title: Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.
abstract: Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.
authors.affiliation: Department of Respiratory Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
authors.forename: Jin-Yin
authors.initials: JY
authors.lastname: Zhao
authors.affiliation: Department of Respiratory Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
authors.forename: Zhi-Xia
authors.initials: ZX
authors.lastname: Chen
authors.affiliation: Department of Respiratory Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China2Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhong
authors.affiliation: Department of Respiratory Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
authors.forename: Long-Yun
authors.initials: LY
authors.lastname: Li
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
authors.forename: Li-Cheng
authors.initials: LC
authors.lastname: Liu
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
authors.forename: Xiao-Xu
authors.initials: XX
authors.lastname: Hu
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Chen
authors.affiliation: Department of Respiratory Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
authors.forename: Meng-Zhao
authors.initials: MZ
authors.lastname: Wang
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription
chemical_list: C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion