Abstract 5123: Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay
Authors: Cao Ru , Lea Kris , Jasti Madhu , Schageman Jeff , Hanif Khalid , Li Yanchun , Gu Jian , Bagai Varun , Kshatriya Priyanka , Wikman Harriet , Loges Sonja , Bramlett Kelli 
Published: August 2019

Currently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants.

Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems.

In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly,

additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor.

Overall, this study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models.

Citation Format: Ru Cao, Kris Lea, Madhu Jasti, Jeff Schageman, Khalid Hanif, Yanchun Li, Jian Gu, Varun Bagai, Priyanka Kshatriya, Harriet Wikman, Sonja Loges, Kelli Bramlett. Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5123.