The Identification and Real-time Quantitative PCR Detection of EEF1G/ALK Fusion Gene in a Pediatric Patient with Anaplastic Large Cell Lymphoma
Objective To identify the novel partner for ALK fusion gene in a pediatric patient with anaplastic large cell lymphoma,and to establish a real-time quantitative PCR method.Methods The second-generation sequencing based on anchored multiple PCR was used to identify the partner genes of the ALK fusion gene in the inguinal lymph nodes of the studied pediatric patient.The method of real-time quantitative PCR was established to detect the expression levels of EEF1G/ALK fusion gene,and the repeatability,sensitivity and specificity of the method were validated.EEF1G/ALK fusion gene expression levels were detected based on these methods.Results Second-generation sequencing was used to identify EEF1G as the partner gene of the ALK fusion gene,and Sanger sequencing verified that the fusion occurred at EEF1G Exon 6 and ALK Exon 20.The minimum limit of quantitation of the real-time quantitative PCR method was 40 copies per PCR reaction.The method showed a good reproducibility that the intra-assay CV of the lower positive specimens and the higher positive specimens were 0.52％and 0.17％,respectively;The inter-assay CV were 1.32％and 1.14％,respectively.The method was specific that other ALK positive specimens were detected as negative.EEF1G/ALK quantification of lymph node puncture tissue for recurrence was 138.92％,while the peripheral blood was 0.0039％,and the other samples were negative.Conclusion The rareness of EEF1G/ALK fusion gene is identified and confirmed in this pediatric patient.The established real-time quantitative PCR method of EEF1G/ALK fusion gene has a good reproducibility,high sensitivity and good specificity,which is suitable for MRD monitoring.
Anaplastic large cell lymphomaEEF1G/ALK fusion geneNext-Generation SequencingReal-time quantitative PCR
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