The Molecular Heterogeneity of NRAS Variants in Thyroid Nodules

Introduction: NRAS variants are the most common mutations in thyroid nodules with (B)ethesda III/IV cytology. This study aims to characterize co-occurring variants, molecular signatures, and hallmarks of cancer pathways in nodules with NRAS variants.

Methods: The Afirma database was assessed for the most common NRAS hot spot mutations, co-occurring mutations, BRAF-RAS score (BRS), thyroid differentiation score (TDS), ERK expression, and activity levels of hallmarks of cancer pathways.

Results: Of 76,710 Afirma GSC-suspicious or BV/VI nodules, 12.6% had NRAS mutations. Of those, 79.7% were NRAS:p.Q61R and 19.4% were NRAS:p.Q61K. 421 nodules (3.8%) had co-occurring mutations, EIF1AX being the most common (1%, about twice the rate seen alone). Of 872 nodules with TERT promoter mutations profile, 7.8% had concurrent NRAS+TERT mutations (more than twice the rate of TERT alone). The presence of NRAS (+/- co-mutations) was highly RAS-like on the BRS. NRAS+TERT had significantly lower TDS scores and higher ERK expression than NRAS alone. Hallmarks of cancer pathways analyzed across 2,055 NRAS+ nodules revealed two distinct clusters: one with higher inflammatory pathway activity, and another with higher DNA repair, estrogen response, and MYC activity. The latter had a higher rate of BV/VI nodules and risk of vascular invasion. NRAS:p.Q61R was equally distributed across the two clusters.

Conclusions: NRASp.Q61R/K comprises 99% of reported NRAS variants. TERT is the most frequent co-mutation and, when present, is associated with a lower TDS and higher ERK expression. Among NRAS:p.Q61R/K nodules, there are two different clusters of samples based on the activity of hallmarks of cancer pathways. Further studies correlating these clusters with clinical and pathological outcomes are necessary.