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Ruili Yu,1 Tingyi Sun,1 Xianwei Zhang,1 Zhen Li,1 Yang Xu,2 Kaihua Liu,2 Yuqian Shi,2 Xue Wu,2 Yang Shao,2,3 Lingfei Kong1 1Department of Pathology, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 2Geneseeq Research Institute, Nanjing Geneseeq Technology Inc., Nanjing, Jiangsu, People’s Republic of China; 3School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of ChinaCorrespondence: Lingfei KongDepartment of Pathology, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, 7 Weiwu Road, Zhengzhou, 450003, Henan Province, People’s Republic of ChinaTel +86 138 0386 8286Email lfkong9@163.comPurpose: TP53 is the most frequently mutated gene in gastric cancer and it can be potentially used for gastric cancer diagnosis and screening. However, standardized clinical approaches that could accurately and cost-effectively detect TP53 mutations in gastric cancer are largely lagged behind.Patients and Methods: We conducted next-generation sequencing (NGS) analysis of 425 cancer-related genes in 42 gastric cancer patients in our cohort. A 1313-patient cohort derived from the cBioPortal database was used for validation. We performed immunohistochemistry (IHC) staining with four commonly used p53 antibodies, and the NGS results were used as the gold standard to optimize the IHC threshold for each antibody.Results: By NGS analysis, we found that around 80% of gastric cancer patients in our cohort harbored TP53 alterations. Genetic alterations of BRCA1/ 2 or KMT2B were mostly exclusive with TP53 mutations, so were the MSI status or low grade of tumors. These results were further validated using the data from cBioPortal. We then used the NGS-derived TP53 status to optimize four commonly used IHC antibodies for detecting TP53 mutations. We showed that all antibodies could achieve more than 93% accuracy when proper IHC positivity thresholds were used, especially for the SP5 antibody that could reach 100% sensitivity and specificity with the 20% threshold.Conclusion: Our results indicated that exclusivity between TP53 and BRCA mutations could be potentially used as a cost-effective way to predict BRCA status. Also, setting proper IHC thresholds for each specific antibody is critical to accurately detect TP53 mutations and facilitate disease diagnosis.Keywords: TP53, gastric cancer, next-generation sequencing, co-mutation, immunohistochemistry threshold