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Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples

INTRODUCTION AND OBJECTIVES: 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 hybridizati...

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Autores principales: Kurobe, Masahiro, Kojima, Takahiro, Nishimura, Kouichi, Kandori, Shuya, Kawahara, Takashi, Yoshino, Takayuki, Ueno, Satoshi, Iizumi, Yuichi, Mitsuzuka, Koji, Arai, Yoichi, Tsuruta, Hiroshi, Habuchi, Tomonori, Kobayashi, Takashi, Matsui, Yoshiyuki, Ogawa, Osamu, Sugimoto, Mikio, Kakehi, Yoshiyuki, Nagumo, Yoshiyuki, Tsutsumi, Masakazu, Oikawa, Takehiro, Kikuchi, Koji, Nishiyama, Hiroyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145148/
https://www.ncbi.nlm.nih.gov/pubmed/27930669
http://dx.doi.org/10.1371/journal.pone.0165109
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author Kurobe, Masahiro
Kojima, Takahiro
Nishimura, Kouichi
Kandori, Shuya
Kawahara, Takashi
Yoshino, Takayuki
Ueno, Satoshi
Iizumi, Yuichi
Mitsuzuka, Koji
Arai, Yoichi
Tsuruta, Hiroshi
Habuchi, Tomonori
Kobayashi, Takashi
Matsui, Yoshiyuki
Ogawa, Osamu
Sugimoto, Mikio
Kakehi, Yoshiyuki
Nagumo, Yoshiyuki
Tsutsumi, Masakazu
Oikawa, Takehiro
Kikuchi, Koji
Nishiyama, Hiroyuki
author_facet Kurobe, Masahiro
Kojima, Takahiro
Nishimura, Kouichi
Kandori, Shuya
Kawahara, Takashi
Yoshino, Takayuki
Ueno, Satoshi
Iizumi, Yuichi
Mitsuzuka, Koji
Arai, Yoichi
Tsuruta, Hiroshi
Habuchi, Tomonori
Kobayashi, Takashi
Matsui, Yoshiyuki
Ogawa, Osamu
Sugimoto, Mikio
Kakehi, Yoshiyuki
Nagumo, Yoshiyuki
Tsutsumi, Masakazu
Oikawa, Takehiro
Kikuchi, Koji
Nishiyama, Hiroyuki
author_sort Kurobe, Masahiro
collection PubMed
description INTRODUCTION AND OBJECTIVES: 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. MATERIALS AND 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.
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spelling pubmed-51451482016-12-22 Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples Kurobe, Masahiro Kojima, Takahiro Nishimura, Kouichi Kandori, Shuya Kawahara, Takashi Yoshino, Takayuki Ueno, Satoshi Iizumi, Yuichi Mitsuzuka, Koji Arai, Yoichi Tsuruta, Hiroshi Habuchi, Tomonori Kobayashi, Takashi Matsui, Yoshiyuki Ogawa, Osamu Sugimoto, Mikio Kakehi, Yoshiyuki Nagumo, Yoshiyuki Tsutsumi, Masakazu Oikawa, Takehiro Kikuchi, Koji Nishiyama, Hiroyuki PLoS One Research Article INTRODUCTION AND OBJECTIVES: 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. MATERIALS AND 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. Public Library of Science 2016-12-08 /pmc/articles/PMC5145148/ /pubmed/27930669 http://dx.doi.org/10.1371/journal.pone.0165109 Text en © 2016 Kurobe et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kurobe, Masahiro
Kojima, Takahiro
Nishimura, Kouichi
Kandori, Shuya
Kawahara, Takashi
Yoshino, Takayuki
Ueno, Satoshi
Iizumi, Yuichi
Mitsuzuka, Koji
Arai, Yoichi
Tsuruta, Hiroshi
Habuchi, Tomonori
Kobayashi, Takashi
Matsui, Yoshiyuki
Ogawa, Osamu
Sugimoto, Mikio
Kakehi, Yoshiyuki
Nagumo, Yoshiyuki
Tsutsumi, Masakazu
Oikawa, Takehiro
Kikuchi, Koji
Nishiyama, Hiroyuki
Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title_full Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title_fullStr Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title_full_unstemmed Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title_short Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples
title_sort development of rna-fish assay for detection of oncogenic fgfr3-tacc3 fusion genes in ffpe samples
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145148/
https://www.ncbi.nlm.nih.gov/pubmed/27930669
http://dx.doi.org/10.1371/journal.pone.0165109
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