In recent years, the detection of gene fusions has become a pivotal aspect of cancer diagnostics and treatment planning. Gene fusions, which result from structural rearrangements in the genome, can drive oncogenesis by creating aberrant proteins that promote tumor growth and survival. Accurate and timely identification of these fusions is essential for guiding precision oncology. However, existing detection methods often face challenges such as limited sensitivity, specificity, or applicability across diverse cancer types. Addressing this critical need, a new study published in BMC Cancer introduces a cutting-edge whole transcriptome sequencing (WTS) assay designed for comprehensive and reliable detection of gene fusions in clinical cancer specimens.
The innovative WTS assay developed by Zhao et al. offers a highly sensitive and specific platform that captures not only gene fusions but also critical RNA alterations like MET exon 14 skipping and EGFR VIII mutations, broadening its clinical utility. By leveraging whole transcriptome sequencing rather than targeted panels, this assay examines the entirety of RNA transcripts, thus enabling an unbiased exploration of fusion events across the transcriptome. This holistic approach allows clinicians and researchers to uncover both known and novel fusion transcripts that may impact diagnosis, prognosis, or targeted therapy options.
A significant hurdle in transcriptome analysis is RNA quality. RNA degradation can severely impair sequencing outcomes and fusion detection. To overcome this, the team identified a key quality metric—DV200—representing the percentage of RNA fragments longer than 200 nucleotides. They established a threshold of DV200 ≥ 30% to ensure sufficient RNA integrity for accurate fusion calling. This parameter provides a practical cut-off for clinical laboratories to assess sample suitability and enhance assay performance, addressing one of the major technical barriers in RNA sequencing from clinical specimens.
Optimization of input RNA quantity and sequencing depth were also focal points in the assay development. The researchers determined that using at least 100 ng of RNA input, combined with a minimum of 80 million mapped reads per sample, maximized the assay’s sensitivity. Additionally, they defined expression thresholds—specifically, fusion transcript levels exceeding 40 copies per nanogram of input RNA—necessary to confidently identify fusion events. These performance benchmarks not only improve detection rates but also help standardize protocols for routine clinical application.
The validation phase of this WTS assay demonstrated robust clinical utility. Out of 63 previously characterized gene fusion-positive samples, the assay correctly identified 62, yielding an impressive sensitivity of 98.4%. Equally important, the assay exhibited 100% specificity by reporting no fusions in 21 known fusion-negative control samples. This combination of high sensitivity and specificity underscores its potential as a reliable diagnostic tool that minimizes both false positives and false negatives, a critical balance in precision oncology.
Reproducibility and repeatability are crucial for any diagnostic assay, particularly in clinical settings where consistency impacts patient outcomes. The research team conducted rigorous repeat testing on multiple samples and observed excellent concordance, affirming the assay’s robustness. An exception was noted with the TPM3::NTRK1 fusion, which was expressed at levels below the established threshold, highlighting ongoing challenges in detecting ultra-low abundance fusions. Nonetheless, these findings suggest that the assay can be confidently implemented for a broad range of fusion detection scenarios.
One of the most striking aspects of this study is the application of the WTS assay to real-world clinical samples from patients with non-small cell lung cancer (NSCLC). Among these samples, 68.9% of identified fusions were classified as potentially actionable, meaning they could guide targeted therapy decisions. This is in contrast to a 20% actionable fusion rate observed in a wider pan-cancer cohort, illustrating the assay’s value in stratifying patients for personalized treatment based on fusion profile.
Beyond actionable fusions, the assay also uncovered numerous gene fusions with potential diagnostic and prognostic significance across various tumor types. These findings expand the understanding of the fusion landscape in oncology and open avenues for further research into novel biomarkers. The ability to systematically profile such fusions across different cancers underscores the versatility and comprehensive capacity of whole transcriptome sequencing.
Technically, the WTS assay employs advanced sequencing technologies coupled with refined bioinformatics pipelines. This integration enables the high-throughput identification of fusion events alongside MET exon 14 skipping and EGFR VIII alterations, which have known clinical implications. The multiplexed detection capability allows simultaneous assessment of multiple RNA abnormalities, thereby streamlining molecular diagnostics into a single, cohesive workflow.
Moreover, by circumventing the limitations of targeted sequencing panels, which test predefined fusion partners, this assay can detect rare or atypical fusion events that may otherwise be missed. This advantage is particularly important in heterogeneous tumors or in cases where emerging or novel fusions could inform therapeutic strategies. The open-ended nature of whole transcriptome sequencing thus sets a new standard for exploratory and diagnostic cancer genomics.
The study also emphasizes the importance of quality control metrics and standardized thresholds in facilitating the translation of WTS from research to clinical use. Parameters like RNA input, DV200 value, and minimal read counts form the backbone of assay reliability, enabling consistent results across laboratories. This technical rigor ensures that the assay’s promising sensitivity and specificity are reproducible in diverse clinical environments.
Importantly, the high sensitivity of the assay not only improves detection rates but also allows for the identification of low-abundance fusions, which could have been overlooked by less comprehensive methods. This sensitivity has significant implications for early diagnosis and monitoring of residual disease, where fusion transcript levels may be minimal yet clinically important.
Looking ahead, the integration of such WTS assays into routine clinical workflows holds promise for transforming cancer care. The ability to capture the full fusion transcriptome in a single test accelerates molecular diagnostics and offers a more complete molecular profile to oncologists. This comprehensive approach supports precision medicine by identifying patients eligible for fusion-targeted therapies, novel clinical trials, or those requiring specialized monitoring.
Furthermore, the inclusion of fusion events with diagnostic and prognostic relevance enriches the molecular characterization of tumors. This may lead to improved risk stratification and treatment personalization, ultimately enhancing patient outcomes. As cancer treatment paradigms increasingly rely on molecularly driven decisions, assays like the one developed here will be instrumental in pushing the boundaries of precision oncology.
In summary, the work by Zhao and colleagues represents a significant advance in cancer genomics. Their novel WTS assay provides a sensitive, specific, and reproducible method for detecting gene fusions and related RNA alterations in clinical cancer specimens. By enabling comprehensive fusion profiling across tumor types, this assay sets a new benchmark for molecular diagnostics and reinforces the transformative potential of whole transcriptome sequencing in oncology.
The implications of this research extend beyond technical development; they embody an important step towards personalized cancer care, where every patient’s tumor transcriptome is utilized to guide optimal therapy. Continued validation and adoption of such assays will likely fuel further discoveries in fusion biology and expand the catalog of actionable alterations accessible through next-generation sequencing technologies.
As precision oncology evolves, tools that combine thorough molecular characterization with clinical applicability will shape the future of cancer diagnosis and treatment. Zhao et al.’s whole transcriptome sequencing assay exemplifies this paradigm, offering a powerful resource for uncovering the complexities of gene fusions and enhancing the therapeutic landscape for cancer patients worldwide.
Subject of Research: Development of a whole transcriptome sequencing assay for detection of gene fusions and related RNA alterations in clinical cancer samples.
Article Title: Development and application of a whole transcriptome sequencing assay for the detection of gene fusions in clinical cancer specimens.
Article References:
Zhao, S., Du, X., Zhang, Y. et al. Development and application of a whole transcriptome sequencing assay for the detection of gene fusions in clinical cancer specimens. BMC Cancer 25, 842 (2025). https://doi.org/10.1186/s12885-025-14186-w
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