Abstract 4145: Determination of the EGFR mutational status on FFPE material from patients with lung cancer using Multiplex PCR followed by sequencing on a compatible 454 sequencing platform.
Authors: van Schooneveld Eleni , De Laere Bram , Salgado Roberto , Vermeulen Peter , Wildiers Hans , Vergote Ignace , Dirix Luc , Van Laere Steven 
Published: August 2014

Abstract
Background: The epidermal growth factor receptor belongs to the ErbB family of cell-surface receptors. Hotspot mutations within this gene occur in 27% of lung cancers and patients carrying mutation(s) likely receive more benefit from EGFR targeted therapy. We have evaluated the feasibility of multiplex PCR targeting known mutational hotspots in EGFR exon 18 till 21, followed by 454 pyrosequencing to determine the EGFR mutational status in patients with lung cancer.
Methods: FFPE samples from 10 lung cancer patients were collected. Four samples were mutant and six carried the wild-type (WT) genotype. A multiplex PCR was performed in which primers had a gene specific sequence, a patient specific ‘bar code’ (MID) and Roche sequencing tags. The unique multiplex identifiers (MIDs) allow appropriate mixing of patient samples and differentiation in the sequence data. Amplicon generation was verified by fragment analysis (GeneScan). Amplicons were purified using AMPure XP beads, quantified by Quant-iT PicoGreen, pooled in equimolar ratios and diluted before emulsion PCR. Sequencing was carried out in 2 runs using Titanium Amplicon chemistry (Lib-A) on a 454 GS Junior. Two samples were analyzed in duplicate to assess reproducibility. Data were analyzed using the amplicon variant analyzer software.
Results: A throughput of 52.159 and 28.769 high quality reads was achieved for the library in the performed runs. Considering the EGFR amplicons, a mean coverage of 837 reads (range 589 - 1350) was obtained. Four variants were detected by sequencing and were 100% concordant with results obtained using a StripAssay in an ISO-certified setting. The variants and frequencies detected were G719X (21,7%), p.L747-S752del (21,46%), p.H773_V774insNPH (11,14%) and p.L861Q (24,11%). In the WT samples no EGFR mutations were detected, suggesting a 100% specificity on the obtained 454 data. Additionally, variant analysis showed 2 different SNPs in exon 20 and 21 (rs1050171 and rs17290559, respectively). These are classified as the synonymous mutations Q787Q and R836R and do not have any clinical significance. The SNPs in exon 20 and 21 were detected in 67% and 17% of the WT samples, respectively. Inter-run reproducibility was established from comparison of 2 WT samples in different sequence runs. Intra-assay reproducibility was demonstrated via analysis of EGFR mutant samples in duplicate.
Conclusions: Our study provides a basis for the implementation of next generation sequencing for the detection of somatic mutations in EGFR in a routine clinical setting. The used multiplex PCR and subsequent 454 sequencing allowed the detection of significant activating or resistance associated EGFR mutations in FFPE material from patients with lung cancer. We obtained 100% concordance with previously obtained results from an accredited laboratory.
Citation Format: Eleni van Schooneveld, Bram De Laere, Roberto Salgado, Peter Vermeulen, Hans Wildiers, Ignace Vergote, Luc Dirix, Steven Van Laere. Determination of the EGFR mutational status on FFPE material from patients with lung cancer using Multiplex PCR followed by sequencing on a compatible 454 sequencing platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4145. doi:10.1158/1538-7445.AM2013-4145