title: Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.
abstract: OBJECTIVE
  Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples.
  
  
  METHODS
  The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.
  
  
  RESULTS
  FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.
  
  
  CONCLUSIONS
  We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors.
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Masahiro
authors.identifier: http://orcid.org/0000-0002-5819-523X
authors.initials: M
authors.lastname: Kurobe
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Astellas Pharma Inc., Tsukuba, Japan.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Shuya
authors.initials: S
authors.lastname: Kandori
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawahara
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshino
authors.affiliation: Tsukuba Clinical Research and Development Organization (T-CReDO), Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ueno
authors.affiliation: Tsukuba Clinical Research and Development Organization (T-CReDO), Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Iizumi
authors.affiliation: Department of Urology, Graduate School of Medicine, Tohoku University, Sendai, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Mitsuzuka
authors.affiliation: Department of Urology, Graduate School of Medicine, Tohoku University, Sendai, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Arai
authors.affiliation: Department of Urology, Graduate School of Medicine, Akita University, Akita, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsuruta
authors.affiliation: Department of Urology, Graduate School of Medicine, Akita University, Akita, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Habuchi
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Matsui
authors.affiliation: Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Ogawa
authors.affiliation: Department of Urology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Sugimoto
authors.affiliation: Department of Urology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kakehi
authors.affiliation: Department of Urology, Hitachi General Hospital, Hitachi, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Nagumo
authors.affiliation: Department of Urology, Hitachi General Hospital, Hitachi, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Tsutsumi
authors.affiliation: Department of Urology, Tsukuba Medical Center Hospital, Tsukuba, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Oikawa
authors.affiliation: Department of Urology, Tsukuba Medical Center Hospital, Tsukuba, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kikuchi
authors.affiliation: Department of Urology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nishiyama
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics; D005260:Female; D005557:Formaldehyde; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008875:Middle Aged; D009368:Neoplasm Transplantation; D050596:Oncogene Fusion / Q000235:genetics*; D016612:Paraffin Embedding; D012313:RNA / Q000235:genetics*; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000235:genetics
chemical_list: D015342:DNA Probes; D008869:Microtubule-Associated Proteins; C120551:TACC3 protein, human; D005557:Formaldehyde; D012313:RNA; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3