BRAF gene mutational analysis of the thyroid and correlation with clinicopathologic findings

Fine needle aspiration biopsy (FNA) cytology has been found to be an effective method for determining the likelihood of malignancy in patients with a thyroid nodule. However, an equivocal cytologic diagnosis is associated with approximately one-fifth of all thyroid FNAs. Recently, an activating point mutation in the BRAF kinase gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC), while being highly specific for this tumor. Studies have suggested that BRAF V600E mutation may serve as a novel marker for PTC and perhaps is associated with a poor clinicopathologic outcome. The primary objective of this thesis was to develop a laboratory method for identifying the BRAF V600E mutation in thyroid tissue samples, for the purpose of refining indeterminate cytologic diagnoses. A secondary objective of this thesis was to investigate the relationship between the presence of the BRAF V600E mutation and various clinicopathologic features of PTC. Forty-two archival thyroid FNAs (19 = indeterminate; 5 = benign; 18 = PTC) matched formalin-fixed, paraffin-embedded (FFPE) surgical samples (5= benign; 37 = PTC) were each evaluated for the BRAF V600E mutation using LightCycler PCR with fluorescent melting curve analysis (LCPRC) and direct DNA sequencing. Wild-type and mutant DNA was used as controls. BRAF V600E mutation analysis confirmed the diagnosis of PTC in 3/19 (15.6%) indeterminate FNA samples. Nine of 19 (47.4%) corresponding FFPE surgical samples collected from the same patients were positive for the BRAF mutation, for a 69% rate of mutation concordance between the sample types. The BRAF V600E activating point mutation was identified in 14/18 (77.8%) malignant FNA samples. Fourteen of 18 (77.8%) surgical samples collected from the same patients were positive for the BRAF mutation, for a 100% rate of mutation concordance between the sample types. The BRAF mutation was not detected in FNA or FFPE samples from 5/5 benign thyroid nodules. There was no significant correlation between BRAF V600E mutation status and the clinicopathologic parameters of age, sex, histologic variant, and tumor stage. In view of the large numbers of thyroid nodules discovered each year that require FNA evaluation, BRAF mutation analysis has the potential to serve as a useful adjunct technique in a significant portion of indeterminate cytologic diagnoses. Due to the limited size of the present study, we were unable to extrapolate the full diagnostic utility of BRAF mutation analysis in patients with a histologic diagnosis of PTC.