Molecular Determinants of Thyroid Nodules with Indeterminate Cytology and RAS Mutations

RAS gene family mutations are the most prevalent in thyroid nodules with indeterminate cytology and are present in a wide spectrum of histological diagnoses. We evaluated differentially expressed genes and signaling pathways across the histological/clinical spectrum of RAS-mutant nodules to determine key molecular determinants associated with a high risk of malignancy. Sixty-one thyroid nodules with RAS mutations were identified. Based on the histological diagnosis and biological behavior, the nodules were grouped into five categories indicating their degree of malignancy: non-neoplastic appearance, benign neoplasm, indeterminate malignant potential, low-risk cancer, or high-risk cancer. Gene expression profiles of these nodules were determined using the NanoString PanCancer Pathways and IO 360 Panels, and Angiopoietin-2 level was determined by immunohistochemical staining. The analysis of differentially expressed genes using the five categories as supervising parameters unearthed a significant correlation between the degree of malignancy and genes involved in cell cycle and apoptosis (BAX, CCNE2, CDKN2A, CDKN2B, CHEK1, E2F1, GSK3B, NFKB1, and PRKAR2A), PI3K pathway (CCNE2, CSF3, GSKB3, NFKB1, PPP2R2C, and SGK2), and stromal factors (ANGPT2 and DLL4). The expression of Angiopoietin-2 by immunohistochemistry also showed the same trend of increasing expression from non-neoplastic appearance to high-risk cancer (p < 0.0001). The gene expression analysis of RAS-mutant thyroid nodules suggests increasing upregulation of key oncogenic pathways depending on their degree of malignancy and supports the concept of a stepwise progression. The utility of ANGPT2 expression as a potential diagnostic biomarker warrants further evaluation.