Afirma Genomic Sequencing Classifier (GSC) and Xpression Atlas Molecular Findings in Bethesda III Thyroid Nodules: Comparative Analysis of Nuclear Versus Other Atypia and Correlation with Histology

Background

• The 2023 Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) divides atypia of undetermined significance (AUS) nodules into two major subcategories: nuclear atypia (AUS-N) and other (AUS-O).
• While studies have reported the malignant risk of AUS thyroid nodules differs according to the nature of atypia, little is known about the difference of molecular profiles between AUS-N and AUS-O thyroid nodules.
• This study aims to investigate the molecular findings between AUS-N and AUS-O thyroid nodules across a large, real-world cohort of fine needle aspiration (FNA) samples and to correlate these findings with histological outcomes.

Design

• A retrospective study of consecutive thyroid FNA samples diagnosed as AUS and sent for Afirma Genomic Sequencing Classifier (GSC) testing.
• Afirma GSC classifier results, Xpression Atlas (expressed variants and fusions) results in GSC-suspicious samples, and tumor specific molecular signatures were analyzed and compared between AUS-N and AUS-O samples.
• A single center, retrospective analysis of histopathology specimens with AUS-N or AUS-O cytology were analyzed as well.

Results

• Between April 2024 – December 2024, there were 5,345 paired cytology and Afirma GSC samples sent through Thyroid Cytopathology Partners (TCP), read as AUS, and stratified as AUS-N or AUS-O (Table 1).
  • AUS-N = 920
  • AUS-O = 4,425
• The Afirma GSC ensemble classifier reported GSC-(S)uspicious results in 58.2% of AUS-N, compared to 28.1% of AUS-O (p<0.01) (Table 2).
• Overall, there were more molecular variants in the AUS-N Afirma GSC-S nodules than in AUS –O (34.6% vs. 27.8%), mainly driven by the proportion of BRAFV600E. There was no difference in the overall proportion of fusions between the two groups though higher risk fusions (RET, NTRK3, ALK) were more common in the AUS-N group (Table 3).
• AUS-N were significantly more BRAF-like on BRAF-RAS score (BRS) and had higher ERK and follicular-mesenchymal transition (FMT) signature expression than AUS-O nodules (Figure 1).
• There were 201 surgically resected AUS, Afirma GSC-S nodules analyzed from the University of Michigan (Table 4).
  • There were significantly more malignancies in the AUS-N (28/82=34.1%) vs AUS-O (18/119=15.1%).
  • There were significantly more Papillary Thyroid Carcinomas (PTC) in the AUS-N (25/27=92.6%) compared to AUS-O (12/18=66.7%).