Alterations of the INK4a-ARF gene locus in pleomorphic adenoma of the parotid gland

Abstract

Pleomorphic adenomas of the parotid gland are benign tumours composed of epithelial and mesenchymal cells. The INK4a-ARF (CDKN2A) locus on chromosome 9p21 encodes two tumour suppressor proteins, p16(INK4a) and p14(ARF), which act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in pleomorphic adenomas, this study analysed alterations of p14(ARF), p16(INK4a), p53, and pRb in these tumours. After microdissecting the different histological components, 42 pleomorphic adenomas of the parotid gland were analysed for INK4a-ARF inactivation by DNA sequence analysis, methylation-specific PCR (MSP), restriction enzyme-related polymerase chain reaction (RE-PCR), mRNA expression, microsatellite analysis, and immunohistochemistry. In addition, microdeletion of p14(ARF) and p16(INK4a) were assessed by differential PCR. The status of p53 and Rb was examined by direct sequencing and immunohistochemistry. Using microdissection, it was possible to examine the tumour components, i.e. epithelial, mesenchymal, and transitional, separately after immunohistochemical identification. Methylation of p14(ARF) was found in 1/42 cases and alterations of p16(INK4a) occurred in 12/42 of pleomorphic adenomas, which correlated with loss of mRNA transcription. Microdeletions or specific mutations of either exon were not detected. Methylation was detected exclusively in the epithelial and transitional components and not within the mesenchymal part of the tumour. p53 mutations were detected in 4/42 adenomas, also occurring solely in the epithelial components of the tumours. pRb was detected immunohistochemically in 40/42 adenomas. In normal, corresponding parotid tissue, p14(ARF), p16(INK4a), p53, and pRb alterations were not observed. The observation that alterations of p14(ARF) and p16(INK4a), and also p53 mutations, occurred exclusively in the epithelial and transitional components of pleomorphic adenoma supports the theory that these areas are prone to malignant transformation to carcinoma in adenoma.