PFKFB2 Promoter Hypomethylation as Recurrence Predictive Marker in Well-Differentiated Thyroid Carcinomas

Abstract

Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. <i>BRAF</i> and <i>TERT</i> 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 <i>BRAF</i> and <i>TERT</i> 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. <i>BRAF</i> (V600E) and <i>TERT</i> promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in <i>PFKFB2</i>, <i>ATP6V0C</i>, and <i>CXXC5</i> were evaluated by bisulfite pyrosequencing. <i>ATP6V0C</i> hypermethylation and <i>PFKFB2</i> hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). <i>CXXC5</i> was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower <i>PFKFB2</i> methylation levels (<i>p</i> = 0.004). No association was observed on comparing <i>BRAF</i> (60.7%) and <i>TERT</i> (3.4%) mutations and prognosis. Lower <i>PFKFB2</i> methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI_95% = 1.1⁻9.5). <i>PFKFB2</i> promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of <i>BRAF</i> and <i>TERT</i> mutations.