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

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.