Background: An accurate preoperative workup of cytologically indeterminate thyroid nodules (ITN) may rule out malignancy and avoid diagnostic surgery for benign nodules. This study assessed the performance of molecular diagnostics (MD) and 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG)-positron emission tomography/computed tomography (PET/CT) in ITN, including their combined use, and explored whether molecular alterations drive the differences in [¹⁸F]FDG uptake among benign nodules. Methods: Adult, euthyroid patients with a Bethesda III or IV thyroid nodule were prospectively included in this multicenter study. They all underwent MD and an [¹⁸F]FDG-PET/CT scan of the neck. MD was performed using custom next-generation sequencing panels for somatic mutations, gene fusions, and copy number alterations and loss of heterozygosity. Sensitivity, specificity, negative and positive predictive value (NPV, PPV), and benign call rate (BCR) were assessed for MD and [¹⁸F]FDG-PET/CT separately and for a combined approach using both techniques. Results: In 115 of the 132 (87%) included patients, MD yielded a diagnostic result on cytology. Sensitivity, specificity, NPV, PPV, and BCR were 80%, 69%, 91%, 48%, and 57% for MD, and 93%, 41%, 95%, 36%, and 32% for [¹⁸F]FDG-PET/CT, respectively. When combined, sensitivity and specificity were 95% and 44% for a double-negative test (i.e., negative MD plus negative [¹⁸F]FDG-PET/CT) and 68% and 86% for a double-positive test, respectively. Concordance was 63% (82/130) between MD and [¹⁸F]FDG-PET/CT. There were more MD-positive nodules among the [¹⁸F]FDG-positive benign nodules (25/59, 42%, including 11 (44%) isolated RAS mutations) than among the [¹⁸F]FDG-negative benign nodules (7/30, 19%, p = 0.02). In oncocytic ITN, the BCR of [¹⁸F]FDG-PET/CT was mere 3% and MD was the superior technique. Conclusions: MD and [¹⁸F]FDG-PET/CT are both accurate rule-out tests when unresected nodules that remain unchanged on ultrasound follow-up are considered benign. It may vary worldwide which test is considered most suitable, depending on local availability of diagnostics, expertise, and cost-effectiveness considerations. Although complementary, the benefits of their combined use may be confined when therapeutic consequences are considered, and should therefore not routinely be recommended. In nononcocytic ITN, sequential testing may be considered in case of a first-step MD negative test to confirm that withholding diagnostic surgery is oncologically safe. In oncocytic ITN, after further validation studies, MD might be considered. Clinical Trial Registration: This trial is registered with ClinicalTrials.gov: NCT02208544 (August 5, 2014), https://clinicaltrials.gov/ct2/show/NCT02208544.

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