Preoperative mRNA Expression Signatures Associated with 2025 American Thyroid Association (ATA) Risk of Recurrence Categories

Background

Molecular testing refines risk in indeterminate thyroid nodules with (B)ethesda III or IV cytology, yet its preoperative prognostic value remains unclear. This study evaluates the ability of preoperative messenger RNA (mRNA) expression signatures derived from the Afirma Genomic Resource for Intelligent Discovery (GRID) to distinguish tumors with lower versus higher 2025 ATA risk of recurrence.

Methods

A retrospective, single-center study of thyroid cancer cases that underwent Afirma Genomic Sequencing Classifier testing as part of routine clinical care. 2025 ATA risk of recurrence labels were assigned and ATA low and low-intermediate (L-I) risk groups were combined and compared with intermediate-high (I-H) and high-risk groups. Expressed variants and fusions and molecular data from the Afirma GRID were compared between groups. The low/L-I group was used as the reference and logistic regression was used to associate GRID signatures with the ATA risk classification.

Results

A total of 72 cases met inclusion criteria with the following ATA 2025 risk classification: 26 low, 19 low-intermediate, 16 intermediate-high, and 11 high. Of the low/low-intermediate group, 35 (77.8%) samples had B III/IV cytology and 10 (22.2%) had B V/VI cytology. Of the intermediate-high/high group, 20 (74%) had B III/IV cytology and 7 (26%) had B V/VI cytology (p=NS between ATA groups). When evaluating expressed BRAFp.V600E, RAS variants, and ALK/NTRK/RET fusions, there was no significant difference between the ATA risk groups. Amongst GRID signatures, intermediate-high/high risk cancers correlated with increased expression of Apical Junction hallmark (OR 3.91, p < 0.01), and Invasion Signature Score (OR 3.35, p = 0.03), and decreased expression of Immune Content estimation (OR 0.34, p = 0.04). Differences were most pronounced between the low-intermediate and intermediate-high risk groups.

Discussion/Conclusion

In this study, well described mutations, such as BRAFp.V600E, did not distinguish recurrence risk using ATA 2025 criteria, suggesting that detection of variants and fusions alone is insufficient for prognostic stratification. mRNA expression of Apical Junction, Invasion Signature Score, and Immune Content estimation were associated with higher-risk disease. These findings identify transcriptional signatures as potential preoperative prognostic markers that may refine thyroid cancer risk assessment among tumors arising from the same Bethesda cytologic category and with the same expressed variants and fusions. Incorporation of these molecular markers may enhance individualized surgical and surveillance decision-making.