Abstract LB-123: Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer
Authors: Oxnard Geoffrey R. , Thress Kenneth S. , Paweletz Cloud P. , Felip Enriqueta , Cho Byoung Chul , Stetson Daniel , Dougherty Brian , Lai Zhongwu , Morkovets Aleksandra , Vivancos Ana , Kuang Yanan , Ercan Dalia , Cantarini Mireille , Barrett J Carl , Janne Pasi A. 
Published: August 2015

Abstract
Introduction: EGFR T790M is the most common mechanism of acquired resistance to EGFR TKIs in pts with EGFR-mutant lung cancer. AZD9291 is an irreversible, mutant-selective EGFR TKI developed to have potency against both sensiziting EGFR mutations and T790M. In the ongoing phase I study of AZD9291 (AURA, NCT01802632), the response rate in pts with T790M-positive lung cancer was &gt;60%. The molecular mechanism underlying acquired resistance to AZD9291 is not known.
Methods & Results: To explore for mechanisms of resistance to AZD9291, we studied cfDNA extracted from pretreatment and post-progression plasma collected on AURA.Next-generation sequencing (NGS) of cfDNA was first performed on an exploratory cohort of 7 pts. All exons of a 20 gene panel (including EGFR) underwent PCR amplification and NGS using an Illumina HiSeq. In 1 pt, NGS of progression plasma identified a new EGFR C797S mutation in exon 20, not present in pretreatment plasma. Stable expression of C797S in Ba/F3 cells induced a &gt;100-fold increase in IC50 to AZD9291 compared to EGFR activating and T790M mutations alone. To validate the plasma NGS, digital droplet PCR (ddPCR) assays were developed to detect key EGFR mutations including C797S. 15 T790M-positive cases were identified with progression plasma available for analysis. Serial plasma ddPCR showed that both the EGFR activating and T790M mutation levels decreased with AZD9291 treament and increased at progression, with 3 molecular subtypes of resistance apparent. In 6 pts (40%), C797S was detected in addition to T790M; NGS of resistance biopsies from 2 of these pts confirmed presence of acquired C797S. In 5 pts (33%), T790M was detected without evidence of C797S. Intriguingly, in 4 pts (27%), the T790M levels became undetectable with treatment despite high levels of the EGFR activating mutation at progression, suggesting overgrowth of a competing non-T790M resistance mechanism. Further NGS of progression plasma revealed additional evidence of the genomic heterogeneity of resistance. Individual sequencing reads indicate that C797S and T790M can occur either in cis or in trans (i.e. on competing resistant alleles). In the 2 pts with tumor NGS demonstrating C797S, plasma NGS identified both the DNA alteration seen in tumor as well as a second DNA alteration encoding for C797S.
Conclusion: Using complementary assays for genomic analyses of cfDNA, we identified 3 molecular subtypes of acquired resistance to AZD9291, including an EGFR C797S mutation never before reported in pts. Due to the key role of the C797 residue in drug binding, C797S is expected to induce resistance to all irreversible EGFR TKIs currently in clinical development. Plasma NGS revealed substantial genomic heterogeneity and highlights the need for combination therapies to effectively prevent or treat drug resistance in cancer.
Citation Format: Geoffrey R. Oxnard, Kenneth S. Thress, Cloud P. Paweletz, Enriqueta Felip, Byoung Chul Cho, Daniel Stetson, Brian Dougherty, Zhongwu Lai, Aleksandra Morkovets, Ana Vivancos, Yanan Kuang, Dalia Ercan, Mireille Cantarini, J Carl Barrett, Pasi A. Janne. Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-123. doi:10.1158/1538-7445.AM2015-LB-123