Abstract 1502: A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies
Authors: Vandenbroucke Ina , Vermeiren Katrien , Mokany Elisa , Tan Lit Yeen , Lima Nicole , Walker Samantha , Vandercruyssen Geneviève , Claes Bart , Baere Inky D. , Holemans Pascale , Rondelez Evelien , Todd Alison , Maertens Geert , Sablon Erwin 
Published: May 2015

Abstract
Introduction
The MAPK/ERK pathway is a complex signaling cascade involved in many cancer types. KRAS and BRAF gene mutations are present in a number of cancers, including colon, lung and pancreas, and identification of mutations in these genes is of great importance in clinical diagnostics. Moreover, there is a growing demand for performing multiple tests simultaneously on a single sample and there is an increased need to provide these answers to oncologists in a short timeframe.
Methods
IdyllaTM is a fully integrated and automated molecular diagnostics platform (1) that combines speed and ease of use with high sensitivity and high multiplexing capabilities. Moreover, it overcomes the current time-consuming step of processing formalin-fixed paraffin-embedded tissue (FFPE) samples. After insertion of a single FFPE slice into the cartridge, the complete process of sample preparation, real-time PCR and reporting is fully automated and takes less than 2 hours. We present here a KRAS-BRAF mutations prototype assay that allows the sensitive detection of 13 KRAS mutations and 5 BRAF mutations in one single assay. The assay discriminates the individual mutations at codons 12, 13 and 61 of KRAS and codon 600 of BRAF using novel “Primer Assisted Sequence Switching” (PASS) primers along with “Multi-component Nucleic Acid enzyme” (MNAzyme) detection. These technologies confer advantages for multiplex mutation analysis; PASS primers selectively amplify the target sequences of interest resulting in enhanced specificity between wild type (WT) and mutant, and between mutants, and MNAzymes allow for efficient detection and discrimination of multiple mutations simultaneously.
Results
Several performance characteristics of the IdyllaTM KRAS-BRAF prototype assay were examined: specificity, cross-reactivity, sensitivity and performance on clinical samples. Specificity of mutant versus WT as well as cross-reactivity between individual mutations at each codon was evaluated. Results demonstrated excellent specificity and cross-reactivity for all individual targets, with delta Cq values of &gt;7 between mutants and &gt;12 between mutant and WT. Sensitivity was assessed using cell lines embedded in FFPE containing defined ratios of mutants and dilutions of these in FFPE WT background. The results indicated sensitivities of &lt;1% of mutant allele. The performance of the IdyllaTM KRAS-BRAF prototype assay was evaluated on a set of colon cancer and melanoma FFPE samples characterized by Sequenom MassARRAY, Illumina MiSeq, or Biocartis IdyllaTM BRAF-only assay which demonstrated a &gt;96 % concordance.
Conclusion
The new and fully integrated IdyllaTM KRAS-BRAF prototype assay combines extended multiplexing capabilities with excellent specificity, high sensitivity, ease of use, and short turnaround time for mutation analysis on FFPE samples.
(1) For research use only
Citation Format: Ina Vandenbroucke, Katrien Vermeiren, Elisa Mokany, Lit Yeen Tan, Nicole Lima, Samantha Walker, Geneviève Vandercruyssen, Bart Claes, Inky De Baere, Pascale Holemans, Evelien Rondelez, Alison Todd, Geert Maertens, Erwin Sablon. A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2014-1502