DNA Methylation Dynamics and Prognostic Implications in Metastatic Differentiated Thyroid Cancer

<b><i>Background:</i></b> Distant metastases (DM) are the leading cause of thyroid cancer-related death in patients with differentiated thyroid cancer (DTC). Despite significant progress in understanding DNA methylation in DTC, the methylation landscape of metastatic primary tumors and DM remains unclear. Our primary objective was to investigate DNA methylation dynamics during DTC progression, with a secondary goal of assessing potential clinical implications. <b><i>Materials and Methods:</i></b> We conducted a multicenter retrospective study in patients with DTC who underwent surgery at five university hospitals. We profiled DNA methylation in a discovery series of 97 samples (15 normal tissues, 30 non-metastatic [non-mDTC], and 35 metastatic [mDTC] primary DTC, and 17 paired metastases [lymph nodes and DM]). Results were validated in an independent series of 17 non-mDTC and 13 mDTC. We used receiver operating characteristic curve analysis to evaluate the identified prognostic CpG-signature. <b><i>Results:</i></b> DNA methylation alterations, mostly hypomethylation, increased progressively from primary tumors to DM, both in papillary (PTC) and follicular (FTC) thyroid carcinomas. Compared with normal tissue, non-metastatic primary PTC (non-mPTC) exhibited more hypomethylated than hypermethylated CpGs in contrast to non-metastatic primary FTC (non-mFTC). However, metastatic tumors, both mPTC and mFTC, predominantly exhibited hypomethylated CpGs. The overlap of differentially methylated CpGs (DMe-CpGs) was low between non-mPTC and non-mFTC (14% non-mPTC DMe-CpGs present in non-mFTC) but significantly higher between mPTC and mFTC (60% mPTC DMe-CpGs present in mFTC), underscoring the convergence of epigenetic changes during metastatic progression. The presence of many <i>de novo</i> DMe-CpGs from metastatic primary tumors (83% from mPTC and 40% from mFTC) in DM, including metachronous DM, supports the hypothesis that DM originates from a major subclone of the primary tumor. We identified and validated a 156-CpG signature in primary tumors capable of distinguishing between non-mDTC and mDTC, offering potential prognostic value for DM development regardless of histology. <b><i>Conclusions:</i></b> These results show a progressive increase in DNA methylation alterations, mainly hypomethylation, during PTC and FTC metastatic progression, suggesting a linear model, though the DNA methylation dynamics differs between the two histological types. The analysis of the 156-CpG signature in primary tumors may help identify patients with DTC at high risk for DM, enhancing a more personalized treatment.