Discovery and Validation of RUNX1 DNA Methylation in Differentiating Papillary Thyroid Cancer from Benign Nodules

Although most thyroid nodules can be diagnosed preoperatively by thyroid ultrasonography and fine-needle aspiration biopsy, it remains a challenge to accurately identify malignancy of thyroid nodules when the biopsy is indeterminate. This study aims to explore a novel biomarker to distinguish benign and malignant thyroid nodules. Tissue samples from patients with Stage I&II papillary thyroid carcinoma (PTC) and benign thyroid nodules (BTN) were collected for genome profiling by methylation EPIC 850K array and RNA-Sequencing. Genes with significantly differential DNA methylation and inverse mRNA expression were filtered out. The altered methylation of RUNX1 gene was validated in two independent case-control studies with a total of 699 formalin fixed paraffin-embedded (FFPE) samples using mass spectrometry and calculated by binary logistic regression analysis. Hypomethylation of RUNX1 gene in PTC patients compared to BTN subjects was verified in Validation Ⅰ (140 PTC vs. 189 BTN, ORs ≥ 1.50 per-10% methylation, P ≤ 4.40E-05, for all measurable CpG sites) and Validation Ⅱ (184 PTC vs. 186 BTN, ORs ≥ 1.72 per-10% methylation, P ≤ 2.38E-11, for all measurable CpG sites). Besides, RUNX1 methylation achieved good accuracy in differentiating early-stage PTC from BTN in Validation Ⅰ (AUC: 0.74) and Validation Ⅱ (AUC: 0.79). Gender- and age-stratified analysis revealed RUNX1 hypomethylation as an important risk factor for thyroid disease in younger women. We disclosed a significant association between RUNX1 hypomethylation and PTC, suggesting RUNX1 methylation based on FFPE tissue samples as a potential biomarker for predicting malignancy of thyroid nodules.