Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples.
Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples.
复制标题
用于检测临床胶质母细胞瘤样本中癌基因扩增和 EGFRvIII 突变状态的实用生物信息学 DNA 测序流程。
DOI：
10.1016/j.jmoldx.2019.02.001
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Tsankova,NadejdaM
中科院分区：
文献类型：
--
作者：
Miller,MichaelL;Tome-Garcia,Jessica;Waluszko,Aneta;Sidorenko,Tatyana;Kumar,Chitra;Ye,Fei;Tsankova,NadejdaM
胶质母细胞瘤是一种预后不良的恶性脑肿瘤。胶质母细胞瘤中的致癌突变经常影响受体酪氨酸激酶途径组分，这些组分由于异质性表达而难以定量。EGFRvIII是胶质母细胞瘤中常见的致癌受体酪氨酸激酶突变蛋白，增强肿瘤恶性程度，是一种新兴的肿瘤特异性免疫靶点，强调了对其更容易获得和定量检测的需求。我们使用来自117个脑和371个参考临床肿瘤样本的标准化下一代测序数据来检测商业化Ion AmpliSeq Cancer Hotspot Panel第2版中的焦点基因扩增，并基于EGFR内基因截短区域的相对覆盖率缺失来推断EGFR vIII状态。在胶质母细胞瘤（n= 45）中，检测到EGFR [18（40%）]、PDGFRA[3（7%）]、KIT[2（4%）]、MET[1（2%）]和AKT1 [1（2%）]扩增。关于EGF和PDGFRA扩增，新一代测序和原位杂交之间几乎完全一致。与以前的报道一致，该方法仅在EGFR扩增的胶质母细胞瘤中检测到EGFR vIII [8（44%）]，这一点已通过长距离PCR得到证实。我们的研究提供了一个实用的方法，检测癌基因扩增和大的基因内突变，在临床实施的热点面板，可以量化使用zscores。使用DNA测序对EGFRvIII的有效检测消除了转录物降解的问题，并且所提供的脚本有助于有效地并入实验室的生物信息学管道。
Glioblastoma is a malignant brain tumor with dismal prognosis. Oncogenic mutations in glioblastoma frequently affect receptor tyrosine kinase pathway components that are challenging to quantify because of heterogeneous expression. EGFRvIII, a common oncogenic receptor tyrosine kinase mutant protein in glioblastoma, potentiates tumor malignancy and is an emerging tumor-specific immunotarget, underlining the need for its more accessible and quantitative detection. We used normalized next-generation sequencing data from 117 brain and 371 reference clinical tumor samples to detect focal gene amplifications across the commercial Ion AmpliSeq Cancer Hotspot Panel version 2 and inferEGFRvIIIstatus based on relative coverage dropout of the gene's truncated region withinEGFR. In glioblastomas (n= 45), amplification ofEGFR[18 (40%)],PDGFRA[3 (7%)],KIT[2 (4%)],MET[1 (2%)], andAKT1[1 (2%)] was detected. With respect toEGFRandPDGFRAamplification, there was near-complete agreement between next-generation sequencing andin situhybridization. Consistent with previous reports, this method detectedEGFRvIIIexclusively inEGFR-amplified glioblastomas [8 (44%)], which was confirmed using long-range PCR. Our study offers a practical method for detecting oncogene amplifications and large intragenic mutations in a clinically implemented hotspot panel that can be quantified usingzscores. The validated detection ofEGFRvIIIusing DNA sequencing eliminates problems with transcript degradation, and the provided script facilitates efficient incorporation into a laboratory's bioinformatic pipeline.