Related Experiment Video
Updated: Jan 23, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts
Robyn T Sussman1, Amanda R Oran1, Carmela Paolillo1
1From the Center for Personalized Diagnostics, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia.
Context.—:
Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK.
Objective.—:
To validate an anchored multiplex polymerase chain reaction-based panel for the detection of fusion transcripts in a university hospital-based clinical molecular diagnostics laboratory.
Design.—:
We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei.
Results.—:
Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific.
Conclusions.—:
This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.
Related Concept Videos
02:45Nuclear Fusion
A helium nucleus has a mass that is 0.7% less than that of four hydrogen nuclei; this lost mass is converted into energy during the fusion. This reaction produces about...
01:10Transcription
Transcription is the process of synthesizing RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. Additionally, many other proteins and regulatory sequences are involved in the proper synthesis of messenger RNA (mRNA). Regulation of transcription is responsible for the differentiation of all the different types of cells and often for the proper cellular response to environmental signals.
Transcription Can Produce Different Kinds...
03:00Next-generation Sequencing
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
02:35Transcription Elongation Factors
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA...
02:42Eukaryotic Transcription Activators
The binding domains are capable of recognizing and interacting with regulatory sequences on the DNA. These...
02:42Transcription Attenuation in Prokaryotes
There are several different mechanisms used to attenuate transcription. In ribosome mediated...