Background: Molecular testing for oncogenic gene mutations and chromosomal rearrangements plays a growing role in the optimal management of thyroid nodules, yet lacks standardized testing modalities and systematic validation data. Our objective was to assess the performance of molecular cytology on preoperative thyroid nodule fine-needle aspirates (FNAs) across a broad range of variables, including independent collection sites, clinical practices, and anatomic pathology interpretations.
Methods: Single-pass FNAs were prospectively collected from 806 nodules 1 cm or larger by ultrasonography at five independent sites across the United States. Specimens were shipped in a nucleic acid stabilization solution and tested at a centralized clinical laboratory. Seventeen genetic alterations (BRAF, KRAS, HRAS, and NRAS mutations, PAX8-PPARG and RET-PTC rearrangements) were evaluated by multiplex polymerase chain reaction and liquid bead array cytometry in 769 FNAs that met inclusion criteria. Cytology, histology, and clinical care followed local procedures and practices. All results were double-blinded.
Results: Thirty-two specimens (4.2%) failed to yield sufficient nucleic acid to generate molecular data. A single genetic alteration was detected in 80% of cytology malignant cases, 21% of indeterminate, 7.8% of nondiagnostic, and 3.5% of benign cases. Among 109 nodules with surgical histology reference standard, oncogenic mutations were present in 50% of malignant nodules missed by cytology. There were 14 cancers not identified by cytology or molecular tests, including 5 carcinomas with histologic sizes less than 1 cm (3 multifocal) and 8 noninvasive follicular variants of papillary carcinoma (4 encapsulated). No mutations were detected in 89% of the nodules benign by histopathology with 6 false-positive molecular results in 5 adenomas (2-5.5 cm) and 1 cystic nodule with an incidental papillary microcarcinoma (0.15 cm). The posttest probability of thyroid cancer was 100% for nodules positive for BRAF or RET-PTC, 70% for RAS or PAX8-PPARG, and 88% for molecular cytology overall.
Conclusions: Centralized and standardized molecular testing for genetic alterations associated with a high risk of malignancy efficiently complements the local cytopathologic diagnosis of thyroid nodule aspirates in the clinical setting. Actionable molecular cytology can improve the personalized surgical and medical management of patients with thyroid cancers, facilitating one-stage total thyroidectomy and reducing the number of unnecessary diagnostic surgeries.