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Updated: Mar 7, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from
Umberto Malapelle1, Clara Mayo de-Las-Casas2, Danilo Rocco3
1Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Background:
When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.
Methods:
Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.
Results:
SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.
Conclusions:
SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.
Insights
This study introduces SiRe, a next-generation sequencing panel for analyzing cell-free DNA (cfDNA). SiRe accurately detects clinically relevant mutations in cfDNA, proving feasible for clinical practice.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Cell-free DNA (cfDNA) serves as a non-invasive surrogate for tumor genetic analysis when tissue is unavailable.
- Detecting low-abundance mutated alleles in cfDNA requires highly sensitive sequencing methods.
- Targeted next-generation sequencing (NGS) panels are crucial for identifying clinically relevant mutations in cfDNA.
Purpose of the Study:
- To develop and validate a novel NGS panel (SiRe) for ultra-deep sequencing of cfDNA.
- To assess the analytical performance and clinical utility of the SiRe panel.
- To identify specific gene mutations in cfDNA for various cancer types.
Main Methods:
- Developed the 'SiRe' NGS panel targeting 568 mutations in six key cancer genes (EGFR, KRAS, NRAS, BRAF, cKIT, PDGFRα).
- Evaluated analytical sensitivity using cell line DNA and artificial standards with known mutations.
- Assessed cfDNA from retrospective patient cohorts (n=42, 12, 11) and prospectively from non-small-cell lung cancer (NSCLC) patients (n=79).
Main Results:
- The SiRe panel demonstrated high analytical performance with a 0.01% lower limit of detection.
- Achieved 96.8% concordance with the Taqman-derived assay (TDA) in retrospective cfDNA analysis.
- In prospective NSCLC patients, SiRe detected 8.7% of EGFR mutations at baseline and 42.9% of EGFR p.T790M mutations at progression.
Conclusions:
- The SiRe NGS panel is a highly sensitive and specific tool for cfDNA analysis.
- SiRe exhibits strong analytical performance and feasibility for clinical application.
- This panel facilitates non-invasive genetic profiling of cancers using cfDNA.
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