A testing algorithm for detection of the B-type Raf kinase V600E mutation in papillary thyroid carcinoma

Abstract

The B-type Raf kinase (BRAF) V600E mutation is a useful diagnostic marker for papillary thyroid carcinoma (PTC). We developed a testing algorithm for the BRAF mutation using BRAF immunohistochemistry (IHC) in PTC. Formalin-fixed, paraffin-embedded PTC tissues from 91 patients were immunostained with a BRAF V600E mutation–specific antibody. The immunostaining results were interpreted semiquantitatively by adding the scores of the proportion of positive cells (scored 0 to 5) and staining intensity (score 0 to 3). To validate BRAF IHC, 4 molecular methods were used: direct sequencing, pyrosequencing, peptide nucleic acid clamping polymerase chain reaction, and real-time polymerase chain reaction. The cut-off score of BRAF IHC for predicting the BRAF mutation was 5.5. In 68 cases, where the BRAF IHC score was 6 or more, 89.7% were positive for the BRAF mutation by 2 or more molecular methods and 97.1% by one or more methods. In 13 cases, where the IHC score was between 4 or more and less than 6, 61.5% were positive for the BRAF mutation by 2 or more methods and 69.2% by at least one method. In 10 cases, where the IHC score was less than 4, BRAF mutation was noted in one case only by one molecular method. BRAF V600E IHC scores of less than 4 or 6 or more should not require confirmation by additional tests. However, BRAF V600E IHC scores of 4 or 5 may require confirmation of the BRAF V600E mutation. In addition, BRAF IHC scores 6 or more were more sensitive than any single molecular method; therefore, BRAF IHC may overcome the limitations of using a single type of molecular method.