Eric Wenlong Li^1,2, Ing-Soo Tiong², Clare Gould², Alberto Catalano¹, Hnin Aung³, Louise Seymour³, Deborah White⁴, Ella Thompson², Piers Blombery², Harry Iland¹, Dale Wright⁵

¹Molecular Haematology Laboratory, Royal Prince Alfred Hospital, Sydney, NSW, Australia; ²Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia; ³Department of Cytogenetics, Pathology Queensland, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia, ⁴Precision Cancer Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; ⁵Cytogenetics, Sydney Genome Diagnostics, The Children’s Hospital at Westmead, Westmead, NSW, Australia

Background: Targeted RNA-based next-generation sequencing (RNAseq) is increasingly used in diagnosing oncogenic gene fusions. The lack of practice guidelines and external quality assurance programs poses a challenge to managing the quality of this new diagnostic approach. Method: Four molecular diagnostics laboratories in Australia, utilising either the Archer FusionPlex® or Qiagen Fusion XP® platforms, compared diagnostic results from six samples against reference methods: G-banded karyotype +/- fluorescence in-situ hybridisation or targeted RNAseq, or whole transcriptome sequencing, where available. Results: Three of the four laboratories successfully identified five gene fusions (BCR::ABL1, ETV6::RUNX1, ETV6::SRR, NUP98::HOXD13, KMT2A::USP2). One laboratory failed to identify the novel ETV6::SRR due to a low-confidence fusion call made by the bioinformatics pipeline. All laboratories missed the @IGH::EPOR fusion (identified by whole transcriptome analysis), although one laboratory noted EPOR overexpression. Comparing diagnostic reports revealed differences in the terminology used for variant descriptions (HGVS vs ISCN), the reference transcripts, the inclusion of genomic coordinates, and the pathogenicity classification. Conclusion: Laboratories showed a high level of agreement in identifying gene fusions using targeted RNAseq. However, detecting non-chimeric gene rearrangements resulting in gene dysregulation represents a significant limitation of current platforms. Establishing practice guidelines will help standardise reporting and reduce variations between laboratories.