Molecular Profiling of 50 734 Bethesda III-VI Thyroid Nodules by ThyroSeq v3: Implications for Personalized Management

Abstract

Context: Comprehensive genomic analysis of thyroid nodules for multiple classes of molecular alterations detected in a large series of fine needle aspiration (FNA) samples has not been reported.

Objective: To determine the prevalence of clinically relevant molecular alterations in Bethesda categories III-VI (BCIII-VI) thyroid nodules.

Methods: This retrospective analysis of FNA samples, tested by ThyroSeq v3 using Genomic Classifier and Cancer Risk Classifier at UPMC Molecular and Genomic Pathology laboratory, analyzed the prevalence of diagnostic, prognostic, and targetable genetic alterations in a total of 50 734 BCIII-VI nodules from 48 225 patients.

Results: Among 50 734 informative FNA samples, 65.3% were test-negative, 33.9% positive, 0.2% positive for medullary carcinoma, and 0.6% positive for parathyroid. The benign call rate in BCIII-IV nodules was 68%. Among test-positive samples, 73.3% had mutations, 11.3% gene fusions, and 10.8% isolated copy number alterations. Comparing BCIII-IV nodules with BCV-VI nodules revealed a shift from predominantly RAS-like alterations to BRAF V600E-like alterations and fusions involving receptor tyrosine kinases (RTK). Using ThyroSeq Cancer Risk Classifier, a high-risk profile, which typically included TERT or TP53 mutations, was found in 6% of samples, more frequently BCV-VI. RNA-Seq confirmed ThyroSeq detection of novel RTK fusions in 98.9% of cases.

Conclusion: In this series, 68% of BCIII-IV nodules were classified as negative by ThyroSeq, potentially preventing diagnostic surgery in this subset of patients. Specific genetic alterations were detected in most BCV-VI nodules, with a higher prevalence of BRAF and TERT mutations and targetable gene fusions compared to BCIII-IV nodules, offering prognostic and therapeutic information for patient management.

Keywords: fine needle aspirate; gene fusions; genetic analysis; mutations; thyroid cancer; thyroid nodule.

Figure 1.

Molecular profiles of ThyroSeq v3-positive nodules with different Bethesda categories.

Figure 2.

Molecular profiles of test-positive nodules with different Bethesda categories.

Figure 3.

ThyroSeq v3 Detection of novel RTK fusions by measuring differential expression of the tyrosine kinase (TK) and extracellular domains of the RTK genes. A. Since wild-type RTK genes are not expressed in thyroid follicular cells, no expression or very low expression of both parts of the gene is expected (no or few sequencing reads detected by ThyroSeq v3); B. As a result of gene fusion, the TK domain of the RTK gene is fused with an actively transcribed partner gene leading to an increased expression of the TK domain (high number of sequencing reads), whereas the extracellular domain remains undetectable (no or few sequencing reads).