Abstract 1389: A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer
Authors: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes
Journal: Cancer Research (2016)

Abstract Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens. Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling. Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including &amp;gt;100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at &amp;lt;10% abundance, improved PPV for low-input specimens, and achieved absolute quantification of RNA. The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to &amp;lt;5% abundance, and RNA translocations could be observed in sub-nanogram quantities of fusion-positive FFPE TNA. The unified DNA/RNA panel identified clinically-relevant DNA and RNA variants and maintained similar coverage uniformity to the separate DNA and RNA panels. Analysis of 61 lung adenocarcinoma and 36 squamous cell carcinoma specimens revealed mutation distributions in driver genes and RNA fusions consistent with the known spectrum of variants from each tumor type as previously reported by TCGA and other groups. Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing. Citation Format: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes. A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1389.