Detection of leukemia gene fusions by targeted RNA-sequencing in routine diagnostics

Marie Engvall,Britt-Inger Jonsson,Nicola Cahill,Helene Hallböök,Martin Höglund,Lucia Cavelier

doi:10.1186/s12920-020-00739-4

Marie Engvall, Britt-Inger Jonsson + Show 4 more

Open Access

https://doi.org/10.1186/s12920-020-00739-4

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Abstract

BackgroundWe have evaluated an NGS-based method to detect recurrent gene fusions of diagnostic and prognostic importance in hematological malignancies. Our goal was to achieve a highly specific assay with a simple workflow, short turnaround time and low cost.MethodThe assay uses a commercially available anchored multiplex PCR panel for target enrichment and library preparation, followed by sequencing using a MiSeq instrument. The panel includes all recurrent gene fusions in AML and ALL and is designed to detect gene-specific fusions without prior knowledge of the partner sequence or specific break points. Diagnostic RNA samples from 27 cases with hematological malignancies encompassing 23 different transcript variants were analyzed. In addition, 12 cases from a validation cohort were assessed.ResultAll known fusion transcripts were identified with a high degree of confidence, with a large number of reads covering the breakpoints. Importantly, we could identify gene fusions where conventional methods had failed due to cryptic rearrangements or rare fusion partners. The newly-identified fusion partners were verified by RT-PCR and transcript-specific qPCR was designed for patient-specific follow-up. In addition, 12 cases were correctly assessed in a blind test, without prior knowledge of molecular cytogenetics or diagnosis.ConclusionIn summary, our results demonstrate that targeted RNA sequencing using anchored multiplex PCR can be implemented in a clinical laboratory for the detection of recurrent and rare gene fusions in hematological diagnostic samples.

Highlights

We have evaluated an NGS-based method to detect recurrent gene fusions of diagnostic and prognostic importance in hematological malignancies
In summary, our results demonstrate that targeted RNA sequencing using anchored multiplex PCR can be implemented in a clinical laboratory for the detection of recurrent and rare gene fusions in hematological diagnostic samples
For all cases with known aberrations, as determined by chromosome analysis, fluorescence in situ hybridization (FISH) analysis, reverse transcriptase (RT)-PCR and/or SNParray, the gene fusions could readily be detected by the Archer anchored multiplex PCR and MiSeq sequencing (Table 2)

Summary

Introduction

We have evaluated an NGS-based method to detect recurrent gene fusions of diagnostic and prognostic importance in hematological malignancies. Our goal was to achieve a highly specific assay with a simple workflow, short turnaround time and low cost. Chromosomal rearrangements such as translocations, inversions or deletions, can cause breakpoints within genes leading to gene fusions which code for fusion proteins with altered functionality. The fusion protein has successfully been targeted with specific tyrosine kinase inhibitors, greatly improving the prognosis of CML patients [3]. Another gene fusion that is effectively treatable is the PML-RARA fusion in acute myeloid leukemia (AML). This gene fusion expresses a fusion protein which acts as a transcriptional regulator

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