随着免疫检查点抑制剂（Immune checkpoint inhibitors，ICIs）治疗方法的发展，错配修复缺陷（Mismatch repair deficiency，dMMR）已成为区分可能受益于这些治疗方法的癌症患者的预测性生物标志物，并且被批准成为首个与组织/部位无关的生物标志物。dMMR作为一种预测性生物标志物，具有泛癌性和检测流程简单的优势而被广泛应用。MLH1基因作为MMR（Mismatch repair，MMR）系统的重要基因，突变发生率较高，准确检测癌症患者的MLH1蛋白表达可以反映MMR系统是否缺陷，进而对于指导临床治疗决策来说至关重要。目前，免疫组织化学染色（Immunohistochemistry，IHC）作为检测MLH1蛋白的主要方法，其敏感性和特异性均很高，但是，该方法检测过程中的许多方面（如抗原修复，抗体和试剂的选择，抗体孵育，洗涤和复染）会影响其性能并具有很大的可变性。临床实验室在IHC检测MLH1蛋白的分析过程中仍有很多环节有待提高。为了解决这些局限性，需要质量控制（Quality control, QC）材料进行分析和验证检测过程中的问题，从而使得临床检测标准化。然而，目前现有的QC材料因大批量生产困难、缺乏典型的组织病理结构和无法全面评估IHC过程等原因，不能满足临床实验室IHC检测实现标准化的需求。因此，本研究基于CRISPR/Cas9技术和裸鼠异种移植的方法，制备了一种可持续生产制备并具有典型组织病理结构等特征的新型福尔马林固定石蜡包埋（Formalin-fixed, paraffin embedded, FFPE）样本QC材料。

  在本研究中，我们通过CRISPR/Cas9技术将体外转录的MLH1sgRNA转染至稳定表达Cas9蛋白的GM12878细胞系中，从而制备MLH1蛋白缺失的细胞系。经过一系列方法：Sanger测序、蛋白质免疫印迹（Western blotting，WB）、IHC和高通量测序（Next generation sequencing，NGS）方法筛选和验证MLH1蛋白缺失后，通过异种移植成瘤最终制备成为新型的MLH1蛋白缺失FFPE样本QC材料。为了保证所制备的QC材料具有广泛的临床适用性，采用了苏木素-伊红染色、IHC等方法对其进行验证。

  结果显示，流式分选后成功培养了355株单克隆细胞，其存活率占总分选单克隆细胞的37.0％（355/960）。这些细胞经过Sanger测序后，筛选出了具有MLH1基因突变的细胞株，之后通过WB和IHC鉴定出两株MLH1蛋白缺失的细胞，NGS结果进一步证实了这两株细胞中的MLH1基因突变，形成了终止密码子并终止了MLH1蛋白的表达，最终将其命名为GM12878Cas9_6和GM12878Cas9_10。之后将以上细胞系注射到裸鼠体内形成瘤块进而制备 3 μm厚FFPE 样本，将其进行HE染色后显示，FFPE样本与子宫内膜癌患者的临床病理样本高度一致，其显示出典型的肿瘤组织结构，并具有肿瘤浸润，炎症，出血和组织坏死等病理学特征。此外，组织IHC结果还显示与临床样本一致的表型，即MLH1核染色呈阴性。除此之外，PMS2作为MLH1蛋白的伴侣，FFPE样本中PMS2蛋白核染色也表现为阴性。以上结果证明本实验成功建立了MLH1蛋白缺失的QC材料。

  综上所述，本研究基于CRISPR/Cas9技术成功构建了MLH1蛋白缺失的细胞系，并从分子和蛋白层面进行了验证，其表现出MLH1基因突变和MLH1蛋白的缺失。通过裸鼠异种移植，我们开发了新型的MLH1蛋白缺失的FFPE QC材料，其具有可持续生产制备和典型组织病理结构等优点，适用于临床IHC方法的标准化。除此之外，本研究中所采用的通过CRISPR/Cas9技术编辑细胞系可为MMR系统其他基因敲除细胞系的构建奠定基础，提供新的思路与方法。

  With the development of immune checkpoint inhibitors (ICIs) treatments, mismatch repair deficiency (dMMR) has become a predictive biomarker for distinguishing cancer patients who may benefit from these treatments and approved to be the first biomarker not related to tissue/site. As a predictive biomarker, dMMR is widely used because of its pan-cancer properties and simple detection procedures. As an important gene in the mismatch repair (MMR) system, MLH1 gene has a high mutation rate. Therefore, accurate detection of the MLH1 protein expression in cancer patients can reflect the defect of the MMR system, which is crucial for guiding clinical treatment decisions. At present, the main method for detecting MLH1 protein is immunohistochemical (IHC). Although its sensitivity and specificity are both high, many aspects of the detection process (antigen retrieval, antibody and reagent selection, antibody incubation, washing and counterstaining) will affect its performance and result in substantial variability. In clinical laboratories, much remains have to be improved in the analysis process of IHC to detect MLH1 protein. In order to solve these limitations, quality control (QC) materials are required to analyze and verify the problems in the testing process, so as to standardize clinical testing. However, the current existing QC materials cannot meet the requirements of laboratory IHC testing quality assurance due to difficulties in mass production, lack of typical histopathological structures, and inability to fully evaluate the IHC process. Therefore, based on CRISPR/Cas9 technology and xenotransplantation methods, we developed a kind of well-characterized QC material that is easy to produce and has typical histopathological structures.

  In this study, CRISPR/Cas9 technology was used to transfect in vitro transcribed MLH1sgRNA into Cas9-expressing GM12878 cell line to establish MLH1 protein-deficient cell lines. After screening by Sanger sequencing, Western blotting (WB), IHC and next-generation sequencing (NGS) were applied to verify MLH1 protein deficiency. tumor formation by xenotransplantation was finally prepared into a new type of MLH1 protein-deficient formalin-fixed, paraffin embedded (FFPE) sample QC materials. In order to ensure that the prepared QC material materials have a wide range of clinical applicability, hematoxylin-eosin staining (HE) and IHC are used to verify them.

  The results showed that we successfully cultured 355 monoclonal cells, with a survival rate of 37.0% (355/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and IHC. The NGS results further confirmed MLH1 gene mutation in these two cell lines, which resulted in the formation of stop codons and terminated the expression of the MLH1 protein and were finally named GM12878Cas9_6 and GM12878Cas9_10. After that, the above cell lines were injected into nude mice to form tumor masses to prepare 3 μm thick FFPE samples. The HE staining showed that FFPE samples were highly consistent with the clinicopathological samples of endometrial cancer patients, which showed typical tumor tissue pathological features, such as tumor infiltration, inflammation, hemorrhage and necrosis. Tissue IHC results also showed a consistent phenotype with cell lines and clinical samples, in which MLH1 nuclear staining was negative. In addition, PMS2 protein in FFPE samples also was negative.

  In conclusion, our study successfully established MLH1 protein-deficient cell lines using CRISPR/Cas9 technology, and verified them from molecular and protein level, showing MLH1 gene mutation and MLH1 protein deficiency. By employing xenografting, we developed novel FFPE QC materials with the advantages of sustainable production and typical histological structures that are suitable for the standardization of the clinical IHC method. Our study provides a new direction for research on QC materials used in clinical laboratory testing.