title: An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma.
abstract: To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break-apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break-apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break-apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.
authors.affiliation: Asia & Emerging Markets iMed, AstraZeneca R&D. 199 LiangJing Road, ZhangJiang Hi-Tech Park, Shanghai, 201203, China.
authors.forename: Tianwei
authors.initials: T
authors.lastname: Zhang
authors.forename: Yachao
authors.initials: Y
authors.lastname: Lu
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Ye
authors.forename: Meizhuo
authors.initials: M
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Yin
authors.forename: Paul
authors.initials: P
authors.lastname: Gavine
authors.forename: Zhongsheng
authors.initials: Z
authors.lastname: Sun
authors.forename: Qunsheng
authors.initials: Q
authors.lastname: Ji
authors.forename: Guanshan
authors.initials: G
authors.lastname: Zhu
authors.forename: Xinying
authors.initials: X
authors.lastname: Su
mesh_terms: D002277:Carcinoma / Q000235:genetics*; D002291:Carcinoma, Papillary; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000235:genetics*; D014178:Translocation, Genetic
chemical_list: D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human