Incremental Diagnostic Value of Cell-Block-Based Targeted BRAF and RAS Testing in TBSRTC Category III–IV Thyroid Nodules: A Single-Center Retrospective Study

Background/Objectives: Risk stratification of thyroid nodules classified as categories III–IV in The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) remains challenging. This study evaluated the incremental diagnostic value of cell-block-based targeted molecular testing beyond TBSRTC cytology within the 2023 TBSRTC and WHO 2022 frameworks. Methods: This retrospective study included 126 TBSRTC category III–VI thyroid fine-needle aspiration samples. BRAF exon 15 and KRAS/NRAS exons 2–4 were assessed by bidirectional Sanger sequencing; TERT testing was limited to six selected cases. Histopathology was available for 69 nodules, including 59 TBSRTC category III–IV nodules in the primary diagnostic-performance cohort. Logistic regression, ROC analysis, bootstrap correction, repeated stratified five-fold cross-validation, calibration, and exploratory decision curve analysis were performed. Results: BRAF/KRAS/NRAS mutations were detected in 35 of 126 nodules (27.8%). Among resected nodules, mutations occurred in 23 of 32 malignant (71.9%) and 10 of 37 benign/NIFTP cases (27.0%; p < 0.001); BRAF showed the strongest association with malignancy (50.0% vs. 2.7%; p < 0.001). In the resected cohort, combined cytology and molecular testing yielded an AUC of 0.81 versus 0.70 for cytology alone (bootstrap ΔAUC 0.10; 95% CI, 0.03–0.18). In the surgically verified, surgery-enriched TBSRTC category III–IV subgroup (n = 59), the combined model improved AUC from 0.65 to 0.81 (bootstrap ΔAUC 0.15; 95% CI, 0.01–0.29) and achieved 57.7% sensitivity, 90.9% specificity, 83.3% positive predictive value, and 76.3% accuracy. Conclusions: In the surgically verified TBSRTC category III–IV subgroup, targeted BRAF/RAS testing provided incremental rule-in information beyond cytology but had limited rule-out capability. BRAF V600E was strongly associated with malignancy, whereas non-V600 BRAF and RAS alterations required morphologic and clinical context. Negative molecular results should not be used in isolation to exclude malignancy. External validation is required.