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

Abstract

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non–small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Next Generation Sequencing for Gene Fusion Analysis in Lung Cancer: A Literature Review

“…RNA-based approaches have the overall advantage of allowing an analysis of transcriptionally expressed gene fusions and sequencing is not affected by intronic regions [ 13 ]. In addition, RNA panels, besides gene fusions, allow a contemporary analysis of exon skipping events and expression levels of genes with a clinical value [ 42 ]. Although RNA analysis can be based on hybrid-capture or amplicon-based methods, most studies used the latter ones.…”

“…Moreover, biological material is often scarce, and it is not always possible to purify both DNA and RNA. In the studies evaluated in this review, different extraction methods have been used, both manual and automatized, in some cases RNA and DNA distinct kits were used [ 31 , 35 ]; in other works kits allowing to simultaneously separate DNA and RNA were preferred [ 29 , 30 ] and in some studies the use of total nucleic acids without splitting DNA from RNA was employed [ 42 , 43 ]. The possibility to use the same sections to purify both DNA and RNA can be advantageous, particularly for small biopsies and cytological specimens.…”

“…Moreover, most data have been obtained on FFPE surgical and biopsy specimens and few data are available on RNA from cytological specimens, which are the only available material in about 50% of advanced lung ADC [ 17 ]. Cohen et al reported a 30% dropout rate for cytological specimens and Haynes found that none out of 50 FNA smears had sufficient RNA to perform the analysis [ 39 , 42 ].…”

“…For RNA analysis, 94/109 biopsies and all 110 resections were adequate but no FNA smear had sufficient RNA to perform the test. Moreover, their RNA pool enables also the detection of MET exon 14 skipping and quantification of 23 other mRNA targets, with a potential prognostic value [ 42 ]. In the same way, Song and collaborators developed a single tube dual-template assay together with a specific bioinformatic pipeline.…”

Gene rearrangement detection by next-generation sequencing in patients with non-small cell lung carcinoma

“…Separately prepared samples under specific conditions and the different approaches in DNA and RNA sequencing procedures, which also necessiate differently prepared samples are the main obstacles in simultaneous testing. Recent studies to create an optimal panel for simultaneous DNA and RNA sequencing in NSCLC patients have achieved useful results covering more than hundred RNA and more than fifty DNA targets and this could be an optimal solution 60 . The two NGS platforms currently used in clinical laboratories, are the Illumina and the IonTorrent.…”