The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Using the Infinium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modified DNA from 6 normal adrenocortical tissue samples, 27 adrenocortical adenomas (ACA), and 15 adrenocortical carcinomas (ACC). Genes involved in cell cycle regulation, apoptosis, and transcriptional regulation of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included CDKN2A, GATA4, BCL2, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Gene expression studies of selected hypermethylated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tissues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2'-deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hypermethylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. In conclusion, the current study represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor development.

Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel Lindau (VHL) syndrome. In VHL, only about 30% of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought for. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found in both patients with PCCs and PGLs. Since loss of 11q is a common event in VHLassociated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In this study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation as well as mRNA expression of SDHAF2 and SDHD was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted. LOH was found in more than 50% of the VHL-associated PCCs, and was correlated to a significant decrease (p<0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression.In addition, the lack of mutations and promoter methylation in the investigated samples indicate that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.3

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