BRAF Exon 15 Mutations in Papillary Carcinoma and Adjacent Thyroid Parenchyma: A Search for the Early Molecular Events Associated with Tumor Development

Abstract

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples-only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

Keywords: BRAF exon 15 mutations; BRAF p.V600E; follicular cell atypia; follicular cell hyperplasia; massively parallel sequencing; papillary thyroid carcinoma; psammoma bodies; tumor development.

Figure 1

Histologic appearance of the samples analyzed (a–d). (a) Papillary carcinoma BRAF p.V600E mutated (×100). (b) Psammoma body (arrow) protruding into a dilated lymphatic space (×100); the inset shows the psammoma body at higher magnification and in the plane of focus (×400). BRAF p.V600E is identified after sequencing of DNA extracted from the area containing the psammoma body. (c) Focus of follicular cell atypia (×200) (arrows, inset at higher magnification, ×400). BRAF p.V600M is identified after the sequencing of DNA extracted from the area with follicular cell atypia. (d) Focus of follicular cell hyperplasia with oncocytic change (×200) (arrows, inset at higher magnification, ×400). BRAF p. S607P is identified after the sequencing of DNA extracted from the area with follicular cell hyperplasia.

Figure 2

BRAF exon 15 status in thyroid samples. Bars represent the number of samples that are BRAF p.V600E, BRAF non p.V600E mutated, and BRAF wild type (WT). Percentages are in parenthesis. Two separate samples were analyzed from one follicular adenoma for a total of four samples from three follicular adenomas. Two separate samples were analyzed from one oncocytic follicular adenoma. PTC: papillary thyroid carcinoma; PTC-CL: classic PTC; PTC-TC: PTC, tall cell variant; PTC-FV-INF: PTC follicular variant—Infiltrative; PTC-FV-ENCAPS-CAPSINV: PTC follicular variant—Encapsulated, with capsular invasion; PsB: psammoma bodies; FA: follicular adenoma; FA-ONC: follicular adenoma, oncocytic; ATY: thyroid tissue with follicular cell atypia; HYP: thyroid tissue with follicular cell hyperplasia; and NORM: histologically normal thyroid tissue.

Figure 3

Schematic drawing of BRAF exon 15 mutations. The position and residue substitutions of all mutations identified are shown with respect to the standard BRAF nucleotide, codon, and amino acid residue sequence.

Figure 4

Functional characterization of BRAF p.G593D, BRAF p.A598T, BRAF p.S607F, and BRAF p.S607P compared to BRAF wild type (WT), BRAF p.V600E, BRAF p.K601E, and empty vector (EMPTY). (a) Effect of BRAF mutation on the MAPK signaling pathway: representative Western Blot of ERK, p-ERK, BRAF, and p-BRAF proteins in HEK-293 cells transiently transfected with wild type or mutant BRAF plasmids. Vinculin was used as a loading control. (b) Effect of BRAF mutation on ERK1/2 phosphorylation: densitometric analysis of Western Blots showing the p-ERK/ERK ratio in HEK-293 cells transiently transfected with wild type and mutant BRAF plasmids. HEK-293 cells transfected with BRAF-V600E and BRAF-K601E show increased activation of the MAPK pathway compared to BRAF WT, whereas HEK-293 cells transfected with BRAF-G593D, BRAF-A598T, BRAF-S607F, and BRAF-S607P show similar MAPK activation to BRAF WT. Data have been normalized to nontransfected controls (not shown); data are reported as the mean ± SEM of at least two independent experiments. **: p < 0.005 and ***: p < 0.0001. (c) Effect of BRAF mutation on BRAF phosphorylation: densitometric analysis of Western Blots showing the p-BRAF/BRAF ratio in HEK-293 cells transiently transfected with wild type and mutant BRAF plasmids. There are no significant differences in BRAF phosphorylation levels between the different constructs. Data have been normalized to nontransfected control; data are reported as the mean ± SEM of at least two independent experiments.