The value of NGS-based multi-gene testing for differentiation of benign from malignant and risk stratification of thyroid nodules

Background: Fine-needle aspiration (FNA) biopsy is typically used in conjunction with cytopathologic evaluation to differentiate between benign and malignant thyroid nodules. Even so, the cytology results for 20-30% of thyroid nodules are indeterminate. This study sought to evaluate the usefulness of next-generation sequencing (NGS)-based multi-gene panel testing for risk stratification and the differentiation of benign from malignant thyroid nodules.

Methods: Thyroid nodule samples were obtained from a cohort of 359 patients who underwent FNA. An NGS-based multi-gene panel testing was conducted for these samples, in which single-nucleotide variants (SNVs) and small insertion/deletions (InDels) can be detected in 11 genes and fusion events can be identified in 5 genes. Surgical resection was conducted for 113 patients (113/359), and then histopathology results were obtained.

Results: In comparison to cytology alone, the diagnostic sensitivity of NGS combination cytology increased from 0.7245 (95% CI: 0.6289-0.8032) to 0.898 (95% CI: 0.8223-0.9437); the associated AUC was 0.8303 (vs. Cytology AUC: 0.7622, P < 0.001). BRAF ^V600E was identified in 136 patients, of whom 79 underwent surgery and were diagnosed with papillary thyroid carcinoma (PTC) pathologically. TERT promoter mutations or BRAF/RAS co-mutations with other genes were identified in 5 patients, while 4 patients were diagnosed with malignant thyroid cancer using the pathological method. RAS mutations were identified in 27 patients, while 10 patients underwent surgery, which showed that 3 patients were classified as PTC and 7 cases were benign. In addition, 4 RET fusions, 1 RET activation mutation, and 3 TP53 inactivation mutations were identified in the remaining 8 patients who have not undergone surgery. Negative genetic test results or variants with uncertain significance were identified in 183 patients. Among these patients, 12 malignant thyroid tumors, including 11 PTC and 1 MTC, were diagnosed in 20 patients who received surgery.

Conclusion: Thyroid nodules coupled with BRAF ^V600E, TERT promoter variants, BRAF/RAS co-mutations with other genes, RET fusions, and RET activating mutations were classified as high-risk. Nodules with RAS mutations (NRAS, KRAS, HRAS) and TP53 inactivating mutations were considered to be in the intermediate-risk group, while those with non-pathogenic mutations (negative and variants of uncertain significance) were placed in the low-risk group. When combined with cytopathology, NGS increases the sensitivity of diagnosing benign and malignant thyroid nodules, and the reference is useful for patient risk stratification.