Oral Leukoplakia Samples Research Articles

Investigation of the Vascular-Endothelial Pattern of Expression of DAPK-1 in Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders Through Immunohistochemistry.

Introduction Potentially malignant disorders, like oral lichen planus (OLP) and oral leukoplakia (OL)of several degrees of dysplasia, manifest a significant potential of malignant transformation being a precursor of oral squamous cell carcinoma (OSCC). The role of microvascularization in carcinogenesis is critical; therefore, microvascularization constitutes a major therapeutic target. DAPK-1 constitutes a possible cancer marker, with proven implications in other human cancers, and there isn't any study on its vascular endothelial expression in the oral cavity, particularlyin oral cancer and oral potentially malignant diseases. The present study aims to investigate the vascular endothelial expression of the DAPK-1in paraffin-embedded tissue samples of oral leukoplakia, oral squamous cell carcinoma, and oral lichen planus. Materials and methods The study focuses on the immunohistochemical, vascular-endothelial, expression pattern of biomarker DAPK-1 (NBP2-38468, Novus Biologicals, Centennial, CO, US). Tissue samples were obtained from six cases of oral lichen planus (OLP) (3 of reticular and 3 of erosive form), 30 cases of oral leukoplakia (OL) (10 with no dysplasia, 10 with mild dysplasia, and 10 with moderate/severe dysplasia), 22 cases of OSCC (2 well-differentiated, 17 moderately differentiated, and 3 poorly differentiated), as well as 5 cases of normal oral epithelium. The tissue samples were retrieved from the archives of the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, as well as from St Lukas Hospital of Thessaloniki, Greece, from2004-2019. In accordance with the Research and Ethics Committee guidelines of the Aristotle University, School of Dentistry, and the Helsinki II declaration, the study was conducted. The primary inclusion criteria for the study focused on the presence of sufficient precancerous or cancerous tissue. Conversely, inadequate tissue served as the exclusion criteria.The staining was evaluated exclusively in a quantitative manner. The vascular endothelial staining was evaluated as either positive or negative. If at least one endothelial cell exhibited positive staining, the section was classified as positive. Statistical analysis was carried out using SPSS Statistics v25.0 (IBM Corp., Armonk, NY, US) utilizing Pearson's chi-square or Fisher's exact test, depending on the sample size, to compare OLP to OL, OLP to OSCC, OLP to normal, OL to OSCC, OL to normal, and OSCC to normal. The significance level was established at 0.05 (p=0.05). Results A prevalence of positive OL cases may be noticed. The comparison between OLP and OL yieldedFisher's exact test of p>0.999, OLP and OSCC p=0.389, OLP and normal oral epithelium p>0.999, OL and OSCC p=0.226, OL and normal oral epithelium p>0.999, as well as OSCC and normal oral epithelium p=0.342. Conclusions The role of DAPK in tumorigenesis is already supported bylimited literature. However, its implication in the development of OSCC and oral potentially malignant disorders (OPMDs) has yet to be elucidated. Its elevated expression in OL suggests a role in affecting the microenvironment, the vessels, in particular,surrounding oral potentially malignant lesions, possibly assisting their transition into cancer. The evaluation of the vascular-endothelial immunohistochemical profile of DAPK-1 in OL, OLP, and OSCC requires further studies in more tissue samples to illustrate its possible implications.

Aldehyde Dehydrogenase: An Off-Label Marker of Endothelial Activation in Oral Squamous Cell Carcinoma.

The vascular endothelial (VE) expression of aldehyde dehydrogenase (ALDH) 1/2 family in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cases has not been studied so far. The aim of this study was to illustrate the "off-label" endothelial expression of cancer stem cell (CSC) biomarker, ALDH1/2, adjacent to oral potentially malignant and malignant lesions in order to shed some light on the mechanisms leading to oral carcinogenesis. Materials and methods: The expression of CSC protein-biomarker ALDH1/2 was detected through immunohistochemistry (IHC) in 30 paraffin-embedded samples of OL and 21 samples of OSCC compared to five samples of normal oral mucosa. Statistical analysis was done using SPSS, Pearson Chi-square, and Fischer's exact test. The significance level was set at 0.05 (p≤ 0.05). Results: In oral mucosal vessels, ALDH1/2 was not expressed. It was expressed significantly more in the vessels of OSCCs compared to the OLs (Fisher's exact test, p-value= 0,001). However, when endothelial expression of ALDH1/2 in the vasculature of OLs was compared with that of the normal oral mucosa, no significant change was noticed (Fisher's exact test, p-value=1.000). Discussion: The IHC VE expression of ALDH1/2 in OSCCvasculature but not in OL indicates a possible significantly stronger activation of endothelial cells during carcinogenesis, which could be an indicator of the role of inflammation in the development of field cancerization and of prognostic value for (vascular/lymphatic) metastasis.

The preliminary exploration of immune microenvironment in oral leukoplakia concomitant with oral submucosal fibrosis: A comparative immunohistochemical study.

Oral leukoplakia concomitant with oral submucous fibrosis is a high-risk oral potentially malignant disorder, but little is known about its immune microenvironment. Thirty samples of oral leukoplakia concomitant with oral submucous fibrosis, 30 oral leukoplakia samples, and 30 oral submucous fibrosis samples were collected from two hospitals. Immunohistochemistry was performed to analyze expression of T cell biomarkers [CD3, CD4, CD8, and Forkhead box P3 (Foxp3)], a B cell biomarker (CD20), macrophage biomarkers (CD68 and CD163), an immune inhibitory receptor ligand (PD-L1), and Ki-67. The numbers of CD3+ (p < 0.001), CD4+ (p = 0.018), and CD8+ (p = 0.031) cells in oral leukoplakia concomitant with oral submucous fibrosis were less than those in oral leukoplakia. The number of CD4+ cells (p = 0.035) in oral leukoplakia concomitant with oral leukoplakia was higher than that in oral submucous fibrosis. More CD3+ (p < 0.001), CD4+ (p < 0.001), Foxp3+ (p = 0.019), and CD163+ (p = 0.029) cells were found in oral leukoplakia than in oral submucous fibrosis. Various levels of immune infiltration were observed among oral leukoplakia concomitant with oral submucous fibrosis, oral leukoplakia, and oral submucous fibrosis. Characterization of the immune microenvironment may contribute to personalized immunotherapy.

High density of CD8 T cell and immune imbalance of T lymphocytes subsets are associated with proliferative verrucous leukoplakia.

Oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL) are oral potentially malignant disorders (OPMDs) that microscopically show no or varying degrees of dysplasia. Even sharing clinical and microscopic aspects, PVL shows a more aggressive clinical behaviour, with a malignant transformation rate greater than 40%. Inflammatory infiltrate associated with dysplastic lesions may favour malignant transformation of OPMDs. This study aimed to evaluate the density of T cells and cytokines in dysplastic lesions from OL and PVL patients. Additionally, we evaluated whether soluble products produced in vitro by dysplastic keratinocytes are capable of modulating apoptosis rates and Th phenotype (Th1, Th2, Th17 and Treg) of peripheral blood mononuclear cells. The density of CD3, CD4 and CD8 T cells was assessed by immunohistochemistry. Cytokines and chemokines profile from frozen tissue samples were analysed using the LUMINEX system. Apoptosis rates and Th phenotype modulation were evaluated by flow cytometry. Our results showed an increase in the number of CD8 T cell in the subepithelial region from PVL dysplastic lesions in relation to OL samples. PVL showed increased levels of IL-5 and a decrease in IL-1β and IFN-γ levels compared to OL. Soluble products of PVL and oral carcinoma cell cultures were able to reduce apoptosis rate and promote an imbalance of Th1/Th2 and Th17/Treg. The high-subepithelial density of CD8 T cells and immune imbalance of T lymphocytes subsets probably play an important role in the pathogenesis of PVL and may explain its more aggressive behaviour in relation to OL.

ASSOCIATION OF FOXP3 WITH CLINICAL AND HISTOPATHOLOGICAL ASPECTS OF MALIGNANT TRANSFORMATION OF THE ORAL LEUKOPLAKIA

<h3>Objectives</h3> Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral mucosa. Several factors are associated with increased risk of malignant transformation. The aim was to evaluate the expression of Foxp3+ cells in OL samples and their relationship with clinical and pathologic factors associated with the malignant transformation of OL. <h3>Study Design</h3> Twenty-five samples from patients diagnosed with OL were selected. Clinical data such as age, sex, and location were obtained from archives. The histopathologic degree was obtained, and immunohistochemistry was performed using anti-Foxp3. Ten fields from each slide were obtained and Foxp3+ cells were counted. The mean density was compared between groups and statistical analysis was applied. Normal oral mucosa (NOM) samples were used as control. <h3>Results</h3> Foxp3+ cells were increased in samples of smokers when compared to NOM samples (<i>P</i> < .05). Foxp3+ cells were increased in females (<i>P</i> < .05) and in OL samples with severe epithelial dysplasia when compared with samples of moderate epithelial dysplasia (<i>P</i> < .05). <h3>Conclusions</h3> The study suggests that the number of cells associated with modulation of immune response may be affected by clinical and histopathologic characteristics associated with malignant transformation of OL.

Correlation of H3K9ac and H4K12ac With Cell Proliferation Marker Ki-67 in Oral Leukoplakia: An Immunohistochemical Study.

This study aimed to analyze the immunohistochemical expression of H3K9ac and H4K12ac in oral leukoplakia (OL) and its association with cell proliferation marker Ki-67 and clinicopathologic data. Paraffin-embedded, formalin-fixed tissue samples from 50 OLs and 15 fragments of the normal oral mucosa (NOM) were submitted to immunohistochemical assay using the streptavidin-biotin-peroxidase method. Quantitative analysis of the antigen-antibody reaction was performed by obtaining integrated optical density (IOD) and the percentage of positive nuclei (PPN) with ImageJ software. OL samples presented higher PPN ( P =0.02) and lower IOD values ( P =0.007) for H4K12ac in comparison to NOM. The area under the receiver operating characteristic curve for PPN and IOD values of H4K12ac immunostaining were 0.70 ( P =0.02) and 0.73 ( P =0.007), respectively. No differences were found between OL and NOM for H3K9ac. Cell proliferation marker Ki-67 had a positive correlation with PPN ( P <0.0001) and IOD ( P =0.0007) for H3K9ac expression and with IOD values ( P =0.002) for H4K12ac expression. The present findings suggest that alterations in the acetylation pattern of H4K12 occur in the early stages of oral carcinogenesis and that both H3K9ac and H4K12ac might have a role in the regulation of epithelial cell proliferation of OL.

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.

Human papillomavirus and leukoplakia of the oral cavity: a systematic review.

IntroductionHuman papillomavirus (HPV) infection is mainly a problem of the female reproductive tract. It can occur in the oral cavity as well. Commonly HPV infections are subclinical, without any visible symptoms, and last no longer than 2 years. Usually the clinical manifestation of HPV infection is benign, but in some cases it can also promote malignant transformation. In the paper we have tried to estimate the prevalence of HPV detected in samples of oral leukoplakia (OLK), the most common premalignant lesions of the oral mucosa.AimTo review the current literature to estimate the prevalence of HPV (HPV DNA) detected in samples of oral leukoplakia.Material and methodsWe searched PubMed/Medline, Scopus, and Cochrane Library databases for studies that examined the prevalence of HPV in leukoplakia with HPV DNA detection by polymerase chain reaction.ResultsHPV positive cases in OLK ranged from 0% to 100% in studies. The overall HPV prevalence in leukoplakia was 6.66%. The prevalence of HPV 16 positive cases was 2.95%. The high-risk HPV prevalence was 5.16%, when the low-risk HPV prevalence was 3.32%. When dysplasia is mentioned, HPV was detected in 19.56% of lesions with dysplasia, compared to 38.16% among non-dysplastic lesions.ConclusionsFurther studies should be extended also to include low-risk HPV and compare its prevalence with presence of dysplasia in leukoplakia. Besides, leukoplakia and other premalignant lesions can no longer be treated as one lesion.

Chewing tobacco may act as a risk factor for dysplastic transformation of squamous cells in Oral leukoplakia- A cytochemistry based approach

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.

Evaluating the expression of GLUT-1 in oral leukoplakia.

Aim:The aim of the present study is to analyze the role of GLUT-1 in detection of early alterations occurring in oral leukoplakia. This study was to evaluate the expression of GLUT-1 in normal oral epithelium, the expression of GLUT-1 levels in the tissue samples of oral leukoplakia and to statistically compare the expression of GLUT-1 in normal epithelium and oral leukoplakia.Materials and Methods:The study sample comprised formalin-fixed and paraffin-embedded tissue specimens from 23 cases of histopathologically diagnosed oral leukoplakia and formalin-fixed paraffin-embedded tissue specimens from 10 cases of normal oral mucosa. Sections were mounted on glass slide coated with Aminopropyltriethoxysilane (APES; Sigma chemical co., USA) and processed for subsequent immunohistochemical study to demonstrate GLUT-1.Results:GLUT-1 expression in normal oral mucosa revealed weak positivity in all 10 cases (100%). The oral leukoplakia cases showed immunopositivity in all 23 cases (100%) of which 10 cases (39.14%) demonstrated focal positivity and 13 cases (60.86%) of diffuse positivity. The results were compared statistically using ANOVA test was significant at P = 0.002.Conclusion:The present study shows expression of GLUT-1 in leukoplakia may be used as a reliable marker to identify the high risk group for malignant transformation.

Transglutaminase 3 contributes to malignant transformation of oral leukoplakia to cancer.

Oral leukoplakia (OL) is the most common premalignancy in the oral cavity. The objective of this study was to investigate the biological role of transglutaminase 3 (TGM3) in malignant transformation of OL and its clinical value for predicting oral squamous cell carcinoma (OSCC) risk in patients with OL. Immunohistochemistry was used to measure TGM3 expression in OL samples from 98 patients. Patient clinicopathological and follow-up data were analyzed. The TGM3 biological role in OL cells was investigated in gain-of-function and loss-of-function assays, and the TGM3 downregulated mechanism in OLs was characterized. TGM3 mRNA and protein expressions were frequently downregulated in OL cells and samples. DNA hypermethylation was a mechanism of TGM3 downregulation. TGM3 overexpression and silencing affected the proliferation, colony formation, and apoptosis of OL cells through apoptosis-related protein dysregulations. Lower TGM3 levels were strongly associated with the grade of epithelial dysplasia and OSCC development. Multivariate analyses showed that TGM3 was the independent predictor for malignant transformation of OL. Collectively, these data indicated that TGM3 played an important role in OL malignant transformation and may serve as a predictor to identify OL with OSCC development.

Mature dendritic cell density is affected by smoking habit, lesion size, and epithelial dysplasia in oral leukoplakia samples

ObjectiveTo compare the densities of CD1a + immature and CD83+ mature dendritic cells, and inflammatory infiltrate cells between smokers and non-smokers with oral leukoplakia. Parameters associated with malignant transformation were also evaluated. Design21 smokers and 23 non-smokers diagnosed with oral leukoplakia were obtained. Densities of inflammatory infiltrate cells were calculated in H&E sections. Immunohistochemistry using anti-CD1a and anti-CD83 was performed and densities were calculated. Comparisons and statistical analyses were performed among the groups and parameters as gender, lesion size, site, and presence of cell dysplasia were analyzed. ResultsA lower density of CD83+ cells was observed in smokers compared to non-smokers (P < 0.05). For samples of smokers, a lower density of CD1a + cells, CD83+ cells, and inflammatory infiltrate cells was observed in samples with <10 mm compared to samples ≥10 mm of diameter (P < 0.05), and a lower density of CD83+ cells was also observed between samples without dysplasia compared to samples with dysplasia (P < 0.05). ConclusionIn oral leukoplakia samples, dendritic cell density decreases in the presence of smoking habit, and increases in larger lesions and with epithelial dysplasia. Smoking habit is an external factor that contribute to alteration of the anti-tumoral immune defense system in lesions of oral leucoplakia, reinforcing that smoking elimination is important to control the development of this disease.

CD47 as a potential prognostic marker for oral leukoplakia and oral squamous cell carcinoma.

Cluster of differentiation (CD)47, which acts as a negative indicator for phagocytic cells, is overexpressed on the surface of multiple human solid tumor cell types. Avoiding phagocytosis by CD47 is required for the progression of solid tumors. The present study investigated the expression of CD47 in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and preliminarily explored the impact of CD47 on the proliferation of OSCC cells. A total of 56 tissue samples, including 36 cases of OLK, 10 cases of OSCC and 10 cases of normal oral mucosa (NOM) were selected to detect the expression of CD47 by immunohistochemistry. For subgroup analysis, OLK samples were divided into OLK with low-risk dysplasia (LR-OLK) and OLK with high-risk dysplasia (HR-OLK). The subcellular localization of CD47 was determined by immunofluorescence in three OSCC cell lines (Tca8113, SCC-9 and Cal-27). The effect of CD47 antibody on the proliferation of the Cal-27 cell line was analyzed using the Cell Counting kit-8 assay. CD47 expression in OLK and OSCC lesions was significantly higher than in NOM (P<0.05). Compared with LR-OLK, the expression of CD47 in HR-OLK and OSCC cells was upregulated (P=0.0327 and P=0.0048, respectively). CD47 was highly expressed in OSCC cell lines (Tca8113, Cal-27 and SCC-9) and weakly expressed in normal oral keratinocytes. The proliferation of Cal-27 cells was inhibited by CD47 antibody in a concentration and time-dependent manner. CD47 may be a reliable biomarker for predicting the progression of oral precancer and cancer, and it may serve as an important molecular target for designing a novel therapy for oral cancer.

Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma.

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.

Evaluation of Collagen in Leukoplakia, Oral Submucous Fibrosis and Oral Squamous Cell Carcinomas Using Polarizing Microscopy and Immunohistochemistry

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.

Chemoprevention of oxidative stress-associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety.

Oxidative stress is known to play an important role in oral cancer development. In this study we aimed to examine whether a chemical activator of NRF2, sulforaphane (SFN), may have chemopreventive effects on oxidative stress-associated oral carcinogenesis. We first showed that Nrf2 activation and oxidative damage were commonly seen in human samples of oral leukoplakia. With gene microarray and immunostaining, we found 4-nitroquinoline 1-oxide (4NQO) in drink activated the Nrf2 pathway and produced oxidative damage in mouse tongue. Meanwhile whole exome sequencing of mouse tongue identified mutations consistent with 4NQO's mutagenic profile. Using cultured human oral keratinocytes and 4NQO-treated mouse tongue, we found that SFN pre-treatment activated the NRF2 pathway and inhibited oxidative damage both in vitro and in vivo. On the contrary, a structural analogue of SFN without the isothiocyanate moiety did not have such effects. In a long-term chemoprevention study using wild-type and Nrf2-/- mice, we showed that topical application of SFN activated the NRF2 pathway, inhibited oxidative damage, and prevented 4NQO-induced oral carcinogenesis in an Nrf2-dependent manner. Our data clearly demonstrate that SFN has chemopreventive effects on oxidative stress-associated oral carcinogenesis, and such effects depend on Nrf2 and the isothiocyanate moiety.

Abstract 829: Chemoprevention of oxidative stress-associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety

Abstract Oxidative stress is known to play an important role in oral cancer development. In this study we aimed to examine whether a chemical activator of NRF2, sulforaphane (SFN), may have chemopreventive effects on oxidative stress-associated oral carcinogenesis. We first showed that Nrf2 activation and oxidative damage were commonly seen in human samples of oral leukoplakia. With gene microarray and immunostaining, we found 4-nitroquinoline 1-oxide (4NQO) in drink activated the Nrf2 pathway and produced oxidative damage in mouse tongue. Meanwhile whole exome sequencing of mouse tongue identified mutations consistent with 4NQO's mutagenic profile. Using cultured human oral keratinocytes and 4NQO-treated mouse tongue, we found that SFN pre-treatment activated the NRF2 pathway and inhibited oxidative damage both in vitro and in vivo. On the contrary, a structural analogue of SFN without the isothiocyanate moiety did not have such effects. In a long-term chemoprevention study using wild-type and Nrf2-/- mice, we showed that topical application of SFN activated the NRF2 pathway, inhibited oxidative damage, and prevented 4NQO-induced oral carcinogenesis in an Nrf2-dependent manner. Our data clearly demonstrate that SFN has chemopreventive effects on oxidative stress-associated oral carcinogenesis, and such effects depend on Nrf2 and the isothiocyanate moiety. Citation Format: Aixian Lan, Wenjun Li, Yao Liu, Xinyan Zhang, Shanshan Zhou, Olesya Palko, Hao Chen, Mayanga Kapita, Justin Prigge, Edward Schmidt, Xin Chen, Zheng Sun, Xiaoxin Luke Chen. Chemoprevention of oxidative stress-associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 829.

Clinicopathological features and prognostic implications of Raf kinase inhibitor protein downregulation in tongue squamous cell carcinoma.

Raf kinase inhibitor protein (RKIP) is recognized as a suppressor of metastasis, and the downregulation of RKIP is associated with aggressive events and a poor outcome in a variety of solid tumors. However, the clinical relevance of RKIP expression in tongue squamous cell carcinoma (TSCC) remains unclear. In the present study, the expression of RKIP in 85 pairs of TSCC and corresponding adjacent non-cancerous tissues, 30 matched metastatic lesions from the cervical lymph nodes and 32 oral leukoplakia samples were assessed using immunohistochemical methods. The association between RKIP expression and clinicopathological features was then evaluated. Kaplan-Meier survival analysis and Cox proportional hazards model were used to estimate the effect of RKIP expression on the survival time of patients with TSCC. The results revealed that RKIP expression was dramatically downregulated in TSCC, and to an even greater extent in metastatic lesions. RKIP downregulation was significantly associated with the presence of lymphatic metastasis and the clinical stage of TSCC. Furthermore, patients with low RKIP expression demonstrated a significantly shorter overall survival time. Multivariate analysis indicated that RKIP expression may be an independent prognostic factor in TSCC. In conclusion, the present findings indicate that the lack of RKIP expression is of clinical significance and may serve as a prognostic biomarker in TSCC.

Association between histopathological features of dysplasia in oral leukoplakia and loss of heterozygosity

Oral leukoplakia (OL) dysplasia is graded on the basis of architectural and cytological features, and grade does not correlate well with malignant transformation. Loss of heterozygosity (LOH) profiles have been validated as risk predictors of OL malignant transformation. We aimed to assess whether the histological parameters used to grade dysplasia show different LOH profiles. Areas of epithelial dysplasia of 29 OL samples were microdissected, and LOH was assessed by use of a panel of 11 microsatellite markers located on chromosomes 3, 9, 11, and 17. Dysplasia was graded, and the cytological and architectural parameters were scored. Dysplasia was graded as mild in 18 samples, moderate in nine, and severe in two. The moderate/severe dysplasias and the mild dysplasias did not show different frequencies of allelic loss. Irregular epithelial stratification was associated with LOH at marker D3S1234 (3p14.2). In addition, the presence of drop-shaped rete ridges and premature keratinization in single cells showed associations with LOH at D9S162 (9p22) and P53 (17p13.1), respectively. We provide evidence that architectural and cellular changes in OL have different LOH patterns.

Cyclooxygenase-2 and Ki67 Expression in Oral Leukoplakia: a Clinicopathological Study.

ABSTRACTObjectivesOral leukoplakia is a precancerous lesion of the oral mucosa. The upregulation of Ki67 and cyclooxygenase-2 has been reported in both dysplastic and non-dysplastic tissues. The aim of this clinicopathological study was to investigate the prognostic value of Ki67 and cyclooxygenase-2 expression for oral leukoplakia.Material and MethodsA total of 50 samples were investigated and the study group consisted of 30 oral leukoplakia samples. Samples of 10 intact oral mucosa and 10 squamous cell carcinoma were included as negative and positive control groups, respectively. Epithelial dysplasia was defined as oral intraepithelial neoplasia (OIN) and classified into subgroups 1 - 3. Tissue samples were assessed immunohistochemically for Ki67 and cyclooxygenase-2 expression. Clinicopathological correlations of oral leukoplakia patients were also investigated.ResultsAll OIN 3 patients were non-smokers (P < 0.05), and homogeneous oral leukoplakia lesions also presented OIN. Both cyclooxygenase-2 and Ki67 expression increased with the severity of lesions, which defined different subgroups (P < 0.05), except there was no significant difference between the hyperkeratosis and OIN groups for Ki67 expression.ConclusionsCyclooxygenase-2 and Ki67 expression may have a prognostic value for the malignant transformation of oral leukoplakia.