Vascular endothelial growth factor 936 C/T polymorphism is associated with vascular invasion in oral squamous cell carcinoma.

OBJECTIVE: The objective of this study was to analyze the relation of VEGF 936 C/T polymorphisms in progression of oral squamous cell carcinoma (OSCC) patients and healthy subjects in Taiwan. STUDY DESIGN: We genotyped 218 patients with OSCC, comparing the genotypes and gene frequencies with those of 121 control subjects. VEGF 936 C/T polymorphisms were determined by using polymerase chain reaction and restriction fragment length polymorphism. RESULTS: There were no significant differences in genotype, phenotype, or gene frequency between the OSCC and control groups. Among patients with OSCC, there were also no significant differences in the polymorphism between those with and without cervical lymph node metastases or in survival. However, 21 of the 218 patients had vascular invasion by their OSCC, and these patients were significantly more likely to have a C/C (P = .033) or C/T genotype (P = .026) than were those without vascular invasion. CONCLUSIONS: This study suggests that the VEGF 936 C allele is associated with vascular invasion in OSCC.

Sarcomas and sarcomatoid tumor after radiotherapy of oral squamous cell carcinoma: analysis of 4 cases.

Radiation-induced sarcoma (RIS) or postirradiation sarcoma has been reported rarely as a long-term complication of radiation therapy (RT). We report 4 cases of oral sarcomas or sarcomatoid tumors with a rather short latency period after radiotherapy of the prior OSCC. Histopathological evaluation and immunohistochemical study were performed using a panel of markers including vimentin, cytokeratin, S-100, desmin, myoglobin, HHF-35, p53, and p16. All reported cases were positive for vimentin and negative for cytokeratin. Two cases were positive for myoglobin, desmin, or HHF-35, and were probably myogenic origin. One case was possibly a fibrosarcoma and the subclassification of the other one was not specified. Diverse expression of p53 and p16 was further observed in these 4 cases. Report of the complicated clinical processes and the analysis of genetic markers of these cases provide useful clinical and pathogenetic insights of mesenchymal malignancies associated with a status post OSCC radiation.

Effects of sonic and ultrasonic scaling on the surface roughness of tooth-colored restorative materials for cervical lesions.

This study investigated the effects of sonic and ultrasonic scaling on the surface roughness of five commonly used tooth-colored restorative materials for Class V cavities, including a flowable resin composite (Tetric Flow), a compomer (Compoglass F), a glass ionomer (Fuji II), a resin-modified glass ionomer (Fuji II LC Imp) and a resin composite (Z100). Twenty rectangular block specimens (16 x 6 x 1.5 mm) of each material were cured against matrix strips, then stored in artificial saliva for two months before performing the periodontal instrumentation. Each specimen was divided into two experimental zones, and both scaling treatments were performed on each sample. The surface roughness (Ra) of these materials was determined before and after the different instrumentations, and differences were evaluated with the use of a profilometer. Data were statistically analyzed using repeated measures of ANOVA with Tukey's multiple comparisons and paired t-tests at a significance level of 0.05. Significant increases in surface roughness of all test materials were recorded from both scaling treatments. With the exception of Tetric Flow, ultrasonic scaling had more adverse effects on the surface roughness of all test materials compared to sonic scaling. For the test materials Z100 and Tetric Flow, resin composites showed the least surface changes in both scaling treatments, while Fuji II glass ionomer demonstrated the greatest roughness after instrumentation. More importantly, the mean surface roughness values of several materials after instrumentation were above the critical threshold roughness of 0.2 microm.

Use of methylene blue as a diagnostic aid in early detection of oral cancer and precancerous lesions.

We assessed the sensitivity, specificity, and positive and negative predictive value of methylene blue staining in the diagnosis of oral cancer in 58 patients. The sensitivity was 90%, the specificity 69%, positive predictive value 74%, and negative predictive value 87%. Because of the number of false negatives and false positives we recommend that the diagnosis should always be confirmed by histopathological examination of a biopsy specimen. Methylene blue staining may, however, be useful as a screening tool for oral cancer in large, high-risk groups in a similar way to the more expensive toluidine blue.

Areca nut extract treatment down-regulates involucrin in normal human oral keratinocyte through P13K/AKT activation.

Areca (betel) is an important etiological factor linked to the high prevalence of oral carcinoma and other oral diseases in South Asians. Involucrin is a key component of the cornified envelop and a differentiation marker of keratinocyte. In this study, we found that 5 microg/ml non-toxic areca nut extract (ANE) treatment resulted in the 0.5-fold down-regulation of involucrin and disruption in involucrin distribution in normal human oral keratinocyte (NHOK). Progressive down-regulation of involucrin during oral carcinogenesis was noted. Activation of AKT by 1.7-fold and up-regulation of COX-2 by 2-fold were elicited following ANE treatment in NHOK. Treatment with PI3K/AKT blockers reverted the down-regulation of involucrin. ANE also down-regulated involucrin by 0.6-fold and disturbed both cornified envelope and cell aggregation in calcium-induced differentiated NHOK. However, such phenomena seemed to be independent from the ANE-associated COX-2 activation. The ANE-associated down-regulation of involucrin through AKT pathway could underlie the areca-associated epithelial pathogenesis.

Functional polymorphism in NFKB1 promoter is related to the risks of oral squamous cell carcinoma occurring on older male areca (betel) chewers.

Areca (betel)-chewing is tightly associated with the high prevalence of oral squamous cell carcinoma (OSCC) in Asians. NFKB1 encodes a 105kDa protein that can be processed to produce p50 subunit of nuclear factor-kappaB protein complex. A insertion (ins)/deletion (del) polymorphism (-94ins/delATTG) in NFKB1 promoter, which may drive the ins allele two-fold increase in NFKB1 transcription relative to del allele, was recently found. This study identified that the odds ratio in OSCC carrying ins allelotype were 1.78 relative to controls (56.7 vs 41.8%) in subjects more than 50 years old. L allelotype of Heme oxygenase-1 (HO-1), accounting for a long (GT)(n) repeat in HO-1 promoter, is associated with the risks of areca-related OSCC. Subjects carried both NFKB1 ins and HO-1 L allelotypes had significant risks for various subsets of OSCC. OSCC with lymph node metastasis or advanced stage had significantly higher frequency of NFKB1 ins and HO-1 L allelotypes. This study suggested that the functional NFKB1promoter polymorphism could be valuable for assessment of cancer risk.

Association of GST genotypes with age of onset and lymph node metastasis in oral squamous cell carcinoma.

BACKGROUND: Environment-gene interaction in oral carcinogenesis is well demonstrated by phase I and II enzymes that are involved in the metabolism of carcinogens. This study investigated the association of glutathione S-transferase (GST)T1 and GSTM1 genotypes of phase II enzyme genes with risk for, age of onset, and neck lymph node metastasis (LNM) in areca-associated oral squamous cell carcinoma (OSCC). METHODS: A total of 114 OSCC male patients and 100 male controls were recruited. All subjects were areca users and tobacco smokers. DNA was obtained from peripheral blood samples. Genotyping of GSTT1 (non-null/null) and GSTM1 (non-null/null) was determined by polymerase chain reaction (PCR) analysis using specific primers that only amplify non-null alleles. RESULTS: No association was found between GST genotype and the risk of OSCC based on case-controls. Patients with the GSTT1 null genotype were older at onset (P = 0.03). Those with the GSTM1 null genotype had a higher incidence of neck LNM than those with the GSTM1 non-null genotype (P = 0.01). Patients with the GSTM1/GSTT1 null genotype appeared to have later onset and a higher incidence of neck LNM than those carrying the opposite genotype. CONCLUSION: The GST genotypes may be important markers for the age of onset and risk of metastasis in OSCC. The data also suggest that the various GST isoforms may be differentially involved in development or progression of OSCC.

Frequent microsatellite alterations of chromosome locus 4q13.1 in oral squamous cell carcinomas.

BACKGROUND: Studies have revealed that losses of chromosome 4q24-25 regions are frequent in cancers including head and neck squamous cell carcinoma. Our previous comparative genomic hybridization analysis showed extensive losses of chromosome arm 4q in oral squamous cell carcinoma (OSCC). METHODS: To be more precise in mapping the potential regions of allelic losses and to understand the microsatellite instability (MSI) on 4q involving in oral pathogenesis, we performed allelotypings using eight polymorphic markers. Microsatellite analyses were first performed on 100 randomly selected controls to confirm the high informative rates of markers. Twenty OSCC tissues were microdissected from surgical specimens for microsatellite alterations (MA) analysis. RESULTS: MA was observed in 95% OSCC cases. The most eminently altered locus was 4q13.1 (75%), followed by 4q22.2 and 4q32.1 (55%). Allelic losses also occurred most frequently on these loci. Thirty-five percent cases had MA spanning 4q13.1 to 4q21.1. MSI occurred in 35% OSCC, at a lesser extent compared with allelic losses. The most common locus for MSI was 4q21.2 (20%). In addition, 4q MSI was significantly associated with the lymph node metastasis of OSCC (P = 0.01). So far, most tumor suppressor genes on 4q have not been specified. CONCLUSION: Our results were additive to previous findings and proposed novel scenario of suppressor loci located at 4q13.1-21.1 whose inactivation could be important for progression of OSCC.

The involvement of K(v)3.4 voltage-gated potassium channel in the growth of an oral squamous cell carcinoma cell line.

BACKGROUND: In our previous study, an A-type voltage-gated K(+) channel, K(v)3.4, was found more frequently expressed in oral squamous cell carcinoma (OSCC) when compared with non-cancerous matched oral tissue. An OSCC cell line, OECM-1, was found to have moderate level of K(v)3.4 expression. METHODS: To further elucidate the roles of K(v)3.4 for the involvement of neoplastic process, we amplified K(v)3.4 coding sequence by reverse transcriptase polymerase chain reaction (RT-PCR), constructed an expression vector carrying this sequence and then stably transfected into OECM-1 OSCC cells. RESULTS: We demonstrated the integration and constitutive expression of K(v)3.4 in the cell. A unique A-type current elicited by such expression in OECM-1 cells was defined by patch clamp analysis. This current pattern can be reversibly blocked by an A-type K(+) channel blocker 2 mM 4-aminopyridine (4-AP). The acquisition of K(v)3.4 activity in OECM-1 cells bestowed growth advantage. However, in 3T3 cell, transfected K(v)3.4 caused only limited increase of growth without forming transformation foci. CONCLUSION: The present study established a stable keratinocyte system carrying functional K(v)3.4 and increase of growth, by which the anti-K(v)3.4 modalities for potential OSCC control can be further investigated.

Genome-wide profiling of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.

The increase of voltage-gated potassium channel Kv3.4 mRNA expression in oral squamous cell carcinoma.

BACKGROUND: Potassium channels have been reported to be involved in the proliferation of many types of cells, including tumor cells. The overexpression of the K+ channel and related channel activity are involved in the neoplastic process. METHODS: We examined the expression of an A-type voltage-gated K+ channel, Kv3.4, in oral squamous cell carcinoma (OSCC) and esophageal squamous cell carcinoma (ESCC) compared with non-cancerous matched tissue (NCMT) using RT-PCR analysis. In addition, administration of an A-type K+ channel blocker, 4-aminopyridine (4-AP), and an antisense oligodeoxynucleotide (ODN) directed specifically against Kv3.4 were performed to identify the involvement of Kv3.4 in the growth of OSCC cells. RESULTS: A significantly increase in the frequency of Kv3.4 mRNA expression was identified in OSCC (64%) compared to corresponding NCMT (29%) (P = 0.05). The increase of Kv3.4 mRNA expression was also eminent in ESCC. Growth of OSCC cells was significantly inhibited by 4-AP in a dose-dependent manner at different time point of treatment. In OECM-1 OSCC cells, a significant growth inhibition was noted in antisense ODN-treated cells compared to control cells. CONCLUSION: We provide novel evidences of the increase of Kv3.4 mRNA expression in OSCC. The abrogation of Kv3.4 inhibits the growth of OSCC cells.

Elevated expression of cyclooxygenase (COX)-2 in oral squamous cell carcinoma--evidence for COX-2 induction by areca quid ingredients in oral keratinocytes.

BACKGROUND: Elevated expression of cyclooxygenase (COX)-2 has been demonstrated in several human cancers. Whether COX-2 is up-regulated in areca quid (AQ) related oral squamous cell carcinoma (OSCC) is unknown and the potential of AQ ingredients to induce COX-2 expression has not been studied. METHODS: COX-2 expression was analyzed by immunohistochemistry and RT-PCR in oral tissues. The COX-2 mRNA and protein induction potential of AQ ingredients were analyzed by real-time RT-PCR and Western blotting in normal human oral keratinocyte (NHOK). RESULTS: COX-2 protein expression was significantly higher (P < 0.01) in OSCC (n = 27) as compared to their adjacent non-cancerous matched tissue (NCMT). COX-2 protein was nearly undetectable in control normal oral mucosa. The level of COX-2 mRNA was markedly elevated in 63% (12/19) of OSCC compared to NCMT. Hydroxychavicol induced COX-2 mRNA and protein expression in NHOK. CONCLUSIONS: COX-2 protein as well as mRNA expression were significantly enhanced in OSCC as compared to NCMT. Hydroxychavicol, a unique ingredient in AQ, induced COX-2 expression in NHOK, which highlighted early involvement of COX-2 in AQ-associated oral oncogenesis.

The biphasic differential expression of the cellular membrane protein, caveolin-1, in oral carcinogenesis.

BACKGROUND: The increased expression of Cav-1 is seen in various cancers from prostate, esophagus, colon, breast and pancreas yet the information regarding the correlation between the expression of Cav-1 and oral cancer is blind. Thus, the expression profile of caveolin-1 (Cav-1) in oral carcinogenesis and the correlation to the clinicopathologic covariates are examined in this study. METHODS: Immunohistochemistry was used to detect Cav-1 expression in non-cancerous matched tissues (NCMT; n=12), and tissue from normal oral mucosa (NOM; n=12), oral pre-cancer lesions (OPL; n=17), primary oral squamous cell carcinoma (POSCC; n=47) and metastatic OSCC (MOSCC; n=8). The Cav-1 expression was correlated to the age, site, areca use, stage, size, nodal involvement, and differentiation stage. Western blot was used to confirm the specificity of antibody and to follow changes in Cav-1 expression. RESULTS: The Cav-1 immunoreactivity increased significantly from 8% in NOM and 17% in NCMT to 53% in OPL and 79% in POSCC. In addition, lymph node metastasis (LNM) was present in 62% of Cav-1(+) POSCCs, but only in 10% of Cav-1(-) POSCCs. Remarkably, only 38% of MOSCCs had Cav-1 immunoreactivity. CONCLUSION: An increased Cav-1 expression is seen in the stepwise carcinogenesis from NOM, NCMT, OPL to POSCC. The decrease in expression from the POSCC to MOSCC indicates the value to explore its biphasic functions in oral carcinogenesis. Whether Cav-1 is an important predictor or prognosis for survival still awaits the extension of clinical follow-up.

Cyclin D1 genotype in areca-associated oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignancy in areca-chewing regions, accounting for up to 50% of malignant tumors in some South Asian countries. Amplification and/or over-expression of cyclin D1 (CCND1) is a frequent event in human malignancies, including OSCC. CCND1 G870A polymorphism (codon 242) gives rise to two isoforms of the protein. The objective of the present study was to evaluate if the risk, onset, and prognosis of areca-associated OSCC is related to CCND1 genotypes. METHODS: We analyzed the CCND1 genotype in 70 OSCC cases and 93 control Taiwanese using single-strand conformation polymorphism techniques. RESULTS: Statistical analysis showed that CCND1 genotype had no impact on the risk, onset, or survival of areca-associated OSCC. However, buccal squamous cell carcinoma (BSCC) appeared to be less frequently associated with AA genotype than non-BSCC (P = 0.02). In addition, amplification of CCND1 was significantly more prevalent in OSCC cases (22%) than in control subjects (2%, P < 0.01). CONCLUSION: This study demonstrates that the CCND1 genotype may confer different risks for BSCC and non-BSCC.

Genetic polymorphism of cytochrome P4501A1 and susceptibility to oral squamous cell carcinoma and oral precancer lesions associated with smoking/betel use.

BACKGROUND: The importance of the CYP1A1 polymorphisms at exon 7 (Ile/Val) and 3'-untranslated region (3'-UTR) has been controversial in oral squamous cell carcinoma (OSCC) or head and neck SCC (HNSCC) denoting the value of exploring the correlation between these polymorphisms and risk of betel/smoking associated OSCC. It is also important to evaluate the association between CYP1A1 polymorphisms and susceptibility of oral precancerous lesion (OPL) to confirm the findings in OSCC cases. METHODS: We examined polymorphic prevalence of CYP1A1 at exon 7 (Ile/Val) and 3'-UTR in 106 cases with OSCC, 60 cases with OPL, and 146 controls. DNA isolated from surgical specimens and whole blood was used for PCR-based genotyping. RESULTS: The prevalence of the CYP1A1 A/G genotype (Ile/Val) and G/G genotype (Val/Val) in exon 7 of cases with OSCC (79.2 and 7.6%) and OPL (68.3 and 10%) were significantly higher than in controls (53.4 and 1.4%) (P < 0.0001). The novelty of the present study is that we identified the onset age of OSCC in CYP1A1 A/G genotype to be significantly younger than that in A/A genotype (P < 0.01). No significant difference was seen between cases and controls regarding the polymorphisms at 3'-UTR. CONCLUSION: The findings indicate that the individuals with the CYP1A1 exon 7 containing G allele were at increased risk for OSCC and OPL.

The retinoic acid receptor-beta (RAR-beta) mRNA expression in the oral squamous cell carcinoma associated with betel quid use.

BACKGROUND: Within the abundant retinoids nuclear receptors, abnormally low expression of the RAR-beta has been shown to contribute to neoplastic progression in oral epithelium in western countries. Distinctly different risk factors contributing to oral squamous cell carcinoma (OSCC) in epidemiologically different societies denote the value of exploring the role of RAR-beta expression in OSCC associated with betel quid (BQ) use in our society. METHODS: We examined the cellular expression of RAR-beta using in situ hybridization (ISH) analysis on 38 pairs of surgical specimens of primary OSCC and non-cancerous matched tissues (NCMT) to correlate with their clinico-pathological features including age, sites of tumor, habit of BQ use, stage, size of primary tumor, lymph node metastasis, differentiation. RESULTS: Of all cases analyzed, BQ users were significantly younger than non-BQ users (51.2 +/- 2.1 vs. 60.2 +/- 2.6, P = 0.01). 52% OSCC of BQ users (13/25) and 23% OSCC of non-BQ users (3/13) exhibited the absence of RAR-beta expression. In 17 paired-samples from buccal mucosa (BM), most NCMT and less than half of OSCC exhibited RAR-beta expression (16/17, 94% vs. 8/17, 47%, P = 0.003). The RAR-beta expression was seen in the vast majority of the well-differentiated OSCC and in less than half of the moderately differentiated OSCC only (15/20, 75% vs. 7/18, 39%, P = 0.03). CONCLUSION: A correlation between the loss of RAR-beta expression and more advanced histopathological grade tumors was observed. This study also suggests that the loss of RAR-beta expression is significant in BM OSCC, which preferentially occurs in BQ users.