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.

Background/Objectives: RAS mutations are among the most common genetic alterations in thyroid cancer and are generally associated with less aggressive behavior. However, when co-occurring with TERT (telomerase reverse transcriptase) promoter mutations, known markers of poor prognosis, tumors exhibit markedly more aggressive features. The allele frequency (AF) of RAS may serve as a potential indicator of clonal dominance and the likelihood of additional high-risk mutations, such as TERT mutation. This study aims to assess whether a high RAS AF correlates with the presence of coexisting TERT promoter mutations and other molecular alterations. Methods: A retrospective chart review was performed on 111 patients with thyroid nodules harboring RAS mutations, either alone or in combination with TERT promoter mutations. All patients underwent molecular testing with ThyroSeq v3 and subsequent thyroidectomy at McGill University teaching hospitals. RAS AF was analyzed in relation to TERT mutation status, nodule size, and other molecular alterations including copy number alterations (CNA) and gene expression profiles (GEP). Results: The mean RAS AF was significantly higher in nodules with both RAS and TERT mutations (38.1%) compared to those with RAS mutations alone (22.1%) (p = 0.002). Nodules with coexisting TERT mutations were also significantly larger (mean size: 3.7 cm vs. 2.4 cm; p = 0.005). Malignant nodules, regardless of TERT status, showed a trend toward higher RAS AF than benign nodules (23.0% vs. 16.3%; p = 0.052). Higher RAS AF was also associated with the presence of CNA and/or GEP positivity. Notably, GEP was positive in 100% of nodules with both RAS and TERT mutations, compared to 37.5% in RAS-only nodules (p = 0.002). Conclusions: A high RAS AF increases the likelihood of a TERT promoter mutation and other genetic alterations, highlighting the importance of RAS AF in optimizing patient care and management.