Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay

Abstract

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP’s) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression. Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2017-728