Molecular Landscaping of Thyroid Nodules by Whole Transcriptome RNA Sequencing and Its Correlation

Abstract

Introduction: Thyroid nodules are common, but cytology alone may not reliably classify indeterminate lesions. This study aimed to characterize the transcriptomic and genomic landscape of thyroid nodules using whole-transcriptome RNA sequencing and determine its relationship with the 2023 Bethesda cytological categories to improve diagnostic stratification. Methodology: This observational, hospital-based descriptive study included 63 surgically excised thyroid nodules classified as Bethesda III (n=20), IV (n=29), or V (n=14). RNA extracted from fresh tissue underwent quality assessment, strand-specific library preparation, and paired-end high-throughput sequencing. Bioinformatic analyses included transcript quantification, variant and fusion detection, copy-number analysis, and molecular subtyping. Molecular findings were correlated with cytology, ultrasonography, histopathology, thyroid function, and surgical procedure. Results: The mean age was 51.97 ± 11.60 years, and 55 patients (87.3%) were women. Papillary thyroid carcinoma was identified in 50 cases (79.4%). Mutation profiles, molecular subtypes, and copy-number variations differed significantly across Bethesda categories (all p<0.001). All Bethesda V nodules were BRAF-like, whereas all Bethesda IV nodules were RAS-like. Bethesda III lesions were heterogeneous, with non-BRAF/non-RAS profiles predominating (60%). BRAF V600E and ETV6-NTRK3 were enriched in Bethesda V; HRAS G13R and NRAS Q61R predominated in Bethesda IV; and DICER1, EIF1AX, and EZH1 alterations were confined to Bethesda III. Bethesda V showed frequent 7p gains, whereas 1q gains and 22q losses characterized Bethesda IV. TIRADS category was not associated with Bethesda score (p=0.112), while surgical procedure was significantly associated (p=0.031). Conclusion: Whole-transcriptome RNA sequencing provided clear molecular separation of Bethesda categories and revealed substantial biological heterogeneity within indeterminate nodules. Integrating molecular profiles with cytology may improve malignancy-risk estimation, guide surgical extent and targeted therapy, and reduce unnecessary interventions. Larger multicentre studies with longitudinal outcomes are required before routine clinical implementation.

Keywords: Thyroid nodules; Whole-transcriptome RNA sequencing; Bethesda classification; Gene expression profiling; Molecular subtyping; Next-generation sequencing; Papillary thyroid carcinoma; Precision oncology.