Distinct molecular profiles of indeterminate and malignant thyroid nodules in patients under 21 years of age

Although uncommon, thyroid nodules (TNs) in pediatric and young adult patients carry a higher malignancy risk and often present with a high burden of metastatic disease than in adults. The molecular features underlying this distinct clinical behavior remain unclear. We analyzed Afirma Genomic Sequencing Classifier (GSC) data from 283,621 TNs, comparing patients <21 and ≥21 years old. Cytology (Bethesda), GSC benign (B) vs suspicious (S) calls, and Afirma Xpression Atlas (XA) variant/fusion profiles were evaluated in GSC-S and Bethesda V/VI samples. Genome-wide expression was used to derive pathway signatures and thyroid cancer-related scores: BRAF-RAS score (BRS), ERK signaling score, follicular-to-mesenchymal transition (FMT) score, epithelial-to-mesenchymal transition (EMT) score, and thyroid differentiation score (TDS). Among 2,397 patients <21 years (median age: 18.9; 81.4% female) and 281,224 adults ≥21 years (median age: 59.8; 77.1% female), the <21-year samples showed more Bethesda V/VI cytology (14.5% vs 5.0%; P < 0.0001) and a lower GSC-B rate (43.5% vs 68.8%; P < 0.0001). In GSC-S samples, total variant detection was higher in the <21-year group (45.3% vs 37.4%), with enriched BRAF p.V600E, TSHR, and DICER1 variants, while HRAS variants were more common in adults (all P < 0.01). Gene fusions involving RET, NTRK3, and ALK were enriched in the <21-year group (14.5% vs 5.5%; P < 0.0001). TERT promoter mutations were absent in <21-year GSC-S and Bethesda V/VI samples (vs 4.2% and 9.3% in adults). The GSC-S <21-year cohort showed cell-cycle pathway enrichment. The RET/NTRK/ALK-positive <21-year group demonstrated enrichment of angiogenesis and EMT pathways, higher ERK/EMT/FMT scores, and lower BRS/TDS vs genotyped-matched adults. These molecular differences provide mechanistic insight into the more invasive phenotype in pediatric and young adult TNs.