In this study, real-world clinical results of molecular testing for indeterminate thyroid nodules were analyzed based upon Bethesda categorization, mutation analysis, and microRNA classifier results and were correlated to show the utility of molecular analysis in assessing malignancy risk of indeterminate thyroid nodules. Cytology 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. In 2685 patients clinically tested, PIK3CA, PAX8/PPARγ, and RET/PTC mutations occurred in less than 1%. Of note, 2% had BRAF V600E 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. The study concluded that microRNA classification complements cytology and mutation analysis with the capacity to better differentiate nodules at high risk of malignancy.