Research Papers:

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients

Claudia Vollbrecht _, Annika Lehmann, Dido Lenze and Michael Hummel

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Oncotarget. 2018; 9:18529-18539. https://doi.org/10.18632/oncotarget.24908

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Claudia Vollbrecht1,2,3, Annika Lehmann1, Dido Lenze1,* and Michael Hummel1,2,*

1Charité-Universitätsmedizin Berlin Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Berlin, Germany

2German Cancer Consortium (DKTK), Partner Site Berlin, Berlin, Germany

3German Cancer Research Center (DKFZ), Heidelberg, Germany

*Shared senior authorship

Correspondence to:

Claudia Vollbrecht, email: [email protected]

Keywords: liquid biopsy; cfDNA; NSCLC; T790M; NGS

Received: January 24, 2018     Accepted: March 12, 2018     Published: April 06, 2018


Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy.

We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays.

Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay.

Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

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