The Genomic Landscape of 250,000+ Thyroid Nodules Undergoing Exome-Enriched RNA Sequencing

Introduction

• The Afirma Genomic Sequencing Classifier (GSC) is used to risk stratify and reclassify cytologically indeterminate thyroid nodules to molecularly benign or suspicious.
• The Afirma Xpression Atlas (XA) panel detects 905 variants and 235 fusion pairs from 593 predefined genes using exome-enriched RNA-sequencing.¹
• In 2021, genomic analysis of 50,000+ consecutive thyroid nodules with Bethesda III-VI (BIII-VI) cytology assessed by Afirma GSC was published, showing that almost one-half of BIII/IV Afirma GSC suspicious and most BV/VI nodules had at least one genomic alteration identified, which may optimize personalized treatment decisions.²

Study Aim

We aimed to describe the updated genomic landscape of BIII-BVI thyroid nodules sent for Afirma GSC testing in real world practice, including data on TERTp mutations, when available.

Methods

• Cytologic and molecular differences were assessed in 252,510 consecutive thyroid tumors with BIII-VI cytology undergoing Afirma GSC testing from 2018-2024.
• TERTP was assessed in 8,627 samples from 2023-2024.

Results

• Median patient age was 59.3 years. 76.5% of patients were female, and median nodule size was 2.2 cm. The proportion of parathyroid (PTA) and medullary thyroid carcinoma (MTC) classifier positive lesions were < 1%, while > 78% of nodules referred for testing were Bethesda 3 (Table 1).
• 140,565 (71%) BIII and 23,101 (57%) BIV nodules were classified as GSC-(B)enign.
• Overall, 69% of indeterminate nodules (BIII/ BIV) were classified as GSC-B.
• The proportion of XA variants increased with higher Bethesda category classifications (Table 2). Higher risk variants and fusions were enriched in nodules with BVI cytology, while RAS mutations were predominant in BIII/BIV nodules (Table 2, Figure 1).
• There was a significant decrease in GSC-S rates among BIII/IV nodules from 2018-2021 and reversal of that trend from 2021-2024 (Figure 2) (p < 0.001).

Conclusion

• This large study describes the molecular landscape of ~87,000 samples from suspicious thyroid nodules assessed by the Afirma Genomic Sequencing Classifier.
• Our findings highlight the molecular alterations seen across Bethesda categories in suspicious thyroid nodules:
  • BRAFV600E is the most frequent variant, particularly in BV/VI.
  • NRAS is the most prevalent RAS variant, most common in BIII/IV nodules.
  • TERTP mutations, alone or co-occurring with BRAFV600E were most common in BVI.
  • Fusions were identified in 6% of non-GSC-Benign nodules, most often in BV.
  • Most prevalent fusions involved RET proto-oncogene, seen in Bethesda V/VI > III/IV.
  • PPARY fusions were primarily detected in Bethesda III/VI.
• Temporal shifts in GSC-S rates are of interest and warrant further investigation.