Gene expression analysis by oligonucleotide microarray in oral leukoplakia

Background:  Oral leukoplakias (LP) are the most frequent types of oral pre-cancerous lesions, but there is no accurate assessment of this malignant transformation or even genetic diagnosis of the oral epithelial dysplasia. We need to identify the new genetic diagnosis system of the epithelial dysplasia.
Methods:  Oligonucleotide microarray was used to analyze expression patterns of 29 952 genes in 10 LP patients. We compared the different gene expressions between mild dysplasia cases and severe dysplasia cases.
Results:  Ninety-six genes expressed differentially were selected as candidates for up-regulated in severe dysplasia. Subsequently, we further selected 16 genes with highest differentially expression. By hierarchical clustering analysis, the 10 cases were divided mild dysplasia from severe dysplasia.
Conclusions:  The 16 genes are suggested as biomarker gene sets of efficacy and quickly recognized in the development of oral epithelial dysplasia.     

Reduction of syndecan-1 expression during lip carcinogenesis

Background:  Actinic cheilitis (AC) is an oral pre-cancerous lesion that sometimes develops into lip squamous cell carcinoma (SCC). Syndecan-1, a transmembrane heparan sulfate proteoglycan, modulates cell-proliferation, adhesion, migration and angiogenesis. Malignant epithelial cells often down-regulate their own syndecan-1 production, and are capable of inducing aberrant syndecan-1 expression in stromal cells. The aim of this study was to evaluate the variations in syndecan-1 expression during lip carcinogenesis, in normal lip (NL), AC and well-differentiated lip SCC.
Methods:  Biopsies of NL vermillion ( n  = 19), AC ( n  = 23) and lip SCC ( n  = 24) were stained immunohistochemically for syndecan-1.
Results:  Syndecan-1 expression was significantly reduced in AC and lip SCC as compared to NL ( P  < 0.05), with a significant reduction in lip SCC as compared to AC ( P  < 0.0001). In lip SCC lesions, syndecan-1 expression at the epithelium overlying the tumor was increased when compared to the tumor itself ( P  < 0.03), but was significantly reduced as compared to AC and NL ( P  < 0.001).
Conclusion:  The results showed that epithelial syndecan-1 expression is reduced as lip carcinogenesis progresses (NL>AC>lip SCC), suggesting that syndecan-1 could be a useful marker of malignant transformation in the lip.     

Effects of thalidomide on DMBA-induced oral carcinogenesis in hamster with respect to angiogenesis

Background:  Thalidomide has been shown to have anti-angiogenic effects in pre-clinical models as well as a significant antitumor effect in hematologic tumors. However, the effects of thalidomide on oral pre-malignant lesions and oral carcinogenesis remain unexplored. The authors aimed to investigate the chemopreventive effect of thalidomide on 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters with respect to angiogenesis.
Methods:  Seventy male Syrian golden hamsters were randomly divided into five groups, with two of 20 and three of 10. DMBA solution (0.5% in acetone) was applied topically to the left cheek pouch of male Syrian golden hamsters in group A and B, while animals in group C were painted with acetone, three times a week for 6 weeks. For the next 18 weeks, animals in group B and D received thalidomide daily (40 mg/kg body weight/day) by gavage, animals in group A and C received same volume of saline. Animals in group E received no treatment and served as blank control. At the end of the experiment, animals were killed and tissue samples were collected for examinations.
Results:  Thalidomide significantly decreased the squamous cell carcinoma (SCC) incidence from 57.9 to 11.8%; angiogenesis was inhibited in dysplasia and SCC. The gene expression of vascular endothelium growth factor and tumor necrosis factor (TNF)-α was downregulated.
Conclusions:  Thalidomide has inhibitory effect against the malignant transformation of oral pre-cancerous lesion and angiogenesis during oral carcinogenesis. Such inhibition is related to its modulation of TNF-α.     

The prevalence and distribution of human papillomavirus genotypes in oral epithelial hyperplasia: proposal of a concept

Background:  Evidence is accumulating for the aetiological role of human papillomavirus (HPV) in the pathogenesis of potentially malignant oral mucosal lesions and squamous cell carcinomas.
Methods:  Paraffin tissue sections from 49 patients with 'white patches' of the oral mucosa were investigated histologically, by broad-spectrum PCR followed by genotyping and chromogenic in situ hybridisation (CISH).
Results:  Histologically, 33 flat hyperplasias and 16 papillary hyperplasias were diagnosed. Twenty-two of 28 samples studied (78.6%) were positive for HPV DNA by PCR and six were negative. The following HPV types were detected in decreasing order of prevalence: HPV 35, HPV 6, HPV16, HPV 53, HPV 18, HPV 51 and HPV 55. Seventeen samples (60.7%) contained high-risk HPV DNA. Using CISH, ≥ 1 HPV signals were detected at least in a few epithelial cells in 95% of cases studied. All but one case were positive with the high-risk HPV probe and all HPV infections contained low viral load. Concordant positive results both by PCR and CISH were detected in 14 of 19 cases (73.7%) analysed.
Conclusions:  The high prevalence of HPV infection in hyperplastic 'white patches' of the oral mucosa supports the putative role of HPV at an early stage of oral carcinogenesis. These results further indicate that the majority of white oral mucosal lesions – flat, exophytic, wart-like or papillary proliferations – could be considered as the clinical manifestations of oral HPV infection. This finding has clinical relevance regarding therapy and patient management and may help in elucidating the role of HPV infection in oral carcinogenesis.     

Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers

Objective:  Areca use is the major cause for oral squamous cell carcinoma and oral submucous fibrosis (OSF) in South Asians. Lysyl oxidase (LOX) is a copper-activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The presence of a G to A polymorphism at nucleotide 473 caused a non-conservative Arg158Gln change in the LOX amino acid sequence. OSF is a precancerous lesions characterized by the accumulation of collagen in oral submocousa. The aim of this study was to investigate the relationship between LOX Arg158Gln polymorphism and the risk of OSF.
Method:  PCR-restriction fragment length polymorphisms and direct sequencing was utilized to compare LOX polymorphic allelotype in male areca-chewing controls ( n  = 216) and OSF ( n  = 83) patients.
Results:  There was a borderline of statistically significant difference in Arg158Gln genotype lying between control and OSF patients. However, the G/A+A/A of LOX Arg158Gln in OSF patients older than 50 year was statistically significantly higher than controls older than 50 year (odd's ratio: 4.48; 95% CI = 1.58–12.67).
Conclusion:  The elder OSF patients were increased in LOX Arg158Gln. Our findings may suggest a potential application in risk population selection using LOX polymorphism for preventive intervention of OSF genesis in a subset of areca chewers.     

Physiotherapeutic treatment improves oral opening in oral submucous fibrosis

Background:  In oral submucous fibrosis (OSF) fibrous bands and burning mucosal pain restrict oral opening to limit speech and eating. The pathogenesis of OSF remains unclear, while surgical and pharmacological treatments have limited success, and are often inaccessible in communities using areca nut where OSF is prevalent. Improved outcomes are reported for surgical treatment when followed by physiotherapy. We tested the hypothesis that physiotherapy alone can modify tissue remodelling in OSF to increase oral opening.
Materials and methods:  Fifty-four Nepali OSF patients were managed for 4 months in three randomly assigned groups receiving either: five times daily physiotherapy by inter-positioning tongue spatulas between teeth and adding a new spatula every 5–10 days; local injection of hyaluronidase with steroids; or no active treatment.
Results:  More males presented with OSF than females ( p  < 0.05). All patients reported reduced opening and 47% had mucosal pain. Progressive mucosal involvement was always in the same order, starting with the soft palate, and then progressing to the fauces, unilateral buccal mucosa, bilateral buccal mucosa, floor of mouth and finally lip mucosa ( p  < 0.006). Physiotherapy improved oral opening ( p  < 0.0005), but not oral pain, while no clear improvement was seen in untreated patients as well as patients managed by injection.
Conclusions:  We conclude OSF in the Nepali population progresses in a predictable pattern, and that physiotherapy is effective for increasing the oral opening. We further suggest physiotherapy can be readily used to improve OSF in communities with otherwise limited health resources.     

Genetic analysis of DNA microsatellite loci in salivary gland tumours: comparison with immunohistochemical detection of hMSH2 and p53 proteins

To investigate genetic alterations in salivary gland tumours, microsatellite instability at eight representative loci and loss of heterozygosity (LOH) on chromosome 17 were analysed by polymerase chain reaction amplification. The results were compared with immunohistochemical expression of the hMSH2 and p53 proteins. Microsatellite instability and expression loss of hMSH2 protein were not recognized in the salivary gland tumours, suggesting a low frequency of abnormalities of the mismatch repair system. LOH associated with the p53 gene was detected in approximately one-half of pleomorphic adenomas and salivary carcinomas, which often showed strong p53 immunoreactivity. These features suggest that the p53 gene plays an important role in malignant transformation of salivary gland tumours. The genetic characteristics of pleomorphic adenomas might reflect a low-grade potential for malignant progression.     

Alterations of FHIT and P53 genes in keratocystic odontogenic tumor, dentigerous and radicular cyst

Background:  The purpose of this study was to determine fragile histidine triad (FHIT) and p53 protein expression, and to analyze FHIT and p53 gene status in keratocystic odontogenic tumor (KOT), dentigerous cysts (DC) and radicular cysts (RC).
Methods:  The methods used were immunohistochemistry and molecular genetic methods including loss of heterozygosity (LOH) and gene sequencing.
Results:  FHIT protein expression was different among groups. Aberrant expression was the highest in KOT, then in RC and DC. p53 protein expression was different among groups. LOH in paraffin-embedded specimens was detected in 22.6% and 12.9% for FHIT and p53 respectively. Mutation of p53 gene at codon 237 was observed in only two specimens (one KOT and one DC). Of the six frozen specimens, three exhibited FHIT gene LOH (two RC and one KOT). KOT showed loss of exons 6–7 at FHIT locus and mutation at codon 237 at p53 locus, but this could be a chance result.
Conclusion:  Aberrations of FHIT and p53 genes/proteins could be considered markers responsible for the development of odontogenic lesions.     

Variant haplotypes at XRCC1 and risk of oral leukoplakia in HPV non-infected samples

Background:  One of the mechanisms in human papillomavirus (HPV)-related carcinogenesis is inhibition of DNA repair by HPV oncoprotein. In this study, we investigated whether polymorphisms at XRCC1 , one of the DNA repair loci, could modulate the risk of tobacco-related leukoplakia and cancer in HPV-infected individuals.
Methods:  Tissue DNA from 83 oral cancer, 91 leukoplakia and 100 healthy controls were screened for HPV 16/18 infection and polymorphisms at XRCC1 by PCR–RFLP to estimate the risk of diseases independently and jointly.
Results:  Human papillomavirus infection was significantly associated with increased risk of leukoplakia and cancer (OR = 2.8, 95% CI = 1.2–6.5 and OR = 5.5, 95% CI = 1.6–19, respectively). Independently, genotypes at three polymorphic sites on XRCC1 did not modulate the risk of diseases but pooled variant haplotypes increased the risk of leukoplakia in overall and HPV non-infected (OR = 1.8, 95% CI = 1.2–2.8; OR = 2.2, 95% CI = 1.2–4.0, respectively) samples but not that of cancer.
Conclusion:  The association between variant haplotypes at XRCC1 and risk of leukoplakia is pronounced in non-infected individuals since HPV oncoprotein could inhibit directly the DNA repair activity of XRCC1. But more samples of leukoplakia and cancer are essential to validate these results.     

Human papillomavirus as a risk factor in oral carcinogenesis: a study using in situ hybridization with signal amplification

Introduction:  It is still controversial whether human papillomavirus (HPV) can be considered a risk factor in oral carcinogenesis. The aim of this study was to detect HPV DNA in 50 cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas, using in situ hybridization with signal amplification (CSA-ISH).
Methods:  HPV DNA was assessed in paraffin sections using CSA-ISH with a wide-spectrum biotinylated DNA probe. In HPV-positive cases, genotyping with specific probes to HPV types 6/11, 16/18 and 31/33 was performed.
Results:  The overall prevalence of HPV infection was 24%, markedly higher than that found in the control group. Results showed a discrete proportional relationship in the indices found in leukoplakia with no dysplasia, leukoplakia with dysplasia, and squamous cell carcinoma, but this was not statistically significant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. In genotyping, HPV types 16/18 were the most prevalent, and types 6/11 were only found in groups of mild or no dysplasia.
Conclusion:  The results suggest that HPV is not likely to play a role in the progression of malignant transformation in oral lesions. Nevertheless, the increased prevalence of HPV infection compared to normal oral mucosa and the fact that high-risk HPV types were the most frequently identified do not allow the exclusion of HPV as a risk factor in oral carcinogenesis.     

Oral cancer: diagnostic options as an aid to histology in order to predict patients at high risk for malignant transformation

Oral cancer continues to afflict the general population in growing numbers with a relatively low survival rate. Today, through histology, the potential danger of a pre-cancerous lesion can be detected, yet it does not reflect with certainty its biologic behavior. Recent studies have shown that some pre-cancerous lesions with mild dysplasia may undergo malignant transformation and lesions with severe dysplasia may remain quiet. Our goal is to locate the relatively small population of patients that are at high risk for malignant transformation at an early stage of carcinogenesis so they can be treated effectively. Methods that can identify the molecular and DNA changes may provide the data needed to predict the true malignant potential of a pre-cancerous lesion and its potential for transformation with a higher degree of certainty. The purpose of this article was to review some of the diagnostic options that are being studied and their link to carcinogenesis.     

Loss of heterozygosity at 5q 21–22 (adenomatous polyposis coli gene region) in oral squamous cell carcinoma is common and correlated with advanced disease

We determined the frequency of loss of heterozygosity (LOH) at chromosome 5q21–22 (adenomatous polyposis gene region) in oral SCC from 49 patients using PCR-based assays. Of 43 informative (heterozygous) tumors, 41.9% [95% confidence interval (CI)=27.0, 57.9] contained LOH at 5q21–22. LOH at 5q21–22 was strongly associated with stage at diagnosis: 100% (3/3), 50% (13/26), and 14% (2/14) of tumors from patients with distant metastases, regional spread, and localized disease, respectively, contained this genetic alteration ( P =0.01). There were no statistically significant associations between LOH at 5q21–22 and other patient or tumor characteristics, but LOH was more commonly found in the tumors of heavy smokers, infrequent alcohol consumers, and in tumors containing either p53 mutations or HPV-DNA. In univariate analyses, LOH at 5q21–22 was associated with poor prognosis (hazard ratio=1.8, 95% CI 0.8, 4.5); this relationship did not persist after adjustment for stage of disease (hazard ratio=1.1, 95% CI=0.4, 3.1). These data provide further evidence that inactivation of the APC gene and/or other genes at 5q21–22 is common and may be involved in the development and/or progression of oral SCC. Larger studies are needed to determine whether LOH at 5q21–22 is linked to known oral SCC etiologic factors and/or the prognosis of oral SCC patients, as well as to genetic instability at other loci involved in these malignancies     

Untersuchung der Tumorsuppressoren p16INK4a und p14ARF in Mundschleimhautleukoplakien

The inactivation of p16 and p14^ARF is considered to be an important step in the carcinogenesis of oropharygeal carcinomas. This consideration is supported by the observation of multiple allelic losses in the coding loci of chromosome 9p21 in squamous cell carcinomas and in dysplastic premalignant lesions. The present study hypothesized that comparable alterations already occur in leukoplakia, which are seen as potential predecessors of oral squamous cell carcinomas and that it is possible to differ leukoplakia with from leukoplakia without further malignant transformation. Furthermore we evaluated, whether such leukoplakia show sequence alterations in the genes p16 and p14^ARF, which are capable to cause a limitation in gene function. The results show that the LOH pattern in genes p16 and p14^ARF occur as well in leuplakia with malignant transformation as in leukoplakia, that do not show clinical alterations. The rate of allelic loss did not differ significantly. Overall, the incidence of allelic loss was lower in leuplakia compared to succeeding squamous-cell carcinomas (p?<?0,05). The results further illustrated an increase in LOH patterns in dyplastic leukoplakia, without reaching statistical significance. Significant increases in allelic losses were found in heavy smokers, (p?<?0,05). PCR analysis of the exons 1-alpha, exon 1-beta and exon 2 in leukoplakia, containing LOH patterns did not show genetic alterations. Thus we concluded, that gene deletion and gene mutation have a minor role in the inactivation process of p16 and p14^ARF in oral leukoplakia. Representing an early process in carcinogenesis, gene deletion and mutation occur in leukoplakia with and without malignant transformation. Therefore, taken as a singular parameter they represent an uncertain criteria to assess the potential of malignant transformation. However they could provide information in combination with other genetic factors like chromosomal methylation patterns and histology.     

Fine deletion analysis of 1p36 chromosomal region in oral squamous cell carcinomas

Background:  Squamous cell carcinoma is the most common cancer type of the oral cavity and approximately 50% of the patients succumb to the disease. Unfortunately, few are known about the molecular mechanisms involving in the formation of oral squamous cell carcinoma (OSCC). Recently, it has been reported that 1p36 chromosomal region is deleted in various cancer types and is suspected to harbor various tumor suppressor genes (TSGs). However, limited studies exist on genetics alteration on 1p36 in OSCC and the responsible TSG remained unidentified.
Methods:  To investigate area susceptible to harbor TSG(s) involved in OSCC on 1p36 region, paired normal and tumor tissues of 27 patients with diagnosis of OSCC have been analyzed for loss of heterozygosity (LOH) using nine microsatellite markers based on recent gene mapping.
Results:  LOH was found at least in one locus in 85% of the cases (23 of 27). Interestingly, microsatellite instability was also found in 7% (two of 27) of the cases analyzed. The higher LOH frequencies were found with the markers D1S243 (25%), D1S468 (22%), D1S450 (25%), D1S228 (38%), D1S199 (28%), and D1S1676 (23%).
Conclusions:  Three preferentially deleted regions have been identified in OSCC: region 1 (D1S468-D1S243), region 2 (D1S450-D1S228), and region 3 (D1S199-D1S1676). Multiple candidate TSGs, such as RIZ1, p73, UBE4B, Rap1GAP, EPHB2, and RUNX3, are located in these three areas. The data obtained in this study can be used for further functional analysis of these genes involved in OSCC carcinogenesis.     

The role of basic fibroblast growth factor in oral submucous fibrosis pathogenesis

Background:  Oral submucous fibrosis (OSF) is a chronic fibrotic disease of oral mucosa and oropharynx, induced by betel quid chewing often resulting in restricted mouth opening. The principal cells implicated as a source of extracellular matrix in areas of fibrosis are fibroblasts. Accumulation of connective tissue matrix is secondary to factors such as cytokines and growth factors. The contribution of basic fibroblast growth factor (bFGF) in disease progression and the consequent stromal changes with increase in the severity of OSF was studied.
Methods:  A case series analysis of 30 cases of OSF was carried out for bFGF expression using immunohistochemistry. Connective tissue changes in these cases were corroborated using aldehyde fuchsin and Verhoeff's hematoxylin special stains.
Results:  bFGF immunoreactivity was found to be increased in fibroblasts and in endothelial cells in early OSF cases, while the expression of bFGF in stroma increased notably in advanced fibrosis.
Conclusion:  Increased bFGF expression in early stages of the disease was explainable to an initial injury phase because of areca consumption, followed by cellular activation by chemotactic cytokines and other growth factors with eventual fibrosis occurring as a result of molecular alteration at the cellular level.     

Apoptotic mechanism of paclitaxel-induced cell death in human head and neck tumor cell lines

Background:  Paclitaxel (taxol) is clinically used to treat various human tumors. However, the cellular and molecular mechanism regarding apoptotic effect of paclitaxel on head and neck squamous cell carcinoma (HNSCC) remains elusive.
Methods:  The apoptotic effect and the mechanism of paclitaxel on FaDu hypopharyngeal cancer cell line, OEC-M1 gingival cancer cell line, and OC3 betel quid chewing-related buccal cancer cell lines were investigated by morphological observations, cell viability assay, flow cytometry assay and Western blotting methods.
Results:  Rounded-up cell number increased with membrane blebbing as the treatment of paclitaxel (50–500 nM) increased from 24 to 48 h among these cell lines. In cell viability assay, cell surviving rate significantly decreased from 87 to 27% as the dosage and duration of paclitaxel treatment increased ( P  <   0.05). Flow-cytometry analysis further demonstrated that 50 nM paclitaxel induced G2/M phase cell arrest among these cell lines within 8 h treatment, and then G2/M phase cell fraction significantly decreased as subG1 phase cell fraction significantly increased after 24 h treatment ( P  <   0.05), suggesting that cells underwent apoptosis. Furthermore, the activated caspases-8, -9, -3, -6 and poly ADP-ribose polymerase cleavage could all be significantly detected in FaDu, OEC-M1 and OC3 cells ( P  <   0.05).
Conclusion:  Paclitaxel activated cell cycle arrest and caspase protein expressions to induce apoptosis in HNSCC cell lines.     

Multiple logistic regression analysis of risk factors for carcinogenesis of oral submucous fibrosis in mainland China

Oral submucous fibrosis (OSF), a chronic progressive disorder, is regarded as the premalignant lesion of oral squamous cell carcinoma (OSCC). Its distribution is associated with chewing betel quid (BQ). The objective of the present study was to investigate risk factors for the carcinogenesis of OSF in mainland China. A case-control study was performed in 42 patients with OSCC that originated from OSF and 40 OSF controls. Epidemiological data and information related to risk factors were collected using a short structured questionnaire. Odd ratios (OR) and 95% confidence intervals (CI) were derived from logistic regression analysis. In multivariate analysis, only age, duration of BQ chewing, duration of cigarette smoking, and OSF accompanied by oral leukoplakia or oral lichen planus were associated with significantly increased risk for the malignant transformation of OSF. These findings contribute to current knowledge on the prevention of carcinogenesis of OSF in mainland China.     

MYC gene amplification reveals clinical association with head and neck squamous cell carcinoma in Indian patients

Background:  Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). This study is focused to analyze the correlation between MYC gene amplification and various clinicopathological features and outcome in a cohort of 49 dysplastic and 187 primary head and neck lesions.
Methods:  MYC gene amplification was assessed by differential polymerase chain reaction using primer sets from the MYC gene as target locus and DRD2 gene as the control locus.
Result:  The MYC gene amplification was detected in a total of 23.7% (56/236) head and neck lesions comprising 14.2% (7/49) dysplastic lesions and 26% (49/187) HNSCC samples. The clinicopathological association study between MYC gene amplification with the different clinical parameters like sex, tumor stage, tumor differentiation, lymph node status, tobacco habit and HPV 16/18 status determined significant association of MYC amplification with tumor progression ( P  = 0.009). Kaplan Meier analysis revealed MYC gene has no prognostic significance on survival in HNSCC.
Conclusion:   In conclusion, our results suggest that MYC gene amplification is associated with tumor progression in HNSCC.