Detection of multiple types of cancer driver mutations using targeted RNA sequencing in non–small cell lung cancer

Abstract

Background: DNA-based next-generation sequencing has been widely used in the selection of target therapies for patients with nonsmall cell lung cancer (NSCLC).RNA-based next-generation sequencing has been proven to be valuable in detecting fusion and exon-skipping mutations and is recommended by National Comprehensive Cancer Network guidelines for these mutation types. Methods:The authors developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for the detection of fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels). In total, 1253 formalin-fixed, paraffinembedded samples from patients with NSCLC were analyzed by DNA and RNA panel sequencing in parallel to assess the performance of the RNA panel in detecting multiple types of mutations. Results:In analytical validation, the RNA panel achieved a limit of detection of 1.45-3.15 copies per nanogram for SNVs and 0.21-6.48 copies per nanogram for fusions. In 1253 formalin-fixed, paraffin-embedded NSCLC samples, the RNA panel identified a total of 124 fusion events and 26 MET exon 14-skipping events, in which 14 fusions and six MET exon 14-skipping mutations were missed by DNA panel sequencing. By using the DNA panel as the reference, the positive percent agreement and the positive predictive value of the RNA panel were 98.08% and 98.62%, respectively, for detecting targetable SNVs and 98.15% and 99.38%, respectively, for detecting targetable indels. Conclusions: Parallel DNA and RNA sequencing analyses demonstrated the accuracy and robustness of the RNA sequencing panel in detecting multiple types of clinically actionable mutations. The simplified experimental workflow and low sample consumption will make RNA panel sequencing a potentially effective method in clinical testing.See related editorial on pages 2294-6, this issue.

Uncommon/rare oncogenic drivers in non‐small cell lung cancer: Consensus and contention

“…However, on the other hand, two samples with positive results by DNA-NGS, failed to have RNA-NGS results due to insufficient RNA quality, indicating the higher sample quality requirements of RNA-NGS. Another study conducted head-to-head comparison of DNA-NGS and RNA-NGS in clinical formalin fixed paraffin embedded (FFPE) samples of 1253 NSCLC patients and found that 6% (10/ 168) of patients with negative results of DNA-NGS oncogenic drivers test were rediscovered by RNA-NGS detection (including 7 samples of fusion mutations, 3 samples of MET exon 14 skipping mutations), and these patients were offered targeted therapy [23].…”

“…Using an NGS panel composed of a specific number of genes, typically covering at least 50 genes, is a feasible strategy. If no oncogenic drivers are detected by a DNA-based NGS panel, RNA-based NGS should be considered [23]. In addition to considering the number of genes, it is also important to consider the tiers of somatic variants on the Level of evidence: strong Strength of recommendation: strong In the mid-1990s, with the development of imaging and radiology the multidisciplinary team (MDT) model was established and started to flourish due to its patientcentered core as well as the consequent improved clinical treatment decision.…”

RNA sequencing steps toward the first line

“…In the article in this issue of Cancer by Ju and colleagues, both DNA-based and RNA-based NGS were performed separately with nucleic acid extracted from the same formalin-fixed, paraffinembedded NSCLC samples (N = 1253). 5 Their RNA-based panel covered 30 genes associated with clinically significant fusions plus six housekeeping genes. Because of the fusion-oriented design, some genes with clinically important mutations, such as ERBB2 and KRAS, were not included.…”