ObjectivesReal‐world clinical results of (1) Bethesda categorization, (2) mutation analysis, and (3) a microRNA classifier were correlated to show the utility of molecular analysis in assessing malignancy risk of indeterminate thyroid nodules.MethodsCytology and molecular results of clinically tested thyroid nodules were compared. An additional microRNA threshold was determined based on nodules with known disease status, establishing a 3‐tiered microRNA approach to clinical risk assessments. Expected rate of malignancy given mutation panel and 3‐tiered microRNA approach was validated in an independent cohort of atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm or suspicious for follicular neoplasm (FN/SFN) nodules with surgically derived outcomes.ResultsIn 2685 patients clinically tested, PIK3CA, PAX8/PPARγ, and RET/PTC mutations occurred in less than 1%. Of note, 2% had BRAFV600E mutation and 82% lacked mutations. The maximum expected risk of malignancy in nodules lacking mutations was 9% and 17% for AUS/FLUS and FN/SFN nodules, respectively. Positive microRNA status further increased risk, with the most worrisome status (level‐3) elevating risk to 36% and 54%, respectively. RAS mutations occurred in 15% of nodules tested clinically, including in 8% of those that were cytologically benign. The maximum expected risk of malignancy in nodules with RAS or PAX8/PPARγ mutations was 49% and 65% for AUS/FLUS and FN/SFN nodules, respectively. Positive microRNA status further increased risk, with the most worrisome microRNA status (level‐3) elevating risk to 85% and 91%, respectively.ConclusionsMutation panels alone do not sufficiently risk stratify thyroid nodular disease. microRNA classification complements cytology and mutation analysis with the capacity to better differentiate nodules at high risk of malignancy.

INTRODUCTION: Focused and expanded mutation panels were assessed for the incremental utility of using an expanded panel in combination with microRNA risk classification. METHODS: Molecular results were reviewed for patients who underwent either a focused mutation panel (ThyGenX ® ) or an expanded mutation panel (ThyGeNEXT ® ) for strong and weak oncogenic driver mutations and fusions. microRNA results (ThyraMIR ® ) predictive of malignancy, including strong positive results highly specific for malignancy, were examined. RESULTS: Results of 12 993 consecutive patients were reviewed (focused panel = 8619, expanded panel = 4374). The expanded panel increased detection of strong drivers by 8% (P < .001), with BRAFV600E and TERT promoters being the most common. Strong drivers were highly correlated with positive microRNA results of which 90% were strongly positive. The expanded panel increased detection of coexisting drivers by 4% (P < .001), with TERT being the most common partner often paired with RAS. It increased the detection of weak drivers, with RAS and GNAS being the most common. 49% of nodules with weak drivers had positive microRNA results of which 33% were strongly positive. The expanded panel also decreased the number of nodules lacking mutations and fusions by 15% (P < .001), with 8% of nodules having positive microRNA results of which 22% were strongly positive.CONCLUSIONS: Using expanded mutation panels that include less common mutations and fusions can offer increased utility when used in combination with microRNA classification, which helps to identify high risk of malignancy in the cases where risk is otherwise uncertain due to the presence of only weak drivers or the absence of all drivers.