Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M

Abstract

Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

Fig. 1

Acquired resistance to AZD9291 mediated by acquired EGFR C797S. (a) In the index case (Subject #1), targeted NGS identified an acquired T→A mutation (green) in 1.3% of reads, encoding for an EGFR C797S mutation. Overlapping reads spanning T790 and C797 contain both the T790M and C797S mutations, indicating the two mutations occur in cis on the same allele. (b) Ba/F3 cells harboring one of two EGFR activating mutations (exon 19 deletion or L858R) plus the T790M resistance mutation, either with or without C797S, were treated with either AZD9291 or CO-1686 at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated control cells. Experiments were repeated 3 times, with mean and standard deviation plotted at each concentration. The curves were fitted using a non-linear regression model with a sigmoidal dose response. (c) Ba/F3 cells expressing EGFR del 19/T790M and del 19/T790M/C797S cells were treated with 1.0 μM AZD9291 or CO-1686 for 6 hours. Cell extracts were immunoblotted to detect total or phosphorylated EGFR andtubulin (loading control). (d–f) Representative images from serial plasma ddPCR show three molecular subtypes of acquired resistance to AZD9291 (N/D: not detected). A subset of subjects acquire an EGFR C797S resistance mutation, always in the presence of T790M (d). Other subjects maintain the EGFR T790M mutation without evidence of an acquired C797S (e). The remaining subjects lose the EGFR T790M mutation despite increasing levels of the EGFR activating mutation, converting to T790M− resistance (f).

Fig. 2

NGS of baseline and progression cfDNA demonstrates several different genomic presentations of acquired EGFR C797S. (a) NGS of cfDNA from Subject #2 indicates the acquired C797S G→C mutation (blue) is on a different allele, in trans with the T790M mutation. (b–c) NGS of baseline and progression tumor biopsies (top panels) confirmed the acquired C797S mutation detected with plasma NGS (bottom). Plasma NGS detects the same T→A C797S mutation (green) found in the tumor and additionally detects a second G→C mutation encoding for C797S (blue).