Figure 4: Fluorescent signatures from cancer cell lines with high frequency TKI- sensitizing and TKI- resistant mutations in EGFR exons 18-21. Black lines correspond to reference normal fluorescent signatures; gray lines correspond to fluorescent signatures from a cell line, arrows point to relevant differences indicative of mutations. All samples were processed in triplicate and plotted individually. In every instance EGFR mutations were confirmed by sequencing. Panel A: Each exon 19 deletion generates a unique fluorescent signature. Panel B-E: TKI-related mutations generate distinctive fluorescent signatures (Panel B, exon 18 G719S mutation in cell line SW48; Panel C, exon 20 T790M mutation in cell line NCI-H1975; Panel D, exon 21 L858R mutation in cell line NCI-1975; Panel E, exon 19 ΔL747-E749 deletion in cell line HCC4006). Panel F: Altered fluorescent signature from exon 21 in cell line HCC4006 due to a silent mutation in EGFR codon 836. This data demonstrates that silent mutations that do not affect the protein sequence generate their own fluorescent signature that distinguishes these mutations from true TKI-related mutations.