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Updated: Aug 20, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Molecular characterization of genomic breakpoints of ALK rearrangements in non-small cell lung cancer
Zizong Wang1, Yushuai Han2, Houquan Tao3
1Department of Thoracic Surgery, The Affiliated Hospital of Qingdao University, China.
Abstract:
ALK rearrangement is called the 'diamond mutation' in non-small cell lung cancer (NSCLC). Accurately identifying patients who are candidates for ALK inhibitors is a key step in making clinical treatment decisions. In this study, a total of 783 ALK rearrangement-positive NSCLC cases were identified by DNA-based next-generation sequencing (NGS), including 731 patients with EML4-ALK and 52 patients with other ALK rearrangements. Diverse genomic breakpoints of ALK rearrangements were identified. Approximately 94.4% (739/783) of the cases carried ALK rearrangements with genomic breakpoints in the introns of ALK and its partner genes, and 2.8% (21/739) of these cases resulted in frameshift transcripts of ALK. Meanwhile, 5.6% (44/783) of the ALK rearrangement-positive cases had breakpoints in the exons that would be expected to result in abnormal transcripts. RNA-based NGS was performed to analyse the aberrant fusions at the transcript level. Some of these rearranged DNAs were not transcribed, and the others were fixed by some mechanisms so that the fusion kinase proteins could be expressed. Altogether, these findings emphasize that, when using DNA-based NGS, functional RNA fusions should be confirmed in cases with uncommon/frameshift rearrangement by RNA-based assays.
Insights
Identifying Anaplastic Lymphoma Kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC) is crucial for targeted therapy. DNA-based next-generation sequencing (NGS) requires RNA confirmation for uncommon or frameshift ALK rearrangements to ensure accurate patient selection for ALK inhibitors.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Anaplastic Lymphoma Kinase (ALK) rearrangement, termed the 'diamond mutation', is a key driver in a subset of non-small cell lung cancer (NSCLC).
- Accurate identification of ALK rearrangement-positive patients is critical for effective treatment with ALK inhibitors.
Purpose of the Study:
- To analyze the spectrum of ALK rearrangements in a large cohort of NSCLC patients.
- To evaluate the concordance between DNA-based and RNA-based next-generation sequencing (NGS) for detecting functional ALK fusions.
- To provide recommendations for optimal diagnostic strategies in ALK-rearranged NSCLC.
Main Methods:
- DNA-based next-generation sequencing (NGS) was employed to identify ALK rearrangements in 783 NSCLC cases.
- Genomic breakpoints within ALK and partner genes were characterized.
- RNA-based NGS was utilized to confirm aberrant transcript fusions at the RNA level for selected cases.
Main Results:
- A total of 783 ALK rearrangement-positive NSCLC cases were identified, with EML4-ALK being the most common.
- Diverse genomic breakpoints were observed, with the majority occurring in introns, leading to potential frameshift transcripts in 2.8% of cases.
- RNA analysis revealed that not all DNA rearrangements result in expressed fusion proteins, highlighting the importance of transcript-level confirmation.
Conclusions:
- DNA-based NGS is effective in detecting ALK rearrangements but may not fully capture functional fusion protein expression.
- For cases with uncommon or frameshift ALK rearrangements identified by DNA-based NGS, RNA-based assays are essential to confirm functional RNA fusions.
- Integrating both DNA and RNA analysis ensures accurate patient stratification for ALK-targeted therapies in NSCLC.
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