Abstract B168: Monitoring acquired resistance to EGFR-TKIs and clonal evolution in metastatic NSCLC patients using liquid biopsies
Authors: Knebel Franciele , Bettoni Fabiana , Shimada Andrea , Cruz Manoel , Alessi João Victor , Negrao Marcelo , Muniz David , Reis Luiz Fernando , Katz Artur , Feher Olavo , Saragiotto Daniel , Camargo Anamaria 
Published: August 2018

Abstract
Epidermal Growth Factor Receptor (EGFR) activating mutations predict sensitivity to first- and second-generation anti-EGFR tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, NSCLC invariably develops acquired resistance (AR) to these agents, leading to disease progression. EGFR-T790M secondary mutation is the most frequent AR mechanism in this setting. Patients with EGFR-T790M positive tumors demonstrate impressive response rate to osimertinib, a third-generation EGFR-TKI, but disease progression also occurs. Intratumor heterogeneity and selection of genetic alterations that confer resistance to target therapy have been recognized as crucial in the development of AR to EGFR-TKIs. In the present study, we used liquid biopsies to monitor the appearance and dynamics of genetic alterations known to be associated with EGFR-TKIs AR in circulating tumor DNA (ctDNA) of patients with metastatic NSCLC treated with EGFR-TKIs. A total of 21 patients were recruited for this study, of whom 17 were initially treated with erlotinib, 2 with gefitinib, and 2 with afatinib. Disease progression was observed in 9 patients using erlotinib, 2 patient using gefitinib, and 1 patient using afatinib. The detection of EGFR mutations in these patients was carried out in serial liquid biopsies using droplet digital PCR (ddPCR) at a sensitivity between 0.1-0.5%. Of the 12 patients with disease progression, 7 (58.3%) were positive for the EGFR-T790M mutation in tissue biopsy and for 4 (57.1%) of these patients the EGFR-T790M was also detected by liquid biopsy at disease progression. Of the 7 EGFR-T790M positive patients, 5 received osimertinib, including 2 patients with EGFR-T790M positive liquid biopsy. Patients with EGFR-T790M positive liquid biopsies were followed closely using serial liquid biopsies to monitor the levels of the original EGFR mutation and of the EGFR-T790M resistance mutation. In general, plasma levels of the activating EGFR mutation and of the EGFR-T790M mutation accurately paralleled the clinical and radiologic evolution of the disease. Patients showing sequential increase in both EGFR mutations showed marked disease progression while those with stable levels presented indolent disease. EGFR-T790M plasma levels became undetectable within 1-2 weeks after the start of osimertinib, anticipating radiologic response to the drug. Of note, increases in T790M levels during treatment anticipated AR to osimertinib in one of our patients. This was accompanied by the emergence of a second EGFR resistance mutation (C797S) and a selective amplification of the EGFR-exon19del allele. In conclusion, liquid biopsies for EGFR genotyping can be used as a complementary strategy to tissue biopsies and should be considered the initial approach to identify both EGFR-TKI sensitizing and resistance mutations due to its minimally invasive nature. Also, plasma levels of EGFR mutations accurately paralleled the clinical and radiologic evolution of disease for these patients, allowing early detection of AR to TKIs. Finally, liquid biopsies can also be used to study novel mechanisms of AR to EGFR-TKIs.
Citation Format: Franciele Knebel, Fabiana Bettoni, Andrea Shimada, Manoel Cruz, João Victor Alessi, Marcelo Negrao, David Muniz, Luiz Fernando Reis, Artur Katz, Olavo Feher, Daniel Saragiotto, Anamaria Camargo. Monitoring acquired resistance to EGFR-TKIs and clonal evolution in metastatic NSCLC patients using liquid biopsies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B168.