Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.
Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.
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基于CRISPR/Cas9技术开发肿瘤突变负荷测量新型参考材料
DOI：
10.3389/fonc.2022.845636
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发表时间：
2022
影响因子：
4.7
通讯作者：
中科院分区：
文献类型：
--
作者：
作为影响免疫检查点抑制剂治疗决策的生物标志物，肿瘤突变负荷（TMB）估计的准确性、可靠性和可比性至关重要。为了提高这些测试的一致性和可靠性，提供地面实况数据的合格参考材料至关重要。在这项研究中，我们开发了一套不同TMB值的福尔马林固定和石蜡包埋（FFPE）样品作为TMB估计的新参考材料。通过CRISPR/Cas9技术将MutS Homolog 2（MSH 2）基因和DNA聚合酶抑制剂（POLE）基因中的几个临床相关变体导入人细胞系中，我们首先构建了四种典型的经验证具有超变或超变表型的细胞系。随后通过细胞混合和石蜡包埋，制备新型FFPE样品。证实了我们的新型FFPE样品具有足够的细胞量、高再现性，并且它们可以提供匹配的野生型样品作为遗传背景。双平台全外显子组测序验证表明，我们的FFPE样品也是高度灵活的，因为它们含有跨越临床相关范围（2.0-106.1 mut/Mb）的不同TMB值。在不限制产量和TMB值的情况下，我们基于CRISPR/Cas9编辑的新型FFPE样品适合作为候选参考材料。从实用的角度来看，这些样品可用于TMB评估的确认、验证、内部质量控制和能力验证。
As a biomarker that affects treatment decisions of immune checkpoint inhibitors, the accuracy, reliability, and comparability of tumor mutational burden (TMB) estimation is of paramount importance. To improve the consistency and reliability of these tests, qualified reference materials providing ground-truth data are crucial. In this study, we developed a set of formalin-fixed and paraffin-embedded (FFPE) samples with different TMB values as the novel reference materials for TMB estimation. By introducing several clinically relevant variants in MutS Homolog 2 (MSH2) gene and DNA polymerase epsilon (POLE) gene into human cell lines using CRISPR/Cas9 technology, we first constructed four typical cell lines which verified with hypermutator or ultramutator phenotype. Followed by cell mixing and paraffin embedding, the novel FFPE samples were prepared. It was confirmed that our novel FFPE samples have sufficient quantity of cells, high reproducibility, and they can provide matched wild type sample as the genetic background. The double-platform whole exome sequencing validation showed that our FFPE samples were also highly flexible as they containing different TMB values spanning a clinically relevant range (2.0–106.1 mut/Mb). Without limitations on production and TMB values, our novel FFPE samples based on CRISPR/Cas9 editing are suitable as candidate reference materials. From a practical point of view, these samples can be used for the validation, verification, internal quality control, and proficiency testing of TMB assessment.
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影响因子：
4
作者：
Alexandrov, Ludmil B.;Stratton, Michael R.
通讯作者：
Stratton, Michael R.
影响因子：
3.8
作者：
Park S;Lee C;Ku BM;Kim M;Park WY;Kim NKD;Ahn MJ
通讯作者：
Ahn MJ
DOI：
10.1056/nejmoa1801946
发表时间：
2018-05-31
期刊：
The New England journal of medicine
影响因子：
--
作者：
Hellmann MD;Ciuleanu TE;Pluzanski A;Lee JS;Otterson GA;Audigier-Valette C;Minenza E;Linardou H;Burgers S;Salman P;Borghaei H;Ramalingam SS;Brahmer J;Reck M;O'Byrne KJ;Geese WJ;Green G;Chang H;Szustakowski J;Bhagavatheeswaran P;Healey D;Fu Y;Nathan F;Paz-Ares L
通讯作者：
Paz-Ares L
影响因子：
64.5
作者：
Hugo W;Zaretsky JM;Sun L;Song C;Moreno BH;Hu-Lieskovan S;Berent-Maoz B;Pang J;Chmielowski B;Cherry G;Seja E;Lomeli S;Kong X;Kelley MC;Sosman JA;Johnson DB;Ribas A;Lo RS
通讯作者：
Lo RS
影响因子：
10.9
作者：
Hsiehchen D;Espinoza M;Valero C;Ahn C;Morris LGT
通讯作者：
Morris LGT