基于CRISPR/Cas9技术的肿瘤突变负荷检测参考物质研究

The tumor mutation burden (TMB) is an important biomarker that plays a pivotal role in immunotherapeutic decision making for various kinds of cancer patients. Ensuring accuracy and reproducibility of TMB testing by different targeted next-generation sequencing panel requires reliable reference materials for monitoring assay sensitivity and specificity. Since our previous researches demonstrated that non-specific sgRNAs can lead to many unexpected mutations at any genomic loci after clustered regularly interspaced short palindromic repeat and the CRISPR-associated protein 9 (CRISPR/Cas9) editing in vitro. Hence, we are planning to develop a kind novel reference material by CRISPR/Cas9 technology for various kinds of TMB testing. In our project, CRISPR/Cas9 technology is going to be used to edit several novel cell lines which containing different mutations (high TMB level, medium TMB level, and low TMB level). After subcutaneous inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts can be prepared and tested for suitability as candidate reference materials for tumor tissue TMB testing. Besides, these constructed novel cell lines can also be digested by micrococcal nuclease (MNase) for the preparation of synthetic reference materials for cell-free DNA TMB testing. Without limitations on TMB level and production, our novel synthetic FFPE samples and cfDNA samples based on CRISPR/Cas9 editing, xenografts, and MNase digestion are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of TMB detection. The successful development of these reference materials can provide substantial foundation for improving the quality of TMB research and ensuring the effective clinical application of TMB detection.

肿瘤突变负荷（Tumor mutation burden, TMB）检测现已成为指导肿瘤免疫治疗的重要生物学标志物。目前临床上逐渐有越来越多的TMB检测方法被研发和使用，然而这些不同的检测、分析和计算方法常导致肿瘤患者TMB结果间存在差异，进而影响患者的下一步治疗方案。因此，为了保证TMB的临床检测有效性，研制可靠的且能模拟真实临床样本的TMB参考物质以确保其检测结果的准确性和一致性显得十分关键。我们前期的研究发现，特异性较差的CRISPR/Cas9系统可造成类似TMB的基因组多位点脱靶突变产生。故本研究拟采用CRISPR/Cas9基因编辑、异种移植和体外酶消化模拟细胞凋亡等技术，构建多种具有相同基因背景但含有不同TMB水平的肿瘤组织FFPE样本和血浆游离DNA样本参考物质，建立一种可长期持续生产、模拟真实肿瘤组织或血浆游离DNA特征的新型TMB检测参考物质构建平台。

作为影响肿瘤免疫治疗决策的生物标志物，肿瘤突变负荷（Tumor mutational burden, TMB）检测的准确性、可重复性和可比性至关重要。为了提高TMB检测的一致性和可靠性，最为关键的便是研制可模拟真实临床样本的TMB检测参考物质。在本项目研究中，我们通过使用CRISPR/Cas9技术将MSH2基因和POLE基因中的特定突变序贯引入人类细胞系，首先构建了多种具有“超强突变”特征（超高TMB水平）的新型细胞系。随后通过背景细胞混合、石蜡包埋和/或体外酶消化，我们最终研制了一系列具有不同TMB值的FFPE样本和模拟血浆样本作为新型人工模拟肿瘤组织/血浆游离DNA TMB检测参考物质。研究验证结果表明，本项目制备得到的人工模拟肿瘤组织TMB检测参考物质具有足够的细胞数量、高度的可重复性，并可提供相匹配的野生型样本作为配对遗传背景样本，其样本TMB值可覆盖2.0-106.1mut/Mb。通过将本项目制备的人工模拟肿瘤组织TMB检测参考物质发放至实验室进行高通量测序（大panel）TMB检测室间质量评价，进一步证实了本研究中所制备的TMB检测参考物质的实用性。.总而言之，本项目的研究成果为临床进一步研究肿瘤TMB升高机制提供了一定的科学依据；同时，本研究所制备的TMB检测参考物质为提高国内外TMB检测和研究质量、保证其临床有效应用提供坚实的物质基础。