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RT-PCR assay to detect FGFR3::TACC3 fusions in formalin-fixed, paraffin-embedded glioblastoma samples
Loudy P Priesterbach-Ackley1, Joyce van Kuik1, Bastiaan B J Tops2
1Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Background:
One targeted treatment option for isocitrate dehydrogenase (IDH)-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next-generation sequencing (NGS) is not routinely performed, and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using reverse transcription polymerase chain reaction (RT-PCR) on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin-embedded (FFPE) tissue is available.
Aim:
To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples.
Methods:
Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 (n = 13) were subjected to FGFR3::TACC3 RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS (n = 6), FGFR3 Fluorescence In Situ Hybridization (n = 6), and RNA sequencing (n = 5).
Results:
Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an FGFR3::TACC3 fusion in 7 samples, covering 3 different fusion variants. For 5 of these FF was available, and the presence of the fusion was confirmed through RT-PCR on FF. With RNA sequencing, 1 additional sample was found to harbor an FGFR3::TACC3 fusion (variant not covered by current RT-PCR for FFPE). The frequency of FGFR3::TACC3 fusion in this cohort was 9/353 (2.5%).
Conclusions:
RT-PCR for FGFR3::TACC3 fusions can successfully be performed on FFPE material, with a specificity of 100% and (due to limited primer sets) a sensitivity of 83.3%. This assay allows for the identification of potential targeted treatment options when only formalin-fixed tissue is available.
Insights
Reverse transcription polymerase chain reaction (RT-PCR) can detect fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions in formalin-fixed, paraffin-embedded glioblastoma. This method enables targeted therapy identification when only FFPE tissue is available.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genomics
Background:
- Glioblastoma treatment can target fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions.
- Detecting FGFR3::TACC3 fusions is challenging due to limitations in routine RNA next-generation sequencing and immunohistochemistry.
- Reverse transcription polymerase chain reaction (RT-PCR) is effective on fresh frozen (FF) tissue, but formalin-fixed, paraffin-embedded (FFPE) tissue is often the only available sample type.
Purpose of the Study:
- To develop and validate an RT-PCR assay for detecting FGFR3::TACC3 fusions in FFPE glioblastoma samples.
- To establish a reliable method for identifying patients eligible for targeted therapies using FFPE tissue.
Main Methods:
- Immunohistochemical staining for FGFR3 was performed on 353 glioblastoma samples.
- FGFR3::TACC3 RT-PCR was conducted on FFPE tissue from 13 samples with FGFR3 overexpression, using 5 primer sets.
- Additional analyses included next-generation sequencing (NGS), Fluorescence In Situ Hybridization (FISH), and RNA sequencing for fusion-negative samples.
Main Results:
- RT-PCR detected FGFR3::TACC3 fusions in 7 out of 13 FFPE samples, identifying 3 distinct fusion variants.
- Fusion presence was confirmed via RT-PCR on FF tissue for 5 samples.
- RNA sequencing identified one additional fusion, and the overall frequency of FGFR3::TACC3 fusions in the cohort was 2.5% (9/353).
Conclusions:
- RT-PCR is a viable method for detecting FGFR3::TACC3 fusions in FFPE glioblastoma specimens.
- The developed RT-PCR assay demonstrated 100% specificity and 83.3% sensitivity for FFPE samples.
- This assay facilitates the identification of targeted treatment opportunities using archival FFPE tissue.
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