Sensitive genotyping of mutations in the EGFR gene from NSCLC patients using PCR-GoldMag lateral flow device

Xian-Ying Li,Juan-Li Zhu,Qian Liu,Xue-Min Yang,Chao Zhang,Wen-Li Hui,Ya-Li Cui,Qin-Lu Zhang,Ming-Wei Chen

doi:10.1038/s41598-017-08210-8

Abstract

Epidermal growth factor receptor (EGFR) mutations predict better outcomes with EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Most common activating mutations include in-frame deletion in exon 19 and L858R substitution in exon 21, which account for >90% of all EGFR mutations in NSCLC. In this study, a PCR-GoldMag lateral flow assay (PCR-GoldMag LFA) was developed for the visual detection of delE746-A750 and L858R of EGFR mutations. Forty formalin-fixed paraffin-embedded (FFPE) tissue samples of NSCLC patients were analyzed using PCR-GoldMag LFA system and verified by direct sequencing and TaqMan-PCR detection methods. Results showed that EGFR mutations were detected in 34 cases among the 40 samples (85%) by PCR-GoldMag LFA method. Among the 34 cases, 5 cases were simultaneously detected with delE746-A750 in exon 19 and L858R mutation in exon 21. Compared with sequencing, only 4 samples were detected as delE746-A750, which revealed higher sensitivity of PCR-GoldMag LFA detection method than direct sequencing. TaqMan-PCR method verified the L858R mutation and was in 100% agreement with our method. These results indicated that our method has obvious advantages to analyze clinical samples and offers a more sensitive alternative to direct sequencing for the detection of EGFR mutations.

Highlights

Lung cancer is one of the leading causes of death worldwide and is expected to remain a major health problem in the future[1]
Expected PCR fragments are allowed to bind to the PGMNs-anti-digoxin antibody conjugates on the adjacent conjugate pad, forming DNA-PGMNs-antidigoxin antibody complexes. These complexes flow along the strip, and were captured by pre-immobilized streptavidin on the test line (T line) with a result of a red band
The excess PGMNs-anti-digoxin antibody conjugates is captured by goat anti-mouse IgG on the control line (C line), evidencing the work of the system

Summary

Introduction

Lung cancer is one of the leading causes of death worldwide and is expected to remain a major health problem in the future[1]. The new PCR-GoldMag lateral flow device for SNP detection, including MTHFR C677T and Apolipoprotein E polymorphisms, has been validated by sequencing for more than 2,000 genomic DNAs in 6 hospitals in China, and showed a high specificity and sensitivity[21, 22]. This method allows for rare signals to be detected with greater sensitivity, tends to be faster and cheaper, and can be used as “targeted method” for genotyping of EGFR gene. We first demonstrate a PCR-GoldMag LFA for the two most common therapy-related EGFR mutations, delE746-A750 and L858R

Methods

Results

Conclusion