Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients.

Haiyan E Liu,James Che,Jonathan Goldman,Dino Di Carlo,Meghah Vuppalapaty,Melissa Matsumoto,Michael Chiu,Steve Crouse,Clementine Lemaire,Elodie Sollier,James Carroll,Stefanie S Jeffrey,Edward B Garon,Violet R Hanft,Charles Wilkerson,Corinne Renier

doi:10.3389/fonc.2020.572895

Abstract

Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutations, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks, are not always successful and give results relevant to the timepoint of the assay. To detect secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKIs and lead to the administration of a 3rd generation drug, effective and non-invasive monitoring of EGFR mutation status is needed. Liquid biopsy analytes, such as circulating tumor cells (CTCs) and circulating tumor DNA (cfDNA), allow such monitoring over the course of the therapy. The aim of this study was to develop and optimize a workflow for the evaluation of cfDNA and CTCs in NSCLC patients all from one blood sample. Using Vortex technology and EntroGen ctEGFR assay, EGFR mutations were identified at 0.5 ng of DNA (∼83 cells), with a sensitivity ranging from 0.1 to 2.0% for a total DNA varying from 25 ng (∼4 CTCs among 4000 white blood cells, WBCs) to 1 ng (∼4 CTCs among 200 WBCs). The processing of plasma-depleted-blood provided comparable capture recovery as whole blood, confirming the possibility of a multimodality liquid biopsy analysis (cfDNA and CTC DNA) from a single tube of blood. Different anticoagulants were evaluated and compared in terms of respective performance. Blood samples from 24 NSCLC patients and 6 age-matched healthy donors were analyzed with this combined workflow to minimize blood volume needed and sample-to-sample bias, and the EGFR mutation profile detected from CTCs and cfDNA was compared to matched tumor tissues. Despite the limited size of the patient cohort, results from this non-invasive EGFR mutation analysis are encouraging and this combined workflow represents a valuable means for informing therapy selection and for monitoring treatment of patients with NSCLC.

Highlights

Lung cancer is the leading cause of cancer deaths in the United States, among both men and women
In terms of DNA input, the corresponding Epidermal Growth Factor Receptor (EGFR) mutation was successfully detected for each cell line for DNA quantities as low as 0.2 ng, which corresponds to approximately 33 cells (Figure 3A)
Characterization of the cfDNA – circulating tumor cells (CTCs) DNA Workflow (i) On one hand, to validate the cfDNA workflow, HCC827 DNA was spiked into EDTA whole blood at 0.5 ng per 2 mL of blood (Experiment x, Figure 4). cfDNA was extracted from the plasma and tested for EGFR mutations. 19Del was successfully detected as expected

Summary

Introduction

Lung cancer is the leading cause of cancer deaths in the United States, among both men and women. Several studies have shown that distinct mutation patterns in EGFR, including exon 19 deletion and exon 21 L858R substitution, were associated with positive treatment outcomes following first-line TKI therapy with afatinib, erlotinib, gefitinib and dacomitinib. These drugs can be administrated alone or, for a better overall survival, in combination with other treatment options. The monitoring of these actionable mutations throughout the patient care is fundamental for the selection of suitable TKI treatment

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