A novel loop-mediated isothermal amplification method for efficient and robust detection of EGFR mutations

Horiuchi, Sho; Saito, Yuichi; Matsui, Atsuka; Takahashi, Nobumasa; Ikeya, Tomohiko; Hoshi, Eishin; Shimizu, Yoshihiko; Yasuda, Masanori

EGFR mutations in pulmonary adenocarcinoma are associated with sensitivity to TKI therapy. Hence the identification of EGFR mutations has become a standard analysis in the treatment pathway of patients with pulmonary adenocarcinoma. Although there are a number of methods available, there is no standardized approach to satisfy the practical clinical requirements of simplicity, rapid and cheap. Several PCR based methods have previously been used as a routine test for the detection of EGFR status in United States, European Union, Japan and China, including the Scorpion Amplification Refractory Mutation System (ARMS) ® (22) , such as Therascreen PCR assay. This method was approved by the FDA as a standard approach for EGFR gene analysis in lung cancer (fda. gov/medical-devices/recently-approved-devices/therascreenr-fg fr-rgq-pcr-kit-p180043); however, although stable and reliable, this approach has procedural complexities, including complex settings and controls for the temperature at several times using a (24) and Cycleave PCR™ (25) , have been developed in Japan and are commercially used in centralized laboratories. The sensitivity of the PNA-LNA PCR clamp is >97% with 100% specificity (26) and the accuracy of Cycleave PCR is 96.7% (27) , so it was the same for our results as it was those. LAMP is a new PCR method and is considered to be a robust approach for gene analysis as it does not require sophisticated or expensive equipment, such as a thermal cycler necessary for PCR (15) . Therefore, the LAMP method may have potential to decrease the costs of gene analysis. Previous studies have demonstrated the value of LAMP in field of bacteriology and virology (15, 17, 28, 29) ; however, few studies have reported the value of LAMP in oncology. Ikeda et al (30) used LAMP assays to detect EGFR mutations in NSCLC, demonstrating the value of LAMP, but only the L858R mutation was studied. Therefore, the present study aimed to investigate other EGFR mutations, including those in exon 19, exon 21 and other minor mutations using LAMP assays.