A five-gene molecular testing panel refines thyroid nodules diagnosis and risk stratification

Abstract Background Current clinical guidelines recommend molecular testing for the diagnosis of thyroid nodules with indeterminate cytology. However, RNA-based commercial molecular testing kits are expensive and largely inaccessible for patients outside of the US. Methods In this study, we designed a PCR-based 5-gene panel ( BRAF , HRAS , NRAS , TERT , CCDC6 - RET ) and evaluated its performance for the diagnosis and risk stratification of thyroid nodules. Results The fine-needle aspiration (FNA) samples from 354 thyroid nodules were evaluated with the 5-gene panel. Thyroid cancer-associated genomic alterations were identified in 205 of 354 nodules (57.9%), with BRAF V600E being the most prevalent mutation (184/354, 52.0%). 160 nodules from 152 patients underwent surgery immediately after fine-needle aspiration cytology (FNAC), irrespective of molecular testing results. Based on the molecular findings, the remaining 194 nodules were classified as low risk (mutation negative or RAS -mutated), intermediate risk ( BRAF -mutated), and high risk ( BRAF / RAS plus TERT mutations), and clinical follow-up data were collected. We found that patients in the high- and intermediate-risk group were more likely to be diagnosed with thyroid cancer or experience disease recurrence than patients in the low-risk group. For thyroid nodules with indeterminate cytology, this 5-gene panel demonstrated a sensitivity of 86.9%, specificity of 95.0%, positive predictive value of 98.9%, negative predictive value of 59.4%, and overall accuracy of 88.2%. Conclusion Our 5-gene panel provides improved diagnostic accuracy and risk stratification for cytologically indeterminate thyroid nodules, it may serve as an affordable and accessible molecular testing approach to support the management of thyroid nodules.