Molecular Testing in Indeterminate Thyroid Nodules: Current Evidence, Clinical Utility, and Practical Considerations

Indeterminate thyroid nodules classified as Bethesda III (atypia of undetermined significance) and Bethesda IV (follicular neoplasm) remain a diagnostic challenge because cytomorphology alone cannot reliably distinguish benign from malignant disease. Molecular testing has emerged as an adjunct to cytological, sonographic, and clinical assessment to refine preoperative risk stratification and inform management. This narrative review critically summarizes current evidence for major commercially available molecular platforms, including the Afirma Gene Expression Classifier, Afirma Genomic Sequencing Classifier (GSC), ThyroSeq v3, and ThyGeNEXT^®/ThyraMIR^®, with emphasis on diagnostic performance, clinical utility, limitations, and contemporary practice integration. In pivotal validation studies, Afirma GSC demonstrated a sensitivity of 91%, specificity of 68%, positive predictive value (PPV) of 47%, and negative predictive value (NPV) of 96%, whereas ThyroSeq v3 demonstrated a sensitivity of 94%, specificity of 82%, PPV of 66%, and NPV of 97%. In a blinded multicenter validation study of Bethesda III/IV nodules, ThyGeNEXT/ThyraMIR demonstrated prevalence-adjusted sensitivity of 95%, specificity of 90%, PPV of 75%, and NPV of 97% at a standardized disease prevalence of 30%. These estimates should not be interpreted as direct head-to-head comparisons because studies differ in population characteristics, disease prevalence, reference standards, assay generation, and classification of noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Clinical-utility evidence indicates that molecular testing can influence decisions regarding surveillance and surgery and may reduce potentially avoidable diagnostic operations, although the magnitude of benefit varies across assays and clinical settings. The Bethesda System for Reporting Thyroid Cytopathology supports selective molecular testing when results are expected to meaningfully influence management. Molecular testing should therefore be regarded as an adjunct to integrated risk assessment rather than a stand-alone determinant of management, with results interpreted alongside cytological, sonographic, clinical, patient-specific, and institutional factors.