Background: Thyroid cancer is the most common endocrine malignancy, and accurate diagnosis is crucial for effective management. Fine needle aspiration cytology, guided by the Bethesda System for Reporting Thyroid Cytopathology, categorizes thyroid nodules into six categories, with Bethesda III and IV representing indeterminate diagnoses that pose significant challenges for clinical decision-making. Understanding the molecular profiles of these categories may enhance diagnostic accuracy and guide treatment strategies. Methods: This study retrospectively analyzed data from 217 patients with Bethesda III and IV thyroid nodules who underwent ThyroSeq v3 molecular testing followed by thyroid surgery at McGill University teaching hospitals. The analysis focused on the presence of specific molecular mutations, copy number alterations (CNAs), and gene expression profiles (GEPs) within these nodules. The relationship between these molecular findings and the clinico-pathological features of the patients was also examined. Results: This study identified notable differences in the molecular landscape of Bethesda III and IV thyroid nodules. Bethesda IV nodules exhibited a higher prevalence of CNAs and distinct GEPs compared to Bethesda III nodules. Interestingly, the BRAFV600E mutation was found exclusively in Bethesda III nodules, which correlated with more aggressive malignant behavior. These findings underscore the potential of molecular profiling to differentiate between the clinical behaviors of these indeterminate nodule categories. Conclusions: Molecular profiling, including the assessment of CNAs, GEPs, and specific mutations like BRAFV600E, provides valuable insights into the nature of Bethesda III and IV thyroid nodules. The distinct molecular characteristics observed between these categories suggest that such profiling could be instrumental in improving diagnostic accuracy and tailoring treatment approaches, ultimately enhancing patient outcomes in thyroid cancer management.
