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

CONTEXT: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 which inform clinical decision making. OBJECTIVE: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 percent 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 intra-run, inter-run and inter-laboratory 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 7ng DNA input with > 95% sensitivity. Both NPA and PPA were 100% against the reference primer pair. The test remains accurate in 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 NGS-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 GSC suspicious or among Bethesda V/VI nodules.