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Summary: We investigated the promotor hypermethylation status of multiple genes in 49 oral squamous cell carcinomas (OSCC), using the methylation-specific PCR (MSP) assay. The genes examined included p16 INK4a , p14 ARF , RB1, p21 Waf1 , p27 Kip1 , PTEN, p73, 0 6 -MGMT, and GST-P. Detailed clinicopathological data, such as patient age, sex, tobacco use, alcohol consumption, lesion site, degree of tumor differentiation, tumor size, presence of lymph node metastasis, and clinical stage, were collected for all 49 samples. Overall, gene methylation was detected in 46.9% (23/49) of samples and was closely correlated with tobacco use or/and alcohol consumption. Of the genes investigated, p16 INK4a , p14 ARF , 0 6 -MGMT, RB1, PTEN, and p27 Kip1 were found to be methylated in 34.7%, 20.4%, 12.2%, 10.2%, 6.1%, and 4.1% of these 49 tumors, respectively, but methylation of p21 Waf1 , p73, and GST-P was not detected at all. Methylation frequencies were much higher for each gene when computed among informative cases only. Concurrent promotor hypermethylation of p16 INK4a and p14 ARF correlated significantly with tumor size, lymph node metastasis, and stage III/IV advanced OSCC; p14 ARF hypermethylation, in particular, was significantly associated with both lymph node metastasis and late clinical stage. Our results suggest that DNA methylation of multiple genes, especially hypermethylation of the p14 ARF promoter, is common in OSCC and is associated with the use of tobacco and/or alcohol consumption. For this type of cancer, the data further implicates gene methylation as playing an important role in tumor progression. [Copyright &y& Elsevier]

Summary: As combinations of genetic and/or epigenetic alterations occurring during salivary gland carcinogenesis are largely unknown, we here analyzed 36 salivary gland carcinomas (SGCs) for changes in INK4a/ARF, RB1, p21, p27, PTEN, p53, MDM2 and O6-MGMT genes using methylation specific PCR (MSP), loss of heterozygosity (LOH) assays and mutational analysis with immunohistochemistry (IHC), as well as histone H3 and H4 acetylation status. The RB1 gene was found to be the most frequently methylated (41.7% of cases), while methylation of p27Kip1 and O6-MGMT were less frequent 8.3% and 5.6%, respectively). Two other genes, p21Waf1 and PTEN, were unmethylated in the SGCs examined. RB1 methylation significantly correlated with loss of expression as determined by IHC (P=0.03), and also a poor prognosis (P=0.02). p53 mutations were found in 8 cases (22.2%), coupled with p14ARF hypermethylation in two cases. LOH in INK4a/ARF and the RB1 locus was observed in 33.3% and 28.6% of the lesions, respectively. There was no correlation between 9p21 LOH and methylation of the INK4a/ARF gene. Promoter hypermethylation of RB1 coupled with LOH was evident in three samples immuno-negative for RB1. Acetylation of histone H3 and H4 was detected in 6 and 5 cases, respectively. These findings indicate that epigenetic silencing of tumour suppressor genes via promoter hypermethylation might be crucial for salivary gland carcinogenesis, particularly in the RB1 gene. Thus epigenetic events including methylation and acetylation as well as genetic alterations may have important contributions. [Copyright &y& Elsevier]