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Updated: Aug 5, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Amplicon-based DNA and RNA unified NGS for enhanced fusion variant detection in suboptimal real-world NSCLC FFPE
Qin Feng1, Yue Wang1, Mengli Huang2
1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Pathology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
Background:
Timely identification of actionable fusion and exon-skipping events is essential for selecting targeted therapies in non-small cell lung cancer (NSCLC), yet routine RNA-based assays frequently fail in formalin-fixed paraffin-embedded (FFPE) specimens, leading to missed treatment opportunities. Amplicon-based DNA and RNA unified next-generation sequencing (D+R NGS) may enable guideline-recommended profiling from limited or degraded tissue, but its performance in real-world specimens is unclear.
Methods:
The D+R NGS assay uses 20 ng of co-extracted RNA and DNA from FFPE tissue. Library preparation is performed in a single-tube amplicon-based workflow, followed by simultaneous sequencing and automated bioinformatics analysis. Its performance was evaluated in 759 NSCLC FFPE samples. Concordance was assessed against droplet digital polymerase chain reaction (ddPCR) in 46 matched fresh surgical specimens. Detection rates were evaluated in 172 surgical, 168 biopsy, and 142 cytology samples, all processed within one year. Clinical applicability was examined in 231 archived specimens stored for 1-8 years with known reverse transcription polymerase chain reaction (RT-PCR) or DNA-based next-generation sequencing (DNA-based NGS) fusion/skipping results.
Results:
The D+R NGS assay demonstrated exceptional analytical validity, showing 100% concordance with ddPCR in 46 fresh NSCLC samples for 28 mutations and 18 fusions/skipping events, with strong correlations in variant allele frequencies (R = 0.997, p < 0.001) and fusion read counts (R = 0.799, p < 0.001). It achieved high success rates across diverse sample types: 99.4% in surgical, 98.6% in cytology, and 95.2% in biopsy specimens, with robust performance even in samples with tumor areas <3 mm². In challenging low-input RNA scenarios (as low as 5-10 ng), the assay reliably detected fusions, and it identified actionable alterations in 5 of 11 samples that had failed routine quality control (QC) due to insufficient RNA. When applied to 231 archival samples stored for 1-8 years, the assay confirmed 139 known fusions/skipping events (99.3%) and detected 18.2% additional fusions in DNA-NGS-negative cases, including receptor tyrosine kinase (ROS1) fusions and MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping, while maintaining a > 95% success rate despite a decline in read counts over time.
Conclusion:
The amplicon-based D+R NGS method demonstrates high compatibility with diverse NSCLC FFPE samples, overcoming RNA degradation challenges in small-biopsy and long-term archived specimens.
Insights
This study introduces a novel DNA and RNA unified next-generation sequencing (D+R NGS) assay for non-small cell lung cancer (NSCLC). The D+R NGS assay effectively identifies actionable genomic alterations in challenging formalin-fixed paraffin-embedded (FFPE) specimens, improving targeted therapy selection.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Accurate identification of actionable genomic alterations is crucial for non-small cell lung cancer (NSCLC) targeted therapy.
- Routine RNA-based assays often fail with formalin-fixed paraffin-embedded (FFPE) specimens, limiting treatment options.
- Amplicon-based DNA and RNA unified next-generation sequencing (D+R NGS) offers a potential solution for profiling limited or degraded FFPE tissues.
Purpose of the Study:
- To evaluate the performance and clinical utility of an amplicon-based D+R NGS assay.
- To assess the assay's ability to detect actionable fusion and exon-skipping events in diverse NSCLC FFPE samples.
- To determine the assay's effectiveness in overcoming RNA degradation challenges in archival and small biopsy specimens.
Main Methods:
- The D+R NGS assay utilizes 20 ng of co-extracted RNA and DNA from FFPE tissue in a single-tube workflow.
- Performance was evaluated in 759 NSCLC FFPE samples, with concordance assessed against droplet digital polymerase chain reaction (ddPCR) in 46 fresh samples.
- Detection rates and clinical applicability were examined across surgical, biopsy, and cytology samples, including 231 archived specimens stored for 1-8 years.
Main Results:
- The D+R NGS assay showed 100% concordance with ddPCR for mutations, fusions, and skipping events in fresh samples.
- High success rates were achieved across various sample types (surgical, biopsy, cytology), even with small tumor areas and low RNA input.
- The assay successfully identified actionable alterations in samples that failed routine quality control and detected additional fusions in archival specimens, including ROS1 and MET exon 14 skipping.
Conclusions:
- The amplicon-based D+R NGS method is highly compatible with diverse NSCLC FFPE samples.
- This assay effectively overcomes RNA degradation issues in small-biopsy and long-term archived specimens.
- D+R NGS facilitates guideline-recommended molecular profiling, enabling timely targeted therapy selection for NSCLC patients.
