Research On Quality Control Products For MLH1 Gene Mutation Detection Based On CRISPR/Cas9 Technology

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 proteindeficient 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 GM12878Cas9₆ and GM12878Cas9₁0.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.