Abstract 3118: Next generation sequencing assay for detection of gene fusions and exon deletion events in tissue and liquid biopsy samples at very low frequency
Authors: Gottimukkala Rajesh K. , Hyland Fiona C. , Marcovitz Amir , Schageman Jeoffrey , Bagai Varun , Cao Ru , Williams Paul D. , Myrand Scott P. , Gu Jian , Sadis Seth , Bramlett Kelli S. 
Published: September 2020

Abstract
Introduction: Gene fusions caused by chromosomal rearrangements and the exon deletion events caused by aberrant RNA splicing events play a key role in oncogenesis and the progression of cancer. Next generation sequencing using molecular tagged AmpliSeq HD chemistry enables highly sensitive variant detection down to &lt;=0.1% mutant allele fractions. Here we present the Oncomine Precision RNA Assay developed using the Ion AmpliSeq HD Technology for use on both tissue and liquid biopsy samples to detect fusion and exon deletions with very high sensitivity. The assay also includes capabilities for novel fusion detection in driver genes via exon level expression measurements.
Methods: The Oncomine Precision RNA assay can detect &gt;900 known fusion isoforms including those of ALK, ROS1, RET, NTRK1,2,3, FGFR1,2,3, and BRAF and exon deletion events in MET and EGFR genes. The assay also includes amplicons in 8 key driver genes like NTRK1,2,3 to detect novel gene fusions in a partner agnostic manner based on the expression imbalance. The amplicons to detect targeted isoforms are designed strategically around the known break-points of the targeted fusions and exon skipping events. These amplicons generate reads when the target variant is present in the sample and provide complete information of the fusion event including the precise break-point with annotations such as cosmic ID. The unique molecular tags attached on both ends of the reads are used for error correction and this enables us to detect fusions at a very low frequency with high accuracy which is critical in liquid biopsy samples . The assay also includes amplicons to measure the wild-type expression of MET and EGFR genes and the exon deletion events are detected by comparing the expression of the exon-deletion transcript to that of the wild-type transcript.
Results: To test the panel, we used the GenexusTM sequencer to sequence ALK, ROS1, RET,FGFR3 and NTRK1 fusion positive cell lines and correctly identified all the targeted fusions and concordant results with expression imbalance. We sequenced cell-free total nucleic acid(cfTNA) control samples and identified the expected fusions in ALK, RET and ROS1 genes and the MET Exon 14 skipping event. We sequenced 16 FFPE samples with known truth and correctly identified all the 11 ALK and 5 ROS1 fusion isoforms and observed highly concordant results with the expression imbalance in the samples positive for ALK fusion. We sequenced the ALK, RET, NTRK1 and FGFR3 cell lines diluted to 2%, 2%, 5% and 20% respectively with the background of wild-type RNA and detected the expected fusions and observed concordant results with the expression imbalance.
Conclusions: This targeted sequencing assay enables researchers to detect gene fusions and exon deletions in both FFPE and blood samples with &gt;99% sensitivity and high specificity.
For research use only. Not for use in diagnostic procedures.
Citation Format: Rajesh K. Gottimukkala, Fiona C. Hyland, Amir Marcovitz, Jeoffrey Schageman, Varun Bagai, Ru Cao, Paul D. Williams, Scott P. Myrand, Jian Gu, Seth Sadis, Kelli S. Bramlett. Next generation sequencing assay for detection of gene fusions and exon deletion events in tissue and liquid biopsy samples at very low frequency [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3118.