How RNA Sequencing Is Improving Detection of Actionable Gene Fusions in Precision Oncology
Key Takeaways
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RNA sequencing is reshaping precision oncology
Precision oncology is increasingly driven by the ability to identify clinically actionable genomic alterations that influence diagnosis, prognosis, and treatment selection. While DNA-based next-generation sequencing (NGS) has transformed molecular diagnostics over the past decade, important genomic alterations, including many clinically relevant gene fusions, can still remain undetected.
During the MGI Corporate Satellite Symposium at ESHG 2026, Dr. Raquel T. Lima from IPATIMUP (Portugal) shared how her team has successfully implemented RNA sequencing into routine clinical diagnostics to improve the detection of actionable gene fusions in solid tumours. Her presentation demonstrated how RNA sequencing complements existing DNA-based testing, enabling more comprehensive molecular characterization and expanding access to targeted therapies for patients.
Why Gene Fusions Matter in Cancer
Gene fusions are among the most clinically significant genomic alterations in oncology. They arise when chromosomal rearrangements join portions of two different genes, creating abnormal fusion proteins capable of driving cancer development and progression. These alterations are important because they can:
- ✓Drive tumour initiation and progression
- ✓Define distinct molecular tumour subtypes
- ✓Improve diagnostic accuracy
- ✓ Predict response to targeted therapies
- ✓ Qualify patients for precision medicine clinical trials
In many cancers, identifying a single fusion event can fundamentally change both diagnosis and treatment strategy.
The success of targeted therapies such as Imatinib for chronic myeloid leukaemia and Crizotinib for ALK-positive lung cancer illustrates how fusion detection has become a cornerstone of precision oncology. As more tissue-agnostic therapies become available, comprehensive fusion detection is becoming increasingly important across multiple tumour types.
The Limitations of Conventional Fusion Detection
Historically, laboratories have relied on techniques such as karyotyping, fluorescence in situ hybridisation (FISH), RT-PCR, and Sanger sequencing. While these methods remain valuable, they were developed to answer targeted diagnostic questions rather than comprehensively profile complex tumour genomes.
DNA-based NGS significantly improved the ability to detect structural variants by providing broader genomic coverage and higher throughput. However, DNA sequencing still has inherent limitations when identifying gene fusions. Challenges include:
- Large intronic regions that are difficult to sequence
- Complex genomic rearrangements
- Repetitive DNA sequences
- Difficulty determining whether detected rearrangements are biologically active
- Limited ability to identify novel fusion partners
Most importantly, DNA sequencing detects genomic alterations but cannot confirm whether those alterations produce functional RNA transcripts that influence tumour biology.
Why RNA Sequencing Provides Greater Clinical Insight
RNA sequencing addresses many of these limitations by directly analysing expressed RNA transcripts rather than genomic DNA. Instead of inferring whether a structural rearrangement may produce a functional fusion protein, RNA sequencing confirms whether that fusion is actively expressed within the tumour. This provides several important advantages:
- ✓Direct detection of expressed fusion transcripts
- ✓Precise identification of exon-exon fusion junctions
- ✓Discovery of previously unknown fusion partners
- ✓Improved biological interpretation
- ✓Higher confidence in clinically actionable findings
Recent large-scale clinical studies involving more than 2,300 patient samples have demonstrated that RNA sequencing identifies clinically relevant fusions and splice variants that would have been missed using DNA testing alone, leading to improved diagnostic yield and more informed treatment decisions.
Bringing RNA Sequencing into Routine Clinical Diagnostics
At IPATIMUP, RNA sequencing has been introduced to complement an already extensive molecular diagnostics service supporting both adult and paediatric oncology.
As Portugal’s national reference centre for molecular oncology testing, IPATIMUP operates at the intersection of routine diagnostics, translational research, and multidisciplinary clinical decision-making through molecular tumour boards and second-opinion testing. The laboratory already performs high-volume molecular testing using targeted DNA and RNA panels, hybrid-capture sequencing, shallow whole-genome sequencing, and whole-exome sequencing.
However, clinicians continued to encounter patients whose tumours lacked identifiable driver mutations despite strong clinical suspicion that actionable genomic alterations were present. This unmet clinical need, particularly in sarcomas, rare cancers, paediatric tumours, and non-small cell lung cancer, motivated the implementation of comprehensive RNA sequencing.
Implementing RNA Sequencing using MGI Technology
To introduce RNA sequencing into routine practice, the IPATIMUP team selected the MGI sequencing platform based on several practical considerations important for a clinical laboratory. These included:
- Robust analytical performance
- Reliable processing of FFPE tumour samples
- Scalability for increasing clinical workloads
- Cost-effectiveness
- Straightforward integration into existing laboratory workflows
The laboratory developed a complete workflow encompassing:
- 1RNA quality assessment
- 2Ribosomal RNA depletion
- 3Library preparation
- 4DNA Nanoball (DNB) generation
- 5Sequencing on the MGI DNBSEQ platform
- 6Bioinformatics analysis
- 7Clinical interpretation of fusion events
The workflow was specifically optimised for FFPE samples, which remain one of the most challenging specimen types encountered in routine molecular pathology due to RNA degradation.
Clinical Validation Demonstrated Excellent Performance
The team first evaluated the workflow using a validation cohort of fifteen FFPE tumour samples with previously confirmed gene fusions. The results demonstrated:
Following validation, the workflow was applied to a larger discovery cohort comprising 84 FFPE tumour samples that had previously tested negative for gene fusions using existing molecular assays. Performance remained excellent, achieving:
These findings demonstrated that RNA sequencing could be implemented successfully within a real-world diagnostic laboratory while maintaining the robustness required for clinical decision-making.
RNA Sequencing Revealed Clinically Actionable Findings
Perhaps the most compelling result came from the discovery cohort. Among patients who previously had no detectable gene fusion, RNA sequencing identified fusion events in 52% of evaluated cases.
These included 28 confirmed oncogenic fusions, and additional fusions requiring further biological interpretation. Importantly, many of these newly identified fusions carried immediate clinical relevance. Across the study:
- 13 patients received improved molecular diagnosis
- 15 patients became eligible for targeted therapeutic opportunities
Examples included actionable alterations involving ALK, FGFR2, RET, and BRAF. In other cases, fusion detection enabled more accurate classification of challenging tumour types such as sarcomas, gliomas, thyroid cancers, and cholangiocarcinomas, directly influencing diagnostic confidence and patient management
Supporting Precision Oncology Beyond DNA Testing
Rather than replacing DNA sequencing, RNA sequencing serves as a powerful complementary technology.
DNA sequencing remains essential for identifying single nucleotide variants, copy number alterations, and structural rearrangements. RNA sequencing adds another critical layer by determining which genomic alterations are functionally expressed and therefore biologically relevant.
Together, these technologies provide a more complete molecular picture that enables:
- More accurate diagnosis
- Improved tumour classification
- Greater access to targeted therapies
- Better identification of clinical trial opportunities
- More informed multidisciplinary clinical decision-making
As precision oncology continues to evolve, integrating RNA sequencing alongside DNA testing is becoming an increasingly important strategy for comprehensive molecular profiling.
Looking Ahead
The experience at IPATIMUP demonstrates that RNA sequencing can be successfully integrated into routine clinical workflows while delivering measurable improvements in diagnostic yield and clinical actionability. By overcoming many of the limitations of conventional fusion detection methods, RNA sequencing enables laboratories to identify biologically relevant alterations that would otherwise remain hidden.
As targeted therapies continue to expand across oncology, comprehensive RNA-based fusion detection is poised to become an essential component of modern precision medicine, supporting clinicians with the molecular insights needed to deliver increasingly personalised cancer care.
Frequently Asked Questions
- ? Why are gene fusions important in precision oncology? Gene fusions can act as oncogenic drivers, improve tumour classification, predict response to targeted therapies, and identify patients eligible for precision medicine clinical trials.
- ? Why isn't DNA sequencing alone sufficient? DNA sequencing detects structural rearrangements but cannot confirm whether they produce biologically active fusion transcripts. RNA sequencing directly measures expressed RNA, providing greater clinical confidence.
- ? Which tumour types benefit most from RNA sequencing? RNA sequencing is particularly valuable in sarcomas, rare tumours, paediatric cancers, central nervous system tumours, cholangiocarcinoma, thyroid cancer, and selected lung cancers where actionable gene fusions are common.
- ? Can RNA sequencing improve patient treatment? Yes. By identifying clinically actionable fusion events that may be missed by conventional testing, RNA sequencing can expand access to targeted therapies and clinical trials while improving diagnostic accuracy.
- ? Why are FFPE samples challenging? Formalin-fixed paraffin-embedded (FFPE) tissues often contain fragmented and degraded RNA, making sequencing more difficult. Optimised workflows are therefore essential for reliable clinical implementation.
- ? Is RNA sequencing replacing DNA sequencing? No. RNA sequencing complements DNA sequencing by confirming which genomic alterations are functionally expressed, together providing a more comprehensive molecular profile for precision oncology.
