RING observational trial to compare T790M mutation testing in blood by different methodologies

Background: EGFR- mut NSCLC patients benefit from EGFR-TKIs therapy. However, most patients relapse within 1 to 2 years. In 40-60% of the cases, the mechanism of resistance is the emergence of the EGFR p.T790M mutation, and third generation TKIs have shown efficacy in this setting. The availability of new biopsies at relapse to TKIs is limited and guidelines recommend testing for the p.T790M in blood at relapse to TKIs, and rebiopsy only in case of a negative result. Several blood based methodologies for EGFR-mut detection have been developed, including some NGS approaches, but comparison studies are limited. We aim to evaluate the agreement, specificity and sensitivity of methodologies available for T790M testing in liquid biopsies and to determine the best pre-analytical conditions for T790M mutation identification. Trial design: The RING is a non-PAS, non-interventional, cross-sectional, diagnostic study that will enroll 75 patients from different Spanish institutions. Blood samples will be collected at the time of progression to first and second generation TKIs, and sent to the laboratories participating in the study. Three DNA extraction procedures will be tested; Maxwell® RSC cfDNA (Promega), the QIAamp® Circulating Nucleic Acid Kit (Qiagen) and automatic extraction using Qiasymphony® (Qiagen). The presence of the p.T790M in the purified DNA will be tested by 7 methodologies, namely cobas® EGFR Mutation Test v2 (Roche Diagnostics), Therascreen EGFR Plasma RGQ PCR Kit (Qiagen), QuantStudio® 3D Digital PCR System (Applied Biosystems), a 5′-nuclease real-time PCR (Taqman®) assay in presence of PNA, BEAMing digital PCR (Sysmex Inostics), NGS with the Oncomine® panel for Ion Torrent (Thermofisher) and NGS with the Lung Cancer Panel for GeneReader® (Qiagen). The kappa coefficient values and its corresponding 95% confidence intervals (95% CI) will be used to assess the agreement between methodologies and the Intraclass Correlation Coefficients (ICC) and Bland & Altman plots to evaluate the concordance between quantitative methodologies. Finally, we calculate that 40 patients will have a re-biopsy mutation T790M analysis, which will be considered the gold standard to estimate the sensitivity and specificity of each method. Legal entity responsible for the study: Spanish Lung Cancer Group / Grupo Español de Cancer de Pulmon (Slcg/Gecp). Funding: AstraZeneca. Disclosure: All authors have declared no conflicts of interest.