Advancing diagnostic targeted RNA sequencing for haematological malignancies within an Australian sample exchange...

Targeted RNA-based next-generation sequencing (targeted RNA-Seq) is increasingly incorporated into oncogenic gene fusion diagnostics. The absence of external quality assurance programs presents a challenge to the quality management of this novel diagnostic methodology. Four Australian molecular diagnostics laboratories employing Archer FusionPlex or Qiagen Fusion XP platforms compared results from six diagnostic samples with a reference method. Three of four laboratories identified all five gene fusions (BCR::ABL1, ETV6::RUNX1, ETV6::SRR, NUP98::HOXD13, KMT2A::USP2), and one laboratory missed the novel ETV6::SRR fusion. All laboratories missed the IGH::EPOR fusion identified by whole-transcriptome sequencing at a research laboratory; however, they were able to detect EPOR overexpression driven by the gene fusion. Diagnostic report comparison highlighted variations in the use of variant description nomenclature (Human Genome Variation Society vs International System for Human Cytogenomic Nomenclature), reference transcript, inclusion of genomic coordinates, and pathogenicity classification. This study demonstrates high concordance between laboratories in gene fusion identification using targeted RNA-Seq. Detecting non-chimeric gene producing rearrangements involving regulatory elements is a technical limitation of the current platforms. The incorporation of gene expression analysis, with confirmation using orthogonal methods, may help improve diagnostic yield. Developing practice guidelines will help harmonise reporting content and minimise interlaboratory variations.