Abstract
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.
Highlights
The detection of activating epidermal growth factor receptor (EGFR) mutations in a subset of non-small cell lung cancer (NSCLC) patients and the development of corresponding tyrosine kinase inhibitors (TKI) has led to an important expansion of the therapeutic options and to a significant improvement of the clinical outcome of these patients [1, 2]
Primary EGFR mutations derived from the analysis of corresponding tumor tissue of the analyzed 26 liquid biopsies (LB) from 21 patient samples were composed of 15 point mutations (11 patients), 10 indels (9 patients) and one unknown status (Supplementary Table 2)
Due to the advanced tumor stage of the respective NSCLC patients, the acquisition of tissue specimens may be difficult and the minimal-invasive analysis of LB is becoming increasingly attractive as an alternative approach to stratify patients according to their mutational profile
Summary
The detection of activating epidermal growth factor receptor (EGFR) mutations in a subset of non-small cell lung cancer (NSCLC) patients and the development of corresponding tyrosine kinase inhibitors (TKI) has led to an important expansion of the therapeutic options and to a significant improvement of the clinical outcome of these patients [1, 2]. To overcome T790M mediated resistance, third generation TKI (e.g. osimertinib) have been developed that irreversibly bind to the mutated EGF receptor providing a further therapeutic option after treatment with TKI targeting activating EGFR mutations [8,9,10]. Tumor tissue is usually employed for the identification of activating as well as T790M resistance EGFR mutations. The use of tissue and biopsies in particular has several shortcomings. They represent only a snapshot of the entire tumor and cannot capture its entire genomic heterogeneity. Thoracic biopsies show considerable high rates of clinical complications and can be technically challenging or even impossible or clinically not feasible [11, 12]
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