Abstract 4910: Sensitive, specific and highly multiplexed mutation detection for cancer management
Authors: Tan Lit Yeen , Mokany Elisa , Walker Samantha , Lonergan Tina , Todd Alison 
Published: August 2015

Abstract
Mutations in RAS, RAF, EGFR and PIK3CA genes are present in a number of cancers including colon, lung and breast cancers. Importantly, many of these mutations are predictive biomarkers of clinical outcomes for targeted therapies. This has led to a growing demand for detection of multiple mutations simultaneously. Multiplex formats allow a greater amount of information to be obtained from each sample.
PASS primer technology can sensitively and specifically identify the presence of SNPs and somatic DNA changes (point mutations, deletions and insertions) with sensitivity up to 1 in 1,000 (0.1%). PASS primers selectively amplify nucleic acid variants and create amplicons that are markedly different from the parent sequence. The method is superior to ARMS PCR for multiplexing, particularly when mutations are present at the same, adjacent or nearby loci.
PASS primers consist of two target specific regions separated by an insert sequence (IS) which is not complementary to the target. A long 5′ target-specific region anchors the primer and a short 3′ region targets the variant base/s thus directing highly specific binding and extension. During amplification, the IS sequences are incorporated into amplicons, introducing ≥10 base difference between the starting single base variant and the resulting amplicon. This reduces competition between primers during amplification and facilitates robust discrimination between amplicons during detection.
MNAzyme® qPCR is a robust alternative to other probe based real-time qPCR protocols such as TaqMan® and Beacons. MNAzymes are bi-specific, catalytic oligonucleotide complexes which form in the present of target and cleave universal reporter probes. The bi-specificity, and the use of well-characterised universal probes sets (suitable for use with any group of targets), makes MNAzyme® qPCR more amenable to multiplexing. When combined with PASS, MNAzyme® qPCR easily discriminates between amplicons since MNAzymes are tailored to specifically detect both the distinct IS and the original mutation.
PASS MNAzyme® qPCR has been used to facilitate multiplex detection of RAS, RAF, EGFR and PIK3CA mutations. As many as eight mutations located at codons 12 and 13 of RAS have been successfully detected in a single well using three channels on a standard PCR machine. The technology is even more powerful when combined with newer instruments with higher multiplex capabilities such as Biocartis’ Idylla automated platform. A multiplexed KRAS/BRAF assay, which detects 18 mutations, was evaluated using colon cancer and melanoma FFPE samples. The results showed &gt;96% concordance with sequencing and significantly specificity compared to the singleplex Therascreen KRAS test.
Since PASS MNAzyme® qPCR affords greater multiplex capacity, along with high specificity and sensitivity, it provides a superior tool for ascertaining mutations from tumour tissues and is particularly well suited for use with liquid biopsies.
Note: This abstract was not presented at the meeting.
Citation Format: Lit Yeen Tan, Elisa Mokany, Samantha Walker, Tina Lonergan, Alison Todd. Sensitive, specific and highly multiplexed mutation detection for cancer management. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4910. doi:10.1158/1538-7445.AM2015-4910