Serum and salivary interleukin-1ß level in oral precancer: An observational study.

BACKGROUND AND AIM: Precancer biomarkers help in early detection and management of oral potentially malignant disorders (OPMDs). Interleukin-1ß (IL-1ß), 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-1ß 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-1ß 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-1ß levels were significantly (P 0.001) lesser in Group I in comparison to Group II. The salivary IL-1ß levels remained insignificant between both the groups. However, in both the groups, the salivary IL-1ß levels were significantly higher compared to the serum IL-1ß levels. CONCLUSION: We found that the serum IL-1ß level can be considered as a prospective biomarker for dysplasia, whereas salivary IL-1ß alone needs more elaborated studies to account for its application as a potential biomarker in OPMD.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence.

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 H2O2. 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 H2O2-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.

Unraveling the Keratin Expression in Oral Leukoplakia: A Scoping Review.

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.

Induction of Peroxiredoxin 1 by Hypoxia Promotes Cellular Autophagy and Cell Proliferation in Oral Leukoplakia via HIF-1α/BNIP3 Pathway.

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 Prx1flox/flox:Cre+ mice compared with Prx1flox/flox mice. In Prx1flox/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.

Expression of FAP in Oral Leukoplakia and Oral Squamous Cell Carcinoma.

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.

Salivary biomarkers: Effective diagnostic tool for oral leukoplakia and oral squamous cell carcinoma.

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.

Evaluation of salivary endothelin-1 as a biomarker for oral cancer and precancer.

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.

Salivary metabolomics for oral leukoplakia with and without dysplasia.

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.

Potential noninvasive biomarkers for the malignant transformation of oral leukoplakia: A systematic review and meta-analysis.

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, ß2-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.

Comparative Somatic Variant Analysis of a Rare Case with Concurrent Oral Leukoplakia and Oral Submucosal Fibrosis.

Oral leukoplakia (OL) and oral submucosal fibrosis (OSMF) are precancerous conditions with common etiologies but with different risks for oral cancer (OC) progression. In rare cases, both conditions occur in the same patient and provide an opportunity for understanding the common and distinctive variants upon exposure of genetically identical normal cells to the same carcinogen(s). We performed exome sequencing of a patient with OL (hyperplasia, but no dysplasia) and OSMF (grade II) in the opposite cheeks using blood DNA as the reference genome. The overall somatic variant burden was higher in OSMF than OL, but opposite in the case of copy number alterations. OL-specific variants were enriched in genes associated with DNA repair, cell division/cell cycle checkpoint pathways, whereas in OSMF, extracellular matrix-receptor interaction was mainly affected. The proportions of variants in cancer driver genes and cancer driver mutations were similar in both cases indicating no difference in the potential risk associated with the two conditions at the stages sampled. Future studies on rare cases similar to the one described in this report will help in understanding the molecular basis of differences associated with OL and OSMF and shared processes accompanying OC progression.

Podoplanin Expression Independently and Jointly with Oral Epithelial Dysplasia Grade Acts as a Potential Biomarker of Malignant Transformation in Oral Leukoplakia.

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.

Mucoadhesive Nucleoside-Based Hydrogel Delays Oral Leukoplakia Canceration.

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 G0/G1 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 expression and clinical significance of Bmi-1 gene in oral leukoplakia with different prognosis].

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.

DNA damage through oxidative stress is an important event in oral leukoplakia.

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.

Expressions of Interleukin-27 in Oral Lichen Planus, Oral Leukoplakia, and Oral Squamous Cell Carcinoma.

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.

A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia.

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.

Expression of p53, p63, podoplanin and Ki-67 in recurring versus non-recurring oral leukoplakia.

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.

A novel lncRNA LOLA1 may predict malignant progression and promote migration, invasion, and EMT of oral leukoplakia via the AKT/GSK-3ß pathway.

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-3ß 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-3ß pathway.

PD-1 blockade prevents the progression of oral carcinogenesis.

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.

Effect of smoking status and programmed death-ligand 1 expression on the microenvironment and malignant transformation of oral leukoplakia: A retrospective cohort study.

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.