Comprehensive and routine analysis of leukaemia samples
Nanopore sequencing rapidly and accurately identifies gene fusions
Identification of gene fusions is crucial in the context of leukaemia, where prognosis and treatment can vary dramatically depending on the gene fusion present and the speed of detection. Compared to current methods for detecting gene fusions, such as real-time PCR and fluorescence in situ hybridisation (FISH), which take days or even weeks to perform, nanopore sequencing offers the advantages of rapid sequencing with real-time data generation.
The BCR-ABL1 gene fusion is present in nearly all patients with chronic myeloid leukaemia (CML)1. With the aim of rapidly detecting a variety of oncogenic fusion events, including the BCR-ABL1 fusion, Jeck et al. developed an assay based on a modified Anchored Multiplex PCR (AMP) method for library construction. Initial proof of concept experiments in the K562 cell line using gene-specific primers against BCR exons 1 and 2, along with sequencing on the MinION, demonstrated the ability of this assay to precisely delineate the BCR-ABL1 fusion junction (Figure 1)2. Furthermore, real-time analysis enabled confident detection of the fusion within just 5 minutes, with the first fusion read being generated within five seconds.
Based on these promising results, Jeck et al. applied an expanded AMP-based assay, targeting an array of oncogenic fusions, to a number of haematological malignancy specimens. Various fusion events were detected including PML-RARA — the hallmark of acute promyelocytic leukaemia. Sensitivity to this critical fusion was 100% in clinical research samples, even with a 1:10 dilution specimen. When multiplexing four samples on a fresh flow cell, all fusions could be detected within 6 hours of sequencing.
Application of this assay to previously characterised libraries from formalin-fixed, paraffin-embedded (FFPE) sarcoma specimens detected a range of gene fusions with high specificity, demonstrating that the problem of fragmentation and lower DNA quality in FFPE specimens did not impede accurate fusion detection using nanopore sequencing. Furthermore, the fraction of MinION reads mapping to a given fusion was higher than that observed with traditional short-read sequencing technology in all but one case, which the authors suggest is likely due to longer read length. The authors concluded that:
‘…nanopore sequencing has great promise as a broad fusion detection platform in time critical settings or for laboratories with a low volume of specimens for which a (rapid) fusion assay is indicated’2.
1. Druker, B.J. et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. NJEM. 344:1031-1037 (2001).
2. Jeck, W.R. et al. A nanopore sequencing–based assay for rapid detection of gene fusions. J Mol Diagn. S1525-1578(17)30630-X (2018).
3. Jeck, W.R. Nanopore sequencing and rapid fusion testing – a ‘killer app’ in molecular pathology. Presentation. Available at: https://nanoporetech.com/resource-centre/william-jeck-nanopore-sequencing-and-rapid-fusion-testing-killer-app-molecular [Accessed: 11 February 2019]