Analytical Validation of a Telomerase Reverse Transcriptase (TERT) Promoter Mutation Assay

Context Telomerase reverse transcriptase (TERT) promoter-mutated thyroid cancers are associated with a decreased rate of disease-free and disease-specific survival. High-quality analytical validation of a diagnostic test promotes confidence in the results that inform clinical decision-making.Objective This work aimed to demonstrate the analytical validation of the Afirma TERT promoter mutation assay.Methods TERT promoter C228T and C250T variant detection in genomic DNA (gDNA) was analyzed by assessing variable DNA input and the limit of detection (LOD) of variant allele frequency (VAF). The negative and positive percentage agreement (NPA and PPA) of the Afirma TERT test was examined against a reference primer pair as was the analytical specificity from potential interfering substances (RNA and blood gDNA). Further, the intrarun, interrun, and interlaboratory reproducibility of the assay were tested.Results The Afirma TERT test is tolerant to variation in DNA input amount (7-13 ng) and can detect expected positive TERT promoter variants down to 5% VAF LOD at 7 ng DNA input with greater than 95% sensitivity. Both NPA and PPA were 100% against the reference primer pair. The test remains accurate in the presence of 20% RNA or 80% blood gDNA for an average patient sample that typically has 30% VAF. The test also demonstrated a 100% confirmation rate when compared with an external next-generation sequencing-based reference assay executed in a non-Veracyte laboratory.Conclusion The analytical robustness and reproducibility of the Afirma TERT test support its routine clinical use among thyroid nodules with indeterminate cytology that are Afirma Genomic Sequencing Classifier suspicious or among Bethesda V/VI nodules.