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Updated: Aug 31, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Experimentally Deduced Criteria for Detection of Clinically Relevant Fusion 3' Oncogenes from FFPE Bulk RNA
Elizaveta Rabushko1,2, Maxim Sorokin1,2,3, Maria Suntsova1,2
1Laboratory for Clinical and Genomic Bioinformatics, Institute of Personalized Oncology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia.
Abstract:
Drugs targeting receptor tyrosine kinase (RTK) oncogenic fusion proteins demonstrate impressive anti-cancer activities. The fusion presence in the cancer is the respective drug prescription biomarker, but their identification is challenging as both the breakpoint and the exact fusion partners are unknown. RNAseq offers the advantage of finding both fusion parts by screening sequencing reads. Paraffin (FFPE) tissue blocks are the most common way of storing cancer biomaterials in biobanks. However, finding RTK fusions in FFPE samples is challenging as RNA fragments are short and their artifact ligation may appear in sequencing libraries. Here, we annotated RNAseq reads of 764 experimental FFPE solid cancer samples, 96 leukemia samples, and 2 cell lines, and identified 36 putative clinically relevant RTK fusions with junctions corresponding to exon borders of the fusion partners. Where possible, putative fusions were validated by RT-PCR (confirmed for 10/25 fusions tested). For the confirmed 3'RTK fusions, we observed the following distinguishing features. Both moieties were in-frame, and the tyrosine kinase domain was preserved. RTK exon coverage by RNAseq reads upstream of the junction site were lower than downstream. Finally, most of the true fusions were present by more than one RNAseq read. This provides the basis for automatic annotation of 3'RTK fusions using FFPE RNAseq profiles.
Insights
Identifying receptor tyrosine kinase (RTK) fusions in FFPE cancer samples is challenging. This study presents a method using RNAseq to automatically annotate these clinically relevant RTK fusions for targeted cancer therapy.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Receptor tyrosine kinase (RTK) fusions are key cancer biomarkers for targeted therapies.
- Identifying these fusions is difficult due to unknown breakpoints and fusion partners, especially in challenging FFPE samples.
Purpose of the Study:
- To develop and validate a method for identifying clinically relevant RTK fusions in FFPE solid cancer samples using RNA sequencing.
- To establish criteria for distinguishing true RTK fusions from artifacts in RNAseq data.
Main Methods:
- RNA sequencing (RNAseq) data from 764 FFPE solid cancer samples, 96 leukemia samples, and 2 cell lines were analyzed.
- Putative RTK fusions were identified by annotating RNAseq reads and validated using RT-PCR.
- Distinguishing features of confirmed 3'RTK fusions were analyzed.
Main Results:
- 36 putative clinically relevant RTK fusions were identified, with 10/25 validated by RT-PCR.
- Confirmed 3'RTK fusions showed in-frame expression, preserved tyrosine kinase domains, and specific RNAseq read coverage patterns.
- True fusions were typically detected by multiple RNAseq reads.
Conclusions:
- RNAseq analysis of FFPE samples can effectively identify clinically relevant RTK fusions.
- Established criteria enable automatic annotation of 3'RTK fusions from FFPE RNAseq profiles, aiding targeted cancer therapy.
- This method facilitates biomarker discovery and patient stratification for precision oncology.
