Abstract 3650: Multi-pronged approach to establish control standards for somatic mutations in next generation sequencing (NGS) oncology test
Authors: Johnson Christopher , Raghunath Sharanya , Wayne Jackie , Shamo Aimee , Bradley Patrick , Hernandez Moises , Loughmiller David , Gillman Jason , Haslem Derrick , Stone Gary , Nadauld Lincoln , Mishra Pravin J. 
Published: July 2016

Abstract
In recent years, somatic mutation testing via Next Generation Sequencing (NGS) has emerged as a powerful tool to detect targetable mutations in diseases such as cancer. These include mutations such as BRAF V600E, which can lead to targeted drug therapy; therefore, improving the standard of care in cancer diagnostics. To generate clinically meaningful results, we need to define standards that can be applied to laboratories performing somatic testing on tumor samples. With ongoing debate about the use of positive or negative controls in somatic testing assays, we designed an experiment to test the concordance of clinically actionable mutations commonly seen in tumor testing. We use cell line controls and commercially engineered DNA to validate concordance of mutations at specific allelic ratios. Our results depict an experiment design to determine if a genomic test can detect actionable mutations with high levels of accuracy and precision.
In this study, we designed a multi-pronged approach to evaluate the need for control standards by the ICG100 clinical NGS panel. To establish a baseline negative control, we performed concordance testing on the NA12878 cell line. We compared SNPs, insertions and deletions found using our methodology to the variants reports by NIST. We found our results to be concordant with NIST at a sensitivity of 92% and a specificity of 93%. To establish a baseline for positive controls, we utilized commercially engineered DNA which contains variants spiked-in at known allelic frequencies. This allowed us to determine if the ICG100 panel was able to retrieve specific somatic mutations. We were able to detect high confidence somatic mutations, such as BRAFV600E, as well as BRAF V600G which was spiked in at a lower expect allelic frequency of 4%. Moreover, we are able to detect with high confidence mutations such as ALK F1174L, EGFR G719S, PIK3CA H1047R, and MET Y1247D, and we were able to reproduce the results across multiple cell lines. Overall, our results show the need to utilize controls as standard protocol which can help assess the proficiency of a clinical assay. With rapid advances is NGS testing methodologies, there is a need for establishing standardized controls that asses the performance of a somatic tumor testing.
Citation Format: Christopher Johnson, Sharanya Raghunath, Jackie Wayne, Aimee Shamo, Patrick Bradley, Moises Hernandez, David Loughmiller, Jason Gillman, Derrick Haslem, Gary Stone, Lincoln Nadauld, Pravin J. Mishra. Multi-pronged approach to establish control standards for somatic mutations in next generation sequencing (NGS) oncology test. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3650.