Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

Cell Biology and Biotherapy Unit Istituto Nazionale Tumori 'Fondazione G Pascale' IRCCS Naples Italy

Center of Predictive Molecular Medicine CeSI Met University of Chieti Chieti Italy

Centre for Cancer Research and Cell Biology Queen's University Belfast Belfast United Kingdom; The Royal Marsden Surrey United Kingdom

Department of Clinical Biochemistry Aarhus University Hospital Aarhus Denmark

Department of Pathology Radboud University Medical Center Nijmegen the Netherlands

Department of Pathology University of Groningen University Medical Center Groningen Groningen the Netherlands

Department of Public Health and Primary Care Biomedical Quality Assurance Research Unit University of Leuven Leuven Belgium

Faculty Hospital Hradec Kralove Hradec Králové Czech Republic

Genomics and Oncology Roche Molecular Systems Pleasanton California

Hospital Universitario HM Sanchinarro Madrid Spain

Institut fur Haematopathologie Hamburg Germany

Institute for Pathology and Bacteriology Otto Wagner Spital Vienna Austria

Laboratoire Alphabio Marseille France

Molecular Pathology Diagnostic Service Queen Elizabeth Hospital Birmingham United Kingdom

Pathology and Bacteriology SMZO Donauspital Vienna Austria

Roche Sequencing Solutions Pleasanton California

University Clinic of Respiratory and Allergic Diseases Golnik Golnik Slovenia

University of Navarra Clinical University of Navarra Pamplona Spain

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