DNA Methylation Profiling in Pheochromocytoma and Paraganglioma Reveals Diagnostic and Prognostic Markers

Abstract

Purpose: Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors, associated with highly variable postoperative evolution. The scarcity of reliable PPGL prognostic markers continues to complicate patient management. In this study, we explored genome-wide DNA methylation patterns in the context of PPGL malignancy to identify novel prognostic markers.Experimental Design: We retrospectively investigated DNA methylation patterns in PPGL with and without metastases using high-throughput DNA methylation profiling data (Illumina 27K) from two large, well-characterized discovery (n ¼ 123; 24 metastatic) and primary validation (n ¼ 154; 24 metastatic) series. Additional validation of candidate CpGs was performed by bisulfite pyrosequencing in a second independent set of 33 paraffin-embedded PPGLs (19 metastatic).Results: Of the initial 86 candidate CpGs, we successfully replicated 52 (47 genes), associated with metastatic PPGL. Of these, 48 CpGs showed significant associations with time to progression even after correcting for SDHB genotype, suggesting their value as prognostic markers independent of genetic background. Hypermethylation of RDBP (negative elongation factor complex member E) in metastatic tumors was further validated by bisulfite pyrosequencing [Db metastatic-benign ¼ 0.29, P ¼ 0.003; HR, 1.4; 95% confidence interval (CI), 1.1-2.0; P ¼ 0.018] and may alter transcriptional networks involving (RERG, GPX3, and PDZK1) apoptosis, invasion, and maintenance of DNA integrity.Conclusions: This is the first large-scale study of DNA methylation in metastatic PPGL that identifies and validates prognostic markers, which could be used for stratifying patients according to risk of developing metastasis. Of the three CpGs selected for further validation, one (RDBP) was clearly confirmed and could be used for stratifying patients according to the risk of developing metastases. Clin Cancer Res; 21(13); 3020-30. Ó2015 AACR.

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas

“…All participants have adopted, and reported on, NGS-based technologies in their research and/or clinical practice [14][15][16][17][18][19][20][21][22][23][24][25][26] . Discussions took place via conference calls, e-mail communications and file exchanges and one plenary session (at the 14th ENS@T Scientific Meeting on November 20th, 2015, Munich, Germany).…”

Pheochromocytoma: The First Metabolic Endocrine Cancer

“…Previous and recent genetic discoveries in PHEO/PGL research have led to the identification of PHEO/PGL-related unique metabolic abnormalities or pathways involved in oxygen sensing, hypermethylation, DNA repair, up-regulation of specific transporters and/or receptors, and particularly, Krebs cycle enzymes (17–20). These changes are tightly linked to metabolic reprogramming in PHEO/PGL, which points out the metabolic nature of PHEO/PGL, defining this cancer as a metabolic disease.…”

Global DNA Methylation Analysis Identifies Two Discrete clusters of Pheochromocytoma with Distinct Genomic and Genetic Alterations

“…Early studies used sequencing- and PCR-based methods to investigate the methylation status of carefully selected candidate genes2829, while more recent studies have used array-based technologies to investigate genome-wide methylation3031. SDHx -mutated tumours have been found to exhibit a hypermethylator phenotype, due to allosteric inhibition of the demethylating TET family of enzymes by the build-up of succinate.…”

“…Another subset of tumours has been found to exhibit global hypomethylation31. Recently, the methylation level of a number of CpG-sites was proposed as markers of malignancy in PPGL, independent of genetic background30, with high levels of methylation of a specific CpG-residue in the promoter of the RDBP gene pinpointed as a strong candidate biomarker for metastatic disease. In this paper we report the results from a global methylation analysis of 39 tumours from 35 patients and evaluate the set of proposed malignancy markers in this cohort.…”