Background: Fine needle aspiration (FNA) cytology, a diagnostic test central to thyroid nodule management, may yield indeterminate results in up to 30% of cases. The Afirma® Genomic Sequencing Classifier (GSC) was developed and clinically validated to utilize genomic material obtained during the FNA to accurately identify benign nodules among those deemed cytologically indeterminate so that diagnostic surgery can be avoided. A key question for diagnostic tests is their robustness under different perturbations that may occur in the lab. Herein, we describe the analytical performance of the Afirma GSC. Results: We examined the analytical sensitivity of the Afirma GSC to varied input RNA amounts and the limit of detection of malignant signals with heterogenous samples mixed with adjacent normal or benign tissues. We also evaluated the analytical specificity from potential interfering substances such as blood and genomic DNA. Further, the inter-laboratory, intra-run, and inter-run reproducibility of the assay were examined. Analytical sensitivity analysis showed that Afirma GSC calls are tolerant to variation in RNA input amount (5-30 ng), and up to 75% dilution of malignant FNA material. Analytical specificity studies demonstrated Afirma GSC remains accurate in presence of up to 75% blood or 30% genomic DNA. The Afirma GSC results are highly reproducible across different operators, runs, reagent lots, and laboratories. Conclusion: The analytical robustness and reproducibility of the Afirma GSC test support its routine clinical use among thyroid nodules with indeterminant FNA cytology.
Keywords: Afirma GSC; RNA-Seq; analytical verification; clinical robustness; genomics; lab developed test; molecular diagnostics; thyroid cancer.