Cell-Free DNA Next-Generation Sequencing Prediction of Response and Resistance to Third-Generation EGFR Inhibitor

Abstract

We profiled 77 nonesmall-cell lung cancer patients with paired baseline and progression blood samples treated with the third-generation EGFR tyrosine kinase inhibitor (TKI) rociletinib using a broad cell-free circulating DNA (cfDNA) next-generation sequencing (NGS) gene panel. We demonstrated a utility of cfDNA NGS to detect EGFR T790M and predict response comparable to tissue-based tests, even at low allele fractions, and we identified resistance mechanisms. Our findings highlight the genomic heterogeneity observed in disease after progression while receiving therapy with a third-generation EGFR TKI. Introduction: The genomic alterations driving resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs) are not well established, and collecting tissue biopsy samples poses potential complications from invasive procedures. Cell-free circulating DNA (cfDNA) testing provides a noninvasive approach to identify potentially targetable mechanisms of resistance. Here we utilized a 70-gene cfDNA next-generation sequencing test to interrogate pretreatment and progression samples from 77 EGFR-mutated non-small cell lung cancer (NSCLC) patients treated with a third-generation EGFR TKI. Patients and Methods: Rociletinib was evaluated in advanced or metastatic (second line or higher) disease with EGFR T790M-positive NSCLC in the TIGER-X (NCT01526928) and TIGER-2 (NCT02147990) studies. Plasma samples were collected at baseline and at the time of systemic progression while receiving rociletinib. The critical exons in 70 genes were sequenced in cfDNA isolated from plasma samples to elucidate a comprehensive genomic profile of alterations for each patient. Results: Plasma-based cfDNA analysis identified 93% of the initial EGFR activating and 85% of the EGFR T790M resistance mutations in pretreatment samples with detectable tumor DNA. Profiling of progression samples revealed significant heterogeneity, with different variant types (eg, mutations, amplifications, and fusions) detected in multiple genes (EGFR, MET, RB1) that may be driving resistance in patients. Novel alterations not previously described in association with resistance to third-generation TKIs were also detected, such as an NTRK1 fusion. Conclusion: cfDNA next-generation sequencing identified initial EGFR activating and secondary T790M resistance mutations in NSCLC patients with high sensitivity, predicted treatment response equivalent to tissue analysis, and identified multiple novel and established resistance alterations.

EGFR T790M relative mutation purity predicts osimertinib treatment efficacy in non‐small cell lung cancer patients

“…Thus, investigating surrogates that can represent the proportion of T70M‐positive cells and predict osimertinib efficacy is feasible and essential. There has been much interest in the analysis of surrogates, such as T790M AF [22, 24, 33–35], EGFR driver AF [7,22], and T790M RMA level [5–10, 22–24] in plasma to predict response to osimertinib. However, inconsistent associations were observed across these studies.…”

“…The MSAF value for each individual patient was the highest AF of the detected somatic variants in corresponding blood sample. The T790M RMA was calculated as the ratio of T790M AF to EGFR driver AF, according to previous reports [5–10, 22–24]. In patients where EGFR driver mutation was undetected, we assumed the ratio to be 1.0.…”

Third generation EGFR TKI landscape for metastatic EGFR mutant non-small cell lung cancer (NSCLC)

“…The limited reports of resistance to osimertinib given as first-line therapy, reveal MET amplification and EGFR C797S mutation as possible predominant mechanisms [17]. Similarly, there may be significant heterogeneity in resistance mechanisms to rociletinib [21,22] and nazartinib [23]. Synthesizing this data however is problematic, as these studies are diverse with regards to line of therapy, use of tumor tissue or plasma circulating tumor DNA (ctDNA) and method and extent of genomic sequencing or alteration detection.…”

Liquid biopsy using extracellular vesicle–derived DNA in lung adenocarcinoma

“…Also, the mutation detection rate in intrathoracic metastatic diseases (M1a) is much lower (39% for activation mutations, 27% for T790M) [82]. To overcome this low sensitivity, various high technology platforms, such as droplet digital polymerase chain reaction (PCR) [81] or BEAMing (Beads, Emulsion, Amplification, and Magnetics) technology, were introduced by several research institutes [79,82]. Briefly, the BEAMing assay uses individual DNA molecules that are attached to magnetic beads in water-in-oil emulsions and processed by compartmentalized PCR amplification.…”