Figure 1. Model of primary resistance to vemurafenib using PTC patient-derived cells harboring the heterozygous BRAF^V600E mutation and with P16 (CDKN2A) deletion.

A. KTC1 cells are spontaneously immortalized cell derived from the pleural effusion of a BRAF^V600E positive recurrent papillary thyroid carcinoma (PTC). B. Probe design for the detection of P16 (CDKN2A) by fluorescence in situ hybridization (FISH) in KTC1 cells. C. FISH analysis for the detection of P16 (CDKN2A) gene in KTC1 cells. D. Microarray analysis of KTC1 cells (pink). Zoom in view of the CDKN2A gene region of chromosome 9 showing the biallelic deletion of 9p21. The larger 3.0 Mb deletion on one chromosome 9 takes out the CDKN2A gene and the entire segment covered by the orange FISH probe, while the smaller 531 kb deletion also results in deletion of CDKN2A but leaves intact a small portion of the region covered by the FISH probe. This explains why a single small red CDKN2A signal was detected by FISH. All above results were validated by two independent replicate measurements. E. Phase contrast images of KTC1 cells treated with 10 µM vemurafenib or DMSO (vehicle) for 48 hours (hrs) show sub-population of cells resistant to treatment (arrowheads). These results were validated at least by three independent replicate measurements. F. Growth curve based on KTC1 cell count shown as fold change (FC) in the presence of 10 µM vemurafenib or vehicle (DMSO). Angular coefficient (m) values between 0 and 2 days (m1); between 2 and 7 days (m2) are shown: cell death rate was significantly reduced by 6.8-folds beyond 2 days by vemurafenib treatment. These data represent the average ± standard deviation (error bars) of four independent replicate measurements (*p < 0.05, **p < 0.01, ***p < 0.001). G. Representative western blot analysis of KTC1 cells treated with 10 µM vemurafenib at the indicated time points shows that phospho(p)-ERK1/2 protein expression levels are not reduced in surviving cells compared to vehicle-treated cells. These results were validated at least by three independent replicate measurements.