Reduced expression of the p16 gene product (protein), an inhibitor of cyclin-D-dependent protein kinase which regulates cell cycle at the G1/S boundary, is implicated in tumor progression in various neoplasms. Hypermethylation of the p16 promoter gene has recently been suggested to be one of the reasons for the reduced protein expression. To explore the role of p16 in the biological behavior of adenoid cystic carcinomas (ACC), we investigated the immunohistochemical expression of p16 protein in 38 ACC tumors (32 primary, 3 recurrent, and 3 metastatic tumors) and the methylation status of its promoter gene. We also examined their relationships to the histological grade of malignancy. Positive reaction of p16 protein was demonstrated in the nuclei of luminar cuboidal cells in areas with tubular patterns. The reactions were reduced in the areas with solid or large cribriform patterns. The levels of p16 expression correlated with the histological grade of malignancy. Recurrent or metastatic tumors did not differ with respect to histological grades from the original tumor except for 1 case, in which p16 expression was reduced compared to the primary tumor. Methylation-specific PCR demonstrated the hypermethylation status of the p16 promoter gene in 4 of 22 primary tumors (21%), all of which showed negative or low expression of the p16 protein. The study indicated that p16 expression was reduced in ACC cases of higher histological grade of malignancy and that hypermethylation of its promoter gene may be involved in its process in some cases.

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