br Added value of this study br We
Added value of this study
We identified proteins that are differentially expressed in GC tu-mour tissues and in sera, as well as changed protein levels before and after surgery. Using a comprehensive multivariate analysis, we identified a nineteen serum protein signature, including the clinically used biomarker CEA, which provided a much greater di-agnostic accuracy for GC detection compared to CEA used alone. This protein signature also improved the diagnostic capac-ity for GC patients at early stage and patients with high microsat-ellite instability.
Implications of all available evidence
The detection of the nineteen serum protein signature using a mini-panel of multiplex PEA technology or other equivalent ap-proaches would translate into clinical application to improve the GC diagnosis and treatment stratification and bring tangible bene-fits for GC patients in the future.
Gastric cancer (GC) is the fifth most commonly diagnosed cancer and the third leading cause of cancer death with over 1,000,000 new cases and an estimated 783,000 deaths in 2018 . Currently, complete surgical resection remains the major curative therapy for gastric cancer. Despite the development of technologies for diagnosis and treatment, most gastric cancer cases in the western world are usually diagnosed
at middle or advanced stages, resulting in unsatisfactory treatment re-sults [2,3]. Measurement of tumour protein biomarkers in circulating blood is the most commonly used noninvasive method for early detec-tion of malignant cancers, and is proven to harbor considerable value in screening, diagnosis, monitoring, and prognosis of tumours since some important proteins secreted/released into blood may reflect the quanti-tative or qualitative changes of the whole body when undergoing any pathological conditions. The currently used gastrointestinal tumour se-rological protein biomarkers, including carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), and cancer antigen 72-4 (CA72-4), are insufficient for GC diagnosis since their positive rates in GC pa-tients are b40% and lower than 20% in GC patients at early stage [4,5]. Serum RVX-208 I (PGI) and II (PGII) as well as PGI/PGII ratio are used for GC screening and diagnosis in countries with high or moderate risk, especially in Asia . However, its clinical performance remains controversial and results are different among various ethnicities . Therefore, searching for reliable blood tumour markers for early diagno-sis is crucial for early intervention therapy hence for improving the sur-vival of gastric cancer patients.
Technologies developed in recent decades display the ability of large-scale screening of proteins. Most proteomic studies aiming to identify tumour-associated protein markers are based on mass spec-trometry (MS) [4,8]. However, the extremely broad range of blood pro-tein concentrations challenges proteomic analyses. Furthermore, many blood proteins in low abundance are likely to be specific at early stages of disease, but their detection with classical MS techniques is impaired by the predominance of high abundant proteins. In addition, MS always requires substantial amount of sample input, limiting its application for many clinical samples where the materials are insufficient. A recently developed technology for multiple proteins detection – multiplex prox-imity extension assay (PEA; Olink Proteomics™)  – enables the si-multaneous detection of large-scale proteins with high selectivity and sensitivity with minimal sample volume (as little as 1 μl aliquot), and no requirement for complex sample pre-treatment. A schematic illus-tration of multiplex PEA is shown in Supplementary Fig. 1. The PEA and proximity ligation assay (PLA) are both proven to be sensitive and specific [10,11]. The specificity is due to the requirement of dual-recognition of a target by a matched pair of DNA-conjugated antibodies. In PEA, upon antigen-antibody binding, the labeled DNA oligonucleo-tides are brought into close proximity and hybridize to each other. An amplifiable reporter DNA molecule is formed by DNA polymerization, which can subsequently be amplified and quantified by real-time qPCR. The target concentration is proportional to the number of re-porter DNA molecules. A limiting factor of most multiplexed immunoas-says is the cross-reactivity of antibodies, which restricts the degree of multiplexing to below 10-plex. The design of the DNA-assisted affinity-based PEA excludes the detection of products from unmatched antibody pairs, allowing large-scale of multiplexing without loss of se-lectivity and sensitivity. The assay sensitivity of multiplex PEA is re-ported to be comparable with that of standard sandwich single-plex ELISA for each individual protein . Multiplex PEA has been applied for biomarker discovery of many diseases such as cardiovascular dis-eases, type-1 diabetes, and cancer (publications are listed in https:// www.olink.com/data-you-can-trust/publications).