Development And Validation Of The Actionseq (Tm) Test System

Introduction In the constantly changing field of oncology precision medicine, it is exceedingly important to keep diagnostic and therapeutic assays clinically relevant. Next generation sequencing (NGS) panels in oncology are greatly impacted by new findings in clinical actionability. In order to ensure that cancer panels continue to provide the most beneficial results to patients, they must be regularly updated. In keeping with this idea, JAX has launched a new 212 oncology gene panel which focuses on genes and variants with documented actionability, referred to as ActionSeq. Methods Development of ActionSeq included the optimization of a new targeted capture assay. This process included running multiple batches of samples through the assay to determine appropriate DNA input, ligation times, PCR cycles, and pooling conditions. The fully optimized assay was then validated using 24 uncharacterized FFPE samples. The validation was executed in 5 phases: (1) confirm that assay optimizations yielded sufficient wet lab results; (2) LOD u0026 sensitivity (3) inter-assay concordance; (4) intra-assay concordance; (5) specificity and accuracy. Results During development, the standard protocol was optimized using a 200ng input, 30 minute ligation period, 5 cycles of pre-PCR, and the pooling of 4 samples per hybridization reaction. Wet lab processing results of the first validation batch can be seen in Table 1. The inter- and intra-assay concordances were found to be ≥ 96% for variants and 100% for CNVs. The sensitivity was calculated to be 98.92% at a LOD of 3% for SNVs, 100% at a LOD of 8% for INDELs, 100% at a LOD of 6 copies for CNV amplifications, and 100% at a LOD of 0 copies for CNV deletions. The specificity and accuracy were found to be 100% for all mutation types. Conclusion Based on the success of this validation ActionSeq has been incorporated into the JAX clinical test menu. This addition accomplished the goal of providing a more clinically relevant (actionable) somatic tumor profiling assay to patients and clinicians. Citation Format: Samantha Helm, Vanessa Spotlow, Aleksandra Ras, Kevin Kelly, Guruprasad Ananda, Sara Patterson, Honey V. Reddi. Development and validation of the ActionSeq TM test system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2017-757