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RNA-Seq for the detection of gene fusions in solid tumors: development and validation of the JAX FusionSeq™ 2.0 assay

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Abstract

Whole transcriptome sequencing (RNA-Seq) has gained prominence for the detection of fusions in solid tumors. Here, we describe the development and validation of an in-house RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions, in formalin-fixed paraffin-embedded (FFPE) specimens, using seventy tumor samples with varying fusion status. Conditions were optimized for RNA input of 50 ng, shown to be adequate to call known fusions at as low as 20% neoplastic content. Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability. Performance analysis of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility. This clinically developed and validated RNA-Seq-based approach for fusion detection in FPPE samples was shown to be on par if not superior to off-the-shelf commercially offered assays. With gene fusions implicated in a variety of cancer types, offering high-quality, low-cost molecular testing services for FFPE specimens will serve to best benefit the patient and the advancement of precision medicine in molecular oncology.

Key messages

A custom RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions,

  • Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability.

  • The assay can be performed with low RNA input and neoplastic content.

  • Performance characteristics of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility.

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Availability of data and materials (data transparency)

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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  1. DanielBergeron, Harshpreet Chandok and Qian Nie have equal contribution.

Authors and Affiliations

  1. Clinical Genomics Laboratory, The Jackson Laboratory, Farmington, CT, 06032, USA

    Daniel Bergeron, Harshpreet Chandok, Qian Nie, Matthew Prego, Melissa Soucy, Kevin Kelly, Guruprasad Ananda, Andrew Hesse & Honey V. Reddi

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Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Daniel Bergeron, Harshpreet Chandok, Qian Nie, and Honey V Reddi. The first draft of the manuscript was written by Daniel Bergeron, Harshpreet Chandok, Qian Nie, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Honey V. Reddi.

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Not applicable. All human specimens used in this study were obtained from commercial sources as listed in the material and methods section of this manuscript. As such no informed consent or waiver for participation was required. DNA/RNA from PDX animals was obtained from the PDX core. The lab was not involved in the care of animals.

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Bergeron, D., Chandok, H., Nie, Q. et al. RNA-Seq for the detection of gene fusions in solid tumors: development and validation of the JAX FusionSeq™ 2.0 assay. J Mol Med 100, 323–335 (2022). https://doi.org/10.1007/s00109-021-02149-0

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