Abstract 2290: Quick turnaround NGS assay for the characterization of biomarkers in FFPE tissue
Authors: Unselt Desiree , Frady Lauren , Pufky John , Torres Fernando , Sims Jennifer , Mason Jennifer 
Published: June 2022

Abstract
Background: Genomic profiling of a tumor to characterize clinically relevant variants has become critically important for cancer diagnosis and prognosis, as well as in driving therapeutic decision making. As such, there is a growing need for rapid inline assessment of somatic variants in genes of key interest in oncology. The Oncomine™ Precision Assay on the Ion Torrent Genexus Sequencer (OPAGX) is an amplicon-based automated next-generation sequencing (NGS) assay that enables quick turnaround genomic profiling of biomarkers across 50 genes from solid tissue. The complete workflow, including library prep, templating, sequencing, and final report generation, can be completed in less than 30 hours for up to 32 formalin-fixed, paraffin-embedded (FFPE) samples. This assay uses AmpliSeq™ HD technology which includes unique molecular tags for sequencing error correction and increased sensitivity for low frequency variant detection. The assay’s detection of hotspot mutations (substitutions, insertions, deletions, copy number variants (CNVs), and fusions) was evaluated for FFPE tissue.
Method: Fully automated library preparation and sequencing was completed in the Genexus™ Integrated Sequencer using 10 ng of DNA derived from FFPE tissue or from characterized control samples. Following gene specific amplification, AmpliSeq™ HD technology is used to label amplicons with dual molecular barcodes. The resultant libraries are bead normalized, pooled, and sequenced on a GX5™ chip to a targeted depth of 3,000,000 reads per sample. Sensitivity and positive predictive value were examined by evaluating the correctness of calling true positive variants compared to false negative and false positive variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate control samples. Specificity and accuracy were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively.
Results: Accuracy and specificity were 99.9% for SNVs and 100% for insertions and deletions above 1% allele frequency (AF). Sensitivity and intra-run precision were 100% for SNVs and insertions and deletions above 1% and 2% AF, respectively. Inter-run precision and PPV were 100% for SNVs and insertions and deletions above 1% AF. The estimated AF LoD was 3.7% for SNVs. CNV gain calls were 100% for MET and EGFR and loss calls were 100% for PTEN (homozygous and heterozygous) and CDKN2A. Additionally, fusion calling for RET and ROS1 were 100%.
Conclusions: OPAGX is a quick turnaround NGS assay enabling precision medicine through automated multi-variant genomic profiling to inform decision making; performance was characterized with analysis of hotspot mutations in FFPE samples in a variety of tumor types, demonstrating utility of implementation in testing of targeted biomarkers across multiple indications.
Citation Format: Desiree Unselt, Lauren Frady, John Pufky, Fernando Torres, Jennifer Sims, Jennifer Mason. Quick turnaround NGS assay for the characterization of biomarkers in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2290.