Peng Rongxue, Lin Guigao, Li Lin, Li Jinming
National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, China.
Front Oncol. 2022 Apr 28;12:845636. doi: 10.3389/fonc.2022.845636. eCollection 2022.
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 () gene and DNA polymerase epsilon () 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.
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。为提高这些检测的一致性和可靠性，提供真实数据的合格参考材料至关重要。在本研究中，我们开发了一组具有不同TMB值的福尔马林固定石蜡包埋（FFPE）样本，作为TMB估计的新型参考材料。通过使用CRISPR/Cas9技术将几个与临床相关的错配修复蛋白2（）基因和DNA聚合酶ε（）基因变体引入人类细胞系，我们首先构建了四个具有高突变或超高突变表型的典型细胞系。随后进行细胞混合和石蜡包埋，制备了新型FFPE样本。证实我们的新型FFPE样本细胞数量充足、重现性高，并且可以提供匹配的野生型样本作为遗传背景。双平台全外显子测序验证表明，我们的FFPE样本也具有高度灵活性，因为它们包含跨越临床相关范围（2.0 - 106.1突变/Mb）的不同TMB值。基于CRISPR/Cas9编辑的新型FFPE样本不受生产和TMB值的限制，适合作为候选参考材料。从实际角度来看，这些样本可用于TMB评估的验证、核查、内部质量控制和能力验证。
