GENES, CHROMOSOMES & CANCER 00:000–000 (2012) Comprehensive DNA Methylation Analysis of Benign and Malignant Adrenocortical Tumors Annabelle L. Fonseca, 1,2 Johan Kugelberg, 1,2,3 Lee F. Starker, 1,2 Ute Scholl, 4 Murim Choi, 4 Per Hellman, 5 Go ¨ran A ˚ kerstro ¨m, 5 Gunnar Westin, 5 Richard P. Lifton, 4 Peyman Bjo ¨ rklund, 1,2,5 and Tobias Carling 1,2,6 * 1 Department of Surgery,Yale University School of Medicine, New Haven,CT 2 Yale Endocrine Neoplasia Laboratory,Yale University School of Medicine, New Haven,CT 3 Faculty of Health Sciences, Link˛ping University, Link˛ping, Sweden 4 Department of Genetics,Yale University School of Medicine, New Haven,CT 5 Department of Surgical Sciences,Uppsala University,Uppsala, Sweden 6 Cancer Genetics and Genomics Program,Yale Cancer Center,Yale University School of Medicine, New Haven,CT 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 Infin- ium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modi- fied 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 hypermethy- lated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD , SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tis- sues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2 0 -deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hyperme- thylated 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 devel- opment. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION Adrenocortical tumors (ACT) are common. Most of these tumors are benign non-functioning adrenocortical adenomas (ACA), and are being increasingly found incidentally through diagnostic imaging for a non-adrenal-related reason, hence the term ‘‘adrenal incidentaloma’’ (Wandoloski et al., 2009). Adrenocortical carcinomas (ACC), on the other hand, are rare malignancies, with an estimated prevalence of 4–12 per million adults (Bertherat and Bertagna, 2009). Functioning ACAs (overproducing either aldosterone or corti- sol) may cause significant morbidity, and ACC is associated with poor prognosis and has an overall 5-year survival of 37–38% (Icard et al., 2001; Abiven et al., 2006). In addition, the histopatho- logical distinction between ACA and ACC may be difficult in the absence of widely invasive or metastatic disease (Weiss, 1984; Aubert et al., 2002; DeLellis, 2004; Pohlink et al., 2004; Bili- moria et al., 2008; de Reynie `s et al., 2009). A bet- ter method of distinguishing benign from malignant ACTs is therefore needed and atten- tion has turned toward a search for molecular markers (Soon et al., 2008). The molecular pathogenesis of both benign and malignant ACT is incompletely clarified. We recently demonstrated that somatic KCNJ5 muta- tions occur frequently in aldosterone producing ACA (Choi et al., 2011), but has not been identi- fied in other ACTs. Identification of molecular markers of adrenocortical carcinoma has been the focus of much recent research. Some suggested markers of malignant adrenocortical tumors include insulin-like growth factor 2 (IGF2) over Additional Supporting Information may be found in the online version of this article. Supported by: NIH (Yale Clinical and Translational Science Award UL1 RR024139). *Correspondence to: Tobias Carling, Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, 333 Cedar Street, TMP202, PO 208062, New Haven, CT 06520, USA. E-mail: tobias.carling@yale.edu Received 12 September 2011; Accepted 10 May 2012 DOI 10.1002/gcc.21978 Published online in Wiley Online Library (wileyonlinelibrary.com). RESEARCH ARTICLE V V C 2012 Wiley Periodicals, Inc.