靶向测序技术检测成人型胶质瘤多靶点拷贝数变异的研究

Study of detection of multi-target copy number variation in adult-type gliomas by targeted sequencing

目的 建立一种成人型胶质瘤临床样本多靶点(1号、19号、7号、10号染色体,EGFR、CDKN2A/B基因)拷贝数变异并行的检测方法.方法 基于二代测序技术建立拷贝数靶向测序方法,采用5个经全外显子测序已明确拷贝数变异信息的临床样本为标准样本,通过检测1号、19号、7号、10号染色体,EGFR、CDKN2A/B基因拷贝数的多种变异情况,评估拷贝数靶向测序方法的准确性;通过原位荧光杂交方法评估这一检测方法对CDKN2A/B基因杂合性和纯合性缺失的鉴别能力;通过回顾性收集中国脑胶质瘤基因组图谱计划(CGGA)数据库中的77例临床样本,对这一方法的临床实用性进行评估.结果 所建立的拷贝数靶向测序方法可以准确检测出5个标准样本中1号、19号、7号、10号染色体,EGFR、CDKN2A/B基因拷贝数的变异情况.通过与原位荧光杂交检测结果进行比对,发现拷贝数靶向测序方法可准确鉴别CDKN2A/B基因的纯合性缺失和杂合性缺失.针对CGGA数据库中临床样本的检测结果显示,采用拷贝数靶向测序方法可完成对77例临床样本的1号、19号、7号、10号染色体,EGFR、CDKN2A/B基因的检测需求,根据检测结果可将77例样本按照2021版世界卫生组织中枢神经系统肿瘤分类方法进行精准分类和分级.结论 基于二代测序技术建立的拷贝数靶向测序方法可以同时检测胶质瘤样本多靶点(1号、19号、7号、10号染色体,EGFR、CDKN2A/B基因)拷贝数的变异,适合作为临床分子病理学常规检测技术.

Objective To establish a method for parallel detection of copy number variation of multiple targets(chromosomes 1,19,7 and 10,EGFR,CDKN2A/B gene)for adult gliomas.Methods A method of detection of multi-target copy number variation by second-generation sequencing technology was established in this study.Five clinical samples with known copy number variation information by whole-exon sequencing were used as standard samples to evaluate the accuracy of the established detection method.The in situ fluorescence hybridization(FISH)method was used to evaluate the ability of the detection method to distinguish the heterozygosity loss and homozygosity loss of CDKN2A/B gene.A total of 77 clinical samples from the Chinese Glioma Genome Atlas Project(CGGA)were retrospectively collected to evaluate the clinical practicability of the detection method.Results The established targeted copy number sequencing method could be used to accurately detect copy number variations of multiple variants of EGFR,CDKN2A/B,chromosomes 1,19,7,and 10 in 5 standard samples.Compared with the FISH assay results,the copy-number targeted sequencing method was able to accurately distinguish the CDKN2A/B homozygous and heterozygous deletions.The established method met the testing requirements of chromosomes 1,19,7,and 10,EGFR,and CDKN2A/B in 77 clinical samples.According to the detection results,77 samples could be used for accurate classification and grading according to the 2021 World Health Organization classification system of the central nervous system tumors.Conclusion The copy number variation targeted sequencing method based on the second-generation sequencing technology can simultaneously detect the copy number variation of multiple targets(chromosomes 1,19,7 and 10,EGFR,CDKN2A/B)in glioma samples,which is suitable for routine clinical molecular pathological detection.