Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. BRAF and TERT mutations have been extensively related to prognosis in thyroid cancer. In this study, the methylation levels of selected CpGs (5-cytosine-phosphate-guanine-3) comprising a classifier, previously reported by our group, were assessed in combination with BRAF and TERT mutations. We evaluated 121 WDTC, three poorly-differentiated/anaplastic thyroid carcinomas (PDTC/ATC), 22 benign thyroid lesions (BTL), and 13 non-neoplastic thyroid (NT) tissues. BRAF (V600E) and TERT promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in PFKFB2, ATP6V0C, and CXXC5 were evaluated by bisulfite pyrosequencing. ATP6V0C hypermethylation and PFKFB2 hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). CXXC5 was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower PFKFB2 methylation levels (p = 0.004). No association was observed on comparing BRAF (60.7%) and TERT (3.4%) mutations and prognosis. Lower PFKFB2 methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI_95% = 1.1⁻9.5). PFKFB2 promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of BRAF and TERT mutations.

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