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Analytical Validation of a Telomerase Reverse Transcriptase (TERT) Promoter Mutation Assay.
Priyanka C Iyer1, Ramona Dadu1, Anna Barque2
1Department of Endocrine Neoplasia and Hormonal Disorders, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
The Journal of Clinical Endocrinology and Metabolism
|March 5, 2024
Summary
The Afirma TERT promoter mutation assay is analytically validated for detecting TERT mutations in thyroid cancer. This robust test demonstrates high accuracy and reproducibility, supporting its clinical use for indeterminate thyroid nodules.
Area of Science:
- Molecular Diagnostics
- Oncology
- Genetics
Background:
- Telomerase reverse transcriptase (TERT) promoter mutations are linked to poorer outcomes in thyroid cancer.
- Accurate diagnostic tests are crucial for informed clinical decisions in thyroid nodule management.
- Analytical validation ensures the reliability of diagnostic test results.
Purpose of the Study:
- To perform comprehensive analytical validation of the Afirma TERT promoter mutation assay.
- To assess the assay's performance characteristics, including sensitivity, specificity, and reproducibility.
Main Methods:
- Evaluated TERT promoter C228T and C250T variant detection across varying genomic DNA inputs.
- Determined the limit of detection (LOD) for variant allele frequency (VAF).
- Assessed negative and positive percent agreement (NPA/PPA), analytical specificity against interfering substances (RNA, blood gDNA), and assay reproducibility (intrarun, interrun, interlaboratory).
Main Results:
- The Afirma TERT assay reliably detects variants down to 5% VAF at 7-13 ng DNA input with >95% sensitivity.
- Achieved 100% NPA and PPA against a reference primer pair, with accuracy maintained in the presence of RNA and blood gDNA.
- Demonstrated 100% concordance with an external next-generation sequencing assay, confirming high analytical specificity and reproducibility.
Conclusions:
- The Afirma TERT test exhibits robust analytical performance and reproducibility.
- These validated characteristics support the assay's routine clinical application for thyroid nodules with indeterminate cytology.
- The assay is suitable for use in conjunction with Afirma Genomic Sequencing Classifier results or for Bethesda V/VI nodules.
