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Abstract 5996: Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid


 Introduction: Recently, liquid biopsy has begun to be adopted for treatment selection and drug-resistance assessment for cancers. However, currently liquid biopsy is mostly focused on analyzing cell-free DNA (cfDNA). Though analyzing RNA provides more accurate analysis for functional gene fusions, little is known about the stability and availability of cfRNA (cell free RNA). Here we report our one-tube robust cfTNA (cell free total nucleic acids) assay to analyze ctDNA and ctRNA simultaneously.
 Methods: We assessed Pillar’s cell free TNA panel performance on 2 confirmed fusion positive and 5 fusion negative samples that were serially diluted to obtain a total of 88 samples including replicates. An input of 10ng cfTNA was used for fusion positive samples with fusion frequency ranging from 0.2% to 20%. A range of 5 to 30ng input was used for fusion negative samples. The amount of input cfRNA was estimated as equal to the cfDNA input. Total nucleic acids were extracted and cfRNA was reverse transcribed. Amplicons targeted the specific breakpoints of 36 known fusion transcripts. Fusion positive samples were diluted for library prep to achieve fusion frequency of 0.5%, 1%, 4%, 5% and 20%. Novaseq was used for sequencing with an average depth of 200,000X and downsampled by 25%. Pillar Variant Analysis Toolkit (PiVAT®) was used to map and call fusions.
 Results: Among dilutions of >=4%, PiVAT achieved >=99% sensitivity and >=99.9% specificity in calling RNA fusions from 10ng input. Even at extremely low dilutions of <=1%, PiVAT achieved >= 50% sensitivity.
 Conclusion: Pillar’s cell free TNA panel offers a simple, rapid, and reliable workflow that enables detection of a broad range of clinically relevant gene fusions. Detection of fusion events in cell-free samples from noninvasive liquid biopsies represents an important step forward in cancer diagnosis and monitoring.
 Citation Format: Tejashree Modak, Jilong Li, Sean Polvino, Yue Ke, Zhaohui Wang, Alex V. Kotlar. Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid [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 5996.