Development and validation of ColoScape: A new colorectal cancer mutation detection assay.

e24189 Background: Colorectal cancer is a highly preventable disease. Herein we report the development and validation of ColoScape, a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated 'drive' mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3). Methods: The high sensitivity of ColoScape is achieved by xenonucleic acid (XNA) probe technology. XNA probes are backbone modified oligomers with natural nucleoside bases (A,T, C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with significantly higher binding affinity. XNA probes bind to the selected wild-type sequences at the respective genetic loci in the target genes. Variant alleles at these loci are selectively amplified since the XNA does not bind tightly to the variant sequence and allows DNA polymerase to copy the mutant allele but no the wild-type allele. For selected mutation sites, primers and FAM-labeled TaqMan probes were designed and tested with the selected XNA oligomers. An internal PCR control selected in the Human Beta-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters were established on colorectal cancer patients DNA extracted from FFPE and plasma ctDNA materials. Results: At least 0.5% allelic frequency mutation in the presence of excess wild-type DNA background can be detected by Coloscape for target mutations in APC (exon 15), KRAS (exon 2),BRAF V600 (exon15) and CTNNB1 (exon 3) in 5-10 ng of input DNA/well. No cross reactivity was observed with wild-type up to 320ng purified gDNA and up to 20ng FFPE DNA per reaction. Intra-assay, inter-assay, lot-to-lot and operator variation comparison showed CV between 3% and 8%. Excluding pre-cancer samples, the assay clinical specificity and sensitivity were 95% and 100% respectively. Pre-cancer detection sensitivity was 60% (6 out of 10 FFPE samples) and 62.5% for stool samples. For tested FFPE clinical samples, assay specificity and sensitivity were 95% and 91% respectively while assay clinical specificity and sensitivity were both 100% for plasma samples. Conclusions: The ColoScapeColorectal Cancer Mutation Detection assay is a sensitive tool intended to facilitate research in colon cancer development, early detection, disease recurrence monitoring and therapeutic intervention.