Abstract
The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced non-small-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow for sensitive and simultaneous detection of multiple mutations in cell-free DNA (cfDNA) with a minimum of extract needed and at lower cost. Patients were screened for the EGFR tyrosine-kinase inhibitor-sensitizing mutations Ex19Del, L858R, L861Q, G719S, and S768I using a novel ddPCR pentaplex assay. Patients who tested positive subsequently were monitored during treatment for the EGFR-sensitizing mutation and two EGFR-resistance mutations, T790M and C797S, using a ddPCR monitor triplex assay. The ddPCR multiplex assays enabled reliable detection of each mutation with a fractional abundance of at least 0.1%. For six patients, longitudinal data were analyzed and the ddPCR results provided a good reflection of the course of the disease and radiologic response. This study confirms that ddPCR on cfDNA supports the diagnosisand therapy selection, and shows that ddPCR multiplex assays on cfDNA could be a valuable additional diagnostic tool for therapy monitoring of non-small-cell lung cancer patients.
Highlights
Address correspondence to Remco de Kock, M.Sc., Clinical Laboratory, Catharina Hospital Eindhoven, Michelangelolaan 2, 5623 EJ, Eindhoven, the Netherlands
Six lung cancer patients who tested positive for the L858R, Ex19Del, S768I, or L861Q EGFR tyrosine-kinase inhibitor (TKI)-sensitizing mutation were monitored during treatment
Based on the EGFR screening triplex described recently,[14] an EGFR screening pentaplex was developed for the simultaneous detection of the Ex19Del, L858R, L861Q, G719S, and S768I mutations using the EGFR Multiplex cell-free DNA (cfDNA) Reference Standard Set (Horizon Discovery)
Summary
The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced nonesmall-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow for sensitive and simultaneous detection of multiple mutations in cell-free DNA (cfDNA) with a minimum of extract needed and at lower cost. Targeted therapy aimed at EGFR tyrosine-kinase inhibitor (TKI)-sensitizing mutations has improved the outcome of eligible nonesmall-cell lung cancer (NSCLC) patients compared with chemotherapy.[1] most TKI-treated patients eventually develop resistance mechanisms, 50% of which is caused by the T790M mutation.[2,3] These patients can be treated with second-line TKIs (eg, osimertinib). DdPCR can be used for multiplexing, enabling the detection of various targets in parallel with a minimum of ctDNA extract required and with relatively low exploitation costs.[14]
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