Abstract 563: Evaluation of EGFR mutation analysis performed by Sanger sequencing versus real-time PCR (EGFR RGQ PCR Kit)

Abstract

Abstract EGFR mutation analysis of exons 18, 19 and 21 is a standard analysis in routine molecular pathology for prediction of response of EGFR-tyrosine kinase inhibitors (EGFR-TKI) in non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the mutation detection using two widely used mutation analysis systems. For this purpose we analyzed exons 18, 19 and 21 of EGFR in 718 NSCLC with both nested PCR followed by direct Sanger sequencing and the EGFR RGQ PCR Kit (Qiagen). 694/718 samples were amplifiable and 676 samples allowed interpretable results by both methods. We found mutations in 74/676 (10,1%) samples by Sanger sequencing and in 91/676 (13,0%) using the RGQ Kit. There were 10/676 (1,5%) cases showing mutations only by Sanger Sequencing but not by the RGQ Kit. These mutations comprised four common EGFR-TKI sensitivity mutations in Exon21 (p.L858R (n = 2) and p.L861Q (n = 2)) as well as a complex deletion-insertion in exon 19 (n = 1). The other 5 mutations detected only by Sanger sequencing were uncommon and very rare mutations located in exon 18 and 21 which cannot be detected by the RGQ Kit due to its primer design. Vice versa, we found mutations in 21/676 samples using the RGQ Kit but not with Sanger sequencing. 20 of these mutations were common EGFR-TKI sensitivity mutations in exon 19 (deletions or complex delins, n = 15) and in exon 21 (p.L858R, n = 5) as well as a “p.G719X” mutation (n = 1) in exon 18. According to these data which includes mutations only in exons 18, 19 and 21 but not in exon 20 the mutation detection rate is higher using the RGQ-Kit than using the Sanger sequencing method (13,0% vs. 10,1%) especially in detecting common EGFR mutations associated with EGFR-TKI sensitivity. However the RGQ Kit fails in detecting uncommon and rare EGFR mutations for that this Kit is not designed and may produce non-informative results in some cases when ΔCT values are out of valid ranges whereas nested PCR followed by Sanger sequencing may still work. Citation Format: Wolfgang Dietmaier, Irene Schardt, Petra Rümmele, Eva Geissinger, Arndt Hartmann, Robert Stöhr. Evaluation of EGFR mutation analysis performed by Sanger sequencing versus real-time PCR (EGFR RGQ PCR Kit). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 563. doi:10.1158/1538-7445.AM2015-563