Clinical Application of Genetic Sequencing of Early Gastric Cancer and Gastric Adenoma Patients

Overview

Because advanced gastric cancer shows poor prognosis, it is important to detect early gastric cancer or precancerous gastric adenoma patients who have a cure rate of 95% or more. Moreover, a large part of early gastric cancer can be completely resected by endoscopic resection, thus ensuring a very high quality of life for patients. However, there are currently no markers that can be used for diagnosis of early gastric cancer or gastric adenoma. In addition, the biggest problem after endoscopic resection of early gastric cancer is metachronous recurrence of the cancer, which requires repeated endoscopic resection or additional surgical gastrectomy. However, there are no discovered markers for prediction of recurrence. Liquid biopsy is a method of obtaining body fluids such as gastric juice or effusion through an endoscopic inlet during gastroscopy or colonoscopy and blood. Based on the advanced analysis method, liquid biopsy reveals more genetic information than tissue biopsy. Therefore, it is highly likely to become an essential factor in future personalized medicine. Therefore, this study was designed to identify whether tumor's molercular profiling based on tissue or blood could be used for prediction of prognosis and diagnosis of early gastric cancer and precancerous gastric adenoma.

1. Acquisition of tumor samples : Tissue and 10ml of blood will be collected for genomic examination of those histologically diagnosed with early gastric cancer or precancerous gastric adenoma lesion, and additional tissue and blood will be collected at 2 months, 1 year after treatment, and recurrence. 2. Suitability of tmor samples : Tumor areas (\> 60%) are dissected under microscopy from 4-μm-thick unstained sections by comparison with an H\&E stained slide, and genomic DNA was extracted using a Qiagen DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After extraction, we measured concentrations and 260/280 and 260/230 nm ratios using a spectrophotometer (ND1000, Nanodrop Technologies, ThermoFisher Scientific, MA, USA). Each sample was then quantified with an Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Samples of genomic DNA with more than 10 ng measured by the Qubit fluorometer were subjected to AmpliSeq library preparation. To identify "druggable" CNVs, we used a 21-gene nCounter CNV assay. 3. Method of tumor molecular profiling * Ion Torrent PGM Amplieq Cancer Panel 2.0 : We use the Ion AmpliSep Cancer Panel V2(Ion Torrent) to detect frequent somatic mutations that were selected based on literature review. It examines 285 mutations in 50 commonly mutated oncogenes and tumor supressor genes. First, 10ng of DNA from each 89 FFPE tumor samples underwent single-tube, multiplex PCR amplication using the Ion AmpliSepCancer Primer Pool and the AmpliSepKit reagents (Life Technologies).Treatment of the resulting amplicons with FuPa Preagent partially digest the primers and phosphorylate the amplicons. The phosphorylate the amplicons are ligated to Ion dapters and purfied. For barcoded library preparation, we substitite barcoded adapters from the Ion XpressTM Library Kit. The ligated DNA undergose nick translation and amplication to complete the linkage between adapters and amplicons and to generate sufficient material for dowmstream template treparation. Two rounds of Agencourt® AMPure® XP Reagent binding at 0.6 and 1.2 bead- to- sample volum ratios removed input DNA and unincorporated primers form the amplicons. The final library molecules are 125-300bp in size. We then transfer the libries to the Ion OneTouchTM system for automated template preparation. Sequencing is performed on the Ion PGMTM sequencer according to the manufacturer's instructions. We use IonTorrent Software for automated data analysis. * nCounter copy Number Variation CodeSets : We custom designed a 21-gene CNV assay as outlined in a previous study. The 21 genes included AURAKA, CCND1, CCNE1, CDK4, CDK6, CDNK1A, CDNK2A, EGFR, ERBB2, ERBB3, FGFR1, FGFR2, IGFR1R, KLF5, KRAS, MDM2, MET, MITF, MYC, PIK3CA, and TNIK. For detection of CNVs, nCounter Copy Number Variation CodeSets were used with 200 ng purified genomic DNA. DNA was fragmented via AluI digestion and denatured at 95°C. Fragmented DNA was hybridized with the codeset of 21 genes in the nCounter Cancer CN Assay Kit (Nanostring Technologies) for 18 h at 65°C and processed according to the manufacturer's instructions. The nCounter Digital Analyzer counted and tabulated the signals of reporter probes. Quantified data were analyzed using NanoString's nSolver Analysis Software. * Immunohistochemistry : RET, ATM, PD-L1, FGFR2, MLH, EGFR, ALK, ROS, TRKA. MET, HER2, PTEN loss (EGFR,CCNEI in selected cases) * Cancer Scan : targeted deep sequencing at Samsung Genome Institute : Illumina HiSeq2000/2500-based, MiSeq NGS targeted sequencing * ASFA™ Spotter 4. Statistics : Analysis of genetic research is primarily technical. Data are presented using summary statistics tables and graphs. The total disease-free survival period is calculated by the Kaplan-Meier method through medical records of subjects who have undergone endoscopic resection.