Investigation of PTEN promoter methylation in ameloblastoma.

BACKGROUND: Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene. Inactivation of PTEN has been reported in various types of cancers. PTEN promoter methylation possibly underlies PTEN inactivation, which results in tumorigenesis. The aim of this study was to investigate whether PTEN promoter methylation contributes to PTEN inactivation in ameloblastoma and its associated protein expression. MATERIAL AND METHODS: In total, 20 fresh-frozen ameloblastoma samples were evaluated for PTEN promoter methylation using methylation-specific polymerase chain reaction (MS-PCR). A subset of 10 paraffin-embedded ameloblastoma samples was examined for PTEN expression through immunohistochemistry. Four primary cultured ameloblastoma cells were investigated for PTEN promoter methylation and PTEN transcriptional expression via reverse transcription PCR. RESULTS: PTEN promoter methylation was detected in 65% (13/20) of the ameloblastoma samples. Of 10 ameloblastoma samples, 4 exhibited reduced PTEN expression. Of 5 samples with methylated PTEN, 3 (60%) were associated with loss of PTEN expression. However, PTEN expression was detected in 4 (80%) of 5 samples with unmethylated PTEN. In addition, 3 (75%) of 4 primary ameloblastoma cell cultures exhibited an inverse correlation between PTEN promoter methylation and PTEN transcription level. CONCLUSIONS: PTEN promoter methylation is found in a number of ameloblastomas but not significantly correlated with loss of PTEN expression. Genetic or epigenetic mechanisms other than PTEN promoter methylation may contribute to PTEN inactivation in ameloblastoma tumor cells.

TRH site-specific methylation in oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) continues to increase each year. Clinical examination and biopsy usually detect OSCC at an advanced stage that is difficult to treat, leading to poor prognosis. DNA methylation pattern is tissue specific and has emerged as a biomarker for the detection of cancers of tissue origin. Herein, we aimed to discover a novel site-specific methylation marker for OSCC. METHODS: We selected OSCC datasets analyzed using the IlluminaHumanMethylation27 BeadChip from the Gene Expression Omnibus repository of the National Center for Biotechnology Information using a bioinformatics approach. From 27,578 CG dinucleotide (CpG) sites, the CpG site with the highest difference in methylation level between healthy and cancerous cells was selected for further validation. A total of 18 mucosal tissue samples were collected from nine healthy controls and nine from OSCC subjects and subjected to microdissection for cell purification, followed by DNA extraction, bisulfite conversion, and pyrosequencing. Additionally, epithelial cells were collected from 2 cohorts including oral rinse from healthy controls, oral rinse and oral swab from OSCC subjects and oral rinse from oropharyngeal squamous cell carcinoma (SCC) were examined for their methylation status using real-time polymerase chain reaction (PCR). RESULTS: Among the 27,578 differentially methylated CpG sites, cg01009664 of the thyrotropin-releasing hormone (TRH) gene showed the greatest difference in methylation level between healthy and cancerous cells. Validation of the TRH gene using pyrosequencing revealed a methylation percentage of 7% ± 3.43% in healthy cells in contrast to 63% ± 19.81% in cancerous cells. Screening of epithelial cells using real-time PCR showed that the DNA methylation level was significantly higher in oral swab and rinse samples collected from OSCC and oropharyngeal SCC subjects than those from healthy controls (p < 0.001). In addition, when using a cutoff at 3.31 ng/µL, the TRH methylation biomarker was able to distinguish OSCC and oropharyngeal SCC subjects from healthy controls with high level of area under the curve, sensitivity and specificity. CONCLUSION: We demonstrated cg01009664 of TRH as a potential biomarker for OSCC and oropharyngeal SCC screening using oral rinse and swab techniques.

Detection of global hypermethylation in well-differentiated thyroid neoplasms by immunohistochemical (5-methylcytidine) analysis.

PURPOSE: While global hypomethylation of DNA has been found in several malignancies, studies on thyroid tumours have shown controversial results using different techniques. To help resolve this issue, we assessed methylation status using two different techniques in papillary thyroid carcinomas (PTC) and follicular adenomas (FA) and carcinomas (FTC), comparing adjacent non-neoplastic thyroid tissue. METHODS: A series of 15 FA, 18 FTC and 17 PTC were assessed by: (1) measurement of methylation levels of long interspersed nuclear elements (LINE-1) using a combined bisulfite restriction analysis polymerase chain reaction protocol and (2) immunostaining with an anti-5-methylcytidine antibody that detects methylated DNA regardless of the DNA sequence. Immunostaining was scored by image analysis. RESULTS: Methylation levels of LINE-1 in FA, FTC and PTC were not significantly different from adjacent normal tissue. There was no significant difference in methylation levels of LINE-1 between FA, FTC and PTC (p = 0.44). By immunohistochemical staining for methylation, the 5-methylcytidine score was significantly higher in tumours than in normal tissue counterparts, for FA (p < 0.001), FTC (p = 0.04) and PTC (p = 0.02). PTC showed the highest 5-methylcytidine expression amongst all tumours which was significantly different from FTC (p = 0.015), but not FA (p = 0.09). There was no correlation in methylation level between LINE-1 and 5-methylcytidine scores for each group and overall. CONCLUSIONS: Well-differentiated thyroid neoplasms (FA, FTC and PTC) were not found by two independent methods to undergo global hypomethylation as part of an oncogenic sequence from normal tissue to carcinoma. Instead, hypermethylation was detected in all types of tumours, implying that this epigenetic event may contribute to oncogenic development of thyroid neoplasms (both benign and malignant).

Tumour necrosis factor-alpha gene polymorphisms and susceptibility to oral lichen planus.

OBJECTIVE: This study is aimed to investigate the association between OLP susceptibility and clinical type in the Thai population and three polymorphisms within the promoter region of the TNF-α at positions -863, -308 and -238 which have putative functional significances. MATERIALS AND METHODS: Genomic DNA from 75 Thai patients with OLP and 154 healthy controls were genotyped for TNF-α polymorphisms-- -863(rs1800630), -308(rs1800629), and -238(rs361525)--using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: We found a higher proportion of TNF-alpha-308 AA genotype (high producer genotype) among OLP patients (5/75; 6.67%) when compared to healthy controls (1/154; 0.65%; OR = 10.93; 95% CI = 1.21-251.9). For other polymorphisms (-863 and -238), we did not find any significant association with OLP development; this was also the case with haplotype analysis (-863/-308/-238). CONCLUSION: TNF-α-308AA may play a relevant role in the susceptibility and severity of OLP in the Thai population. However, further investigation of this study is needed.

LINE-1 methylation difference between ameloblastoma and keratocystic odontogenic tumor.

OBJECTIVE: Global hypomethylation is a common epigenetic event in cancer. Keratocystic odontogenic tumor (KCOT) and ameloblastoma are different tumors but posses the same tissue in origin. Here, we investigated long interspersed nuclear element-1 (LINE-1 or L1) methylation status between ameloblastoma and KCOT. MATERIALS AND METHODS: We studied the methylation levels of the long interspersed nucleotide element-1 (LINE-1) in ameloblastoma and KCOT. After collecting ameloblastoma cells and epithelium lining cells of KCOT by laser capture microdissection from paraffin embedded tissue, combined bisulfite restriction analysis of LINE-1 (COBRALINE-1) was performed to measure LINE-1 methylation levels. RESULTS: The LINE-1 methylation level in KCOT (53.16 +/- 12.03%) was higher than that in ameloblastoma (36.90 +/- 16.52%), with a statistical significance of P = 0.001. The ranges of LINE-1 methylation of both lesions were not associated with either age or sex. CONCLUSION: We found LINE-1 hypomethylation levels between ameloblastoma and KCOT are different. Therefore, global methylations between these tumors are processed differently.

Association of P53 codon 72 polymorphism and ameloblastoma.

OBJECTIVE: To test the hypothesis that P53 codon 72 polymorphism was associated with an increased risk of developing ameloblastoma in the Thai population. MATERIALS AND METHODS: Seventy-eight ameloblastomas and 94 healthy controls were genotyped for the P53 codon 72 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequencies of the Arg/Arg, Arg/Pro and Pro/Pro genotypes were 28.72%, 50.00% and 21.28%, respectively, in the controls; and 44.87%, 51.28% and 3.85%, respectively, in ameloblastomas. Therefore, P53 Arg is an ameloblastoma-susceptible allele [OR (95% CI) = 2.06 (1.28-3.31), P = 0.002]. Sex-adjusted OR (95% CI) is 2.08 (1.29-3.34), P = 0.002; and adjusted OR by clinical type (95% CI) is 2.04 (1.34-3.13), P < 10(-3). Therefore, the increased risk associated with P53 Arg may not be influenced by either the sex of patients or clinical characteristics of the tumours. Moreover, when compared with homozygous P53 Pro, people who carried the Arg allele had a remarkably high risk of developing ameloblastoma [adjusted OR (95% CI) = 7.26 (2.34-23.41), P < 10(-3)]. CONCLUSION: The Arg allele of P53 gene codon 72 may increase susceptibility, and P53 may be important in the aetiology of ameloblastoma.

Anaplastic large cell lymphoma of the palate: A case report.

Anaplastic large cell lymphoma (ALCL) is a very rare subtype of T-cell non-Hodgkin's lymphoma (NHL). Similar to other types of NHL, ALCL primarily involves the nodal areas and sometimes it can involve several extranodal sites such as skin, lung and soft tissue. Primary oral involvement of systemic ALCL is very rare. We report a 55-year-old Thai female with anaplastic lymphoma kinase (ALK)-negative ALCL primarily occurring at the hard palate. The patient was referred to the Department of Oral and Maxillofacial Surgery, Mahidol University, complaining of a swelling on her left palate. An incisional biopsy was performed and revealed a diffuse infiltration of large pleomorphic cells with prominent nuclei and sometimes eccentric horseshoe-shaped nuclei. The tumor cells showed a positivity for CD30, CD2, CD4, CD43 and EMA. A few tumor cells were positive to CD45 and CD3. They were negative for CD5, CD8, CD20, AE1/AE3, HMB45, ALK, TCRαß, TCRγδ, and EBER. The patient reported a decrease in lesionsize after two courses of chemotherapy. However, approximately six months after beginning chemotherapy the tumor metastasized to the nasal cavities and brain. This case represented another rare systemic ALK-negative ALCL case primarily involving the oral cavity.