RT-PCR assay to detect FGFR3::TACC3 fusions in formalin-fixed, paraffin-embedded glioblastoma samples

Abstract

One targeted treatment option for IDH-wildtype glioblastoma focuses on tumors with FGFR3::TACC3 fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next generation sequencing is not routinely performed and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using RT-PCR on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin embedded (FFPE) tissue is available. To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples. Twelve tissue micro-arrays 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 detection of 5 common fusion variants. Fusion negative samples were additionally analyzed with NGS (n=6), FGFR3 FISH (n=6) and RNA sequencing (n=5). 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 one additional sample was found to harbor a 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%). 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.