间变性大细胞淋巴瘤患儿EEF1G/ALK 融合基因的鉴定与检测

The Identification and Real-time Quantitative PCR Detection of EEF1G/ALK Fusion Gene in a Pediatric Patient with Anaplastic Large Cell Lymphoma

目的明确1例伴少见ALK融合基因的间变性大细胞淋巴瘤患儿的分子诊断,建立该融合基因的实时荧光定量PCR检测方法。方法采用基于锚定多重PCR的二代测序技术,鉴定患儿腹股沟肿大淋巴结组织中ALK基因的伙伴基因。建立EEF1G/ALK融合基因实时荧光定量PCR方法,并对方法的重复性、灵敏度和特异性进行性能评价。在此基础上检测此患儿肿瘤组织、骨髓、外周血和脑脊液EEF1G/ALK融合基因的表达。结果经二代测序鉴定该患儿肿瘤细胞EEF1G/ALK融合基因阳性,Sanger测序验证为EEF1G Exon 6和ALK Exon 20的融合。建立的实时荧光定量PCR方法最低定量限为40拷贝/体系;弱阳性标本和强阳性标本的批内精密度CV分别为0.52%和0.17%;批间精密度CV分别为1.32%和1.14%,重复性好;检测其他ALK融合阳性标本结果为阴性,特异性好。复发时送检的淋巴结穿刺组织和外周血的融合基因定量检测结果为138.92%和0.0039%,其余检测为阴性。结论明确此例间变性大细胞淋巴瘤为少见EEF1G/ALK融合基因阳性。建立的EEF1G/ALK融合基因定量检测方法特异性、重复性好,灵敏度高,完全满足融合基因MRD监测的需要。

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 EEF 1 G as the partner gene of the ALK fusion gene,and Sanger sequencing verified that the fusion occurred at EEF 1 G 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.