Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation.

Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.

Copy Number Alterations Predict Development of OSCC from Oral Leukoplakia.

Oral leukoplakia (OLK) is a common type of potentially malignant disorder. Early identification of the malignancy potential leads to a better management of OLK and prediction of development of oral squamous cell carcinoma (OSCC). However, there has been no effective biomarker to assess the risk of malignancy in OLK. Genomic copy number alteration (CNA) is a complex chromosomal structural variation in the genome and has been identified as a potential biomarker in multiple cancers. This study aimed to develop a predictive model for the malignant transformation risk of OLK by copy number analysis. A total of 431 OLK samples with long-term follow-up (median follow-up of 67 mo) from multiple academic centers were analyzed for CNAs. CNA events increased with the severity of hyperplasia, mild dysplasia, moderate dysplasia, and severe dysplasia. More CNA events were present in patients with OLK who later developed OSCC than in those with OLK who did not. By multivariate Cox regression analysis, the OLK of the CNA scorehigh group showed an increased risk of malignant transformation than the CNA scorelow group (P < 0.001). A CNA score model was developed to accurately predict the prognosis (area under the receiver operating characteristic curve [AUC] = 0.879; 95% confidence interval [CI], 0.799-0.959) and was validated using data from 2 external centers (AUC = 0.836, 95% CI, 0.683-0.989; AUC = 0.876, 95% CI, 0.682-1.000), and all of them showed better prediction performances than histopathological grade in assessing the transformation risk of OLK. Furthermore, we performed CNA models among 4 subgroups of OLK with hyperplasia, mild dysplasia, moderate dysplasia, and severe dysplasia and found that CNA score can accurately predict malignant transformation of different subgroups. CNA score may be a useful biomarker to predict malignant transformation of OLK. Subtyping of OLK by the CNA score could contribute to better management of OLK and predicting development of OSCC.

Molecular landscape of proliferative verrucous leukoplakia: a systematic review.

Proliferative verrucous leukoplakia (PVL) is a rare oral potentially malignant disorder characterised by multifocal origin and unpredictable long-term evolution to oral squamous cell carcinoma (OSCC) or oral verrucous carcinoma (OVC). Currently no predictive biomarkers are in clinical use. We aimed to explore the genomic profile of PVL. A total of 685 cases in 26 studies were included in this review. Genomic data were presented in 15% of studies and biomarker analysis was reported in 85% of studies. At first clinical presentation, PVL is characterised by a high loss of heterozygosity (LOH), similar to OSCC, and low copy number alterations (CNA). As these progress, more CNAs and mutations in CDKN2A and alterations to ELAVL1 expression are noted, but no TP53 mutations are identified. There is significantly lower LOH at 17p in early PVL compared with OSCC (p = 0.037). Deletions in chromosomal loci 17q12, 5q31.1 and amplifications in 7q11.2, 7q22 are shared between early lesions and OVC. PVL shows CNAs at 11q31. WNT signalling pathway genes (SUZ12, CTTN and FOLR3) are enriched in CN-altered regions. PVL stroma shows significantly lower α-SMA and higher CD34 expression than OVC and OSCC. The exact genomic landscape is currently unclear, and further studies are necessary to unravel this mystery.

Implication of TIPE2 Expression on the Malignant Transformation of Oral Leukoplakia.

BACKGROUND/AIM: Identification of biomarkers involved in the malignant transformation of oral leukoplakia (OL) is required for the early diagnosis and management of patients with OL. This study aimed to evaluate the functions of tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) expression in the malignant transformation of OL. MATERIALS AND METHODS: The expression levels of TIPE2 and dormant cell markers phospho-ERK and phospho-p38 in a cohort containing 103 surgical specimens from patients with OL were evaluated using immunohistochemistry. The influence of TIPE2 expression on the biological behavior of the immortalized human oral keratinocyte (IHOK) line was investigated in vitro. RESULTS: Increased TIPE2 expression was detected in 40 (38.8%) patients with OL. In a multivariate analysis using clinicopathological variables and TIPE2 expression as cofactors, the presence of dysplasia (p=0.003) and TIPE2 abundance (p=0.019) were identified as independent risk factors for the malignant transformation of OL. Moreover, the in vitro analysis revealed that TIPE2 knockdown can promote the proliferating ability of IHOK; however, the number of apoptotic cells also increased after TIPE2 knockdown in IHOK. Furthermore, TIPE2 expression was significantly associated with phospho-p38 expression, a dormant cell marker, in our cohort (p=0.047). CONCLUSION: TIPE2 expression may contribute to the malignant transformation of OL, and its function may be related to cellular dormancy in OL pathogenesis.

Predictors of malignant transformation in oral leukoplakia and proliferative verrucous leukoplakia: An observational prospective study including the DNA ploidy status.

BACKGROUND: This prospective observational study investigated the determinants of malignant transformation (MT) in localized oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL). METHODS: Demographic, clinical, histological, and DNA ploidy status data were collected at enrolment. Survival analysis was performed (MT being the event of interest). RESULTS: One-hundred and thirty-three patients with OL and 20 patients with PVL entered the study over 6 years (mean follow-up 7.8 years). The presence of OED, DNA ploidy, clinical presentation, and lesion site were associated with MT in patients with OL in a univariate analysis. In a multivariate model, OED was the strongest predictor of MT in patients with OL. Adding DNA ploidy increased the model's predictive power. None of the assessed predictors was associated with MT in patients with PVL. CONCLUSIONS: DNA ploidy might identify a subset OL with low risk or minimal risk of MT, but it does not seem to be a reliable predictor in patients with PVL.

CircHLA-C: A significantly upregulated circRNA co-existing in oral leukoplakia and oral lichen planus.

BACKGROUND: Oral leukoplakia (OLK) and oral lichen planus (OLP) are common precancerous lesions of the oral mucosa. The role of circular RNAs (circRNAs) in OLK and OLP is unclear. The aim of this study was to evaluate the circRNA expression profiles of OLK and OLP, and further explore the potential role of circRNAs in the pathogenesis of these two diseases. METHODS: High throughput sequencing technology was performed to detect the differentially expressed circRNA in OLK (n = 6), OLP (n = 6), oral squamous cell carcinoma (n = 6), and normal oral mucosa tissues (n = 6). Expression of selected circRNAs was validated by qRT-PCR, enzyme tolerance assay, and Sanger sequencing. Expanded sample size validation was done in 20 tissue pairs. The biological processes and signal pathways involved in differential circRNA were analyzed by GO and KEGG enrichment. TargetScan and MiRanda were used to predict miRNAs downstream of circRNA and draw competitive endogenous RNA network diagram. RESULTS: Forty-nine circRNAs were significantly altered in OLK and OLP, including 30 upregulated and 19 downregulated circRNAs. The five selected circRNAs were validated by qRT-PCR, Sanger sequencing, and RNase R assay. GO and KEGG analyses indicated that the upregulated circHLA-C may be involved in the biological process of immune function of OLK and OLP. Bioinformatics analysis indicated that circHLA-C may be involved in the progression of OLK and OLP as a ceRNA. In validation with expanded sample size, PCR results showed that circHLA-C expression was significantly upregulated in OLK and OLP. ROC analysis indicated that circHLA-C has potential diagnostic value with good accuracy and specificity. CONCLUSION: Our study revealed that circHLA-C is the most significantly upregulated circRNA co-existing in OLK and OLP, and we preliminarily discuss the role of circHLA-C in the etiopathogenesis and progression of OLK and OLP.

Comprehensive bioinformatics analysis combined with experimental validation to screen biomarkers for malignant transformation of oral leukoplakia.

Oral leukoplakia (OLK) is the most common potentially malignant disorders in the oral cavity. This study aimed to screen the key genes of OLK malignant transformation using the Gene Expression Omnibus (GEO) database and experiments. In this study, the GEO database was employed to screen OLK malignant transformation-related genes, which were subsequently identified with a series of bioinformatic analyses. External validation showed that the model based on LAPTM4B, NR3C1, and COX6A1 had high accuracy in diagnosing OLK malignant transformation. Furthermore, the DMBA-induced potentially malignant disorders and OSCC models in vivo and real-time PCR experiment in vitro further verified the database analysis results. In conclusion, three key genes (LAPTM4B, NR3C1, and COX6A1) were screened as potential biomarkers for the diagnosis and treatment of OLK malignant transformation.

Whole genome DNA methylation and mutational profiles identify novel changes in proliferative verrucous leukoplakia.

OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. STUDY DESIGN: Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. RESULTS: In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. CONCLUSIONS: Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.

Label-Free Proteomics of Oral Mucosa Tissue to Identify Potential Biomarkers That Can Flag Predilection of Precancerous Lesions to Oral Cell Carcinoma: A Preliminary Study.

Oral squamous cell carcinomas are mostly preceded by precancerous lesions such as leukoplakia and erythroplakia. Our study is aimed at identifying potential biomarker proteins in precancerous lesions of leukoplakia and erythroplakia that can flag their transformation to oral cancer. Four biological replicate samples from clinical phenotypes of healthy control, leukoplakia, erythroplakia, and oral carcinoma were annotated based on clinical screening and histopathological evaluation of buccal mucosa tissue. Differentially expressed proteins were delineated using a label-free quantitative proteomic experiment done on an Orbitrap Fusion Tribrid mass spectrometer in three technical replicate sets of samples. Raw files were processed using MaxQuant version 2.0.1.0, and downstream analysis was done via Perseus version 1.6.15.0. Validation included functional annotation based on biological processes and pathways using the ClueGO plug-in of Cytoscape. Hierarchical clustering and principal component analysis were performed using the ClustVis tool. Across control, leukoplakia, and cancer, L-lactate dehydrogenase A chain, plectin, and WD repeat-containing protein 1 were upregulated, whereas thioredoxin 1 and spectrin alpha chain, nonerythrocytic 1 were downregulated. Across control, erythroplakia, and cancer, L-lactate dehydrogenase A chain was upregulated whereas aldehyde dehydrogenase 2, peroxiredoxin 1, heat shock 70 kDa protein 1B, and spectrin alpha chain, nonerythrocytic 1 were downregulated. We found that proteins involved in leukoplakia were associated with alteration in cytoskeletal disruption and glycolysis, while in erythroplakia, they were associated with alteration in response to oxidative stress and glycolysis across phenotypes. Hierarchical clustering subgrouped half of precancerous samples under the main branch of the control and the remaining half under carcinoma. Similarly, principal component analysis identified segregated clusters of control, precancerous lesions, and cancer, but erythroplakia phenotypes, in particular, overlapped more with the cancer cluster. Qualitative and quantitative protein signatures across control, precancer, and cancer phenotypes explain possible functional outcomes that dictate malignant transformation to oral carcinoma.

Elucidating the Genetic Landscape of Oral Leukoplakia to Predict Malignant Transformation.

PURPOSE: Oral leukoplakia is the most common oral potentially malignant disorder with an annual malignant transformation rate of 1% to 5%. Consequently, oral leukoplakia patients have a 30% to 50% lifetime risk to develop oral squamous cell carcinoma. Although risk factors for malignant transformation of oral leukoplakia have been investigated, no definitive risk stratification model has been proposed. Next-generation sequencing can elucidate the genetic landscape of oral leukoplakia, which may be used to predict the risk for malignant transformation. EXPERIMENTAL DESIGN: We investigated a retrospective cohort of 89 oral leukoplakia patients, and analyzed their oral leukoplakia lesions for the presence of genomic copy-number alterations and mutations in genes associated with oral squamous cell carcinoma. RESULTS: In 25 of 89 (28%) patients, oral squamous cell carcinoma developed during follow-up. Seventy-nine of 89 (89%) oral leukoplakias harbored at least one genetic event. Copy-number alterations were present in 61 of 89 (69%) oral leukoplakias, most commonly gains of chromosome regions 8q24 (46%) and 20p11 (20%) and loss of 13q12 (19%). Mutations were present in 59 of 89 (66%) oral leukoplakias, most commonly in TP53 (28%), FAT1 (20%), and NOTCH1 (13%). Genetic data were combined with the presence of dysplasia to generate a prediction model, identifying three groups with a distinct risk for malignant transformation. CONCLUSIONS: We provide an extensive description of genetic alterations in oral leukoplakia and its relation to malignant transformation. On the basis of our data we provide a model for the prediction of malignant transformation of oral leukoplakia using dysplasia and genetic markers.

SNAI2 promotes the malignant transformation of oral leukoplakia by modulating p-EMT.

OBJECTIVE: Snail family transcriptional repressor 2 (SNAI2) is a key regulator of partial epithelial-mesenchymal transition (p-EMT) and is associated with tumorigenesis. Whether SNAI2 promotes oral leukoplakia (OLK) malignant transformation by modulating p-EMT is unclear. MATERIALS AND METHODS: This study utilized two clinical datasets (GSE26549 and GSE85195) from the Gene Expression Omnibus database, cytological experiments, and a 4-nitroquinoline 1-oxide-induced mice model to explore the role of SNAI2 in OLK malignant transformation. RESULTS: The clinical cohort found SNAI2, as a risk factor (HR = 2.50, 95% CI: 1.08-5.79, p = 0.033), could promote OLK malignant transformation (p = 0.012). Cytological experiments indicated that SNAI2 overexpression promoted DOK cell proliferation, invasion, migration, and increase the protein expression of p-EMT relative signatures, whereas SNAI2 silencing has opposite effects. Furthermore, the mice model and clinical datasets demonstrated the expression of SNAI2 and p-EMT relative signatures were increased with OLK malignant transformation. And SNAI2 was strongly correlated with p-EMT. Besides, co-expressed genes of SNAI2 were also enriched in p-EMT relative biological processes and signaling pathways. CONCLUSIONS: p-EMT plays a significant role in promoting the OLK malignant transformation. As an important regulator of p-EMT, SNAI2 could be a target to block the OLK malignant transformation.

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.

Understanding precancerous lesions of the oral cavity†.

Precancerous lesions provide insight into tumor development as well as prognostication, since distinguishing high-risk from benign disease will stratify clinical management. In a recent issue of The Journal of Pathology, Ghosh et al performed comprehensive genomic characterization of the precancerous lesion leukoplakia, comparing RNA and DNA with peripheral blood, normal mucosa, and squamous cell carcinoma (SCC) of the gingivobuccal region of the oral cavity from the same 28 individuals. The data paint a picture of increasing mutation and early caspase-8 inactivation on the background of inflammation with decreasing immune surveillance in the progression from benign leukoplakia to SCC. This research points to an opportunity for disease intercept at the premalignant niche prior to the development of malignancy. © 2022 The Pathological Society of Great Britain and Ireland.

Recurrent copy number alterations involving EGFR, CDKN2A, and CCND1 in oral premalignant lesions.

BACKGROUND: A major challenge in the management of patients with oral leukoplakia is the difficulty to identify patients at high risk of developing oral squamous cell carcinoma. Our knowledge about genomic alterations in oral leukoplakia, and in particular those that progress to oral squamous cell carcinoma, is scarce and there are no useful biomarkers that can predict the risk of malignant transformation. METHODS: Using a novel, custom-made tissue microarray including 28 high-risk oral leukoplakias and the corresponding oral squamous cell carcinomas from 14 cases that progressed to cancer, we assayed copy number alterations involving the oral squamous cell carcinoma driver genes CDKN2A, CCND1, EGFR, and MYC by fluorescence in situ hybridization. The copy number alterationss were correlated with clinicopathological data from all patients. RESULTS: Copy number alterations were identified in 14/24 oral leukoplakias, analyzable for one or more of the oral squamous cell carcinoma driver genes. EGFR was the most frequently altered gene in oral leukoplakias with amplification/gain in 43.5% followed by loss of CDKN2A (26.1%), gains of CCND1 (26.1%), and MYC (8.3%). Losses of CDKN2A were more common in oral leukoplakias progressing to oral squamous cell carcinoma compared to those that did not. Copy number alterations were more common in oral squamous cell carcinomas than in oral leukoplakias. CONCLUSIONS: Our findings demonstrate that copy number alterations involving the oral squamous cell carcinoma drivers CDKN2A, EGFR, and CCND1 occur in oral leukoplakias and suggest a possible role for these genes in the development and/or progression of subsets of oral leukoplakias.

Significance of p53 overexpression in the prediction of the malignant transformation risk of oral potentially malignant disorders: A systematic review and meta-analysis.

OBJECTIVES: To evaluate the current evidence in relation to the predictive value of p53 overexpression as a biomarker of the malignant transformation risk in oral potentially malignant disorders (OPMD). MATERIAL AND METHODS: We searched PubMed, Embase, Web of Science and Scopus for studies published before July-2021, not restricted by date or publication language, with longitudinal design and assessing p53 overexpression by immunohistochemistry. We evaluated the quality of primary-level studies using QUIPS tool. We carried out meta-analyses, examined inter-study heterogeneity through subgroup and meta-regression analyses, and performed sensitivity and small-study effects analyses to test the stability and reliability of results. RESULTS: Twenty four studies (1,210 patients) met inclusion criteria. P53 overexpression was associated with a statistically significant about 2 fold risk (RR = 1.88, 95 %CI = 1.39-2.56, p < 0.001). Leukoplakia maintained this significant relationship after subgroup meta-analysis (p = 0.002). Regarding the immunohistochemical technique, better results were obtained by the subgroups using anti-p53 DO7 antibody (p = 0.001), at high concentration (dilution < 1: 100, p < 0.001), incubated for long periods (overnight, p = 0.02), and at low temperature (4 °C, p = 0.007). Furthermore, meta-regression analysis showed that the association between p53 overexpression and higher oral cancer risk was independent of the presence and/or severity of epithelial dysplasia (p > 0.05). CONCLUSION: Our systematic review and meta-analysis supports the assessment of p53 overexpression in the prediction of the malignant transformation risk of OPMD.

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 survivin in oral leukoplakia and oral lichen planus.

BACKGROUND: Up to one third (3-33%) oral squamous cell carcinomas develop from potentially malignant lesions. Most common potentially malignant lesions (PML) are leukoplakia and lichen planus. It is very important to diagnose PML at early stage to prevent malignant transformation of lesion. Survivin is a smallest member of the inhibitor of apoptosis (IAP) family of proteins. Survivin plays an important role in apoptosis regulation. High expression of survivin is an early event during oral carcinogenesis and it acts as a tool for the identification of precancerous lesions at higher risk of progression into invasive carcinoma. The aim of the study was to evaluate the expression of survivin in oral leukoplakia, oral lichen planus. METHODS: Fifteen patients with oral lichen planus and 15 patients with oral leukoplakia were selected as subjects for the present study and 15 patients with normal oral mucosa were selected as controls and were evaluated for expression of survivin. All sections were H&E stained and were studied immunohistochemically for the expression of survivin. RESULTS: Expression of survivin was evaluated based on the percentage of cells expressing surviving, as well as grading. Significant survivin expression was detected in oral leukoplakia and oral lichen planus (P<0.001). CONCLUSIONS: Expression of survivin in potentially malignant lesions indicates the potential risk of malignant transformation.

MicroRNAs and their role in the malignant transformation of oral leukoplakia: a scoping review.

BACKGROUND: MiRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level and have been associated with malignant transformation of oral epithelial precursor lesions such as oral leukoplakia. The aim was to perform a scoping review of the contemporary literature about the different roles of miRNAs during the malignant transformation of oral leukoplakia. MATERIAL AND METHODS: We conducted a systematic search with the following MeSH terms: 'oral leukoplakia', 'carcinoma in situ', 'microRNAs', 'mouth neoplasms' and 'epithelial-mesenchymal transition' in PubMed/MEDLINE, EMBASE and SpringerLink. RESULTS: Fifteen articles were included for analysis, among which in vivo and in vitro articles were included. A total of 21 different miRNAs were found to be involved in the malignant transformation process of oral leukoplakia. Regarding their possible effects, 6 miRNAs were classified as oncogenic, 5 as tumour suppressors and 10 were related to epithelial-mesenchymal transition, invasion and migration. CONCLUSIONS: Based on the current review, we concluded that miRNAs-21, 345, 181-b and 31* seem to be potential markers of malignant transformation of oral leukoplakia. However, further clinical prospective studies are needed in order to validate their utility as prognostic biomarkers.

A Systematic Review of MicroRNA Signatures Associated with the Progression of Leukoplakia with and without Epithelial Dysplasia.

Oral cancer is a significant public health issue, being the eighth most common cancer worldwide with over 300,000 cases diagnosed annually. Early diagnosis and adequate management of oral potentially malignant disorders (OPMDs) before transformation into oral squamous cell carcinoma (OSCC) is critical to reduce deaths, morbidity, and to improve overall prognosis. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of protein expression and implicated in the control of numerous cellular pathways and impacting physiological, developmental, and pathological processes. Dysregulation of miRNAs has been reported in many cancers and has been demonstrated to play a critical role in cancer initiation, progression, apoptosis, invasion and metastasis. This systematic review provides a comprehensive summary of the prevailing literature on miRNA signatures in OPMDs, specifically leukoplakia with or without oral epithelial dysplasia, and their utility in predicting malignant transformation into OSCC. Eighteen articles describing 73 unique and differentially expressed microRNAs met the criteria for inclusion in this review. We reviewed the characteristics and methodology for each of these studies and assessed the sensitivity and specificity of the studied miRNAs in predicting malignant transformation. This systematic review highlights the significant interest in miRNAs and their tremendous potential as prognostic markers for predicting the malignant transformation of OPMDs into OSCC.

Association Between X-Ray Repair Cross-Complementing Group 1 Arg399Gln Polymorphism and Risk of Oral Leukoplakia: A Meta-analysis.

Aims: Previous studies have have reported inconsistent results regarding the association of the XRCC1 polymorphism Arg399Gln with oral leukoplakia (OLK) risk. This study was designed to assess the existing evidence of this association using a meta-analytic approach. Materials and Methods: The literature was searched using multiple databases, including PubMed, EMBASE, and China National Knowledge Infrastructure (CNKI), through October 22, 2020. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of any associations. Results: A total of 671 Indian cases and 1009 Indian controls from seven case-control studies were included in the meta-analysis. The overall analysis revealed that the AA genotype was associated with a significantly increased OLK risk compared with the GG+GA genotypes (OR = 1.51, 95% CI = 1.10-2.06). In the subgroup analysis stratified by tobacco use, a significant association was found in the mixed group (OR = 1.51, 95% CI = 1.12-2.56), but not in the tobacco-using group or the no tobacco use group. In the OLK subtype subgroup analysis, a significantly increased risk was found in the hyperplastic subgroup (OR = 5.01, 95% CI = 1.39-18.11), whereas no associations were found in the dysplastic or mixed subgroups. Conclusions: The results of this meta-analysis suggest that the XRCC1 Arg399Gln polymorphism may significantly contribute to susceptibility to OLK in the Indian population.