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

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 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