High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods.
High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods.
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与目前的常规方法相比，靶向二代测序在临床肿瘤标本方差检测方面具有较高的性能
DOI：
10.1186/s13046-017-0591-4
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发表时间：
2017-09-07
期刊：
影响因子：
--
通讯作者：
Li F
中科院分区：
文献类型：
--
作者：
Su D;Zhang D;Chen K;Lu J;Wu J;Cao X;Ying L;Jin Q;Ye Y;Xie Z;Xiong L;Mao W;Li F
背景下一代测序技术（NGS）正越来越多地应用于辅助癌症分子诊断。然而，仍然需要基于大量临床样本来验证NGS检测DNA变异的准确性，特别是对于DNA重排和拷贝数变异（CNV）。本研究的目的是建立靶向NGS的基本参数，为临床诊断提供依据，并与传统的分子诊断方法进行比较，了解靶向NGS的优势。通过临床样本进行以下确认。对扩增难治性突变系统（ARMS）、荧光原位杂交（FISH）和免疫组化（IHC）检测的多种变异体进行靶向NGS评价，以确定灵敏度。结果在测序深度为500×时，检测单个核变异（SNVs）和小插入/缺失（Indels）的最高灵敏度分别可达99%和98.7%，在20%的癌细胞中，CNV检测达到最高水平。通过一个大型临床样本队列对灵敏度和特异性进行了以下确认。对于临床样品中的SNV和indel检测，靶向NGS可以以100%的灵敏度和特异性鉴定所有热点突变。在ALK融合检测中，约86%的IHC鉴定病例可通过靶向NGS进行鉴定，并且靶向NGS检测到的所有ALK融合均通过IHC进行确认。对于HER 2扩增，14例经靶向NGS鉴定的HER 2扩增病例均经FISH证实，约93.3%的Her-2 IHC（3+）病例经靶向NGS鉴定。最后，本文建立的靶向NGS平台已准确检测了215例NSCLC患者的EGFR热点突变。使用大量患者样本的常规方法的比较证实了靶向NGS在检测DNA改变上的高优先级。
BackgroundNext generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis.MethodsGenomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity.ResultsAt sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99% and 98.7% respectively, and in 20% of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100% sensitivity and specificity. OnALKfusion detection, about 86% IHC-identified cases could be identified by targeted NGS and allALKfusion detected by targeted NGS were confirmed by IHC. ForHER2-amplification, 14HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3% of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detectedEGFRhotspot mutations in 215 NSCLC patients.ConclusionsDNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.
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DOI：
10.1038/nrclinonc.2016.96
发表时间：
2017-01
期刊：
Nature reviews. Clinical oncology
影响因子：
--
作者：
Lopez JS;Banerji U
通讯作者：
Banerji U
DOI：
10.1002/wsbm.1159
发表时间：
2012-07
影响因子：
7.9
作者：
Lefebvre, Celine;Rieckhof, Gabrielle;Califano, Andrea
通讯作者：
Califano, Andrea
影响因子：
4.6
作者：
Wallander, Michelle L.;Geiersbach, Katherine B.;Layfield, Lester J.
通讯作者：
Layfield, Lester J.
影响因子：
12.3
作者：
Fisher S;Barry A;Abreu J;Minie B;Nolan J;Delorey TM;Young G;Fennell TJ;Allen A;Ambrogio L;Berlin AM;Blumenstiel B;Cibulskis K;Friedrich D;Johnson R;Juhn F;Reilly B;Shammas R;Stalker J;Sykes SM;Thompson J;Walsh J;Zimmer A;Zwirko Z;Gabriel S;Nicol R;Nusbaum C
通讯作者：
Nusbaum C
影响因子：
15.8
作者：
Greulich H;Chen TH;Feng W;Jänne PA;Alvarez JV;Zappaterra M;Bulmer SE;Frank DA;Hahn WC;Sellers WR;Meyerson M
通讯作者：
Meyerson M