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Updated: Nov 20, 2025
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Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
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Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing
Nisha S Ramani1, Keyur P Patel2, Mark J Routbort2
1From the Department of Pathology (Ramani, Broaddus, Chen, Rashid, Lazar, Roy-Chowdhuri), The University of Texas MD Anderson Cancer Center, Houston.
Archives of Pathology & Laboratory Medicine
|January 25, 2021
Summary
RNA-based next-generation sequencing (NGS) assays are crucial for tumor profiling. Assay success depends on RNA quality, quantity, and preanalytical factors like specimen age and necrosis.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genomics
Background:
- RNA-based next-generation sequencing (NGS) is increasingly utilized for comprehensive molecular profiling of solid tumors.
- Evaluating factors influencing clinical assay performance is essential for reliable diagnostics.
Purpose of the Study:
- To assess preanalytical factors impacting the success rate of RNA-based NGS fusion detection assays.
- To identify key variables affecting RNA quality and yield in tumor specimens.
Main Methods:
- A retrospective review of 767 cases analyzed by a targeted RNA-based NGS assay for fusion detection.
- Analysis of RNA extraction from formalin-fixed, paraffin-embedded tissues and cytology smears.
- Correlation of testing success with specimen type, RNA yield, necrosis, decalcification, and block age.
Main Results:
- Successful NGS fusion testing was achieved in 90.9% of specimens.
- Success rates positively correlated with RNA yield and negatively with specimen necrosis, decalcification, and block age >2 years.
- Clinically relevant fusions were identified in 7.2% of successfully sequenced cases, with similar rates for core needle biopsy and cytology samples.
Conclusions:
- The success of RNA-based NGS testing is multifactorial, significantly influenced by RNA quality and quantity.
- Identifying and mitigating preanalytical factors that affect RNA quality and yield can optimize NGS testing success rates in clinical practice.
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