Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistant non-small cell lung cancer

Abstract

Purpose: Tumors from 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer patients that develop resistance to gefitinib or erlotinib will contain a secondary EGFR T790M mutation. As most patients do not undergo repeated tumor biopsies we evaluated whether EGFR T790M could be detected using plasma DNA.

Experimental design: DNA from plasma of 54 patients with known clinical response to gefitinib or erlotinib was extracted and used to detect both EGFR-activating and EGFR T790M mutations. Forty-three (80%) of patients had tumor EGFR sequencing (EGFR mutant/wild type: 30/13) and seven patients also had EGFR T790M gefitinib/erlotinib-resistant tumors. EGFR mutations were detected using two methods, the Scorpion Amplification Refractory Mutation System and the WAVE/Surveyor, combined with whole genome amplification.

Results: Both EGFR-activating and EGFR T790M were identified in 70% of patients with known tumor EGFR-activating (21 of 30) or T790M (5 of 7) mutations. EGFR T790M was identified from plasma DNA in 54% (15 of 28) of patients with prior clinical response to gefitinib/erlotinib, 29% (4 of 14) with prior stable disease, and in 0% (0 of 12) that had primary progressive disease or were untreated with gefitinib/erlotinib.

Conclusions: EGFR T790M can be detected using plasma DNA from gefitinib- or erlotinib-resistant patients. This noninvasive method may aid in monitoring drug resistance and in directing the course of subsequent therapy.

Figure 1

Detection of EGFR T790M using WAVE/Surveyor and SARMS. A, detection of EGFR T790M from the H1975 (EGFR L858R/T790M) cell line (top) and plasma DNA from patient 35 (bottom). Exon 20 of EGFR was amplified by PCR, the resulting product digested with Surveyor and analyzed using the WAVE-HSsystem (Materials and Methods). In the presence of EGFR T790M, two fragments (asterisk)are generated by Surveyor digestion (solid lines)fromthe positive control (H1975) and patient 35. The wild-type control (A549; dashed line)isuncut. B,SARMS analysis of EGFR T790M. Included are positive and negative control DNA samples and plasma DNA from patients 35 and 37. The horizontal dotted line represents the threshold. DNA from the negative control and patient 37 do not amplify above the threshold whereas DNA from the positive control and patient 35 both cross the threshold in the linear portion of the assay. Fluorescence was measured quantitatively in relative fluorescence units.