DNA Methylation-based Risk Stratification and Classification of Pediatric Thyroid Carcinoma

Abstract Purpose: Accurate assessment of invasiveness in pediatric thyroid carcinomas is essential to prevent unnecessary surgery and avoid surgery-associated complications. DNA methylation, a proven molecular biomarker for cancer classification, holds promise for stratifying thyroid cancer risk. The objectives were to determine the epigenetic hallmarks of pediatric thyroid carcinomas and to investigate whether DNA methylome profiling is a feasible approach for pre-operative risk stratification of this pediatric disease. Experimental Design: We interrogated genome-wide DNA methylation profiles from two separately processed cohorts of pediatric thyroid carcinoma. The reference cohort included 100 samples, consisting of 87 well-differentiated primary tumors—77 papillary and 10 follicular thyroid carcinomas—and 13 matched lymph node metastases. To predict oncogenic drivers and tumor invasiveness, defined by the presence of nodal metastasis, we trained two classifiers on the reference cohort and then evaluated their performance on a second validation cohort of 84 samples, including 83 primary tumors and one lymph node metastasis. Results: We identified distinct methylation patterns associated with tumor invasiveness and key driver mutations, including BRAF p.V600E, RAS-like mutations, kinase fusions, and DICER1 mutations. The differentially methylated regions reflect inflammatory stress, disrupted thyroid development and function, implicating AR, Hippo, and AP-1 signaling. Leveraging these epigenetic signatures, we developed and validated two methylation-based classifiers that accurately predict tumor invasiveness and oncogenic mutation subgroups. Conclusions: In pediatric thyroid carcinoma patients, DNA methylation assays accurately predict tumor invasiveness and driver mutations. Our findings highlight the clinical value of DNA methylation profiling for risk stratification and classification of pediatric thyroid cancers.