What is ThyroSeq v3?
ThyroSeq v3 is a next-generation sequencing molecular test that analyzes 112 genes for multiple types of genetic alterations (point mutations, insertions/deletions, gene fusions, copy number alterations, and abnormal gene expression) to help classify indeterminate thyroid nodules as benign or malignant, thereby guiding surgical decision-making. 1
Technical Specifications
ThyroSeq v3 is a DNA- and RNA-based assay that uses a genomic classifier (GC) to separate malignant from benign thyroid lesions 1. The test detects over 100 genetic alterations including:
- Point mutations: BRAF, RAS, TERT, DICER1 1
- Gene fusions: NTRK1/3, BRAF, RET fusions 1
- Copy number alterations: 22q loss 1
- Abnormal gene expression patterns 1
The analytical requirements include a minimal nucleic acid input of 2.5 ng and a minimal acceptable tumor content of 12% 1.
Clinical Application
Primary Use Case
ThyroSeq v3 is specifically designed for indeterminate thyroid nodules on fine-needle aspiration (FNA) specimens - those classified as Bethesda III or IV categories where cytology alone cannot determine malignancy 2. Since approximately 30% of thyroid FNA samples are inconclusive, this test helps avoid unnecessary surgery by providing molecular risk stratification 2.
Test Performance Metrics
Validation studies demonstrated:
Real-world institutional data shows more variable performance:
- Positive predictive value (PPV) ranges from 18.8% to 78.2% across different institutions 3, 4
- Negative predictive value (NPV) ranges from 75.9% to 100% 5, 4
- Benign call rate: 48-66% 6, 3
Risk Stratification Categories
ThyroSeq v3 provides results in several categories:
- "Positive" (high-risk or intermediate-high-risk mutations): Associated with 77.5% risk of malignancy (including NIFTP) in real-world practice 4
- "Negative": Low risk for malignancy 6
- "Currently negative": Low-risk mutations insufficient alone for malignant development 6
- "Negative but limited": Nondiagnostic due to low cell count 6
Special Performance in Hürthle Cell Neoplasms
ThyroSeq v3 demonstrates superior performance for Hürthle cell cancers, which historically have been challenging for molecular diagnostics 7, 8. The National Comprehensive Cancer Network recommends ThyroSeq v3 specifically for its ability to rule in and rule out malignancy in Hürthle cell lesions, with:
This represents a significant improvement over older molecular tests that had unacceptably high false-positive rates (86% of patients with suspicious findings underwent unnecessary surgery) 8.
Common Genetic Alterations Detected
RAS-like alterations are the most frequently identified mutations (66% of positive cases), with a 73.4% risk of malignancy and 59% malignant diagnosis when excluding NIFTP 4. This group includes:
- Encapsulated follicular variant papillary thyroid carcinomas 4
- Follicular carcinomas 4
- NIFTP (noninvasive follicular thyroid neoplasm with papillary-like nuclear features) 4
BRAF V600E and high-risk mutations demonstrate 100% risk of malignancy, all diagnosed as classic-type papillary thyroid carcinoma 4.
Clinical Utility and Limitations
Key Advantages
- Avoids unnecessary surgery in patients with negative results 6
- Correctly classifies most papillary, follicular, Hürthle cell lesions, medullary thyroid carcinomas, and parathyroid lesions 1
- No false-negative results reported in some institutional experiences with v3 5
Important Caveats
The positive predictive value for malignancy is lower in real-world practice than validation studies, particularly for RAS-like mutations 5, 3. This means some patients with positive results will have benign nodules on final pathology 5, 3.
TP53 mutations have been identified in benign nodules on resection, suggesting caution in interpretation 5.
The test performs best when integrated into institutional algorithms that consider clinical context, rather than used as a standalone decision tool 6.
Comparison to Other Platforms
ThyroSeq v3 is listed among thyroid-specific panel assays alongside other commercial tests, but is distinguished by its comprehensive analysis of 112 genes across multiple alteration types 2. The National Comprehensive Cancer Network specifically recommends ThyroSeq v3 for Hürthle cell neoplasms over other available molecular tests 7.