Development of an extended version of GALEAS bladder: detection of FGFR3 fusions in urine and associations between genomic alterations and gene expression

Background The latest bladder cancer (BC) urine tests based on multiple genomic and/or epigenomic markers detect BC with high sensitivity and specificity. The GALEAS Bladder (GB) gene panel covers several actionable mutations, including in FGFR3. Objective To modify GB to detect FGFR3 fusions as well as single nucleotide variants and assess its potential as a urine-based companion diagnostic. We also study tissue-urine concordance and associations between FGFR3 genomic alterations and FGFR3 expression. Methods Probes were added to GB covering the 3’-region of FGFR3. The new panel was used to analyse genomic DNA extracted from 158 frozen primary BCs. Mutation concordance was also analysed in 107 tumour-urine pairs. Associations between FGFR3 mutations and expression were analysed in 608 FFPE BCs with the Decipher Bladder transcriptome assay. Results and limitations FGFR3-TACC3 fusions were found in 3/3 positive controls and 4/158 frozen BCs. Fusions were also detectable in genomic DNA from FFPE tumour and urine cell pellet DNA. All fusions were corroborated by PCR and Sanger sequencing. FGFR3 mutation status in urine was 94.4% concordant (95% CI 87.7–97.7%); GB detected mutations in urine with 92.1% sensitivity (95% CI 81.7–97.0%) at 97.7% specificity (95% CI 86.5–99.9). All FGFR3 mutations were associated with increased FGFR3 expression. This is a proof-of-principle study, rather than definitive evidence of utility as a companion diagnostic, with the need for fusion detection validation in larger patient cohorts. Conclusions In addition to BC detection, a modified version of GB demonstrates feasibility for tissue and urine-based detection of FGFR3 fusions.