Design, Optimization, and Multisite Evaluation of a Targeted Next-Generation Sequencing Assay System for Chimeric RNAs from Gene Fusions and Exon-Skipping Events in Non–Small Cell Lung Cancer

Abstract

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 nonesmall 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.

An Integrated Next-Generation Sequencing System for Analyzing DNA Mutations, Gene Fusions, and RNA Expression in Lung Cancer

“…The DNA NGS library covers 55 hotspot regions in 20 genes including EGFR , KRAS , STK11 , PIK3CA , TP53 , and others (Table 2) and is based on previously published methods [25], [26], [27]. Development and validation of the RNA NGS panel are described extensively in previously published work [28]. The panel includes coverage of all DNA and RNA markers recommended by the NCCN NSCLC guideline [29] as part of broad molecular testing, in addition to emerging markers of clinical research value [30].…”

“…The I7 and I5 dual index code pairs were identified within the forward and reverse reads, and read pairs with unexpected code pairs were filtered. This index purity filtering step improves the specificity of the final calls and addresses a known limitation of Illumina's chemistry [28], [33], [34]. A local, gapped alignment to a custom reference transcriptome (inclusive of targeted breakpoint junctions) was performed using bowtie2 v2.0.5 (using the option: --sensitive-local) [35].…”

“…Alignments that did not match the expected amplicon boundaries or contain large gaps were filtered. Fusions and splice variant detection employed an upper-tailed Poisson test statistic, and 3′/5′ expression imbalances were assessed on normalized 3′ and 5′ expression data described in previously published work [28]. Gene expression was normalized by the geometric mean of endogenous control genes ( TBP , RAB5C , and GGNBP2 ).…”

NARASIMHA: Novel Assay based on Targeted RNA Sequencing to Identify ChiMeric Gene Fusions in Hematological Malignancies

“…Researchers have therefore developed focussed target enrichment strategies (or gene-panels) for detection of CGF. Typically, these panels utilize capture probes 1,2 or multiplexed PCR approaches 3,4 to enrich targets of interest and detect the CGF, if present. These assays too require prior knowledge of the exact sequence of both partners involved in the formation of CGF thus failing to overcome a principal hurdle of being unable to detect a CGF involving a promiscuous gene where recombination with several partners is known to occur (for e.g., KMT2A-rearranged leukemia) 5 .…”

“…2 Homi Bhabha National Institute (HBNI), Mumbai, India. 3 Pediatric Haematolymphoid Disease Management Group, Tata Memorial Centre, Mumbai, India. 4 Department of Cytogenetics, ACTREC, Tata Memorial Centre, Navi Mumbai, India.…”