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Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Tumor genome sequencing has emerged as a powerful tool for identifying biomarkers for targeted cancer therapies. While DNA sequencing is a well-established method and considered a gold standard, RNA sequencing (RNA-seq) can identify anomalies in gene transcription, regulation of gene expression, and gene fusions, which have critical diagnostic and therapeutic impacts. Tempus Labs is CLIA-certified, CAP-accredited and offers several clinically validated NGS assays for solid tumor and hematological malignancy testing, including the xT targeted 648-gene oncology panel and the xE whole-exome ~20,000 gene panel. The Tempus whole-transcriptome assay by hybrid-capture NGS has been enhanced to improve coverage, focusing on fusion genes and RNA expression calls. We clinically and analytically validated fusion calls for gene rearrangement detection and clinical reporting, and analytically validated RNA expression counts for research purposes only. For RNA fusion (translocation) calling, we sequenced 96 tumor samples, including formalin-fixed, paraffin-embedded (FFPE) tissue, whole blood, and bone marrow samples from 12 cancer types; 82 of these samples contained reportable fusions. Of these, 86 samples were positive for RNA fusions, including 62 positive samples with targeted fusion breakpoints (i.e., utilizing custom probes designed to the breakpoints and spiked-in at hybridization) and 24 positive samples with untargeted fusion breakpoints (i.e., utilizing no custom probes). Targeted fusion accuracy was evaluated against the first version of the RNA sequencing assay. The first version of this assay was evaluated with cell line controls. In addition, validation included a specificity test and a rolling validation of fusions via DNA sequencing (positive controls). The concordance for targeted fusions was 100%, with 99.9% sensitivity and 99.9% specificity. For untargeted fusions, the overall concordance was 97%, with 97% sensitivity. For RNA expression calling, we conducted a clinical linearity study using 88 samples and testing 18 genes to measure concordance between qPCR ΔCT values and normalized gene expression levels. Of these, 15/18 genes met the established acceptance criteria of R > 0.75. In conclusion, the whole-transcriptome by hybrid-capture NGS assay offers clinically and analytically validated unbiased detection of common and novel gene rearrangements, as well as analytically validated gene expression data for comprehensive research analyses. Citation Format: Jun Hu, Jerod Parsons, Brittany Mineo, Josh SK Bell, Jenna Malinauskas, Joshua Drews, Jack Michuda, Calvin McCarter, Rasika Dhond, Jack C. Tyndall, Nithya Sreenivasan, Sheeri Hanjra, Shannon Gallagher, Ankit Jambusaria, Naihui Zhou, Catherine Igartua, Nike Beaubier, Richard A. Blidner, Robert Tell. Comprehensive validation of RNA sequencing for clinical NGS fusion genes and RNA expression reporting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2239.

Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2017-1764

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 >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 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 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.

Various strategies have been employed to achieve control over delivery of siRNA molecules to intended target cells. Photocaging is one specific class of modifications for silencing oligonucleotides that block their bioactivity until exposure to near-ultraviolet light. These caged RNAi effectors enable both spatial and temporal targeting of a dosed release of gene silencing agents by directed light exposure that photocleaves the cage moieties. Herein we compare the photochemical properties of cage compounds and strategies for their use, attached either randomly or site-specifically, to demonstrate various forms of gene expression regulation in vitro and in vivo. This light-controllable strategy has potential applications for precisely probing developing biological systems and eventually enabling targeted gene-silencing therapeutics.

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2'-fluoro modifications elicit RNAi. In this study, we demonstrate that fully-2'-fluorinated nucleic acids can be generated for RNAi studies through either custom solid-phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single-stranded and hybridized fully-2'-fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully-2'-fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully-2'-fluorinated nucleic acids can be successfully generated, and fully-2'-fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully-2'-fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully-2'-fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single-stranded fully-2'-fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

Various chemical modifications to RNA have been incorporated in attempts to improve their pharmacological properties for RNAi interference (RNAi). Recent studies have shown that small interfering RNA (siRNA) containing 2'-fluoro modifications can elicit gene silencing through RNAi. Despite developments in using chemical modifications for increased stability, safety, and efficiency of these therapeutics, they still face challenges of spatial and temporal targeting. One potential targeting strategy is to use photocaging techniques, which involve the covalent attachment of photolabile compounds to the effector nucleic acid species that block bioactivity until exposed to near UV light. In this study we demonstrate that fully 2'-fluorinated nucleic acids (FNAs) can be caged for photoactivated gene silencing in cell culture and in zebrafish embryos. This strategy combines the improvement in chemical and enzymatic stability associated with 2'-substitutions with the targeting ability of a photoinducible trigger. Statistical alkylation of FNAs with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) improved resistance to enzymatic degradation, reduced RNAi effectiveness, and protected the biological system from toxic doses of the effector. Photo-exposure to 365 nm light partially restored the silencing activity of the 2'-fluoro siRNAs. These results suggest that photocaging may offer control over RNAi therapeutics for spatially and temporally directed activation, while improving enzymatic stability and potentially enabling therapeutic dosing via light dose intensity.