DNA methylation profiling of well-differentiated thyroid cancer uncovers markers of recurrence free survival Veronika Mancikova 1 *, Raquel Buj 2 *, Esmeralda Castelblanco 3 , Luc  ıa Inglada-P erez 1,4 , Anna Diez 2 , Aguirre A. de Cubas 1 , Maria Curras-Freixes 1 , Francisco Xavier Maravall 3 , Didac Mauricio 5,6,7 , Xavier Matias-Guiu 8 , Manel Puig-Domingo 5,6,7 , Ismael Capel 9 , Mar  ıa Rosa Bella 10 , Enrique Lerma 11 , Eva Castella 12 , Jordi Lluis Reverter 5,6,7 , Miguel  Angel Peinado 2 , Mireia Jorda 2 and Mercedes Robledo 1,4 1 Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 2 Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain 3 Department of Endocrinology and Nutrition, University Hospital Arnau de Vilanova, IRBLLEIDA, Lleida, Spain 4 ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain 5 Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Spain 6 Department of Endocrinology and Nutrition, University Hospital Germans Trias i Pujol, Barcelona, Spain 7 Department of Medicine, Autonomous University of Barcelona, Barcelona, Spain 8 Department of Pathology, University Hospital Arnau de Vilanova,University of Lleida, IRBLLEIDA, Lleida, Spain 9 Department of Endocrinology and Nutrition, Hospital de Sabadell, Sabadell, Spain 10 Department of Pathology, Hospital de Sabadell, Sabadell, Barcelona, Spain 11 Department of Pathology, Hospital Santa Creu i Sant Pau, Barcelona, Spain 12 Department of Pathology, University Hospital Germans Trias i Pujol, Badalona, Spain Thyroid cancer is a heterogeneous disease with several subtypes characterized by cytological, histological and genetic altera- tions, but the involvement of epigenetics is not well understood. Here, we investigated the role of aberrant DNA methylation in the development of well-differentiated thyroid tumors. We performed genome-wide DNA methylation profiling in the largest well-differentiated thyroid tumor series reported to date, comprising 83 primary tumors as well as 8 samples of adjacent nor- mal tissue. The epigenetic profiles were closely related to not only tumor histology but also the underlying driver mutation; we found that follicular tumors had higher levels of methylation, which seemed to accumulate in a progressive manner along the tumorigenic process from adenomas to carcinomas. Furthermore, tumors harboring a BRAF or RAS mutation had a larger number of hypo- or hypermethylation events, respectively. The aberrant methylation of several candidate genes potentially related to thyroid carcinogenesis was validated in an independent series of 52 samples. Furthermore, through the integration of methylation and transcriptional expression data, we identified genes whose expression is associated with the methylation Key words: well-differentiated thyroid cancer, methylation, BRAF, RAS, prognostic markers Abbreviations: AKT3: v-akt murine thymoma viral oncogene homolog 3; BRAF: v-raf murine sarcoma viral oncogene homolog B1; CIMP: CpG island methylator phenotype; COL4A2: collagen type IV alpha 2; DLEC1: deleted in lung and esophageal cancer 1; EI24: etoposide induced 2.4 mRNA; FA: follicular adenoma; FDR: false discovery rate; FTC: follicular thyroid carcinoma; fvPTC: papillary thyroid carcinoma follicular variant; KLK10: kallikrein-related peptidase 10; NIS: sodium-iodine symporter; PcG: PolyComb Group; PTC: papillary thyroid carcinoma; RARb2: retinoic acid receptor b2; RASSF1: RAS association domain family protein 1; RET: “rearranged during transfection” protooncogene; RFS: recurrence-free survival; TIMP3: tissue inhibitor of metalloproteinase-3; TSHR: thyroid-stimu- lating hormone receptor; WT1: Wilms tumor 1 Additional Supporting Information may be found in the online version of this article. *V.M. and R.B. contributed equally to this work Grant sponsor: Fondo de Investigaciones Sanitarias; Grant numbers: PI11/01359; FIS PI11/02421 and PI11/01354; Grant sponsor: The Fundaci on Mutua Madrile~ na; Grant number: AP2775/2008; Grant sponsors: the Comunidad de Madrid S2011/BMD-2328 TIRONET, “la Caixa”/CNIO international PhD programme (V.M. and A.A.d.C.), CIBERER (L.I.-P.) and Spanish Ministry of Economy and Competitiveness (FPI program) (R.B.); Grant sponsor: Spanish Ministry of Economy and Competitiveness; Grant number: SAF2011/23638; Grant sponsor: Fondo de Investigaciones Sanitarias; Grant number: PI12/00236 (M.C.); Grant sponsor: Technical Support Staff program of the Spanish Ministry of Economy and Competitiveness; Grant number: PTA2011-5655-I (A.D.) DOI: 10.1002/ijc.28703 History: Received 14 Oct 2013; Accepted 19 Dec 2013; Online 31 Dec 2013 Correspondence to: Mercedes Robledo, Spanish National Cancer Center (CNIO), Madrid, Spain, Tel.: 134-917-328-000, Fax: 134-912-246- 972, E-mail: mrobledo@cnio.es or Mireia Jorda, Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain, Tel.: 134-935-543-050, Fax: 134-934-651-472, E-mail: mjorda@imppc.org Cancer Genetics Int. J. Cancer: 00, 00–00 (2014) V C 2013 UICC International Journal of Cancer IJC