The primary occurrence of BRAFV600E is a rare clonal event in papillary thyroid carcinoma

Context: BRAF^V600E is considered a primary event, a negative prognostic marker, and a site for pharmacological intervention in papillary thyroid carcinoma (PTC). We asked whether BRAF^V600Ecan occur as a subclonal event in PTC and whether this and other oncogenes can coexist in the same tumor. Study Design: We determined by pyrosequencing the percentage of mutant BRAF, NRAS, and KRAS alleles in a series of conventional PTC. We also analyzed the BRAF mutation status in PTC cell clones in culture. Results: BRAF^V600E alleles were present in 41 of 72 PTC (56.9%) in the range 44.7 to 5.1% of total BRAF alleles. In four PTC samples, mutant BRAF alleles were about 50%, being therefore compatible with a clonal heterozygous mutation. In 27 PTC samples, BRAF^V600E alleles were in the range of 25 to 5.1%. This finding was confirmed after exclusion of the presence of a large contamination by lymphoreticular cells and by the analysis of PTC cells selected by laser capture. Analysis of clones derived from a single cell confirmed the presence of two distinct PTC populations with wild-type or mutated BRAF. Simultaneous subclonal BRAF and KRAS mutations were demonstrated in two PTC. Conclusions: These data demonstrate that clonal BRAF^V600E is a rare occurrence in PTC, although frequently this cancer consists of a mixture of tumor cells with wild-type and mutant BRAF. These results suggest that BRAF mutation in PTC is a later subclonal event, its intratumoral heterogeneity may hamper the efficacy of targeted pharmacotherapy, and its association with a more aggressive disease should be reevaluated.