Oral Leukoplakia Samples Research Articles

Nested PCR for detection of HSV-1 in oral mucosa.

BackgroundIt has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC).Material and MethodsWe investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing.ResultsHSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant.ConclusionsWe observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected. Key words:HSV-1, nested PCR, PCR.

E-cadherin as a potential biomarker of malignant transformation in oral leukoplakia: a retrospective cohort study.

BackgroundNumerous attempts have been made to establish and develop tumor markers that could determine the susceptibility of normal tissues to transform into cancerous ones. To determine whether altered expression patterns of E-cadherin could be an early event in the progression of potentially malignant disorders to oral squamous cell carcinoma, this study aimed to assess the relationship between the immunoexpression of E-cadherin and the different degrees of epithelial dysplasia in oral leukoplakia.MethodsSurgically excised specimens from patients with oral leukoplakia (n = 31), oral cavity squamous cell carcinoma with cervical lymph node metastasis (n = 12) and normal oral mucosa (n = 9) were immunostained for E-cadherin. Oral leukoplakia samples were distributed into low and high risk group according to a binary system for grading oral epithelial dysplasia. Comparative analyses between E-cadherin expression and microscopic features (WHO histological grading and epithelial dysplasia) were performed by Pearson Chi-square test (P < 0.05).ResultsDifferences in E-cadherin expression were observed between normal oral mucosa and low risk oral leukoplakia (P = 0.006), low and high risk oral leukoplakia (P = 0.019), and high risk oral leukoplakia and oral cavity squamous cell carcinoma with cervical lymph node metastasis (P = 0.0001). In addition, as epithelia undergo dysplastic changes, the risk of malignant transformation increases, and there is a reduction or loss of E-cadherin expression by keratinocytes. Reduced E-cadherin expression was an early phenomenon and it was observed in moderate-severe dysplasia, showing that the loss of epithelial cohesion may be an indicator of progression to oral cavity squamous cell carcinoma.ConclusionsE-cadherin could be used as a novel biomarker to identify lesions with potential risk for malignant transformation, which may provide opportunities for prophylactic interventions in high risk patient groups.

Increased VEGFR2 and MMP9 protein levels are associated with epithelial dysplasia grading

The present study aimed to compare levels of VEGFR2 and MMP-9 among control, epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC) groups. We analyzed 48 patients with oral leukoplakia (OL), 20 patients with OSCC and 21 patients without OL and OSCC. Immunohistochemistry of VEGFR2 and MMP9 were performed and compared among groups. Analysis of tissue immunolocalization of VEGFR2 and MMP-9 assumed non-parametrical distribution and comparison between groups was performed using the Mann–Whitney and Kruskal–Wallis statistical tests. VEGFR2 and MMP9 immunoexpression appeared to correlate with the degree of dysplasia and was observed to increase in lesions with more severe dysplasia as compared to those with lower degrees of dysplasia. Immunoreactivity of MMP-9 was lower in the OL samples compared to the OSCC samples (p=0.004). We observed no difference in VEGFR2 protein levels between OL and OSCC samples. A positive correlation was found between VEGFR2 and MMP-9 in OL samples (r=+0.452, p=0.001), however, no correlation was found in OSCC samples (r=−0.042, p=0.861). In conclusion, the results of the current study suggest that expression of MMP9 and VEGFR2 is associated with ED grading and MMP9 levels are increased in OSCC.

Relationship between microRNA expression levels and histopathological features of dysplasia in oral leukoplakia

Increased expression of microRNAs (miRNAs), miR-21, miR-345, and miR-181b has been demonstrated in oral leukoplakia (OL) that progresses to oral squamous cell carcinoma (OSCC), suggesting a miRNA signature with potential prognostic value. On the basis of these findings, this pilot study aimed to investigate the cytological and histopathological features that are used to grade oral dysplasia and determine associations with the expression of these 3 potentially cancer-related miRNAs. We also compared the expression levels of these miRNAs in OL with normal oral mucosa and OSCC. We evaluated miRNA expression by qPCR in 22 samples of OL demonstrating different grades of dysplasia, as well as 17 cases of OSCC, and 6 samples of normal oral mucosa. We associated the miRNAs expression profiles with cytological and histopathological features of OL. OSCC cases showed increased expression of all 3 miRNAs when compared with OL and normal oral mucosa. Increased expression of miR-21 was also observed in OL when compared with normal oral mucosa. We found a higher expression of miR-21 and miR-181b in OL that presented with an increased number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromasia. Increased expression of miR-21 was also detected in OL with abnormally superficial mitosis. Higher expression of miR-345 was observed in OL with an increased number and size of nucleoli or increased nuclear/cytoplasmic ratio. In conclusion, the present study shows that some cytological and histopathological parameters used to grade dysplasia are associated with altered miRNA expression.

The relevance of EGFR overexpression for the prediction of the malignant transformation of oral leukoplakia

The present study evaluated the relevance of EGFR overexpression in prediction of malignant transformation of oral leukoplakia (OLP). The retrospective study comprised paraffin-embedded tissue samples of OLP that transformed into oral squamous cell carcinoma (OSCC) (n=53) and tissue samples of OLP that did not transform into OSCC (n=45) during a follow-up period of 5 years. EGFR overexpression was assessed immunohistochemically. A significantly different expression rate of EGFR was determined between transformed and non-transformed OLP (p=0.017). A statistically significant increase of EGFR expression for low dysplasia lesions in group I compared to group II was proven (D0, p=0.013; D1, p=0.049). By calculation of ROC curve and determination of highest Youden index the optimal threshold value [cut-off point (COP) = 44.96] for distinguishing the transformed from non-transformed lesions was estimated (critical expression rate of EGFR). Using the determined COP the correlation between high-risk lesions and the detection of increased expression rates were significant (p=0.001). In the future, the assessment of EGFR overexpression in OLP may allow identifying OLP lesions with an increased risk of malignant transformation that may have been regarded harmless when only the grade of dysplasia had been taken into account.

Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity

Introduction: Expression of p53 gene is one of the common findings in premalignancies and malignancies of the oral cavity. The aim of the study was to know whether P53 protein is overexpressed in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. The aim of the present study was to determine the overexpression of P53 protein in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. Materials and methods: Eighty-five histopathologically diagnosed. formalin-fixed, paraffin embedded tissue samples were divided into study group and control group. The study group was further subdivided into three groups: Group 1 consisted of 25 samples of oral submucous fibrosis: group 2 consisted of 25 samples of oral leukoplakia and group 3 consisted of 25 samples of oral squamous cell carcinoma. The control group consisted of 10 normal oral mucous membrane tissue samples. The samples were subjected to immunohistochemisty using indirect immunoenzyme Labelled StreptAvidin Biotin (LSAB) method. The results were analyzed statistically by Chi-square (X 2 ) test of significance Results: 8125 samples of group 1.5;25 samples of group 2. 8125 samples of group 3 and none of the samples of the control group were positive for P53 protein. P53 staining was seen mostly in the basal layer of the samples of group I while in the samples of groups 2 and 3 the staining was more diffuse. Conclusion: Among P53 may be considered a tumor marker as a definite number of samples were positive for P53

Evaluation of MAGE-A expression and grade of dysplasia for predicting malignant progression of oral leukoplakia

The risk of the malignant transformation of oral leukoplakia (OLP) is difficult to predict by histopathology. Melanoma-associated antigen-A (MAGE-A) expression is restricted to malignant cells and may be useful for the more accurate estimation of the potential malignant transformation of pre-malignant lesions. The aim of the present study was to investigate whether the expression of MAGE-A can be used to predict the malignant transformation of OLP. Paraffin-embedded tissue samples of OLP from 74 patients followed-up for at least 5 years were included. A total of 24 progressing and 50 non-progressing OLP, 18 corresponding tumor and 30 healthy mucosa specimens were analysed for MAGE-A 1, 3, 4, 6 10 and 12 expression by nested real‑time RT-PCR and graded for dysplasia. In total, 46% of the progressing lesions expressed at least 1 out of the examined MAGE-A antigens, whereas no expression was detected in any of the non-progressing OLP and normal specimens. The correlation between malignant transformation and MAGE-A expression was statistically significant (p=0.00001). Furthermore, 42% of the progressing OLPs without dysplasia (D0) expressed at least 1 antigen. The correlation between the grade of dysplasia and MAGE-A staining in the malignant transformation group was not significant (p=0.08). The detection of at least 1 MAGE-A antigen may allow the identification of high-risk lesions that may progress into carcinoma with time. Therefore, the investigation of MAGE-A expression should be assessed in order to obtain a more accurate evaluation of the potential cancer risk of OLP.

Detection of MAGE-A Expression Predicts Malignant Transformation of Oral Leukoplakia

The study aimed at checking if MAGE-A expression in oral leukoplakia (OLP) lesions is related to malignant transformation. The 48 samples of OLP that transformed to oral squamous cell carcinoma (OSCC) (group 1) and 50 samples of OLP that did not transform to OSCC (group 2) were included in the study. The expression of MAGE-A was restricted to group 1. The correlation between malignant transformation and MAGE-A occurrence in OLP was statistically significant (p < .0001). Detection of MAGE-A may allow identifying OLP with a high risk of malignant transformation giving a view to a new approach to prevention of oral cancer.

Differences in the expression of five senescence markers in oral cancer, oral leukoplakia and control samples in humans

Oncogene-induced senescence (OIS) may be a response to oncogenic activation, acting as a natural barrier against carcinogenesis at a premalignant stage. Thus, numerous cells in premalignant lesions enter senescence, but none or few in malignant tumours. This event could be due to the loss of senescence pathway effectors, including p16 (INK4a)-pRb or ARF-p53. The aim of this study was to characterize and compare the expression of certain senescent markers between oral precancer and cancer tissue samples. The expression of cyclin D1, Rb, maspin, p53 and mouse double minute 2 (MDM2) was analyzed in 20 paraffin-embedded tissue samples of normal oral mucosa (NOM), 14 samples of oral leukoplakia without dysplasia (OLD-), 11 samples of leukoplakia with dysplasia (OLD+) and 15 samples of oral squamous cell carcinoma (OSCC) by immunohistochemistry in tissue arrays. The expression of p16-pRb pathway markers, cyclin D1, maspin and Rb, was more frequent in OLD+ samples than in OSCC samples, although a statistical significance was only observed for maspin (P=0.036). Cyclin D1 expression was also significantly more frequent in OLD- samples vs. NOM samples. For the ARF-p53 pathway, the expression of p53 and MDM2 was significantly more frequent in the OLD- samples compared to in the NOM ones. These findings may indicate a role for cellular senescence in oral carcinogenesis, considering maspin as a reliable senescence marker and prognostic factor in oral premalignant lesions.

Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: An immunohistochemical study

The aim of this study was to semiquantitatively analyze the immunohistochemical expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in leukoplakias using a panel of monoclonal antibodies recognizing epitopes of CD44s and of the variant exons v5 and v6. To evaluate the efficacy of CD44s and CD44 v5, v6 immunoexpression as possible molecular markers in detecting high-risk leukoplakias when screening for this oral precancer. Samples of oral leukoplakia (40 cases) and of normal mucosa (10 cases) were evaluated. Oral leukoplakia was graded into: hyperkeratosis without dysplastic change (8 cases), mild dysplasia (13 cases), moderate dysplasia (10 cases), and severe dysplasias (9 cases). Expression of CD44s,v5, v6 was analyzed by immunohistochemistry in a semiquantitative manner. Three areas of epithelium were scored B, S, and C, i.e., stratum basale, stratum corneum, and stratum spinosum, respectively in leukoplakias. Scoring of all specimens followed a two-parameter system, which implemented percentage of positive cells and staining intensities. Statistical analyses for each parameter of all groups and normals, mean, and standard deviation were calculated by using computer software package EPISTAT. In normal epithelium CD44s, CD44v5, and CD44v6 were expressed as membranous proteins localized on the surface of epithelial cells. Both basal and spinous layer of epithelia expressed strong positive staining of CD44s, v5, v6 which then gradually faded into the negative staining of the superficial keratin layer. Profile of CD44s and v5 revealed that the mean levels of stratum B, S, and C in normal cases were comparable to the study cases and by Student 't' test P>0.05 not significant. There was, however, a statistically significant decrease in the expression of v6 with increasing grades of dysplasias when compared with normal mucosa. Among CD44s and its variant isoforms,v5, v6, in this study, variant isoform v6 may serve as a marker in detecting high-risk leukoplakias.

Immunohistochemical expression of DNA methyltransferases 1, 3a and 3b in oral leukoplakias and squamous cell carcinomas

Over-expression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers and precancerous lesions. Objective To investigate DNMT1, DNMT3a and DNMT3b enzymes in oral squamous cell carcinoma (SCC) and leukoplakia, and their relationship with histopathologic/clinical parameters. Study design Immunohistochemistry was carried out to evaluate the three DNMTs in 60 samples of oral SCC and 37 samples of oral leukoplakia. Results DNMT3a immunoreactivity in the three groups of oral SCC (39.8%) was significantly higher than in control (22.6%) (ANOVA, Student–Newman–Keuls test, P < 0.05), but not when compared to oral leukoplakia groups (28.2%). For DNMT1 and DNMT3b, there were no statistically significant differences between oral SCC groups (65% and 74.7%), oral leukoplakia groups (68.3% and 70.9%) and control (65.4% and 76.5%). There was a significantly higher mean percentage of DNMT1 immunoreactivity in non-smokers (ANOVA, P = 0.048), and a higher DNMT3a immunoreactivity in alcohol users (ANOVA, P = 0.01). Conclusions Higher DNMT3a immunopositivity may be associated with oral SCC and alcohol use, whilst lower levels of DNMT1 may be related with smoking habit. However, there was a significantly higher mean percentage of DNMT1 immunoreactivity in non-smokers (ANOVA, P = 0.048), and a higher DNMT3a immunoreactivity in alcohol users (ANOVA, P = 0.010).

Hypermethylation of the retinoic acid receptor-beta(2) gene in head and neck carcinogenesis.

Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation. RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

Frequent microsatellite alterations at chromosomes 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment

To better understand genetic alterations in oral premalignant lesions, we examined 84 oral leukoplakia samples from 37 patients who had been enrolled in a chemoprevention trial. The samples were analyzed for two microsatellite markers located at chromosomes 9p21 and 3p14. Loss of heterozygosity (LOH) at either or both loci was identified in 19 of the 37 (51%) patients. Of these 19 patients, seven (37%) have developed head and neck squamous cell carcinoma (HNSCC) while only one of 18 (6%) of patients without LOH developed HNSCC. Our data suggest that clonal genetic alterations are common in oral premalignant lesions; that multiple genetic alterations have already occurred in oral premalignant lesions, allowing at least a focal clonal expansion; and that losses of the 9p21 and 3p14 regions may be related to early processes of tumorigenesis in HNSCC. These genetic alterations in premalignant tissues may serve as markers for cancer risk assessment.