153P - Real-time PCR and digital PCR approach for detecting EGFR status in plasma of patients with NSCLC

Date 15 April 2016
Event European Lung Cancer Conference 2016 (ELCC) 2016
Session Poster lunch
Topics Biomarkers
Lung and other Thoracic Tumours
Presenter Marat Gordiev
Citation Journal of Thoracic Oncology (2016) 11 (supplement 4): S57-S166. S1556-0864(16)X0004-4
Authors M. Gordiev1, D. Sakaeva2, M. Blokhina2, A. Nikitin3
  • 1Molecular-diagnostic Laboratory, Kazan Cancer Centre, 420000 - Kazan/RU
  • 2Chemotherapy, Republican Clinical Oncology Center, 450054 - Ufa/RU
  • 3Genetic Laboratory, Federal Research and Clinical Center, FMBA, 115 682 - Moscow/RU

Abstract

Background

The aim of this study was to compare mutation status in FFPE tissue and plasma samples and the evaluation diagnostic characteristics of real-time wild-type blocking PCR (LNA-clamp) assay and digital PCR approach (dPCR).

Methods

The study included 89 patients with advanced NSCLC with known tissue mutation status. L858R, del19 and T790M mutations was determined in corresponding blood plasma samples. Isolation and analysis of mutations of EGFR (L858R, deletions of the gene EGFR) from tissue was performed by Qiagen FFPE DNA kit (Germany). For dPCR detection of mutations L858R, T790M and del19 in plasma we used a mixture of probes and primers at final concentrations of 900 and 600 nM, respectively. We used different concentrations plasmid positive controls (0%, 0.5% and 5%). The PCR mixture was prepared by 3D Digital PCR Master Mix according to the manufacturer's instructions. The detection was carried out using the system QuantStudio® 3D Digital PCR System (ABI, USA). For real-time PCR was used a thermal cycler Rotor-Gene 6000 (Qiagen, Germany), PCR was performed in a final volume of 20 µl, reaction mixture contained 70 mM Tris-HCl (pH 8.8), 16.6 mM ammonium sulphate, 0.01% Tween-20, 2 mM magnesium chloride, 200 nM of each dNTP, 500 nM of primers, 250 nM of fluorescent probe, 1000 nM of blocking probe, 1.5 units Taq-DNA polymerase. Probes used in fluorescent dyes – FAM and VIC, quenchers – BHQ-1 and BHQ-2. Sets of oligonucleotides were designed and produced by “Testgen Ltd” (Russia).

Results

By comparing of the EGFR mutations status in plasma and tumor samples concordance was 88.7% [95% CI 85%, 92%], sensitivity – 83.3% [95% CI 76%, 90%], specificity – 100%, PPV – 100%. The analytical sensitivity of dPCR was 0.1% Analytical specificity – 99.5%. Analytical sensitivity for real-time PCR was 0.2%, the analytical specificity – 99.7%.

Conclusions

Digital PCR has demonstrated the best analytical sensitivity, wherein concordance with real-time PCR was 100%.

Clinical trial identification

Legal entity responsible for the study

Kazan Clinical Oncology Center, Kazan, RU

Funding

Kazan Clinical Oncology Center, Kazan, RU

Disclosure

All authors have declared no conflicts of interest.