Probes for BRAF

BACKGROUND: The BRAF(V600E) mutation is the most common genetic alteration in papillary thyroid carcinoma (PTC). The aim of this study is to analyze the clinicopathologic correlations of the BRAF(V600E) mutation, BRAF V600E immunohistochemistry (IHC) and BRAF RNA in situ hybridization (ISH) in PTC. METHODS: This study included 467 patients with PTC who underwent surgical resection. We studied the BRAF(V600E) mutation using real-time PCR and BRAF V600E and BRAF RNA ISH using tissue microarray (TMA). RESULTS: The frequencies of a positive BRAF(V600E) mutation by real-time PCR, positive BRAF V600E IHC, and high BRAF RNA ISH were 84%, 86%, and 70%, respectively, in PTC. Conventional PTC had higher positive rates in all three tests than other histologic types. The BRAF(V600E) mutation, BRAF V600E IHC, low ΔCt, and high BRAF RNA ISH were significantly associated with lymph node metastasis. The BRAF(V600E) mutation was significantly associated with positive immunostaining for BRAF V600E mutant protein (P<0.001) overall, with high BRAF RNA ISH only in the follicular variant (P=0.035). No significant correlation was noted between BRAF V600E IHC and BRAF RNA ISH. The sensitivity of BRAF V600E IHC for the BRAF(V600E) mutation was 95%, and the specificity was 61% overall, 96% and 54% in the conventional type, and 85% and 70% in the follicular variant. CONCLUSIONS: Our results showed that positive BRAF V600E IHC significantly correlated with the BRAF(V600E) mutation. This suggests its clinical utility as a screening tool for the BRAF(V600E) mutation. In addition, a high BRAF RNA ISH score could be a candidate marker of aggressive behavior in BRAF(V600E) mutation-positive cases of PTC.

BRAF kinase has an essential role in intracellular signaling, facilitating signal transduction from membrane receptors to the nucleus following epithelial growth factor receptor (EGFR) activation (30). The co-inhibition of MEK- and BRAF kinases have improved outcomes in some _BRAF_-mutated malignities; however, most cases still develop some form of resistance (31-33). While BRAF kinase is normally deactivated in healthy tissues through a negative feedback loop, mutations in the _BRAF_ gene result in persistent activation of downstream cell signaling in the MAPK pathway, leading to uncontrolled cell growth and proliferation (34-36). Others showed in Caucasian lung cancer patient cohorts that smoking status was associated with a non-V600E mutation (20,37,38). In contrast, a study on an East Asian LADC cohort and a meta-analysis of 16 studies found the V600E subtype more common in non-smokers. The same studies showed associations with decreased chemosensitivity and worse prognosis (39,40). This is in line with our results, where increased BRAF RNA expression was also associated with non-smoker patient history and detrimental OS. In our cohort, most patients showed strong diffuse BRAF RNA signals that cannot be explained by potential V600E mutations since its frequency hardly reaches 3-5% in LADC patients. This might mean that WT BRAF can still be overexpressed in the absence of mutation in the _BRAF_ gene due to alternative intracellular signaling pathways or the tumor microenvironment. This is underlined by the fact that the gene’s protein product is also detectable by IHC with at least moderate staining intensity in 80% of samples, according to the HPA database. Activation of the BRAF-MAPK pathway is always preceded by the interaction between the guanosine-nucleotide-binding protein RAS and a Receptor Tyrosine Kinase (RTK). Because BRAF selectively binds to active RAS (41), hypothetically, increased RAS activation and RTK function might have a positive upstream effect on BRAF signaling, even in the absence of activating mutations.