Abstract 643: An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of KRAS/BRAF/NRAS mutations in colorectal cancer

Abstract

Abstract RAS and BRAF gene mutation assessment is essential for therapy decision in metastatic colorectal cancer patients with anti-EGFR monoclonal antibody treatment. However the currently approved assays in clinic is mainly focusing on tissue samples with detecting sensitivity lower than 1% in 10ng genomic DNA background. The emerging analysis of ctDNA offers advantages in clinical diagnosis as a feasible method, which requires much more sensitive methodology due to low abundance in plasma samples. Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection fourteen mutations of KRAS/BRAF/NRAS in an 8-tube PCR strip. The reagent and format of mutation detection were particularly optimized to reach high specificity and sensitivity. Performance evaluation showed that the assay is remarkably sensitive with lower detection limit of 0.1% in a background of 10ng wild-type genomic DNA. A total of 136 CRC FFPE samples were screened by using the assay. Sixty-two samples were positive for KRAS/BRAF/NRAS mutations, and fifty-eight were confirmed by the Sanger sequencing. The four discrepant samples were tested further by ddPCR and the results revealed that the allelic frequencies in these samples were as low as 0.1% to 5%. The evaluation of ctDNA detection is being carried in metastatic colorectal cancer patients. The multiplex allele-specific real-time PCR assay provides a rapid, reliable and low-cost diagnostic tool for accurate detection of KRAS/BRAF/NRAS mutations for clinical application. Citation Format: Jianxin Zhai, Youmin Wu, Xuan Luo, Xinsheng Li, De-Hua Yu. An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of KRAS/BRAF/NRAS mutations in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 643.