Introduction: Next generation sequencing technologies and platforms have been evolving rapidly over the last decade with improved throughput and cost-effectiveness. The application of various NGS assays using both genome wide or targeted sequencing has been widely employed in cancer research to characterize tumor biomarker profiles including SNV/indels, CNV, and fusion in various sample types. Here we will present the performance of OncomineTM Precision Assay developed at Thermo Fisher Scientific, which is compatible with both solid tumor and liquid biopsy sample types. It is the very first automated NGS assay developed with Ion Torrent Genexus system to provide a streamlined workflow directly from sample to results with minimal hands-on time. Methods: The assay employs a novel AmpliSeq™ HD library technology to provide optimal sensitivity and specificity for challenging sample types including degraded FFPE or cell-free nucleic acid from plasma. For library preparation, 10 ng DNA or RNA was used for FFPE and 20 ng cfTNA was used for plasma samples. Ion Torrent Genexus system provides an interface to guide the user to plan the run following a defined workflow according to sample type, and performs library preparation, templating and sequencing automatically. Results: The OncomineTM Precision Assay includes focused genetic content with clinical relevance including 45 hotpot genes (SNV/Indel), 14 CNVs, and 19 structural variants (fusion and exon variants). This study evaluated the assay performance using a set of controls for FFPE or cfDNA from positive cell line material or commercial sources such as Horizon discovery and SeraCare, which contain various SNV/Indels on several important gene loci (EGFR, KRAS, NRAS, BRAR, and PIK3CA), CNV (EGFR, MET amplification and pTEN deletion), and fusion variants across ALK, ROS1, RET, and NTRKs. The results showed that all the expected SNV variants were consistently detected with sensitivity at ≥95% for FFPE with 5% AF and ≥85% for cfDNA samples down to 0.1% LOD. For fusion variants and CNV targets including gain and loss, both sensitivity and PPV at ≥90% could be achieved for FFPE and cfTNA samples. Furthermore, a cohort of positive FFPE tissue was also analyzed and detected expected variants that had been previously confirmed through an orthogonal NGS test. Lastly, a set of matched FFPE and plasma samples was also evaluated using the assay and demonstrated high concordance between FFPE and plasma from each subject. Conclusion: Overall the study demonstrates that the OncomineTM Precision Assay provides an automated and sensitive NGS solution for efficient and effective mutation assessment using both FFPE and liquid biopsy samples. Citation Format: Ru Cao, Jian Gu, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Bo Ding, Khalid Hanif, Kelli Bramlett. Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research [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 215.
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.
Introduction: We describe the development and performance of a new sample-to-report targeted sequencing solution for testing solid tissue cancers using the Genexus Integrated Sequencing System and accompanying software. The assay is designed for research applications from either formalin fixed paraffin embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy samples. The Genexus Integrated Sequencer is a fully automated system requiring minimal touch points and hands on time allowing a novice user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types in less than two days. Methods: The Oncomine Precision Assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biological variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. With about 15 minutes of hands on time, a run is set-up using pre-filled reagent strips for a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: Reported here are the results generated from an early external test site along with development data. The Oncomine Precision Assay is designed to detect somatic variants in 50 unique genes testing all major variant types important in the oncology research. The content was selected based on published accounts of target actionability and prevalence across multiple cancer types. All major variant types are targeted including SNVs, insertions, deletions, copy number variation, fusion transcripts and alternate splice forms. Data is shared from an external test lab using the Oncomine Precision Assay on the Genexus Integrated Sequencer with control and research samples. Results from both multiplexed FFPE and liquid biopsy runs are presented. Data demonstrates use of a single assay and system to effectively call variants from both FFPE and liquid biopsy sample types with a turn-around time of less than 30 hours. Conclusion: The Oncomine Precision Assay and Genexus Integrated Sequencer enable detection of key oncology variants in 50 genes using either solid tissue or liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in less than 2 days with minimal hands-on time and touch-points from the user. Many features of the automated system increase success rates by ensuring the appropriate reagents are properly installed before a run. The user friendly, highly automated, and fully optimized sample to answer system described here has great potential for targeted oncology sequencing in the research setting. Citation Format: Jian Gu, Ru Cao, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Varun Bagai, Khalid Hanif, Jose L. Costa, Kelli Bramlett. Demonstration of the genexus integrated sequencing system with the oncomine precision assay [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 213.
The presence of certain chromosomal rearrangements and the subsequent fusion gene derived from translocations has been implicated in a number of cancers. Hundreds of translocations have been described in the literature recently but the need to efficiently detect and further characterize these chromosomal translocations is growing exponentially. The two main methods to identify and monitor translocations, fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) are challenging, labor intensive, the information obtained is limited, and sensitivity is rather low. Common sample types for these analyses are biopsies or small tumors, which are very limited in material making the downstream measurement of more than one analyte rather difficult; obtaining another biopsy, using a different section or splitting the sample can raise issues of tumor heterogeneity. The ability to study mutation status as well as measuring fusion transcript expression from the same sample is powerful because you're maximizing the information obtained from a single precious sample and eliminating any sample to sample variation. Here we describe the efficient isolation of two valuable analytes, RNA and DNA, from the same starting sample without splitting, followed by versatile and informative downstream analysis. This methodology was applied to FFPE and degraded samples as well as fresh tissues and cells. DNA and RNA were recovered from the same non-small lung adenocarcinoma sample and both mutation analysis, as well as fusion transcript detection was performed using the Ion Torrent PGM™ platform on the same Ion 318™ chip. Using 10ng of DNA and 10ng of RNA input, we applied the Ion AmpliSeq™ Colon and Lung Cancer panel to analyze over 500 COSMIC mutations in 22 genes and the Ion AmpliSeq™ RNA Lung Fusion panel to detect 40 different fusion transcripts. Citation Format: Angie Cheng, Varun Bagai, Joey Cienfuegos, Natalie Hernandez, Mu Li, Jeff Schageman, Richard Fekete, Rosella Petraroli, Alexander Vlassov, Susan Magdaleno. Hotspot mutation and fusion transcript detection from the same non-small lung adenocarcinoma sample. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3580. doi:10.1158/1538-7445.AM2014-3580
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