Does addition of BRAF V600E mutation testing modify sensitivity or specificity of the Afirma Gene Expression Classifier in cytologically indeterminate thyroid nodules?

OBJECTIVE: The purpose of this study was to determine the frequency of BRAF mutation in cytologically indeterminate thyroid nodules and to investigate whether adding the BRAF test improves diagnostic accuracy of the Afirma Gene Expression Classifier (GEC). DESIGN: BRAF V600E mutational status was determined for DNA extracted from cytologically benign (n = 40), indeterminate (n = 208), and malignant (n = 48) fine-needle aspiration specimens previously categorized by GEC as molecularly Benign or Suspicious. Analytical performance of the BRAF assay was assessed to establish reproducibility and limits of detection. Molecular testing results were correlated with blinded expert histopathological diagnoses. RESULTS: The BRAF assay detected mutations reproducibly to 2.5% mutant allele frequency. The prevalence of BRAF mutations in cytologically benign specimens was 2 of 40 (5.0%, 95% confidence interval [CI], 0-16) and in cytologically malignant specimens was 36 of 48 (75.0%, 95% CI, 60-86). In the cytologically indeterminate category, 10.1% of specimens were BRAF+: 2 of 95 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (2.1%, 95% CI, 0-7); 1 of 70 as follicular neoplasm or suspicious for follicular neoplasm (1.4%, 95% CI, 0-9); and 18 of 43 as suspicious for malignancy (41.9%, 95% CI, 27-58). All BRAF+ specimens were classified as Suspicious by the GEC. CONCLUSIONS: BRAF mutations are uncommon in nodules with atypia of undetermined significance or follicular lesion of undetermined significance or follicular neoplasm or suspicious for follicular neoplasm cytology. Most cytologically indeterminate nodules that proved to be malignant were also BRAF-, and all nodules that were false-negative by GEC were also BRAF-. Similarly, all BRAF+ specimens were also GEC Suspicious. Neither GEC test sensitivity nor specificity was improved by addition of BRAF mutation testing.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

OBJECTIVE: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.