Figure 1: Exome sequencing identifies ^V600EB-RAF amplification as a candidate mechanism for BRAFi resistance.

(a) Copy-number variations (CNVs) called from whole-exome sequence data on two triads of gDNAs using ExomeCNV and chromosome 7 as visualized by Circos (outer ring, genomic coordinates (Mbp); centromere, red; inner ring, log ratio values between baseline and disease progression (DP) samples' average read depth per each capture interval; scale of axis for patient no. 5 –5 to 5 and for patient no. 8 –2.5 to 2.5). Two patients whose melanoma responded to and then progressed on vemurafenib. The genomic region coded orange (magnified views shown in the center of Circos maps) represents the location of B-RAF (chr7:140,424,943-140,524,564), which shows an average log ratio value of 1.14 (2.2-fold gain; patient no. 5) and 3.8 (12.8-fold gain; patient no. 8). (b) B-RAF immunohistochemistry on paired tissues derived from the corresponding patients as in a (scale bar, 50 μM). (c) Validation of ^V600EB-RAF copy-number gain by gDNA Q–PCR (black and red by B-RAF primer set 1 and 2, respectively) and recurrence across distinct patients (positives highlighted in orange). PMN, peripheral mononuclear cells; HDF, human dermal fibroblasts for diploid gDNAs. (d) B-RAF V600 mutant to WT ratio increases with disease progression or acquisition of B-RAFi resistance mediated by mutant B-RAF copy-number gain. Chromatograms from Sanger sequencing for melanoma samples from patients who acquired B-RAFi resistance based on distinct molecular alterations: ^V600EB-RAF copy-number gain, ^V600EB-RAF truncation, N-RAS mutation or RTK overexpression.