Integrative Epigenomic and Transcriptomic Profiling Define Malignancy- and Cluster-Specific Signatures in Pheochromocytomas and Paragangliomas

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors primarily involving the adrenal medulla and its associated paraganglia, with heterogeneous clinical behavior and complex molecular drivers. This study aimed to characterize DNA methylation and gene expression patterns in PPGLs to understand the molecular differences between tumor subtypes and malignancy. We performed an integrative analysis of DNA methylation (Illumina EPIC 850K) and gene expression profiles (Affymetrix microarrays) in 24 PPGLs, comparing these with The Cancer Genome Atlas (TCGA) data, to delineate cluster- and malignancy-specific epigenetic patterns. Comparison between pseudohypoxic Cluster I and kinase-signaling Cluster II tumors revealed 13 differentially methylated CpG sites, with a specific CpG within <i>DSCAML1</i> showing hypermethylation in Cluster II accompanied by increased expression, suggesting context-dependent gene body methylation effects. Benign versus malignant comparisons identified 101 differentially methylated CpGs, including hypermethylated CpG in <i>BAIAP2L1</i> and hypomethylated CpG in <i>SHANK1</i> in malignant tumors. Pathway enrichment of differentially methylated genes revealed alterations in Notch signaling, adherens junctions, cytoskeletal regulation, and intracellular transport. Gene expression analysis demonstrated partial overlap between clusters, with malignant tumors exhibiting distinct transcriptional profiles involving RNA processing, metabolism, and adhesion pathways. Correlation between methylation and expression was generally limited, emphasizing that methylation-dependent gene regulation is a locus-specific and context-dependent regulation. These findings illustrate a complex interplay between epigenetic modifications and transcriptional programs in PPGLs, enhancing our understanding of molecular heterogeneity and tumor classification, and identifying candidate biomarkers and therapeutic targets for malignant progression.