We collected surgical stomach cancer tissue samples from 9 patients who underwent surgical operations between 2012 and 2015. Frozen tissue from patients who agreed to preoperative tissue collection was stored in a −70°C deep freezer.
We choose 4 SRC-GC and 5 WD-GC cases. The cancer tissue was cut into 5 µm sections, and H&E staining was performed. H&E-stained slides were used to identify the morphology and location of gastric cancer tissue within normal gastric tissue. Afterward, the remaining tissue was cut at 10-20 µm thickness. We performed tissue collection by laser capture microdissection (LCM; Veritas LCM2110, Molecular Device Corporation, CA, USA) and compared these slides with the H&E slides (Figure 1A-C ) according to the manufacturer’s recommendations. After tissue collection, we extracted RNA using a Total Purification Kit (Norgene Bioteck Corp, Thorold, Ontario, Canada) according to the manufacturer’s recommendations.
We collected 9 surgical samples in total, 6 WD-GC samples and 3 SRC-GC samples, and we found that 1 WD-GC sample was severely degraded. Ultimately, we compared 5 WD-GC samples with 3 SRC-GC samples (Figure 1D-F ). We performed transcriptome sequencing, and we compared expression profiles between clinically comparable samples using transcriptome resequencing data. mRNA sequencing was performed with next-generation sequencing technology by Macrogen, Inc. (Seoul, Republic of Korea). We performed gene enrichment and functional annotation analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resources (https://david.ncifcrf.gov ) with the Ingenuity Pathway Analysis system (Qiagen, Hilden, Germany), and we determined the top 10 different genes in terms of the biologic process, cellular component, and molecular function categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway differences between SRC-GC and WD-GC (Figure 2 ).19 (link)
We generated a schematic illustration of the results of the cancer driver variant analysis. We filtered 170,602 variants and 16,460 genes, and we ultimately extracted 52 variants and 30 cancer driver genes. We performed variant analysis and network analysis with Insilicogen Inc. software (Yongin-si, Korea). We found 16,460 genes with 170,602 variants by comparing IGC (A group) and DGC (B group) samples. The genes were filtered by the 1000 Genomes Project, ExAC, NHLBI ESP exomes (with the parameters African American and European American), and Allele Frequency Community (with the parameters common variants and allele frequency below 0.1) resources. Common variants were filtered to include only pathogenic variants (according to the ACMG guidelines classification). We selected more than 2/3 of the samples in the SRC-GCB group and more than 2/5 of the samples in the WD-GC group and filtered the results by biological context (metastasis, signet ring adenocarcinoma or signet ring cell primary gastric adenocarcinoma). Finally, we found cancer driver variants with a frequency greater than 0.01% in the COSMIC and TCGA databases (Figure 3 ; Table 1 ).
We choose 4 SRC-GC and 5 WD-GC cases. The cancer tissue was cut into 5 µm sections, and H&E staining was performed. H&E-stained slides were used to identify the morphology and location of gastric cancer tissue within normal gastric tissue. Afterward, the remaining tissue was cut at 10-20 µm thickness. We performed tissue collection by laser capture microdissection (LCM; Veritas LCM2110, Molecular Device Corporation, CA, USA) and compared these slides with the H&E slides (
We collected 9 surgical samples in total, 6 WD-GC samples and 3 SRC-GC samples, and we found that 1 WD-GC sample was severely degraded. Ultimately, we compared 5 WD-GC samples with 3 SRC-GC samples (
We generated a schematic illustration of the results of the cancer driver variant analysis. We filtered 170,602 variants and 16,460 genes, and we ultimately extracted 52 variants and 30 cancer driver genes. We performed variant analysis and network analysis with Insilicogen Inc. software (Yongin-si, Korea). We found 16,460 genes with 170,602 variants by comparing IGC (A group) and DGC (B group) samples. The genes were filtered by the 1000 Genomes Project, ExAC, NHLBI ESP exomes (with the parameters African American and European American), and Allele Frequency Community (with the parameters common variants and allele frequency below 0.1) resources. Common variants were filtered to include only pathogenic variants (according to the ACMG guidelines classification). We selected more than 2/3 of the samples in the SRC-GCB group and more than 2/5 of the samples in the WD-GC group and filtered the results by biological context (metastasis, signet ring adenocarcinoma or signet ring cell primary gastric adenocarcinoma). Finally, we found cancer driver variants with a frequency greater than 0.01% in the COSMIC and TCGA databases (
