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Updated: Aug 1, 2025
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Detection of multiple types of cancer driver mutations using targeted RNA sequencing in non-small cell lung cancer
Sheng Ju1,2, Zihan Cui1,2, Yuanyuan Hong3
1Department of Thoracic Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou, China.
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, paraffin-embedded 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.
Insights
This study introduces an RNA sequencing panel for detecting actionable mutations in non-small cell lung cancer (NSCLC). The RNA panel demonstrated high accuracy and robustness, outperforming DNA sequencing for certain mutations, offering a promising tool for clinical testing.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- DNA-based next-generation sequencing (NGS) is standard for NSCLC targeted therapy selection.
- RNA-based NGS is recommended for detecting fusion and exon-skipping mutations in NSCLC.
- Current methods may miss certain actionable mutations crucial for treatment decisions.
Purpose of the Study:
- To develop and validate an RNA-based hybridization panel for detecting actionable driver oncogenes in solid tumors.
- To assess the performance of the RNA panel in identifying fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels) mutations.
- To compare the diagnostic yield of RNA panel sequencing against DNA panel sequencing in non-small cell lung cancer (NSCLC) patients.
Main Methods:
- Development of an RNA-based hybridization panel targeting actionable oncogenes.
- Optimization of experimental and bioinformatics pipelines for mutation detection.
- Parallel DNA and RNA panel sequencing of 1253 formalin-fixed, paraffin-embedded NSCLC samples.
Main Results:
- The RNA panel achieved high sensitivity for SNVs and fusions.
- Identified 124 fusion events and 26 MET exon 14-skipping events in NSCLC samples.
- Detected 14 fusions and 6 MET exon 14-skipping mutations missed by DNA sequencing, with high positive predictive values (98-99%).
Conclusions:
- RNA panel sequencing accurately and robustly detects clinically actionable mutations in NSCLC.
- The simplified workflow and low sample requirement make RNA panel sequencing a viable option for clinical diagnostics.
- RNA sequencing offers a complementary approach to DNA sequencing for comprehensive mutation profiling in cancer.
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