Is lung cancer genetic heterogeneity responsible for resistance to EGFR tyrosine kinase inhibitors? Contribution of digital PCR

Abstract
Introduction: Activating EGFR mutations are predictive biomarkers for a good response and survival to EGFR tyrosine kinase inhibitors (EGFR-TKI). However, ultimately, every patient progress. Several resistance mechanisms have been described, among which the concept of tumor heterogeneity is one of the most important. We studied tumor heterogeneity of the most common gene alterations described for lung cancer, and their role in resistance to EGFR-TKI. Patients and methods: On the one hand, 20 patients with concomitant KRAS and EGFR mutations routinely diagnosed in our laboratory were selected. These mutations were confirmed with qPCR-HRM and Sanger sequencing for each available section and block. EGFR mutations heterogeneity on blocks surfaces were studied using IHC. Deep biopsy cores were performed for EGFR and KRAS mutations heterogeneity sequencing analysis. The percentage of each EGFR and KRAS mutation was assessed using nano-droplets digital PCR (ddPCR ™). On the other hand, 7 EGFR-TKI-resistant patients with activating EGFR mutation were selected for qPCR- HRM and sequencing study of all molecular resistance factors known to date. Results: EGFR and KRAS mutations were different for each patient’s block or section studied. EGFR mutations IHC labelling is heterogeneous and well correlated with molecular biology results. Deep biopsy cores analysis shows a greater heterogeneity for KRAS mutations versus EGFR mutations. For each sample, mutation rates are different for KRAS and/or EGFR as shown using ddPCR. Discussion: This study demonstrates the concept of tumor heterogeneity in a clinical context using highly sensitive techniques. These results also support the hypothesis for the existence of several tumor clones within with a selection and adaptation mechanism under treatments pressure.