Abstract B38: Evaluation of the OnTarget™ system for highly sensitive mutation detection.
Authors: Walker Russell M. , Dunaj Sara , Chan Dalin , Badola Sunita 
Published: August 2014

Abstract
Background: Effective non-invasive methods for patient characterization and monitoring during treatment are desired. Cell-free circulating tumor DNA in plasma is elevated in cancer patients, and can be a valuable tool for evaluating mutation profile over time. Low mutant allele fraction in a limited amount of low quality DNA makes it very challenging to accurately characterize plasma samples. Therefore, a highly sensitive and robust testing method is required for genetic characterization of circulating tumor DNA.
The goals of this study were to:
• Evaluate assay performance and limit of detection of the Boreal Genomics OnTarget™ system 18-mutation panel using human germline DNA
• Assess the relative sensitivity of the OnTarget™ system for KRAS G13D and PIK3CA H1047R mutation detection using cell line derived DNA
• Compare assay performance and sensitivity with multiple DNA input amounts
Materials and methods: DNA was extracted from normal healthy human blood and HCT116 (KRAS G13D and PIK3CA H1047R heterozygous mutant) cell line pellets. The cell line DNA was diluted with the human blood DNA to produce samples with known ratios of mutant and wild-type alleles. Samples were run with the OnTarget™ mutation detection technology platform 18-mutation panel. The platform utilizes oligo probes and a gel based system to enrich targeted mutant fragments from a DNA sample, followed by PCR amplification and next generation sequencing with the Illumina MiSeq™ platform. The 18-plex panel tests mutant allele percentage of 18 common cancer mutations in the BRAF, EGFR, KRAS, and PIK3CA genes.
Results: Repeat testing of germline DNA from multiple human donors was conducted to determine the background variability (noise) of the 18-plex assay. The mean mutant allele percentage for 10 of the 18 mutations assessed was under 0.001%. 7 of the mutations were under 0.01%, with one other mutation was just over 0.01%. Comparing mutant allele percentages of the titrated cell line DNA samples assessed limit of detection of two of the assays at the 300ng input level. The PIK3CA H104R assay detected mutant DNA over the background levels at the 0.01 and 0.001% input amount of mutant DNA. The KRAS G13D assay signal was over background at the 0.01% input, but within the assay noise at the 0.001% input. When testing the same samples at 30ng of DNA input, all mutation detection was similar, except the PIK3CA mutation was not detected at the 0.001% input amount.
Summary: The OnTarget™ mutation enrichment system followed by NGS provides high sensitivity mutation profiling of DNA with high multiplexing capabilities. The low DNA input requirement and limit of detection make it a useful platform for testing of cell-free circulating plasma DNA.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B38.
Citation Format: Russell M. Walker, Sara Dunaj, Dalin Chan, Sunita Badola. Evaluation of the OnTarget™ system for highly sensitive mutation detection. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B38.