Clinical Implication of Highly Sensitive Detection of the BRAF V600E Mutation in Fine-Needle Aspirations of Thyroid Nodules: A Comparative Analysis of Three Molecular Assays in 4585 Consecutive Cases in a BRAF V600E Mutation-Prevalent Area

Abstract

Detection of the BRAF V600E mutation in fine-needle aspiration cytology (FNAC) specimens may increase the value of FNAC. The objectives of the study was to compare the diagnostic performance of BRAF assays that differ in sensitivity and to examine the associations between the BRAF V600E mutation status and the clinicopathological features in papillary thyroid carcinoma (PTC). Three molecular assays were performed in all subjects and compared with regard to FNAC and histology results. We evaluated 4585 consecutive patients who were found to have malignant or indeterminate thyroid nodules by ultrasonography. All FNAC samples were tested for the BRAF V600E mutation using conventional Sanger sequencing, dual-priming oligonucleotide-PCR, and mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The detection sensitivities of the three molecular assays for the BRAF V600E mutation were 20, 2, and 0.1%, respectively. Compared with conventional Sanger sequencing (n = 673), dual-priming oligonucleotide-PCR and MEMO sequencing detected more tumors with the BRAF V600E mutation (n = 919 and n = 1044, respectively), especially tumors with a benign, indeterminate, or nondiagnostic cytology. All BRAF-positive tumors that were histologically examined were shown to be PTC, regardless of cytology results. The clinical sensitivities of the three assays for detecting PTC were 54.8, 74.4, and 79.7%, respectively. BRAF V600E mutations in microcarcinomas (≤ 10 mm) were detected more efficiently as the detection sensitivity of the assay increased (P < 0.001). Tumor size correlated significantly with multifocality, extrathyroidal extension, and lymph node metastasis (P = 0.003, P < 0.001 and P < 0.001, respectively), but the BRAF V600E mutation status was not associated with any of those features. Highly sensitive and specific molecular assays such as MEMO sequencing are optimal for detecting the BRAF mutations in thyroid FNAC because these techniques can detect PTC that might be missed by cytology or less sensitive molecular assays.