Methylation of the p16^INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16^INK4A and p14^ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed, succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16^INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P < 0.0043) and SDHB mutation (P < 0.002). p16^INK4A mRNA expression showed moderate suppression in malignant cases (P < 0.05). In contrast, very little p14^ARF promoter methylation was seen and there was no significant difference in p14^ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16^INK4A, and not p14^ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16 ^INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.