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.

Robert P. Hasserjian¹

¹Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

 

Acute myeloid leukemia (AML) is an aggressive myeloid neoplasm originating from mutated hematopoietic stem cells, which displays arrested maturation resulting in an accumulation of myeloid blasts in the bone marrow and blood.  AML is highly heterogeneous in terms of its biology and clinical behavior, due in large part to the varied portfolios of mutations that in combination drive myeloid proliferation and arrested maturation.  The updated classifications of AML put forth in 2022--the International Consensus Classification (ICC) and 5^th edition World Health Organization Classification (WHO5)--recognize this diversity through several distinct disease groups and specific entities within AML. Many of these entities are defined by a single recurrent genetic driver, while another large group of AML bears one or more gene mutations or cytogenetic aberrations that are strongly associated with myelodysplastic syndromes (MDS). TP53 mutation defines a major subgroup of AML in the ICC that is highly resistant to current therapies.  Accurate diagnosis of classification of AML according to the new ICC and WHO5 schemes requires correctly identifying and enumerating blasts or blast equivalents as well as correctly interpreting genetic testing results.

 

References:

 

1.     Weinberg OK, Porwit A, Orazi A, Hasserjian RP, Foucar K, Duncavage EJ, Arber DA. The International Consensus Classification of acute myeloid leukemia. Virchows Arch. 2023 Jan;482(1):27-37.

2.     Chandra DJ, Lachowiez CA, Loghavi S. Practical considerations in clinical application of WHO 5th and ICC classification schemes for acute myeloid leukemia. Blood Rev. 2024 Mar;64:101156.