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Objective: To identify potential salivary biomarkers for the diagnosis and monitoring of disease progression in oral squamous cell carcinoma and oral leukoplakia., Materials and Methods: An advance search from PubMed and Hindawi was performed with keywords; oral leukoplakia/oral squamous cell carcinoma, salivary biomarker and diagnosis/prognosis. An additional search of articles was done through a manual search from the Google Scholar database., Results: Twenty studies involving salivary biomarkers as diagnostic tools for oral squamous cell carcinoma and/or oral leukoplakia were identified. A narrative review was carried out., Conclusion: Single or multiple salivary biomarkers reported by most studies have shown great potential as diagnostic tools for oral squamous cell carcinoma and oral leukoplakia. However, the validation of sensitivity and specificity should be carried out to ensure the accuracy of the biomarkers. Furthermore, a standardised method for saliva collection should be established to prevent variability in the expression of biomarkers., (© 2023 Wiley Periodicals LLC.)

Hypoxia is a key trigger in the transformation of oral leukoplakia into oral cancer. However, it is still too early to determine the role of hypoxia in the development of oral leukoplakia. Prx1, an antioxidant protein, upregulated by hypoxia, regulates cellular autophagy in leukoplakia. This study aimed to understand the mechanisms by which hypoxia induces Prx1 expression during autophagy in oral leukoplakia. We used an experimental model of tongue epithelial hyperplasia induced by 4-nitroquinoline-1-oxide (4NQO) and dysplastic oral keratinocytes. Prx1 knockdown DOK cells, Leuk-1 cells and control cells were harvested, and cell proliferation was assayed using the Cell Counting Kit-8. Several hypoxia and autophagy-related proteins were examined using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and western blotting in cells and mouse tongue tissues. In addition, the ultrastructure of the cells was observed by transmission electron microscopy. Hypoxia induces cell proliferation, autophagic vesicles and the expression of Prx1, BNIP3, LC3II/I and Beclin-1 in DOK and Leuk-1 cells. However, these effects were all attenuated by Prx1 knockdown. Histologically, 4NQO induced epithelial hyperplasia in the tongue mucosa. The expression of proliferation marker PCNA, autophagy-related proteins LC3B and Beclin-1, as well as HIF-1α/BNIP3 was significantly lower in the tongue tissues of Prx1 flox/flox:Cre+ mice compared with Prx1 flox/flox mice. In Prx1 flox/flox:Cre+ mice, an increased expression of HIF-1α/BNIP3, LC3B and Beclin-1 was detected in epithelial hyperplasia tongue tissues compared to normal tissues. The current study suggests that Prx1 may promotes cell proliferation and autophagy in oral leukoplakia cells via the HIF-1α/BNIP3 pathway., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Oral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear., Methods: This study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H 2 O 2 . In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2., Results: Clinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H 2 O 2 -induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest., Conclusion: Prx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest related to this work., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Context: Oral cancer is highly prevalent in India. Lack of awareness and delay in diagnosis and treatment of patients with oral cancer leads to high mortality and poor survival of patients. Salivary endothelin-1 is proposed as a prospective biomarker for oral squamous cell carcinoma., Aims: Aim of the study was to evaluate salivary level of endothelin-1 in oral cancer and precancer as a biomarker., Settings and Design: We planned a case control study to evaluate salivary level of Endothelin-1 in oral cancer and precancer as a biomarker., Materials and Methods: A total of 72 subjects were taken in study out of which 24 cases were of histopathologically confirmed premalignat oral lesion (oral leukoplakia and oral submucous fibrosis), 24 cases were of histopathologically confirmed oral squamous cell carcinoma, and 24 cases of healthy age and gender matched controls without any addiction to tobacco in any form from a tertiary care hospital were taken. Saliva was collected from all following standard guidelines and estimation of salivary endothelin-1 was done by ELISA., Statistical Analysis Used: SPSS software version 15., Results: Salivary endothelin-1 values of controls ranged between 0.09 and 1.88 pg/ml while that of premalignant cases ranged between 1.16 and 16.135 pg/ml and of SCC cases ranged between 2.567 and 22.98 pg/ml., Conclusions: Salivary endothelin-1 is raised in oral squamous cell carcinoma compared to premalignant and controls therefore, shows capability to differentiate between premalignant lesion and oral cancer. So, it could be used as a biomarker for early diagnosis., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Objective: This study aimed to investigate the potential of fibroblast activation protein (FAP) as a biomarker in the progression of oral leukoplakia (OLK) carcinogenesis. This was achieved by evaluating FAP expression at different levels of the organisation, namely oral normal mucosa (NM), OLK, and oral squamous cell carcinoma (OSCC)., Materials and Methods: Altogether, 88 paraffin-embedded tissue samples were examined, including 55 cases of OLK, 13 cases of OSCC, and 20 cases of NM (control group). An exhaustive investigation was performed to examine FAP expression in NM, OLK, and OSCC tissues via immunohistochemistry (IHC). The relationship between FAP expression and clinical pathologic characteristics was analysed. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB) also proved the expression of FAP in NM, OLK, and OSCC cells. Aberrant FAP expression in OLK and OSCC was explored using in vitro experiments., Results: Immunohistochemical results showed that high FAP expression was significantly correlated with histopathologic grade (P = .038) but not correlated with age, sex, or region (P = .953, .622, and .108, respectively). The expression level of FAP in NM tissues (0.15 ± 0.01) was minimal, whereas it was observed in OLK (0.28 ± 0.04) and OSCC (0.39 ± 0.02) tissues with a noticeable increase in expression levels (P < .001). The expression level of FAP in OLK with severe abnormal hyperplasia (S-OLK) tissues (0.33 ± 0.04) was significantly higher than in OLK with mild abnormal hyperplasia (MI-OLK, 0.26 ± 0.02) and OLK with moderate abnormal hyperplasia (MO-OLK, 0.28 ± 0.03) tissues (P < .001 and P = .039, respectively). The results of RT-PCR illustrated that the relative expression of FAP mRNA in OLK cells (2.63 ± 0.62) was higher than in NM cells (0.87 ± 0.14), but lower than in OSCC cells (5.63 ± 1.06; P = .027 and .012, respectively). FAP expression was minimal in NM cells (0.78 ± 0.06), modest in OLK cells (1.04 ± 0.06), and significantly elevated in OSCC cells (1.61 ± 0.09) based on the results of WB (P < .001)., Conclusions: Significant variations in FAP expression were observed in NM, OLK, and OSCC tissues and cells. These findings revealed that FAP may be a reliable biomarker for the early diagnosis and evaluation of OLK carcinogenesis., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Intermediate filaments are one of three polymeric structures that form the cytoskeleton of epithelial cells. In the epithelium, these filaments are made up of a variety of keratin proteins. Intermediate filaments complete a wide range of functions in keratinocytes, including maintaining cell structure, cell growth, cell proliferation, cell migration, and more. Given that these functions are intimately associated with the carcinogenic process, and that hyperkeratinization is a quintessential feature of oral leukoplakias, the utility of keratins in oral leukoplakia is yet to be fully explored. This scoping review aims to outline the current knowledge founded on original studies on human tissues regarding the expression and utility of keratins as diagnostic, prognostic, and predictive biomarkers in oral leukoplakias. After using a search strategy developed for several scientific databases, namely, PubMed, Scopus, Web of Science, and OVID, 42 papers met the inclusion and exclusion criteria. One more article was added when it was identified through manually searching the list of references. The included papers were published between 1989 and 2024. Keratins 1-20 were investigated in the 43 included studies, and their expression was assessed in oral leukoplakia and dysplasia cases. Only five studies investigated the prognostic role of keratins in relation to malignant transformation. No studies evaluated keratins as a diagnostic adjunct or predictive tool. Evidence supports the idea that dysplasia disrupts the terminal differentiation pathway of primary keratins. Gain of keratin 17 expression and loss of keratin 13 were significantly observed in differentiated epithelial dysplasia. Also, the keratin 19 extension into suprabasal cells has been associated with the evolving features of dysplasia. The loss of keratin1/keratin 10 has been significantly associated with high-grade dysplasia. The prognostic value of cytokeratins has shown conflicting results, and further studies are required to ascertain their role in predicting the malignant transformation of oral leukoplakia.

Background and Aim: Precancer biomarkers help in early detection and management of oral potentially malignant disorders (OPMDs). Interleukin-1β (IL-1β), a biomarker, is known to be altered in oral submucous fibrosis (OSMF) and oral leukoplakia (OL). Therefore, we evaluated and compared the serum and salivary IL-1β levels in patients with OSMF/oral leukoplakia and in gender- and age-matched healthy individuals., Materials and Methods: An in vivo, prospective, observational study was conducted on 40 subjects. Subjects were divided into two groups with 20 individuals in each group, that is, Group I: OSMF/oral leukoplakia and Group II: control group. Salivary and serum IL-1β levels were quantitatively estimated using enzyme-linked immunosorbent assay (ELISA). The statistical tests used were unpaired t-test and Chi-square test., Results: The serum IL-1β levels were significantly (P 0.001) lesser in Group I in comparison to Group II. The salivary IL-1β levels remained insignificant between both the groups. However, in both the groups, the salivary IL-1β levels were significantly higher compared to the serum IL-1β levels., Conclusion: We found that the serum IL-1β level can be considered as a prospective biomarker for dysplasia, whereas salivary IL-1β alone needs more elaborated studies to account for its application as a potential biomarker in OPMD., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Purpose: Oral leukoplakia (OL) is a common potentially malignant oral disorder. Therefore, there is a need for simple screening methods for OL before its transformation into oral cancer. Furthermore, because invasive open biopsy is the sole method to determine if an OL lesion is dysplastic, there is also a clinical need for non-invasive methods to differentiate dysplastic OL from non-dysplastic OL. This study aimed to identify salivary metabolites that can help differentiate patients with OL from healthy controls (HC) and also dysplastic OL from non-dysplastic OL., Material & Methods: Whole unstimulated saliva samples were collected from patients with OL (n = 30) and HCs (n = 29). The OL group included nine patients with dysplastic OL and 20 with non-dysplastic OL. Hydrophilic metabolites in the saliva samples were comprehensively analyzed through capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate patients with OL from HCs and dysplastic OL from non-dysplastic OL., Results: Twenty-eight metabolites were evidently different between patients with OL and HCs. Finally, three metabolites (guanine, carnitine, and N-acetylputrescine) were selected to develop the MLR model, which resulted in a high area under curve (AUC) of the receiver operating characteristic (ROC) to differentiate patients with OL from HCs (AUC = 0.946, p < 0.001, 95% confidential interval [CI] = 0.889- 1.000). Similarly, two metabolites were evidently different between patients with dysplastic and non-dysplastic OL. Finally, only one metabolite (7-methylguanine) was selected in the MLR model, which revealed a moderate discrimination ability for dysplastic and non-dysplastic OL (AUC = 0761, p = 0.027, 95% CI = 0.551-0.972)., Conclusion: Our candidate salivary metabolites showed potential not only to discriminate OL from HC, but also to discriminate dysplastic OL from non-dysplastic OL., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Background: The rising cancer incidence in patients with oral leukoplakia (OL) highlights the importance of identifying potential biomarkers for high-risk individuals and lesions because these biomarkers are useful in developing personalized management strategies for OL patients. This study systematically searched and analyzed the literature on potential saliva and serum biomarkers for OL malignant transformation., Methods: PubMed and Scopus were searched for studies published up to April 2022. The primary outcome of this study was the difference in biomarker concentrations in saliva or serum samples from healthy control (HC), OL and oral cancer (OC) populations. Cohen's d with 95% credible interval was calculated and pooled using the inverse variance heterogeneity method., Results: A total of seven saliva biomarkers were analyzed in this paper, including interleukin-1alpha, interleukin-6 (IL-6), interleukin-6-8, tumor necrosis factor alpha (TNF-α), copper, zinc, and lactate dehydrogenase. IL-6 and TNF-α exhibited statistically significant deviations in comparisons between HC versus OL and OL versus OC. A total of 13 serum biomarkers were analyzed, including IL-6, TNF-α, C-reactive protein, total cholesterol, triglycerides, high-density lipoproteins, low-density lipoproteins, albumin, protein, β2-microglobulin, fucose, lipid-bound sialic acid (LSA), and total sialic acid (TSA). LSA and TSA exhibited statistically significant deviations in comparisons between HC versus OL and OL versus OC., Conclusion: IL-6 and TNF-α in saliva have strong predictive values for OL deterioration, and LSA and TSA concentration levels in serum also have the potential to serve as biomarkers for OL deterioration., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Some oral squamous cell carcinomas (OSCCs) originate from preexisting oral potentially malignant disorders (OPMDs). Oral leukoplakia (OLK) is the most common and typical OPMD in the clinic, so treatment for it is essential to reduce OSCC incidence. Local chemotherapy is an option other than surgery considering the superficial site of OLK. However, there are no standardized drugs applied to OLK, and traditionally used chemotherapeutic drugs revealed limited efficacy for lack of adhesion. Hence, there is a growing demand to prepare new agents that combine mucoadhesion with an anti-OLK effect. Here, an isoguanosine-tannic acid (isoG-TA) supramolecular hydrogel via dynamic borate esters was successfully fabricated based on isoG and TA. Previously reported guanosine-TA (G-TA) hydrogel was also explored for an anti-OLK effect. Both gels not only exhibited ideal adhesive properties but also integrated anti-OLK activities in one system. In vitro cell viability indicated that isoG and TA inhibited the proliferation of dysplastic oral keratinocytes (DOKs). The in vivo OLK model evidence revealed that both gels showed potential to prevent OLK canceration. In addition, the probable anti-DOK mechanisms of isoG and TA were investigated. The results indicated that isoG could bind to adenosine kinase (ADK) and then affected the mammalian target of rapamycin (mTOR) pathway to inhibit DOK proliferation. TA could significantly and continuously reduce reactive oxygen species (ROS) in DOKs through its antioxidant effect. ROS plays an important role in the progression of cell cycle. We proved that the low level of ROS may inhibit DOK proliferation by inducing G 0 /G 1 arrest in the cell cycle. Altogether, this study innovatively fabricated an isoG-TA hydrogel with ideal adhesion, and both isoG and TA showed in vitro inhibition of DOKs. Moreover, both isoG-TA and G-TA hydrogels possessed potential in delaying the malignant transformation of OLK, and the G-TA hydrogel showed a better statistical effect, providing an effective strategy for controlling OLK.

The present study aimed to detect the expression of interleukin-27 (IL-27) in tissues of oral lichen planus (OLP), oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC) and to investigate the possible role of IL-27 in the above diseases and whether it was involved in the onset and development of the tumor. Paraffin tissues from patients with OLP, OLK, and OSCC were collected, and the expression of IL-27 in the above tissues was detected by immunohistochemical (IHC) staining. Parameters were obtained from the images by the Image-Pro Plus (IPP) image analysis software, and statistical analysis was performed using relevant data. The expressions of IL-27 were significantly higher in specimens with OLP, OLK, and OSCC than those in the healthy group. In OLP, the expression of IL-27 was positively correlated with the degree of lymphocyte infiltration and basal cell liquefaction while independent of the clinical type. In OLK, the expression of IL-27 was positively correlated with abnormal epithelial cell proliferation. In OSCC, the expression of IL-27 was correlated with the degree of squamous cell differentiation and was independent of gender, TNM stage, and lymphatic metastasis. The expressions of IL-27 were significantly higher in tissues with severe OLP and OLK than that in the control group, while similar to that in highly differentiated OSCC. The expressions of IL-27 were significantly elevated in tissues with OLP, OLK, and OSCC, suggesting that IL-27 might be involved in the development of these diseases and play a role in the carcinogenesis of oral precancerous lesions., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Our aim was to evaluate the expression of biomarkers, CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin in oral leukoplakias (OL) and to assess their potential for prediction of malignant transformation (MT). We analyzed the expression of CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin by immunohistochemistry in 52 OL, comprised of 41 low-grade (LG) dysplasia and 11 high-grade (HG) cases. Twelve healthy normal tissues (NT) were also included. Univariate and multivariate analysis were performed to evaluate any association with MT. Variable expression among the studied markers was observed, with a significant increase of high expression from NT to LG and HG cases in CD44v6 (p = 0.002), P53 (p = 0.002), P73 (p = 0.043), and podoplanin (p < 0.001). In multivariate analysis, cases with high podoplanin score showed a significant increased risk of MT (HR of 10.148 (95% CI of 1.503−68.532; p = 0.017). Furthermore, podoplanin combined with binary dysplasia grade obtained a HR of 10.238 (95% CI of 2.06−50.889; p = 0.004). To conclude, CD44v6, p53, p73, and podoplanin showed an increasing expression along the natural history of oral carcinogenesis. Podoplanin expression independently or combined with dysplasia grade could be useful predictive markers of MT in OL.

Oral squamous cell carcinoma (OSCC) is often preceded by a white patch on a surface of the mouth, called oral leukoplakia (OL). As accelerated telomere length (TL) shortening in dividing epithelial cells may lead to oncogenic transformation, telomere length measurement could serve as a predictive biomarker in OL. However, due to high variability and lack of a universal reference, there has been a limited translational application. Here, we describe an approach of evaluating TL using paired peripheral blood mononuclear cells (PBMC) as an internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50 male OL patients and 44 male healthy controls (HC). Relative TL was measured by quantitative PCR. TL of each OL or healthy sample was normalized to the paired PBMC sample (TL ratio). In OL patients, the mean TL ratio was significantly smaller not only in the patch but also in distal normal oral tissue, relative to healthy controls without a high-risk oral habit. Dysplasia was frequently associated with a subgroup that showed a normal TL ratio at the patch but significantly smaller TL ratio at a paired normal distal site. Our data suggest that evaluation of TL attrition using a paired PBMC sample eliminates the requirement of external reference DNA, makes data universally comparable and provides a useful marker to define high-risk OL groups for follow-up programs. Larger studies will further validate the approach and its broader application in other premalignant conditions., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Objective: To detect the expression of Bmi-1 in oral leukoplakia (OL) cells and tissues, and analyze its role and clinical significance in the malignant transformation of OL. Methods: Immunohistochemistry was used to evaluate Bmi-1 expression in OL samples from 109 patients (51 males, 58 females, age range: 18-74 years) who were treated in the Department of Stomatology, the Affiliated Wuxi People's Hospital of Nanjing Medical University and the Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, between 1996 and 2018. The correlation between Bmi-1 expression level and clinicopathological parameters and prognosis in patients with OL was analyzed. The mRNA and protein expression levels of Bmi-1 gene in normal oral mucosal epithelial cells, OL cells, oral squamous cell carcinoma (OSCC) cells, OL tissues and paired adjacent normal tissues were detected by real-time PCR and Western blotting. The effects of Bmi-1 on the proliferation, colony formation and apoptosis were investigated by silencing expression of Bmi-1 in OL cell lines Leuk-1. Results: The protein level of Bmi-1 in OL tissue with severe and mild dysplasia was statistically different (6 819±994 vs 4 713±372, P =0.017). The OSCC-free survival rate of OL patients with high Bmi-1 expression was 65.5% (36/55), which was lower than that of OL patients with low Bmi-1 expression (88.9%, 48/54, P =0.003). Multivariate Cox proportional analysis indicated that Bmi-1 expression was the independent predictor for malignant transformation of OL ( HR =2.522, 95% CI : 1.128-5.640, P =0.024). The mRNA level of Bmi-1 in OL specimens was 0.455±0.120, which was higher than that in paired adjacent normal tissues (0.063±0.009, P =0.014). The Bmi-1 mRNA level in malignant transformed OL specimens was (1.405±0.397), which was higher than that in untransformed OL specimens (0.145±0.017, P <0.001). After transfection of Bmi-1-shNC and Bmi-1-shRNA2 adenovirus into OL cell line Leuk-1, there were significant differences in the number of clone formation (824±40 vs 414±38, P =0.002) and apoptosis rate (17.7%±2.3% vs 36.0%±2.0%, P =0.004). Conclusions: The up-regulation of Bmi-1 expression promotes the malignant biological behavior of OL cells. Bmi-1 expression can be used as a predictor for malignant transformation of OL.

Objective: To evaluate the oxidative DNA damage, through 8-hydroxy-2'-deoxyguanosine (8-OHdG), and its repair by base excision repair pathway [Redox factor-1 (Ref-1); X-ray Repair Cross Complementing-1 (XRCC-1)] in different epithelial dysplasia degrees in oral leukoplakia., Design: Forty-four cases of oral leukoplakia and 10 normal oral mucosa were quantified for 8-OHdG, Ref-1, and XRCC-1 through immunohistochemistry., Results: Cytoplasmic 8-OHdG and nuclear XRCC-1 were significantly associated with multiple synchronous lesions (p = 0.048; p = 0.034, respectively). Nuclear Ref-1 was significantly associated with oral leukoplakia on the tongue (p = 0.027). A significantly gradual cytoplasmic 8-OHdG expression increase was observed between normal oral mucosa and epithelial dysplasia (p < 0.05). Nuclear Ref-1 expression was significantly lower (p < 0.01) in non-dysplasia/mild dysplasia, while its cytoplasmic expression was significantly higher in non-dysplasia/mild dysplasia compared to moderate/severe dysplasia and normal oral mucosa (p = 0.03; p < 0.0001, respectively). A significantly higher cytoplasmic XRCC-1 expression was observed in non-dysplasia/mild and moderate/severe dysplasia compared to normal oral mucosa (p < 0.0001; p < 0.0001, respectively). All epithelial dysplasia degrees showed a correlation between nuclear and cytoplasmic expression of these proteins (p < 0.05)., Conclusions: 8-OHdG formation may not play a role in the development of multiple synchronous oral leukoplakias. However, it is related to the severity of the epithelial dysplasia. The subcellular level of Ref-1 implies different roles according to the degree of epithelial dysplasia. Cytoplasmic XRCC-1 expression indicates a possible failure of the DNA repair mechanism and occurs in early morphological stages of epithelial dysplasia., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Oral leukoplakia (OL), a potentially malignant disorder, recurs in 40% of cases after surgical removal. Recurrence is a risk factor for malignant transformation. We aimed to examine the prognostic significance of four biomarkers related to cell proliferation: p53, p63, podoplanin (PDPN) and Ki-67 in predicting recurrence. Formalin-fixed-paraffin-embedded specimens from excised OL (n = 73, 33 recurrent; 40 non-recurrent) were collected in a prospective study. Immunohistochemistry was used to visualise expression of p53, p63, PDPN and Ki-67. Image analysis software was used for quantification of p53-, p63- and Ki-67-expressing cells, while PDPN was analysed visually. The expression of all four proteins were higher in recurrent compared with non-recurrent OL, only expression of p53 was statistically significant. In uni- and multivariable Cox regression analyses of individual markers, expression of p63 was significantly associated with higher recurrence risk (p = 0.047). OL with a combined high expression of both p53 and p63 had a significantly higher risk to recur [Log Rank, p = 0.036; multivariate Cox, HR: 2.48 (1.13-5.44; p = 0.024)]. Combination of p53 and p63 expression may be used as a prognostic biomarker for recurrence of OL., (© 2021. The Author(s).)

Although dysregulation and dysfunction of long noncoding RNAs (lncRNAs) have been implicated in malignant behavior of oral squamous cell carcinoma (OSCC), whether aberrant lncRNAs play a role in the carcinogenesis of oral leukoplakia (OL) as the best-known precursor of OSCC remains undetermined. Differentially expressed lncRNAs in the occurrence and progression of OL were studied by microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We found a novel key lncRNA n386251 that we named LOLA1 (lncRNA oral leukoplakia progressed associated 1) in the OL progression. The results of qRT-PCR revealed that LOLA1 aberrant expression was validated in tissue samples and cell lines from the normal oral mucosa, OL to OSCC. Fluorescent in situ hybridization showed that LOLA1 expression localized predominately at the cytoplasm of Leuk1 cells. Cell function assays showed that LOLA1 significantly influenced cell migration, invasion, and epithelial-mesenchymal transition (EMT) protein expression. Potential mechanism experiments revealed that AKT/GSK-3β signaling was involved in the regulatory mechanism of LOLA1 in OL progression. Remarkably, Kaplan-Meier analysis revealed that LOLA1 overexpression could predict malignant events of OL progression to OSCC. In conclusion, the current study for the first time profiled and validated the key lncRNAs related to OL progression. Importantly, we demonstrated that a novel lncRNA LOLA1 upregulation was associated with OL malignant progression, suggesting LOLA1 may be a predictive biomarker. Moreover, LOLA1 may promote migration, invasion, and EMT process in OL malignant progression via AKT/GSK-3β pathway., (© 2021 Wiley Periodicals LLC.)

MUC1 and MUC4 are two transmembranous proteins, which have been seen to express aberrantly in various human neoplasms and advocated as independent prognostic markers. Till now no extensive studies have been carried out on combined expression of MUC1 and MUC4 in oral leukoplakia and Oral squamous cell carcinoma. This study is an endeavour to evaluate Immunohistochemical coexpression of MUC1 and MUC4 in Oral Leukoplakia and Oral squamous cell carcinoma and furthr establish them as prognostic markers. Immunohistochemical analysis of MUC1 and MUC4 was done on 24 cases of Oral squamous cell carcinoma, 24 cases of leukoplakia and 12 normal oral mucosal tissues. Chi square test and one way ANOVA test were employed for statistical analysis. Normal oral mucosa and leukoplakia group showed higher frequency of negative immunoexpression compared to oral squamous cell carcinoma group. Furthur in Oral squamous cell carcinoma group, higher frequency of double positive coexpression in well and moderately differentiated oral squamous cell carcinoma and single positive coexpression in poorly differentiated oral squamous cell carcinoma was obtained. A definite rise of immunoexpression of MUC1 and MUC4 was observed from normal oral mucosa to leukoplakia to oral squamous cell carcinoma indicative of their contribution as diagnostic and prognostic markers., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Background: DNA damage is accumulated in the cells over time as the result of both exogenous and endogenous factors. The objective of this study was to analyze the immunohistochemical expression of the repair proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC)., Materials and Methods: Paraffin blocks were selected from the archives of the Laboratory of Hospital Clinico Universitario de Santiago de Compostela, Spain. The sample was composed of 16 cases of OL without dysplasia, 14 cases of OL with dysplasia, and 15 cases of OSCC. The patients' clinical data were collected and immunohistochemical analysis was performed for MLH1, MSH2, MRE11, and XRCC1. The data were submitted to the χ2 and the Kruskal-Wallis (P≤0.05) tests., Results: MSH2 was overexpressed in OSCC (P=0.020) and was positive in 100% of patients with OL with dysplasia or OSCC (P=0.019). Positivity for MLH1 was significantly associated with comorbidity (P=0.040), especially in patients who presented with 2 or more pathologies (P=0.028). XRCC1 positivity was also associated with comorbidity (P=0.039). No significant associations were found for the MRE11A expression. Although the simultaneous positivity for the 4 markers was observed in presence of comorbidities (P=0.006)., Conclusions: This study supports the effect of the overexpression of MSH2 protein in samples of OL with dysplasia and OSCC, most notably in patients who present with comorbidities and negativity for OL without dysplasia., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the head and neck with a poor prognosis. Oral cancer development is a multistep process involving carcinogenesis. Though significant advances in cancer immunotherapy over the years, there is lack of evidence for T-cell exhaustion during oral carcinogenesis. Clinical specimens from healthy donors and patients diagnosed with oral leukoplakia (OLK) or OSCC were collected for immunohistochemical staining with PD-L1, CD86, CD8, PD-1 and CTLA-4 antibodies. Meanwhile, chemically induced mouse models of oral carcinogenesis were constructed with 4-nitroquinolone-N-oxide induction. Exhaustion status of T cells was measured by flow cytometry for spleens and by multiplex immunohistochemistry for formalin-fixed paraffin-embedded lesions in multiple stages of oral carcinogenesis. The efficacy of PD-1 blockade with or without cisplatin treatment was evaluated on the mice in precancerous and OSCC stages. We observed higher expression of PD-1 in the human OLK and OSCC tissues compared with the normal, while low expression CTLA-4 in all oral mucosa tissues. Animal experiments showed that the exhausted CD4+ T cells existed much earlier than exhausted CD8+ T cells, and an increased ratio of stem-like exhausted T cells and partially exhausted T cells were detected in the experimental groups. Besides, the expression of immune checkpoint markers (PDCD1, CTLA4 and HAVCR2) was strongly positively correlated with cytokines (IFNG and IL-2). In summary, T-cell exhaustion plays a vital role in oral carcinogenesis, and PD-1 blockade can prevent the progression of oral carcinogenesis., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Tobacco smoking is associated with an increased risk of oral leukoplakia and head and neck cancer. Although it has recently been reported that the establishment of an immunosuppressive microenvironment in oral potentially malignant disorders may lead to malignant transformation, it is unclear whether the microenvironments of oral potentially malignant disorders differ according to smoking status. We examined differences in programmed death-ligand 1 (PD-L1) expression and subepithelial CD163+ TAM and CD8+ cell/lymphocyte counts in the microenvironment of oral leukoplakia of smoking and non-smoking patients and investigated their associations with malignant transformation. Pathology reports and original biopsy request forms from 1995-2015 were retrospectively reviewed. Lesions clinically characterized as white plaques/lesions of the oral mucosa and pathologically diagnosed as oral epithelial dysplasia were included. Immunohistochemistry was performed to evaluate PD-L1 expression and subepithelial CD163+/CD8+ cell counts. The significance of prognostic factors in predicting malignant transformation was determined using Cox regression analysis. Statistical significance was defined as P<0.05. In total, 200 patients with oral leukoplakia were selected. The mean age at diagnosis was higher in non-smoking patients (n = 141; 66.9 years) than in smoking patients (n = 59; 60.5 years). The 5-year cumulative malignant transformation rate was higher in non-smoking patients than in smoking patients (9.3% vs. 3.0%, respectively). Oral leukoplakia was associated with significantly higher PD-L1 expression and increased numbers of subepithelial CD163+ cells in the non-smoking group compared with the smoking group. Non-smoking-related oral leukoplakia with positive PD-L1 expression was associated with a 6.97-fold (95% confidence interval: 2.14-22.7) increased risk of malignant transformation. The microenvironment of oral leukoplakia differed according to smoking status. A combination of smoking status and PD-L1 expression may predict malignant transformation in oral leukoplakia patients. This study highlights the importance of understanding the interaction between smoking and the microenvironment in oral leukoplakia., Competing Interests: The authors have declared that no competing interests exist.

The use of chewing tobacco is a severe risk factor for oral mucosa related diseases including cancer in India as well as USA, although its relationship with Oral Leukoplakia (OL) or related carcinogenicity is still not clear. This work chose two oncogenic pathway proteins- the Epidermal Growth Factor Receptor and the WNT pathway among leukoplakia patients and established their correlation with the individuals' tobacco chewing habit. 89 fresh patients with OL were selected for the work. The samples were classified based on the individual's tobacco chewing habit. The divided samples were then immunostained with antibodies for both of the EGFR as well as WNT pathway proteins. The samples were further classified based on their proliferation status and the expression of these oncoproteins was also observed. In order to compare the cytological data with histological data, 30 OL patients undergoing biopsy were chosen and immunohistological analysis was performed for the same pathways. Results showed overexpressing EGFR and WNT pathway proteins in all OL samples. Structurally atypic cells had a tendency to overexpress these oncoproteins. However the immunocytochemistry data could not confirm any positive effect of chewing tobacco on the OL's proliferative state. Statistical data from the immunfluorescence finally revealed the overexpression of both EGFR and WNT pathway proteins on the proliferative population establishing chewing tobacco as a positive risk factor for the onset of OL. Data from biopsy samples followed the same trend of protein expression seen in the cytological samples. Dysplastic zones showed huge overexpression of EGFR and WNT pathway proteins among tobacco chewers. In conclusion, this is the first time report showing the effect of chewing tobacco on the EGFR and WNT pathway in OL and its possible role as a potential risk factor for its proliferative type., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Oral squamous cell carcinoma (OSCC) usually originates from oral potentially malignant disorders (OPMD), such as oral leukoplakia (OLK) and oral lichen planus (OLP). Identifying biomarkers for the early diagnosis and evaluation of malignant transformation in OPMD could improve the survival rate of OSCC patients., Objective: The present study aimed to screen for potential salivary biomarkers for evaluating the malignant transformation of OPMD., Methods: Salivary proteases from OLK and OSCC patients or healthy donors and proteases in cultural medium from DOK and Cal-27 cells were detected with a human protease array kit. The concentrations of the salivary Kallikrein 5 (KLK5) and urokinase-type plasminogen activator (uPA) proteases were measured by ELISA. Receiver operating characteristics (ROC) to determine the potential value of these proteases in clinical diagnosis were calculated using SPSS software. Immunohistochemistry was used to detect the KLK5 and uPA expression in the oral organizations., Results: The salivary protease spectrum was different among patients with OLK and OSCC and healthy donors. KLK5 and uPA levels in saliva tended to increase as the disease progressed (healthy < OPMD [OLK and OLP] < OSCC). ROC curves showed the optimum diagnostic cutoffs for KLK5 as a biomarker for OLK, OLP, and OSCC were 5.97, 6.03, and 9.45 pg/mL, respectively, while the cutoffs for uPA were 17.19, 17.26, and 20.96 pg/mL. Their combined analysis showed a higher sensitivity for the differential diagnosis of disease. Furthermore, higher levels of KLK5 and uPA were observed in OSCC tissues than in OLK and OLP., Conclusions: Salivary KLK5 and uPA are potential biomarkers for evaluating OLK and OLP malignant transformation and early diagnosis of OSCC.

Background: Stathmin is an intracellular phosphoprotein that controls the microtubule dynamics by further regulating proper attachment and alignment of chromosomes in a dividing cell. Thus, any mutation or aberrantly expressed protein that reduces the fidelity of spindle assembly will enhance chromosomal instability contributing to aneuploidy. Oral Squamous Cell Carcinoma is an extensively studied malignancy that occurs due to accumulated genetic changes due to carcinogens. The current study is done to evaluate the stathmin role and its expression in OSCC and Oral epithelial dysplasia (OED)., Objective: The aim of the present study is to evaluate the role of stathmin in OSCC and Oral dysplasia and also to correlate the expression of Stathmin with respect to the different histopathological grades of OED and OSCC., Materials and Methods: 30 neutral buffered formalin fixed, paraffin embedded (FFPE) tissues of Oral Leukoplakia/OED and 30 FFPE tissues of OSCC were subjected to immunohistochemistry with stathmin antibody. Five fields of each case with 300 cells were examined and a mean percentage of positive-stained slides were determined. The percentages were recorded accordingly with their respective histological grades. The results were analysed statistically., Results: The results of the present study demonstrated higher mean values of stathmin in tissues with OSCC (2.50) compared to leukoplakia (2.11) and normal tissues (0.00) with a high level of statistical significance (0.0001). There is also an increase in the percentage levels of stathmin with increase in the histological grade of differentiation in OSCC as well as leukoplakia., Conclusion: The present study found a statistical correlation between increased grades of the disease with expression levels of stathmin. This confirms that stathmin expression can contribute to disease progression and that stathmin might have a potential role as an early diagnostic biomarker and can be a therapeutic target for OSCC. , .

Saliva is a highly versatile biological fluid that is easy to gather in a non-invasive manner-and the results of its analysis complement clinical and histopathological findings in the diagnosis of multiple diseases. The objective of this review was to offer an update on the contribution of salivary biomarkers to the diagnosis and prognosis of diseases of the oral cavity, including oral lichen planus, periodontitis, Sjögren's syndrome, oral leukoplakia, peri-implantitis, and medication-related osteonecrosis of the jaw. Salivary biomarkers such as interleukins, growth factors, enzymes, and other biomolecules have proven useful in the diagnosis and follow-up of these diseases, facilitating the early evaluation of malignization risk and the monitoring of disease progression and response to treatment. However, further studies are required to identify new biomarkers and verify their reported role in the diagnosis and/or prognosis of oral diseases.

Various endogenous mitochondria-associated antioxidants protect mitochondria from oxidative stresses such as Superoxide Dismutase 2 (SOD2), Catalase, Glutaredoxin 2 (GLRX2), educed Glutathione (GSH), Glutathione Peroxidase (GPx), and Thioredoxin 2 (TXN2). They protect mitochondria from Reactive Oxygen Species (ROS). Excess ROS causes mitochondrial DNA damage and respiratory chain dysfunction leading to carcinogenesis. In an earlier study we found complex alterations of mitochondrial antioxidants in different stages of oral squamous cell carcinoma. Here, we profiled mitochondrial antioxidants in different oral potentially malignant disorders such as oral leukoplakia (OL), oral lichen planus (OLP), and oral submucous fibrosis (OSMF). Mitochondria was prepared from oral tissues from OL, OLP, and OSMF patients. Biochemical assays and immunoblotting were performed to investigate expression of various mitochondrial antioxidants. Catalase was expressed as Control> OL > OSMF > OLP. GLRX 2 was expressed as OLP> OL >Control >OSMF. GPX1 and GPX4 were expressed as Control >OSMF> OLP > OL. GSH increased in OL and OSMP, but decreased in OLP. SOD2 was expressed as Control >OSMF> OLP > OL. PRX3 was expressed as OL > OLP > OSMF > Control. TXN2 expression was nearly the same in all groups except OL, which showed elevated expression. We conclude that endogenous mitochondria-associated antioxidants show different levels of expressions in various oral potentially malignant disorders.

Objective: To evaluate the prevalence of HPV DNA detection in fresh tissue from oral leukoplakia by Linear Array assay, and its correlation with p16 INK4a immunoexpression in the northwest region of the São Paulo state, Brazil., Patients and Methods: Fifty patients diagnosed with oral leukoplakia were included in the study. Sociodemographic, clinicopathologic and lifestyle data, fresh tissue and formalin fixed paraffin embedded (FFPE) tissue samples were collected. The fresh tissue was stored at -80 °C and then submitted to further viral DNA detection by the Linear Array method. Immunohistochemical analysis for the p16 INK4a expression was performed., Results: Of the 50 patients included in the study, 62 % were men, and the age ranged from 25 to 82 years. Twenty-three (46 %) were elderly, 46 % were middle-aged adults, and only 12 % were young adults. Most patients were smokers (76 %), 14 % were former smokers, and 10 % were non-smokers. Most patients (56 %) were current drinkers, 22 % were ex-drinkers and 22 % were non-drinkers. Thirty-two percent of the lesions presented some degree of dysplasia. No lesions were positive to HPV by Linear Array detection. Thirty (60 %) OL had p16-low immunoexpression and 20 (40 %) had p16-high immunoexpression., Conclusion: HPV was not identified in the population studied. The high p16 INK4a immunoexpression is not dependent on HPV in oral leukoplakia. Broader epidemiological studies are required to clarify the geographic variability in the prevalence of HPV in head and neck squamous cell carcinoma and oral potentially malignant lesions., Competing Interests: Declaration of Competing Interest The authors declare that there is no potential conflict of interest regarding this work., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Screening for oral squamous cell carcinomas (OSCCs) and oral premalignant lesions may decrease the devastating morbidity and mortality associated with the disease. This has led to widespread research for the identification of molecular-based biomarkers. Among them, survivin is a recently characterized protein which is a member of the inhibitor of apoptosis family. The aim of this study is evaluating the expression of survivin in oral leukoplakia, oral lichen planus, and OSCC compared with normal mucosa., Materials and Methods: The retrospective study consisted of twenty cases of oral leukoplakia, oral lichen planus, and OSCC in the age group of 20-70 years. Twenty cases of normal mucosa made up the control group. Immunohistochemical staining was performed with the use of survivin polyclonal antibody. Grades of expression of survivin were evaluated. Kruskal-Wallis test was used for statistical analysis., Results: The expression of survivin was higher in OSCC (80%) when compared to oral leukoplakia (70%), oral lichen planus (45%), and normal mucosa (35%). The variation in the expression of survivin between the samples was statistically significant with P = 0.015 (Kruskal-Wallis test significant at 0.01 level)., Conclusion: It is concluded that survivin can be identified as a useful tool for the identification of potentially malignant disorders at higher risk for progression into invasive carcinoma., Competing Interests: None

Dysplastic leukoplakia (LP) of the oral cavity is a potentially malignant condition for oral squamous cell carcinoma (OSCC), early detection of which remains an unmet clinical need. In an effort to develop non-invasive biomarker based method for early detection of the disease, differential proteomic profiling was carried out with the saliva from patients with risk habits and diagnosed with LP and those with lymph node negative and positive OSCC in comparison to healthy controls with risk habits. Ninety three proteins were observed at elevated level (≥1.5 fold), and 30 were prioritized based on a scoring system comprising of confidence of identification, presence in the various specimen groups, functional relevance, and their secretory potential. Verification was carried out in independent patient cohorts for 8 selected, representative, upregulated proteins using ELISA. Three of them CD44, S100A7, and S100P were significantly altered in patients with LP as well as OSCC and can be regarded as a panel of biomarker candidates for early detection of the malignancy. Other members may also be investigated in a targeted manner to expand the portfolio of biomarkers for early detection. The mass spectrometry data are available via ProteomeXchange with identifier PXD015722. SIGNIFICANCE: There is an unmet clinical need for non-invasive, biomarker based methods for the improved early detection and the subsequent management of oral cancer. The study represents differential proteome profiling of the saliva of patients with oral dysplastic leukoplakia (LP) - a potentially malignant lesion, patients diagnosed with oral squamous cell carcinoma (OSCC), and healthy controls to identify potential markers for the purpose of early detection of malignancy. From among the matched and prioritized proteins with elevated levels in the saliva of patients with LP and those with OSCC, eight were verified. Three of them - CD44, S100A7 and S100P appeared promising candidates as biomarkers for early detection of the neoplastic predisposition and may form the basis of clinical assays for this purpose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: Enormous attempts have been made to develop and establish markers that determines the susceptibility of potentially malignant tissues to transform to oral cancer. E - cadherin encoded by CDH1 gene is a protein which plays an important role in cellular adhesion. This study aimed to assess the relationship between the expression of E- cadherin and different grades of epithelial dysplasia in oral leukoplakia., Materials and Methods: Tumour biopsies from fifty leukoplakia patients was collected. Half of the tissue was sent for histopathological examination and other half was subjected to see E - cadherin expression by real time PCR., Results: On assessing, the expression of E - cadherin was found to be high in samples with mild dysplasia followed by samples with moderate dysplasia. Samples with severe dysplastic feature showed least expression of E - cadherin. All statistical analyses were performed using Statistical Package for Social science (SPSS) and was proven that there is significant decrease in the expression of E - cadherin as the degree of dysplasia increases with a p value 0.001 and confidence interval 95%., Conclusion: We conclude that loss of E - cadherin can be used as a tumour marker that could determine the susceptibility of normal and potentially malignant tissues to transform into oral cancers. To generalise our results, further prospective studies with a large sample size using quantitative real time PCR to read the gene expression should be carried out at multi centre levels., .

Proliferative verrucous leukoplakia (PVL) is a premalignant condition of the oral mucosa with > 70% chance of progression to squamous cell carcinoma (SCC), while lacking the common risks and behavior seen in non-PVL oral squamous carcinogenesis. PVL follows a multi-stage slow, relentless and usually multifocal expansion of surface epithelial thickening that over time takes on a verrucous architecture, eventually leading to verrucous carcinoma and/or dysplasia followed by "conventional" SCC, a process that takes years and is notoriously difficult to manage. As mucosal surfaces and carcinomas arising at these sites, are colonized by microorganisms, host receptors for microbial products have received attention as potential contributors to carcinogenesis. Studies show that microbial pattern recognition toll-like receptor (TLR)2 in various epithelial cells is upregulated in premalignant lesions and in malignant cells and can activate oncogenic pathways. Because of the highly progressive nature of PVL, we examined TLR2 expression in well-characterized PVL samples by immunohistochemistry. We found that, similar to epithelial dysplasia and SCC, PVL keratinocytes throughout the epithelial thickness showed diffuse TLR2 expression even in early stage lesions prior to onset of dysplasia. In contrast, oral mucosal samples in the absence of hyperorothokeratosis or dysplasia, expressed TLR2 primarily in the basal and parabasal layers. Given the high rates of PVL transformation and the previously established pro-cancer role of high TLR2 expression in malignant oral squamous cells, it is important to determine how its' expression and functions are regulated in the oral squamous epithelium, and what is the specific TLR2 role in carcinogenesis.

Objective: The study aimed to evaluate total antioxidant capacity as well as levels of various enzymatic and non-enzymatic antioxidants, and oxidative stress markers in saliva of patients with oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL). Material and methods: Twenty patients with OSCC, 20 patients with OL and 20 healthy subjects were enrolled into this prospective study. Total Antioxidant Capacity (TAC), activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) as well as levels of total glutathione (tGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, uric acid (UA), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) were estimated in saliva using appropriate biochemical methods. Results: The activity of SOD was significantly higher in OSCC group in comparison with OL and control groups. The levels of GSH were markedly lower in OSCC and OL patients as compared to the control group. Likewise, we found that GSH/GSSG ratio was markedly lower in the OSCC and OL groups. Levels of some biomarkers were influenced by clinical staging of OSCC and OL as well as by sociodemographic factors. Conclusions: The results of this pilot study suggest that salivary activity of SOD is higher in OSCC patients, whereas levels of GSH and GSH/GSSG ratio are lower in saliva of patients with OSCC and OL. Clinical staging of OSCC and OL, as well as some sociodemographic factors may also influence salivary antioxidant status.

Background: Oral cancer is one of the life threatening disease which requires an availability of a biomarker for its early detection and also for effective treatment strategies. The current study is done to evaluate the efficacy of one such biomarker i.e. TNF- α as an indicator for oral precancer and oral cancer. Objectives: To evaluate the efficacy of Tumour necrosis factor - alpha (TNF)-α as a salivary biomarker in histopathologically diagnosed cases of oral leukoplakia and Oral squamous cell carcinoma. To correlate the levels of TNF- α with varying histologic grading in Oral Squamous Cell Carcinoma and dysplasia grading in Oral leukoplakia or Hyperkeratosis. Materials and Methods: The study group included 90 subjects that were divided into three groups. OSCC (n=30), leukoplakia (n=30) and controls (n=30). Cases were selected based on inclusion and exclusion criteria of the study. Salivary samples were then collected from all three groups. Salivary levels of TNF-α were estimated using Enzyme Linked Immunosorbent Assay (ELISA). The data on concentration gradients obtained were subjected to appropriate statistical analysis. Results: The results of the present study demonstrated higher levels of salivary TNF-α in individuals with OSCC compared to leukoplakia and healthy control subjects with a high level of statistical significance. ROC curve analysis along with diagnostic parameter calculation also revealed that salivary TNF-α to be a better medium for detecting OSCC. There is also an increase in the salivary TNF-α levels with increase in the histological grade of differentiation in OSCC as well as leukoplakia. Conclusion: The present study concludes that salivary TNF – α can be used as a prognostic biomarker of OSCC. In view of the elevated levels of TNF – α in saliva of individuals with severe dysplasia, it can also be used to monitor the malignant transformation to leukoplakia to OSCC.

Background: Metabolomics is the study of metabolome which describes the full repertoire of small molecules, and the analysis of salivary metabolomics may help in identifying tumor-specific biomarkers for early diagnosis and prediction of tumor progression. The aim of the study was to evaluate the clinical utility of salivary metabolites in oral leukoplakia and oral squamous cell carcinoma., Methods: Salivary metabolomic profile of patients diagnosed with oral leukoplakia (n = 21) and oral squamous cell carcinoma (n = 22) was compared with apparently normal controls (n = 18) using Q-TOF-liquid chromatography-mass spectrometry. MassHunter profile software and Metlin database were used for metabolite identification. ANOVA to identify the regulation of metabolites between the three groups, t test (P < 0.05) to signify the changes between two groups, and chi-square test (P < 0.05) to indicate the presence or absence of metabolites in the study participants of the three groups were performed., Results: Significant upregulation of 1-methylhistidine, inositol 1,3,4-triphosphate, d-glycerate-2-phosphate, 4-nitroquinoline-1-oxide, 2-oxoarginine, norcocaine nitroxide, sphinganine-1-phosphate, and pseudouridine in oral leukoplakia and OSCC was noted. Downregulated compounds in the diseased groups included l-homocysteic acid, ubiquinone, neuraminic acid, and estradiol valerate., Conclusion: A range of salivary metabolites were significantly altered in oral leukoplakia and oral squamous cell carcinoma. Further, it is necessary to evaluate the clinical utility of the individual metabolites in preventing malignant transformation of oral leukoplakia and to improve prognosis of oral squamous cell carcinoma., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The aim of this prospective work was to examine oral leukoplakia for their podoplanin expression to determine whether podoplanin expression is associated with the degree of dysplasia., Materials and Methods: We took biopsy samples from 50 patients with oral leukoplakia in 2013. The preparations studied by immunohistochemistry were analyzed in correlation with the degree of dysplasia and other clinicopathological variables., Results: The Chi-square test showed a significant correlation between podoplanin expression and the degree of dysplasia according to the squamous intraepithelial neoplasia (SIN) classification (p = 0.033). Also, a significant association between age grouping and podoplanin expression was found. We were able to show that the distribution is the same for both age groups in relation to the score of podoplanin expression (p = 0.003)., Conclusion: In a comparable retrospective work of our working group, it could be shown that podoplanin is a reliable predictive marker for the assessment of the risk of malignant transformation. The present work was able to substantiate the assumption that podoplanin not only plays an important role in the context of malignant degeneration but also exerts a major influence in advance., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Oral leukoplakia (OL) is a potentially malignant oral mucosal disorder. A casual association between OL, oral squamous cell carcinoma (OSCC) and human papillomavirus (HPV) infection has been suggested, but no conclusive evidence has been presented. p16, a tumour-suppressor protein, is used as a surrogate marker for HPV infection. The aim of this study was to investigate how overexpression of p16 correlates with HPV infection in OL and in OSCC., Patients and Methods: Seventy-four patients with OL and 13 with OSCC with p16 overexpressed, were analyzed by immunohistochemistry visualizing p16 and a real-time polymerase chain reaction (PCR) assay targeting HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 56, 58 and 59., Results: Overexpression of p16 was observed in 18% of patients with OL. None of the HPV subtypes were detected by PCR analysis in patients with OL. In the p16-positive OSCC specimens, 38% were also HPV16-positive., Conclusion: Overexpression of p16 was not found to be a reliable biomarker for HPV infection in patients with OL and OSCC., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

PIWIL2 is a human Argonaute protein, which is guided by small RNAs to its targets, plays a role in germ cell maintenance and has been proposed to be expressed in precancerous stem cells and tumor stem cells. However, the significance of PIWIL2 expression in oral cancer and precancerous lesions has not been investigated. In this study, we analyzed the expression of the stem cell protein PIWIL2 in oral squamous-cell carcinoma (OSCC) and in premalignant oral leukoplakia (OL) with predominant expression in malignant and premalignant tissues. In the evaluated patients, we found that PIWIL2 was associated significantly with OSCC prognosis and OL. Furthermore, PIWIL2 was found to be expressed in tumor epithelial cells and macrophages in the tumor microenvironment, which are not derived from enlarged lymph nodes. Cytological experiments confirmed that the human squamous cell carcinoma cell line SCC-25, can promote the PIWIL2 and Nanog level in THP-1 cells, which are extensively used to study the modulation of monocytes and macrophages. Our findings showed that PIWIL2 can predict effectively OSCC prognosis and OL with a high risk of OSCC development and substantiate the deduction that cancer stem(-like) cells in oral cancer have the ability to reconstitute the heterogeneity of the bulk tumor and contribute to poor outcome and immunosuppression.

Objective: This study aimed to investigate the expression and relationship of microtubule-associated protein 1 light chain 3 (LC3) and mammalian target of rapamycin (mTOR) in normal oral mucosa, oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC). This work also analyzed the relationship between expression levels and clinical factors. This study evaluated the clinical value of LC3B and mTOR as indices to determine the carcinogenic potential of OLK., Methods: Immunohistochemistry was used to detect the expression of LC3B and mTOR in 20 cases of normal oral mucosa, 120 cases of OLK, and 30 cases of OSCC. The clinical data of 120 patients with OLK were analyzed. The relationships between expression levels and clinical factors were investigated., Results: In normal oral mucosa, OLK and OSCC, the positive rates of LC3B expression were 85.0%, 65.8% and 33.3% (P<0.05), whereas the positive rates of mTOR expression were 20.0%, 48.3% and 76.7% (P<0.05). The expression levels of LC3B and mTOR were correlated and related to clinical typing of OLK (P<0.05)., Conclusions: LC3B and mTOR can be used as molecular biomarkers for early detection of OLK.

Objective: This study was conducted to correlate and compare the immunoexpression of sex-determining region Y-box 2 (SOX-2) in oral leukoplakia (OL) lesions with that in normal buccal mucosa (control)., Materials and Methods: In this observational study, OL with low-risk (n = 34) and high-risk (n = 33) dysplasia and control samples (n = 25) were subjected to immunohistochemical analysis for SOX-2. In the epithelium, SOX-2 positive and negative cells, as well as semiautomatic segmentation of the immunopositive nuclear area were counted. Statistical tests included chi-square, one-way analysis of variance, Tukey, and Games-Howell. The level of significance was 5%., Results: Groups with OL lesions (low and high-risk) showed higher mean numbers of SOX-2 positive cells (63.47 ± 25.70 and 68.18 ± 21.17) compared to the control group (45.85 ± 27.38) (p = 0.00). Groups with OL lesions (low and high-risk) exhibited higher mean positive nuclear area (0.24 ± 0.47 and 1.09 ± 2.06) compared to the control group (0.00 ± 0.01) (p = 0.01)., Conclusion: Oral leukoplakia lesions showed a higher expression of SOX-2, suggesting its contribution to the pathogenesis of OL., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Introduction: Oral leukoplakia is defined as a mucous membrane disorder characterized by white patches that cannot be scraped off. Leukoplakia is the most frequent, potentially premalignant oral mucosa disorder and a good candidate for chemopreventive therapies. Pioglitazone activates peroxisome proliferator-activated receptor gamma (PPARγ), which forms a complex with nuclear cofactors and regulates gene expression of a variety of cell-cycle proteins and is currently being tested preclinically and clinically in aerodigestive cancer prevention., Methods: In the present study, we hypothesized that pioglitazone would decrease proliferation of human leukoplakia cells (MSK Leuk1) and transformed bronchial epithelial cells (BEAS-2B) through regulatory changes of G1 checkpoint protein regulators, p21 and cyclin-D1. MSK Leuk1 and BEAS-2B cells were treated with pioglitazone and assayed for cell proliferation and p21 transcriptional activity., Results: We discovered pioglitazone significantly inhibited cell proliferation in a dose-dependent fashion. We also observed p21 protein induction after treatment with pioglitazone, which was preceded by measurable increases in p21 mRNA induction., Conclusions: We conclude the PPARγ activator, pioglitazone, can activate p21, which is associated with decreased proliferation in 2 aerodigestive preneoplastic cell lines. In addition, the p21 gene may be a potential hypothesis-driven biomarker in translational studies of pioglitazone as a chemoprevention agent for aerodigestive cancer.

Notch1 signaling is essential for tissue development and tumor progression. This signaling pathway has also been implicated in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). However, the role of Notch1 expression in OL and its malignant transformation is unknown. This study aimed to examine the Notch1 expression patterns by immunohistochemistry (IHC) in a cohort of 78 Chinese patients with OL and to analyze the relationship between the patterns and progression of OL to OSCC. Strong Notch1 staining was observed in 10 (13%) of the 78 OL patients, but it was not associated with any of the clinicopathological parameters. However, we observed membranous Notch1 expression in 24 (31%) of the OL samples. Membranous Notch1 expression was significantly associated with the severity of dysplasia (P<0.001) and development of OSCC (P=0.003). By multivariate analysis, membranous Notch1 expression was found to be the only independent factor for OSCC development in the patient population (P=0.019). Among the 24 patients with membranous Notch1 expression, 11 (46%) developed OSCC compared to 8 (15%) of the 54 patients without such expression (P=0.001, determined by log‑rank test). Furthermore, we established a 4‑nitroquinoline‑1‑oxide (4NQO)‑induced murine OSCC model and studied the Notch1 expression patterns in different stages of carcinogenesis. We observed that the extent of expression of membranous Notch1 increased during carcinogenesis. These data indicated a relationship between membranous Notch1 expression and OSCC risk in patients with OL and suggested that membranous Notch1 served as a biomarker for assessing OSCC risk.

Purpose: Recent studies have emphasized the role of podoplanin in oral lesions at risk of malignant transformation. We investigated a group of oral leukoplakias (OLs) to determine a possible relation between altered podoplanin expression and dysplasia, and to compare the results with those obtained by other, widely used biomarkers., Materials and Methods: The population consisted of 40 consecutive patients with a clinical and histological diagnosis of OL. Thirty-two OLs did not show dysplasia, whereas eight lesions presented with dysplasia. Immunohistochemical expression of podoplanin, p53 and Ki67 was analyzed in all samples., Results: All three biomarkers were positive in seven of eight dysplastic OLs. Among the 32 OLs without dysplasia, Ki67 and p53 showed positive values in 21 and 10 samples respectively, whereas podoplanin was positive in only one case. Multiple logistic regression showed that podoplanin was the most powerful variable (Chi square 9.77; p < .01) statistically related to the presence of dysplasia. In addition, podoplanin showed a higher specificity value (96.87%) than Ki67 (34.37%) and p53 (68.75%)., Conclusion: Podoplanin seems to be a reliable means of discriminating lesions with epithelial dysplasia and could be introduced in routine practice as a marker to discriminate OLs at risk of developing cancer., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Objectives: Picrosirius red and MMP are capable of degrading extracellular matrix proteins, expressed in lesions such as squamous cell carcinomas. The present study was undertaken with an aim to analyze and compare changes in collagen using Picrosirius red staining under polarizing microscopy and immunohistochemical staining using anti MMP-13 in samples of oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. Materials and Methods: A total of 70 slides were prepared and divided into 3 groups. Group I comprised 10 slides of normal gingival tissue, Group II 40 slides of potentially malignant disorders and Group III 20 slides of well differentiated oral squamous cell carcinoma. Half the slides for each group were stained with Picrosirius red stain and the remainder with antibodies to MMP-13. Rerults: In Group II, MMP-13 connective tissue expression was greater in OSMF as compared to leukoplakia. Group III showed elevated expression among 70% of cases. Picrosirius red staining in Group II cases, showed higher staining Yellow-Orange andGreen-Yellow mature fibers in OSMF than leukoplakia cases while in Group III, 50% OSCC cases showed Green-yellow stained immature thin fibers. Conclusion: In future, therapeutic measures targeted against MMP-13 may inhibit collagenolysis to some extent and delay spread of tumors. An easy and reliable method to determine the state of the stroma in such cases may be Picrosirius red staining with polarizing microscopy., (Creative Commons Attribution License)

Objective: To compare the methylation profiles in tissues of oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC) with healthy tissues of oral mucosa, in order to identify the role of DNA methylation played in tumorigenesis. Methods: DNA samples extracted from tissues of 4 healthy oral mucosa, 4 OSCC and 4 OLK collected from patients of the Department of Oral Medicine, Capital Medical University School of Stomatology were examined and compared using Methylation 450 Bead Chip. The genes associated with differentially methylated CpG sites were selected for gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. Results: Multiple differentially methylated CpG sites were identified by using the above mentioned assay. Hypermethylation constitutes 86.18% (23 290/27 025) of methylation changes in OLK and hypomethylation accounts for 13.82% (3 734/27 025) of methylation changes. Both hypermethylated and hypomethylated CpG sites were markedly increased in OSCC tissue compared with OLK tissue. The majority of differentially methylated CpG sites were located outside CpG islands, with approximately one-fourth in CpG shores flanking the islands, which were considered highly important for gene regulation and tumorigenesis. Pathway analysis revealed that differentially methylated CpG sites in both OLK and OSCC patients shared the same pathway enrichments, most of which were correlated with carcinogenesis and cancer progression (e.g., DNA repair, cell cycle, and apoptosis). Conclusions: In the present study, methylation-associated alterations affect almost all pathways in the cellular network in both OLK and OSCC. OLK and OSCC shared similar methylation changes whether in pathways or genes, indicating that epigenetically they might have the same molecular basis for disease progression.

Background: We have previously shown that gene expression profiles of oral leukoplakia (OL) may improve the prediction of oral cancer (OC) risk. To identify new targets for prevention, we performed a systematic survey of transcripts associated with an increased risk of oral cancer and overexpressed in OC vs normal mucosa (NM)., Methods: We used gene expression profiles of 86 patients with OL and available outcomes from a chemoprevention trial of OC and NM. MET expression was evaluated using immunohistochemistry in 120 OL patients, and its association with OC development was tested in multivariable analysis. Sensitivity to pharmacological Met inhibition was tested invitro in premalignant and OC cell lines (n = 33) and invivo using the 4-NQO model of oral chemoprevention (n = 20 mice per group). All statistical tests were two-sided., Results: The overlap of 693 transcripts associated with an increased risk of OC with 163 transcripts overexpressed in OC compared with NM led to the identification of 23 overlapping transcripts, including MET. MET overexpression in OL was associated with a hazard ratio of 3.84 (95% confidence interval = 1.59 to 9.27, P = .003) of developing OC. Met activation was found in OC and preneoplastic cell lines. Crizotinib activity in preneoplastic and OC cell lines was comparable. ARQ 197 was more active in preneoplastic compared with OC cell lines. In the 4-NQO model, squamous cell carcinoma, dysplasia, and hyperkeratosis were observed in 75.0%, 15.0%, and 10.0% in the control group, and in 25.0%, 70.0%, and 5.0% in the crizotinib group (P < .001)., Conclusion: Together, these data suggest that MET activation may represent an early driver in oral premalignancy and a target for chemoprevention of OC.

Oral leukoplakia is the most common premalignant lesion of the oral cavity and is associated with development of oral squamous cell carcinoma. Certain changes at cellular and molecular level are important indicators for transformation into carcinoma. Podoplanin, a transmembrane glycoprotein is involved in the cytoskeletal remodeling and increased motility of the cell which helps in determining the malignant potential of oral leukoplakic lesions. The objective of the study was to determine immunohistochemically the expression of podoplanin in homogenous (HOL) and nonhomogenous oral leukoplakia (NHOL) and to compare the expression with clinicopathological parameters. Study group included 15 cases each of HOL, NHOL and control group included 15 healthy volunteers. Both tissues were immunohistochemically stained for podoplanin (D2-40) antibody. No statistical significant difference was observed between the study and control groups for expression of podoplanin but significant difference was observed on comparison of podoplanin scores between HOL and NHOL. Statistical significant difference was observed when the podoplanin expression in the epithelium and the lymphatic vessel density were correlated with the histologic grading of HOL and NHOL. Expression of podoplanin was greater in NHOL as compared with HOL, this supports the fact that NHOL has a greater risk of malignant transformation when compared with HOL. Podoplanin expression, lymphangiogenesis, and lymphatic vessel density increased with increasing grades of dysplasia, suggesting that cellular modeling and motility is increased as the grade of dysplasia advances. Thus suggesting podoplanin can be used as a prognostic marker to determine the malignant potential in oral leukoplakias.

Introduction and Aims: The possible reason suggested for epithelial atrophy in oral submucous fibrosis (OSMF) is ischemia. Dysregulation in the epithelial proliferation and maturation is also thought to be a cause. The β1 integrin identifies the oral epithelial stem cells. The changes induced by the arecanut on these cells may result in epithelial alterations. The aim of this study is to evaluate the stem cells distribution and percentage by assessing the β1 integrin expression., Materials and Methods: The study included normal oral mucosa (15 cases) and disease group (97 cases). The disease group was further subdivided into early (29 cases), moderate (34 cases), advanced OSMF (18 cases) and oral squamous cell carcinoma(OSCC) associated with OSMF (16 cases). The tissues were stained for β1 integrin antibodies. The positive cells and staining intensities were analysed to determine the staining index, and statistically evaluated using KW test statistics., Results: β1 integrin was observed in retepegs region and the percentage of positive cells was 14%- 30% in the control. In OSMF, the β1 integrin positivity was observed in basal and suprabasal layers, and the percentage was ranged from 2%-71%. β1 integrin expression in OSCC was observed both in central and peripheral cells and ranged from 17%-85%. On comparison, the difference in staining index among normal, OSMF and carcinomas was significant at p<0.01. The stem cells percentage was increased both in OSMF and carcinomas. The non-dysplastic epithelium of OSMF with severe atrophy showed lowest percentage. It is inferred that absence of stem cells and proliferation may attribute for the atrophy., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Objective: To assess the level of maturation and proliferation of epithelial cells and the correlation with immunocytochemical expression of adhesion (E-cadherin) and cell differentiation (involucrin) markers., Methods: Cytopathological samples were obtained from four groups of patients: control (CG, n=30); alcohol/tobacco (ATG, n=31), leucoplakia (LG, n=31), and squamous cell carcinoma (SCCG, n=22). Cytopathological smears were collected from all groups for AgNOR, Papanicolaou and immunocytochemical staining., Results: There was an increase in anucleated cells in ATG compared to CG and in LG compared to lesion-free groups (P<.05). In addition, there was a higher rate of intermediate cells in lesion-free groups than in LG (P=.001). When these findings were correlated with positive E-cadherin expression, there was a smaller number of anucleated and intermediate cells (P<.05). The proliferation rate was higher in the SCCG than in the CG (P<.05) and in the ATG compared to LG (P<.05). Moreover, cell proliferation increased in the presence of positive E-cadherin expression in the ATG and LG. No statistically significant results were obtained for involucrin analysis., Conclusion: Cytopathology combined with quantitative techniques such as Papanicolaou, AgNOR, and immunocytochemical expression of E-cadherin detects changes associated with oral carcinogenesis. The innovative approach used in this study allows assessing the expression of cell adhesion (E-cadherin) and differentiation (involucrin) markers by means of oral mucosal cytopathology. The E-cadherin imunocytochemical expression indicated changes associated with the oral carcinogenesis process. An increase in cell proliferation rate in oral squamous cell carcinoma group was associated with the lower immunoexpression of E-cadherin. Cytopathology combined with quantitative techniques and immunocytochemical expression of E-cadherin may detect early alterations associated with oral carcinogenesis., (© 2017 John Wiley & Sons Ltd.)