Plasma-derived candidate biomarkers for detection of gallbladder carcinoma

Sudeshna Kar,Anil Kumar Agarwal,Surbhi Goyal,Javed Akhtar,Ravindra Varma Polisetty,Ankit P Jain,Poonam Gautam,Puja Sakhuja,Ratna Priya,Vaishali Jain,Geeta Chauhan,Amit Javed,Ravi Sirdeshmukh

doi:10.1038/s41598-021-02923-7

Abstract

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types—5′-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC.

Highlights

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis
Three of the tumor-associated proteins, which are present in the plasma in free state, were verified in individual plasma samples by quantitative Enzyme‐linked immunosorbent assay (ELISA)
We found a significant increase in extracellular vesicles (EVs) proteins in advanced stages of GBC in comparison to early stage GBC (Supplementary Fig. S1C)

Summary

Introduction

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC. Abbreviations GBC Gallbladder carcinoma EVs Extracellular vesicles GSD Gallstone disease XGC Xanthogranulomatous cholecystitis iTRAQ Isobaric tags for relative and absolute quantitation ELISA Enzyme-linked immunosorbent assay MME Neprilysin NT5E 5′-Nucleotidase isoform 2 ANPEP Aminopeptidase N ROC Receiver operating characteristic curve AUC Area under the ROC curve NTA Nanoparticle tracking analysis SCX Strong cation exchange LC–MS Liquid chromatography–mass spectrometry FDR False discovery rate PSMs Peptide spectral matches STRING Search tool for the retrieval of interacting genes/proteins. Liu et al analyzed serum levels of 49 inflammatory proteins using Luminex bead-based assay and reported significant association of eight proteins with overall survival of GBC patients[10]

Methods

Results

Conclusion