Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing: results of the RING observational trial

Abstract

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80-0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86-0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.

Trial registration: ClinicalTrials.gov NCT03363139.

Keywords: NGS; circulating free DNA; epidermal growth factor receptor; non-small-cell lung cancer; osimertinib; tyrosine kinase inhibitor.

Fig. 1

Venn diagrams showing concordance among NGS‐based methodologies and PCR‐based platforms for T790M detection. L2 NGS: Oncomine™ Pan‐Cancer Cell‐Free Assay performed in laboratory 2. L3 NGS: GeneRead™ QIAact Lung DNA UMI Cancer Panel performed in laboratory 3. L2 dPCR: QuantStudio^®3D Digital PCR System, performed in laboratory 2. L3 TaqMan in‐house 5‐nuclease real‐time PCR assay in presence of PNA carried out in laboratory 3. L1 BEAMing OncoBEAMEGFRperformed in laboratory 1.

Fig. 2

Comparison of MAFs obtained by NGS‐based platforms. (A) Passing‐Bablok regression showing close concordance between the two methods for the assessment of T790M MAFs and Bland–Altman plot showing low level of bias between both methods for quantifying T790M allele frequency. (B) Passing–Bablok regression and Bland–Altman plot showing the agreement between NGS‐based platforms for the quantification ofEGFR‐sensitizing mutations.