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3020.Full-Text.Pdf Published OnlineFirst March 30, 2015; DOI: 10.1158/1078-0432.CCR-14-2804 Biology of Human Tumors Clinical Cancer Research DNA Methylation Profiling in Pheochromocytoma and Paraganglioma Reveals Diagnostic and Prognostic Markers Aguirre A. de Cubas1, Esther Korpershoek2, Lucia Inglada-Perez 1,3, Eric Letouze4, Maria Curras-Freixes 1, Agustin F. Fernandez 5,Inaki~ Comino-Mendez 1, Francesca Schiavi6, Veronika Mancikova1, Graeme Eisenhofer7,8, Massimo Mannelli9, Guiseppe Opocher6,10, Henri Timmers11, Felix Beuschlein12, Ronald de Krijger13, Alberto Cascon1,3, Cristina Rodríguez-Antona1,3, Mario F. Fraga5,14, Judith Favier15,16, Anne-Paule Gimenez-Roqueplo15,16,17,18, and Mercedes Robledo1,3 Abstract Purpose: Pheochromocytoma and paraganglioma (PPGL) are these, 48 CpGs showed significant associations with time to rare neuroendocrine tumors, associated with highly variable progression even after correcting for SDHB genotype, suggesting postoperative evolution. The scarcity of reliable PPGL prognostic their value as prognostic markers independent of genetic back- markers continues to complicate patient management. In this ground. Hypermethylation of RDBP (negative elongation study, we explored genome-wide DNA methylation patterns in factor complex member E) in metastatic tumors was further the context of PPGL malignancy to identify novel prognostic validated by bisulfite pyrosequencing [Dbmetastatic-benign ¼ 0.29, markers. P ¼ 0.003; HR, 1.4; 95% confidence interval (CI), 1.1–2.0; P ¼ Experimental Design: We retrospectively investigated DNA 0.018] and may alter transcriptional networks involving (RERG, methylation patterns in PPGL with and without metastases using GPX3, and PDZK1) apoptosis, invasion, and maintenance of high-throughput DNA methylation profiling data (Illumina 27K) DNA integrity. from two large, well-characterized discovery (n ¼ 123; 24 met- Conclusions: This is the first large-scale study of DNA methy- astatic) and primary validation (n ¼ 154; 24 metastatic) series. lation in metastatic PPGL that identifies and validates prognostic Additional validation of candidate CpGs was performed by markers, which could be used for stratifying patients according bisulfite pyrosequencing in a second independent set of 33 to risk of developing metastasis. Of the three CpGs selected for paraffin-embedded PPGLs (19 metastatic). further validation, one (RDBP) was clearly confirmed and could Results: Of the initial 86 candidate CpGs, we successfully be used for stratifying patients according to the risk of developing replicated 52 (47 genes), associated with metastatic PPGL. Of metastases. Clin Cancer Res; 21(13); 3020–30. Ó2015 AACR. Introduction hereditary syndromes associated with germline mutations in at least 15 genes and has been extensively discussed elsewhere (1, 2). Pheochromocytoma and paraganglioma (PPGL) are rare neu- Extensive clinical and genetic characterization over the last 25 roendocrine tumors that arise from the adrenal medulla and years has greatly improved our knowledge about the genetic basis paraganglial system, respectively. PPGLs can arise as part of underlying PPGLs. Fortunately, application of high-throughput 1Hereditary Endocrine Cancer Group, Spanish National Cancer CNB-CSIC, Cantoblanco, Madrid, Spain. 15INSERM, UMR970, Par- Research Centre (CNIO), Madrid, Spain. 2Department of Pathology, is-Cardiovascular Research Center (PARCC), Paris, France. Erasmus MC Cancer Institute, University Medical Center, Rotterdam, 16FacultedeM edecine, Universite Paris Descartes, Sorbonne Paris the Netherlands. 3Centro de Investigacion Biomedica en Red de Cite, Paris, France. 17Service de Gen etique, Assistance Publique Enfermedades Raras (CIBERER), Madrid, Spain. 4Programme Hopitaux^ de Paris, Hopital^ Europeen Georges Pompidou, Paris, Cartes d'Identite des Tumeurs, Ligue Nationale Contre Le Cancer, France. 18Rare Adrenal Cancer Network-Cortico Medullosurr enale Paris, France. 5Cancer Epigenetics Laboratory, Institute of Oncolo- Tumeur Endocrine, Institut National du Cancer, Paris, France. gy of Asturias (IUOPA), HUCA, University of Oviedo, Asturias, Spain. 6Familial Cancer Clinic, Veneto Institute of Oncology, Padova, Italy. Note: Supplementary data for this article are available at Clinical Cancer 7Institute of Clinical Chemistry and Laboratory Medicine, Tech- Research Online (http://clincancerres.aacrjournals.org/). € 8 nische Universitat Dresden, Dresden, Germany. Medical Clinic III, J. Favier and A.-P. Gimenez-Roqueplo contributed equally to this article. TechnischeUniversitat€ Dresden, Dresden,Germany. 9Department of Experimental and Clinical Biomedical Sciences, University of Flor- Corresponding Author: Mercedes Robledo, Hereditary Endocrine Cancer ence, Florence, Italy. 10Department of Medicine, University of Group, Human Cancer Genetics Programme. Centro Nacional de Investiga- Padova, Padova, Italy. 11Internal Medicine, Radboud University Med- 12 ciones Oncologicas, CNIO), Melchor Fernandez Almagro 3, Madrid 28029, ical Centre, Nijmegen, the Netherlands. Department of Internal Spain. Phone: 34-91-2246947; Fax: 34-91-2246923; E-mail: Medicine IV Campus Innenstadt, University-Hospital, Ludwig-Max- [email protected] imilians-University of Munich, Munich, Germany. 13Department of Pathology, Josephine Nefkens Institute, Erasmus MC University doi: 10.1158/1078-0432.CCR-14-2804 Medical Center, Rotterdam, the Netherlands. 14Department of Immunology and Oncology, National Center for Biotechnology, Ó2015 American Association for Cancer Research. 3020 Clin Cancer Res; 21(13) July 1, 2015 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst March 30, 2015; DOI: 10.1158/1078-0432.CCR-14-2804 DNA Methylation Profiling in PPGL. Novel Prognostic Markers In this study, we explored DNA methylation patterns in Translational Relevance metastatic PPGL using two large, well-characterized cohorts of Pheochromocytoma and paraganglioma (PPGL) are rare PPGL collected through a multi-institutional collaboration tumors that often present highly variable, postoperative out- within the European Network for the Study of Adrenal Tumors comes. Current therapeutic options for metastatic PPGL are (ENS@T). Candidate CpGs associated with metastatic PPGL very limited. Therefore, novel prognostic markers for meta- were identified using whole-genome DNA methylation profiles static PPGL are urgently needed. In this study, we investigated, obtained from a discovery series containing 24 metastatic (4 identified, and validated novel prognostic and predictive SDHB) PPGLs. These data were replicated for 52 of these markers for PPGLs presenting metastases using whole-genome candidate CpGs in a first validation series, which comprised DNA methylation profiling data from two large, well-charac- whole-genome DNA methylation data (27K Illumina) contain- terized discovery and primary validation series of tumors. Even ing 154 PPGL, including 24 metastatic tumors (10 SDHB). after correcting for SDHB genotype, 48 of these CpGs showed Interestingly, significant associations with time to progression significant associations with time to progression, indicating (TTP) were found for a large proportion of these CpGs. Among potential utility as novel molecular predictive markers. Our these CpGs, hypermethylation of the RDBP (negative elonga- findings suggested that aberrant DNA methylation might tion factor complex member E) gene in metastatic PPGLs was affect nervous system development and transcriptional regu- further validated in a third series by bisulfite pyrosequencing lation networks in PPGL with metastases, providing potential [Dbmetastatic-benign ¼ 0.29, P ¼ 0.003; HR, 1.4; 95% confidence clues for future therapeutic strategies. Specifically, these anal- interval (CI), 1.1–2.0; P ¼ 0.018] of 33 formalin-fixed, paraffin- yses indicated RDBP hypermethylation in metastatic tumors, embedded (FFPE) tissues. Bioinformatics analyses suggested which we validated in another second independent series, that RDBP hypermethylation might contribute to metastatic disrupted the expression of various key molecules that we also disease by altering transcriptional networks, including those observed in transcriptomic analyses. involved in response to apoptotic stimuli, invasion, and main- tenance of DNA integrity. Our results suggest that these CpGs may represent potential markers of malignancy and also serve as prognostic predictors independent of genetic background. technologies to study PPGL has enhanced our understanding of Materials and Methods tumor biology and has already led to novel therapeutic strategies, which may soon be applied in routine clinical practice (3–7). Patient samples However, lack of reliable markers for metastatic disease and/or According to standard protocols, all patients underwent tumor their limited predictive value continue to complicate the clinical resective surgery 4 to 6 weeks after diagnosis of PPGL. All fresh- management of patients with PPGL. Therefore, identifying and frozen tissues were immediately frozen in liquid nitrogen and validating novel prognostic/predictive markers for high-risk stored at À80 C. PPGL was confirmed by pathologic examina- patients with PPGL is of paramount importance for appropriate tion. All samples included in this study contained at least 85% patient management and also help guide therapeutic strategies tumor cells. A PPGL was considered metastatic when the patient (5, 7). presented metastases at non-chromaffin sites distant from the Previous cytogenetic,
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