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Development and Validation of an RNA Sequencing Assay for Gene Fusion Detection in Formalin-Fixed, Paraffin-Embedded Tumors
- Source :
- The Journal of Molecular Diagnostics. 23:223-233
- Publication Year :
- 2021
- Publisher :
- Elsevier BV, 2021.
-
Abstract
- RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh-frozen tumors; however, RNA-seq is much more challenging to use with formalin-fixed, paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and six known fusions from five cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra-assay and interassay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which found that this is a false-negative result for the DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, 20 (76.9%) of which were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could complement DNA-based next-generation sequencing assays.
- Subjects :
- 0301 basic medicine
Tissue Fixation
Serial dilution
Sequence analysis
Biology
Pathology and Forensic Medicine
Fusion gene
03 medical and health sciences
chemistry.chemical_compound
symbols.namesake
0302 clinical medicine
Formaldehyde
Neoplasms
Humans
Gene
Sanger sequencing
Paraffin Embedding
Base Sequence
Sequence Analysis, RNA
Reproducibility of Results
RNA
Molecular biology
030104 developmental biology
chemistry
Cell culture
030220 oncology & carcinogenesis
symbols
Molecular Medicine
Gene Fusion
DNA
Subjects
Details
- ISSN :
- 15251578
- Volume :
- 23
- Database :
- OpenAIRE
- Journal :
- The Journal of Molecular Diagnostics
- Accession number :
- edsair.doi.dedup.....338c16ead6e99f492dcf5306bdffc886