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

Abstract

Background: 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. Method: The 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. Result: All 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. Conclusion: 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.

Improved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing

“…This information is indispensable when developing patientspecific assays to determine disease dissemination and minimal residual disease, for example, in patients with lymphoma and leukemia. 24,[46][47][48][49] This is especially valuable when rare fusions are identified for which no predesigned (commercial) diagnostic test is available.…”

“…Nevertheless, current implementations of targeted RNA-seq are usually based on gene fusion panels, designed to capture a specific set of gene fusion events for a specific tumor type. 22-24…”

CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management

“…To date, the next-generation sequencing (NGS)-based methods such as whole genome sequencing (WGS) [34,35], whole transcriptome sequencing (WTS) [36][37][38], and targeted RNA sequencing (RNA-Seq) [39][40][41], with the enormous power of precise detection of all known and even novel translocations and/or fusions simultaneously, have been implemented as a diagnostic tool for hematologic malignancies including inv(16)/t(16;16) AML.…”

“…: numbers; NGS: next-generation sequencing; WGS: whole genome sequencing; WTS: whole transcriptome sequencing; RNA-Seq: RNA sequencing. * Following the criteria published by Duncavage et al[34] (e.g., CNAs > 5 Mb; and SV: >100 Kb); ** following the report by Stengel et al[37]; *** providing the RNA-Seq platform is partner gene-unrestricted[40]; } the WGS may be unable to distinguish inv(16) from t(16;16); }} all nine cases with cryptic AC16As in this cohort would have not been detected by WGS. }}} Detection of ACAs by WGS would depend on the size of clone(s) with each ACA on each specimen.…”

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

“…In recent years, various NGS based myeloid neoplasm panels developed in-house and commercially have been integrated into routine clinical practice [10][11][12][13]. Panels developed inhouse can differ substantially between laboratories in many aspects including gene content, the analysis of genes in a panel, sequencing library preparation chemistry, sequencing platform, and variant types detected [14][15][16][17][18][19][20]. Among many others, amplicon-based commercial panels, e.g.…”