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Updated: Apr 30, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Detection of gene rearrangements in targeted clinical next-generation sequencing
Haley J Abel1, Hussam Al-Kateb2, Catherine E Cottrell2
1Department of Genetics, Washington University, St. Louis, Missouri.
Abstract:
The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.
Insights
Targeted next-generation sequencing accurately detects ALK and KMT2A gene rearrangements in clinical cancer samples, matching fluorescence in situ hybridization (FISH) performance. This method offers enhanced efficiency and detailed information for molecular oncology testing.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Recurrent gene rearrangements are crucial for cancer risk stratification and treatment.
- Targeted next-generation sequencing (NGS) shows potential for identifying these rearrangements.
- The clinical utility of NGS for rearrangement detection remains largely unproven.
Purpose of the Study:
- To evaluate the sensitivity and specificity of targeted NGS for detecting ALK and KMT2A rearrangements in a clinical setting.
- To compare NGS-based detection with the established fluorescence in situ hybridization (FISH) method.
- To assess the performance of different publicly available software tools for NGS rearrangement analysis.
Main Methods:
- Analysis of 7 ALK-rearranged cancers, 6 KMT2A-rearranged leukemias, and 77 negative cases previously tested by FISH.
- Utilized publicly available software tools: Breakdancer, ClusterFAST, CREST, and Hydra for rearrangement detection.
- Compared NGS results with FISH findings for validation.
Main Results:
- Breakdancer and ClusterFAST correctly identified all 7 ALK and 6 KMT2A rearrangements.
- No false positives were observed in the 77 negative cases using Breakdancer or ClusterFAST.
- Identified a novel ALK noncanonical breakpoint potentially impacting targeted therapy response.
Conclusions:
- Targeted NGS demonstrates sensitivity and specificity comparable to FISH for detecting clinically relevant gene rearrangements.
- NGS offers finer-scale information and increased efficiency for molecular oncology diagnostics.
- NGS is a viable and powerful tool for clinical laboratory identification of gene rearrangements in cancer.
