250P - EGFR testing from circulating cell-free DNA: Ready for routine practice

Date 28 September 2014
Event ESMO 2014
Session Poster Display session
Topics Translational Research
Basic Principles in the Management and Treatment (of cancer)
Presenter Marc Denis
Citation Annals of Oncology (2014) 25 (suppl_4): iv58-iv84. 10.1093/annonc/mdu326
Authors M.G. Denis1, A. Vallee1, C. El Kouri2, H. Lacroix3, M. Marcq4, A. Bizieux5, J. Bennouna6, J. Douillard7
  • 1Department Of Biochemistry And Molecular Biology, Nantes University Hospital, 44093 - Nantes/FR
  • 2Medical Oncology, Catherine de Sienne Institute, 44202 - Nantes/FR
  • 3Department Of Oncology, Nantes University Hospital, Nantes/FR
  • 4Pneumology, La Roche/Yon hospital, La Roche/Yon/FR
  • 5Pulmonology Department, CHD Les Oudairies, La roche sur yon/FR
  • 6Department Of Medical Oncology, Institut de Cancérologie de l'Ouest, 44805 - Saint-Herblain cedex/FR
  • 7Medical Oncology, Centre René Gauducheau (ICO) Institut de Cancerologie de l'Ouest, 44805 - St Herblain CEDEX/FR



Detection of EGFR alterations is critical for predicting the response to tyrosine kinase inhibitors (TKI) in patients with non-small-cell lung cancer (NSCLC). In clinical practice, somatic mutations are detected using formalin-fixed, paraffin-embedded tumor tissues obtained at diagnosis. However, using archival tumor material for mutation testing can be limited by inherent problems, such as insufficient tumor material, low percentage of tumor cells, DNA degradation, and long turnaround time because tracing archival tumor material can be logistically difficult. The aim of our work was to set up and evaluate a routine procedure enabling to detect EGFR alterations in plasma DNA of patients with NSCLC.


Blood was collected in 5-ml EDTA-containing tubes and plasma was obtained by centrifugation (10 min, 2,000 g, 20°C) performed within 3 hours following blood collection. Cell-free DNA was extracted from plasma samples (0.4 ml) using the PureLink® Virus Kit and an iPrep™ Purification Instrument (Life Technologies). Detection of EGFR alterations was performed using the approved Therascreen® EGFR RGQ kit (Qiagen). Samples were tested positive for EGFR mutation when the ΔCt value (Ct of the mutation specific PCR–Ct of the control PCR) was lower than 12 for exon 19 deletions, and below 14 for L858R mutation.


We have included 37 metastatic patients presenting an EGFR mutation in their tumor (exon 19 deletion, n = 20; p.L858R, n = 17). These patients had blood collected before initiation of TKI treatment. The mutation pick-up frequency in plasma was 83.8%, 31 patients being tested positive (exon 19 deletion, n = 18; p.L858R, n = 13). In contrast, no EGFR alteration was detected in samples collected from healthy donors and NSCLC patients with a wild type EGFR (n = 86; specificity 100%). The whole process can be performed in a single day (40 min for DNA extraction; 2h for PCR; a few minutes for data analysis).


Using this simple procedure, EGFR alterations can be detected in plasma of most metastatic NSCLC patients presenting an EGFR mutation in their tumor. Plasma DNA is a useful alternative source of tumor DNA that could be used for EGFR testing in a routine practice within a very short turnaround time, when tumor material is not available. Supported in part by a grant from Astra-Zeneca.


M.G. Denis: Member - European Scientific Advisory Board–Qiagen. All other authors have declared no conflicts of interest.