Abstract 5547: Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations
Authors: Kreston Sarah , Gould Claire , Blair Kylie , Jackson Leisa , Riley Janice , Pestano Gary A. 
Published: June 2023

Introduction: The use of blood-based molecular diagnostics is increasingly becoming routine in clinical oncology practice. The purpose of this study was to evaluate variant resulting concordance for the four major somatic variant classes using two clinical next-generation sequencing (NGS) systems for conducting highly sensitive blood-based analyses. All donor specimens were de-identified remnants from patients previously diagnosed with advanced NSCLC. We utilized two independent panels, systems and bioinformatic pipelines that are focused on clinical testing for key actionable variants. The fifty-gene panel Oncomine Precision Assay (OPA) GX was performed on the Genexus Integrated Sequencer and the 52 gene GeneStrat NGS (GSNGS) test was run as a reference on the Ion GeneStudio S5 PRIME system. The four major classes of mutations evaluated included Single Nucleotide Variants (SNV), Insertions and Deletions (INDEL), copy number amplifications (CNAs) and gene fusions.

Methods: For this study, de-identified reference GSNGS cell-free nucleic acid (cfNA) remnant specimens (n=33 variants) that passed all validated QC bioinformatics thresholds were blinded and tested on the Genexus. DNA concentration was measured by fluorometry, and specimens were diluted to an input of 6.6 ng to 53.4 ng for library preparation. The Genexus system is pre-programmed to process specimens to result generation. Variant calls for all mutation classes were conducted using the on-board bioinformatics pipelines, and results were compared to those generated by the GeneStrat NGS Test. We included specimens harboring somatic variant mutations in KRAS, NRAS, BRAF, EGFR, ERBB2, and FGFR; CNAs; EML4-ALK fusions; and MET exon 14 skipping.

Results: All samples passed the OPA (Genexus) bioinformatic quality control criteria and mapped reads were highly consistent, demonstrating accuracy of calls between both the Ion Torrent platforms, informatic pipelines and panels. We observed 100% concordance in variant calls for SNVs, INDELs and fusions between the two platforms. There was one CNA variant observed on the S5 that was not called by the Genexus workflow, yielding overall concordance of 97% (32 of 33). Of note, 44 additional somatic variants (SNV and INDELs) and 1 additional fusion were detected using the Genexus workflow. This was due to the lower threshold for variant calling on this system.

Conclusion: The high concordance of the independent workflows for the detection of nucleic acid variants in circulation demonstrates the capability of both systems to be used for testing of clinical specimens. Specifically, actionable variants in the four major mutation classes were successfully detected in the reference and test specimen set. As a part of clinical validation, orthogonal testing of variants will need to be conducted.

Citation Format: Sarah Kreston, Claire Gould, Kylie Blair, Leisa Jackson, Janice Riley, Gary A. Pestano. Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5547.