial dysplasia (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Receiver operating characteristic (ROC) analysis revealed that it would be used to identify PVL from COL, AUC = 0.674, with sensitivity and specificity of 0.800 and 0.879. Kaplan-Meier plotter data revealed that patients with high NANOG expression had shorter malignant transformation-free survival than those with low expression (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Multivariate COX analysis further confirmed that high NANOG expression was an independent risk factor for malignant transformation of PVL. ROC analysis also revealed that it would be used to predict the malignant transformation of PVL, AUC = 0.806, with sensitivity and specificity of 0.900 and 0.903. The above resultswere also validated in external case cohort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eNANOG would be a promising diagnostic and prognostic biomarker for PVL.\u003c/p\u003e","manuscriptTitle":"NANOG is a specific biomarker predicting malignant transformation of proliferative verrucous leukoplakia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:12:13","doi":"10.21203/rs.3.rs-7191114/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a7580541-37d3-4c4c-80ec-0868eff5ec20","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-19T09:38:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 16:12:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7191114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7191114","identity":"rs-7191114","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}