Abstract 1391: Development of in vitro multiplex colorectal cancer associated mutation detection assay powered with xenonucleic acid

Abstract

Abstract Precise detection of cancer associated mutations have many advantages including diagnosis and monitoring of the disease and monitoring development of drug resistance. However, unlike germline mutations, somatic mutations in cancer are found at a much lower frequency. For this reason it remains a challenge especially when trying to detect mutations from limited amount of patient tissue biopsy or liquid samples. Currently, detection and genotyping of these mutations is heavily dependent on deep NGS methods. These procedures, however, are costly and time consuming. Here, we developed an alternative high-throughput method that combines DiaCarta’s proprietary xenonucleic acid (XNA) technology that allows target enrichment, and Luminex MAGPIX platform technology that enables simultaneous detection of up to 50 mutation targets. Briefly, multiplex PCR amplification of cancer associated genes is conducted in the presence of XNA. In this reaction, XNA specifically suppresses the wild-type DNA from being amplified and thus, only generates amplicons from the genes that carry mutations. The amplicons are then hybridized in a single tube that contains a set of magnetic beads. Each bead is uniquely color-coded for identification, and each corresponds to a single mutation of interest. The beads are analyzed on Luminex platform for genotyping of the mutations. Using this method, we compared conditions with or without XNA for target amplification. Our preliminary data show that when we tested the cancer oncogene KRAS exon 2 mutation variations, we find that 100% correct allele call was made with XNA for all 7 reference mutations genes. However, in conditions without XNA, the PCR product was composed of mostly wild-type sequences, and thus, only two correct allele calls (28.6%) were made. Consequently we have showed a clear advantage of XNA technology for efficient mutant target enrichment, followed by Luminex MAGPIX platform technology for simultaneous detection of multiple genetic targets. Multiplex capacity of both PCR and Luminex MAGPIX platform allows a significant reduction in the total amount of patient samples needed for precise mutation detection from each assay, especially for the mutations with low allelic frequency. Further development on this assay will provide a more reliable, sensitive and quick method for simultaneously detecting mutations associated with cancer in a single reaction. Citation Format: Michael Y. Sha, Anna J. Lee, Michael J. Powell, Aiguo Zhang. Development of in vitro multiplex colorectal cancer associated mutation detection assay powered with xenonucleic acid [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 1391.