Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.
Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.
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DOI：
10.1371/journal.pone.0165109
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Nishiyama H
中科院分区：
文献类型：
--
作者：
Kurobe M;Kojima T;Nishimura K;Kandori S;Kawahara T;Yoshino T;Ueno S;Iizumi Y;Mitsuzuka K;Arai Y;Tsuruta H;Habuchi T;Kobayashi T;Matsui Y;Ogawa O;Sugimoto M;Kakehi Y;Nagumo Y;Tsutsumi M;Oikawa T;Kikuchi K;Nishiyama H
致癌性FGFR 3-TACC 3融合和FGFR 3突变是膀胱癌（BC）中小分子抑制剂的靶候选物。由于FGFR 3和TACC 3基因非常接近地位于染色体4p16.3上，因此通过DNA-FISH（荧光原位杂交）检测融合不是可行的选择。在这项研究中，我们开发了一种新的RNA-FISH检测使用分支的DNA探针检测FGFR 3-TACC 3融合在甲醛固定石蜡包埋（FFPE）的人BC样品。使用具有人BC细胞系的小鼠异种移植模型开发并验证了RNA-FISH测定。接下来，我们使用104个人BC样品评估了RNA-FISH测定的一致性。在本研究中，原代BC组织作为冷冻和FFPE组织储存。通过RNA-FISH测定在FFPE切片中和通过RT-PCR在冷冻组织中独立地检测FGFR 3-TACC 3融合。我们还通过对从脱蜡FFPE切片中提取的基因组DNA进行靶向测序来分析FGFR 3突变的存在。使用人BC细胞系RT 112和RT 4通过RNA-FISH和RT-PCR在小鼠异种移植物FFPE组织中鉴定FGFR 3-TACC 3融合转录物。据报道，这些细胞系是融合阳性的。在2/60（3%）的非肌肉侵入性BC（NMIBC）和2/44（5%）的肌肉侵入性BC（MIBC）患者中，通过RNA-FISH的FGFR 3-TACC 3融合的信号是阳性的。104例患者的RT-PCR结果与RNA-FISH结果完全一致。在27/60（45%）NMIBC和8/44（18%）MIBC患者中检测到FGFR 3突变。除了1例NMIBC患者外，FGFR 3突变和FGFR 3-TACC 3融合相互排斥。我们开发了用于检测人BC组织的FFPE样品中的FGFR 3-TACC 3融合的RNA-FISH测定。不仅筛查FGFR 3突变，而且筛查FGFR 3-TACC 3融合转录物具有鉴定可以用FGFR抑制剂治疗的其他患者的潜力。
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. 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. 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. 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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影响因子：
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作者：
Raj, Arjun;van den Bogaard, Patrick;Rifkin, Scott A.;van Oudenaarden, Alexander;Tyagi, Sanjay
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Singer, RH
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Guancial, Elizabeth A.;Werner, Lillian;Bellmunt, Joaquim;Bamias, Aristotle;Choueiri, Toni K.;Ross, Robert;Schutz, Fabio A.;Park, Rachel S.;O'Brien, Robert J.;Hirsch, Michelle S.;Barletta, Justine A.;Berman, David M.;Lis, Rosina;Loda, Massimo;Stack, Edward C.;Garraway, Levi A.;Riester, Markus;Michor, Franziska;Kantoff, Philip W.;Rosenberg, Jonathan E.
通讯作者：
Rosenberg, Jonathan E.
影响因子：
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作者：
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通讯作者：
CARBONELL, AW