Investigating an orally available small-molecule inhibitor (vemurafenib) of BRAFV600E in a novel preclinical model of human papillary thyroid cancer.

Abstract

e17014 Background: Patients with BRAFV600E-positive human papillary thyroid cancer (PTC) have poorer prognosis, higher rates of metastases and mortality, and resistance to radioiodine treatment. The goal of this study was to assess the therapeutic efficacy of vemurafenib (a new orally available BRAFV600E selective inhibitor) in a translational therapeutic model of BRAFV600E human nonmetastatic or metastatic PTC. Methods: We have established in vitro cultures of human primary PTC cells with or without heterozygous BRAFWT/V600E, primary normal thyroid (NT) cells, and used previously established human PTC cell lines with BRAFV600E. Immunocytochemistry was used to characterize markers of differentiation. Genotyping (over 500 genes analyzed) by mass spectrometry was performed to determine genetic alterations. Cell viability assays were performed upon different concentrations of vemurafenib: 0.01, 0.1, 1, 5, and 10 µM. Phosphorylation (p) of ERK1/2 (downstream BRAFV600E) was used to measure BRAFV600E activity. Results: We have isolated 8 independent batches of primary human non-metastatic or metastatic PTC cells from total thyroidectomy patients with PTC >1.1 cm, and 4 independent batches of primary NT cells from normal matched thyroid tissue specimens. 62.5% of the isolated batches of PTC cells were heterozygous BRAFWT/V600E and expressed epithelial markers and thyroid differentiation markers (e.g. PAX8, TSH-receptor). The NT cells showed no mutations. BRAFWT/V600E nonmetastatic PTC cells showed decreased viability (IC50: 5 µM) and pERK1/2 levels (IC90: 10 µM) when exposed to vemarufenib, with no toxic effects. PTC cells with BRAFWT, or NT cells showed no change in viability and pERK1/2 levels when exposed to vemarufenib. Importantly, BRAFWT/V600E metastatic PTC cells showed a partial suppression of viability and inhibition of pERK1/2 but only at a higher dose (10 µM) of vemurafenib. Conclusions: We have established the first translational therapeutic model of heterozygous BRAFWT/V600E-PTC. Testing of PTC samples for BRAFWT/V600E and testing in vitro will help predict therapeutic efficacy of Vemurafenib in patients with BRAFWT/V600E-PTC.