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

Tumor tissue samples. The tumor tissues were surgically resected from 189 consecutive patients diagnosed with pulmonary adenocarcinoma by the expert pathologist at The Saitama Cardiovascular and Respiratory Center (Kumagaya, Japan) between January 2016 and October 2017. All pathological diagnosis was determined on the basis of the WHO classification version 8 (19) by an expert pathologist, who normally makes a diagnosis using HE-stained slides using light microscope (Nikon Co., ECLIPSE Ni-u) from a low magnification to a high magnification. The inclusion criteria were as follows: i) Surgically resected tissue of primary lung cancer; ii) pulmonary adenocarcinoma; iii) enough volume materials for molecular testing; and iv) informed written consent from patients. Conversely, cases with no informed consent or less volume sample were excluded. Clinical characteristics of the 59 patients are presented in Table I . The mean patient age was 69.6 years and included 28 males and 31 females. All samples were fixed with 10% buffer formalin at room temperature (24-36 h) to create formalin-fixed, paraffin-embedded (FFPE) tumor blocks at Department of Pathology in Saitama Cardiovascular and Respiratory Center. Hematoxylin-eosin staining was performed by the standard method using Tissue-Tek Prisma (Sakura Finetek Japan Co., Ltd.) according to the manufacturer's protocol. Prior to DNA extraction, the tumor content of each sample was assessed using light microscopy at x10 and x100 magnification, to ensure efficient PCR amplification. After sections were deparaffinized with xylene and hydrated through a graded series of ethanol (100, 100, 85 and 70% ethanol), DNA from the tissue blocks was extracted using the QIAampTM DNA FFPE Tissue kit ® (Qiagen, Inc.) and analyzed using a QIAcube Robot ® (Qiagen, Inc.) according to the manufacturer's protocols (20) .