Abstract 5237: Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers

Abstract

Abstract Objectives: High coverage expression profiling (HiCEP) is an AFLP-based comprehensive gene expression analysis technique invented in Japan. HiCEP has two unique characteristics. First, it can detect low amounts of mRNA with high sensitivity and reliability. Second, HiCEP enables highly quantitative and reproducible mRNA expression analyses. However, it requires complicated processes, including TA cloning of isolated transcripts, to obtain sequence information on detected peaks. We performed next-generation sequencing (NGS)-combined HiCEP and tried to establish a gene expression database of renal cell carcinoma (RCC) cases to identify effective tumor markers. Materials and methods: We collected cancerous and macroscopically non-cancerous regions from 83 RCC cases. Of these, six cases with clear cell RCC were analyzed by HiCEP. Total RNA was extracted from the cancerous and non-cancerous tissues of six clear cell RCC cases, and transcribed to cDNA. The cDNA was synthesized and subjected to digestion with the restriction enzymes MspI or MseI, followed by adapter ligation. Selective PCR by 256 kinds of primer pairs was used to amplify the HiCEP fragments, and products with fluorescently-labeled primer were then analyzed by capillary electrophoresis. HiCEP fragments were sequenced using a next-generation sequencer (ion PGM, Thermo Fisher Scientific). We compared the expression levels of HiCEP peaks in cancerous tissues with those in non-cancerous tissues. Results: We detected several HiCEP peaks in cancerous tissues that showed five times higher expression than in normal tissues. We determined the sequences of the HiCEP fragments by NGS, and developed the first ever cancerous tissue HiCEP fragment database. Conclusion: We successfully established an RCC gene expression database by NGS-combined HiCEP. We are now performing replication analyses and further analyses of blood samples from the same RCC cases to be able to identify diagnostic and prognostic markers of RCC. Citation Format: Makoto Kawaguchi, Hirotaka Matsuo, Ryoko Araki, Seiko Shimizu, Mikiya Takao, Akiyoshi Nakayama, Yosuke Kitamura, Masumi Abe, Keiichi Ito, Nariyoshi Shinomiya. Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5237.