RT-PCR assay to detect fusions in formalin-fixed, paraffin-embedded glioblastoma samples.

Background: One targeted treatment option for isocitrate dehydrogenase ()-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 () fusions. 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 status in FFPE glioblastoma samples.

Methods: Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 ( = 13) were subjected to RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS ( = 6), FGFR3 Fluorescence In Situ Hybridization ( = 6), and RNA sequencing ( = 5).

Results: Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an 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 fusion (variant not covered by current RT-PCR for FFPE). The frequency of fusion in this cohort was 9/353 (2.5%).

Conclusions: RT-PCR for 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.