The Molecular Heterogeneity of <i>NRAS</i> Variants in Thyroid Nodules

OBJECTIVE: 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. STUDY DESIGN: Retrospective database analysis. METHODS: The database of nodules sent for Afirma GSC testing was analyzed for the most common NRAS hotspot mutations, co-occurring mutations, BRAF-RAS score (BRS), thyroid differentiation score (TDS), ERK expression, and activity levels of hallmarks of cancer pathways. RESULTS: Of 87,817 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 (TERTp), 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 score. Hallmarks of cancer pathways analyzed across NRAS+ nodules, with TERTp profiling, revealed 2 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. NRASp.Q61R was equally distributed across the 2 clusters. CONCLUSION: NRAS p.Q61R/K comprises 99% of reported NRAS variants. TERTp is the most frequent co-mutation. Among NRAS p.Q61R/K nodules, there are 2 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.