Targeted Proteomics-Derived Biomarker Profile Develops a Multi-Protein Classifier in Liquid Biopsies for Early Detection of Esophageal Squamous Cell Carcinoma

Background: Early diagnosis of esophageal squamous cell carcinoma (ESCC) remains a challenge due to the lack of specific blood biomarkers. We aimed to develop a serum multi-protein signature for early detection of ESCC. Methods: We selected 70 healthy controls, 30 precancerous patients, 60 stage I patients, 70 stage II patients and 70 stage III/IV ESCC patients from a completed ESCC case-control study in a high-risk area of China. Olink Multiplex Oncology II targeted proteomics panel was used to simultaneously detect the levels of 92 cancer-related proteins in serum using proximity extension assay. Findings: We found that 10 upregulated and 13 downregulated protein biomarkers in serum could distinguish the early ESCC from healthy controls and their serum levels had significant dose-response relationships with ESCC progression. Applying least absolute shrinkage and selection operator (LASSO) regression and backward elimination algorithm, ANXA1 (annexin A1), hK8 (kallikrein-8), hK14(kallikrein-14), VIM (vimentin), and RSPO3 (R-spondin-3) were kept in final model to discriminate early ESCC cases from healthy controls with an area under curve (AUC) of 0.936 (95% confidence interval: 0.899~0.973). The average accuracy rates of the five-protein classifier were 0.861 and 0.825 in training and test data by five-fold cross-validation. Interpretation: Our study suggested that a combination of ANXA1, hK8, hK14, VIM and RSPO3 serum proteins could be considered as a potential tool for screening and early diagnosis of ESCC. Our results, however, need be confirmed in external case-control studies and prospective studies. Funding Statement: This work was supported by National Natural Science Foundation of China (81973116, 91846302, 81573229 and 81502870), National Key Research and Development program of China (2017YFC0907002 and 2017YFC0907003), International S&T Cooperation Program of China (2015DFE32790), European Research Council (682663), Medical and Health Technology Development Plan of Shandong Province (2018WS331). The funding sources had no role in the study design, interpretation of results, writing the manuscript or decision to submit the manuscript for the publication. The corresponding author has full access to all the data and assumes final responsibility for the decision to submit for publication. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: The study protocol was approved by the Institutional Review Boards of the School of Life Sciences, Fudan University (date: February 19, 2009), Qilu Hospital, Shandong University (date: March 8, 2010), and Stockholm Ethical Vetting Board (2018/357-31). The study was carried out in accordance with the approved protocol, and all participants provided written informed consent.