Plasma-derived candidate biomarkers for detection of gallbladder carcinoma

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.

Figure 1

Overall workflow of the study. GBC gallbladder carcinoma, EV extracellular vesicles, iTRAQ isobaric tags for relative and absolute quantitation, ELISA enzyme-linked immunosorbent assay.

Figure 2

(A) Venn diagram showing EV proteins with altered levels in early and advanced stages of GBC. A total of 42, 34, 44 proteins were found to have altered levels in early stage GBC, GBC stage IIIA and stage IVB respectively in comparison to controls (healthy individuals/GSD/XGC). A total of 13 proteins are common among early and advanced stage GBC which may be associated with progression of the disease and a total of 20 proteins are with altered levels only in GBC Stage IVB which may be associated with metastasis. Proteins showing altered levels with ≥ twofold change were considered for the analysis. Altered levels of (B) NT5E (C) ANPEP and (D) MME in early and advanced stages of GBC as observed in quantitative proteomics data. The levels of NT5E and ANPEP were significantly altered (with ≥ twofold change and p value ≤ 0.05) in both early and advanced stage GBC, while MME were significantly altered in early stage GBC. The p values ≤ 0.05, ≤ 0.01, ≤ 0.001 are marked with ‘*’, ‘**’ and ‘***’ respectively. GSD gallstone disease, XGC xanthogranulomatous cholecystitis.

Figure 3

Protein concentration of NT5E, ANPEP and MME in controls and GBC cases using quantitative ELISA. Scatter plot showing NT5E, ANPEP and MME concentration in sonicated plasma samples from discovery cohort (A, D, G), an independent cohort (B, E, H) and combined cohort (discovery + Independent cohort) (C, F, I). Controls include healthy individuals, GSD, XGC cases. A significant increase in the levels of MME was observed in early stage GBC cases, NT5E in advanced stage GBC cases and ANPEP in both early and advanced stage GBC.

Figure 4

ROC curve representing sensitivity and specificity for NT5E, ANPEP and MME. ROC curve showing AUC, sensitivity and specificity for NT5E, ANPEP and MME in plasma samples from discovery cohort (A, D, G), an independent cohort (B, E, H) and combined cohort (discovery + Independent cohort) (C, F, I). AUC for independent cohort was increased for all the three proteins in comparison to the discovery cohort.

Figure 5

IHC analysis to study the expression of NT5E and MME in controls and GBC cases. (A) Representative IHC images showing the expression of NT5E and MME in controls and GBC cases. IHC was performed on formalin-fixed paraffin-embedded (FFPE) tissue microarray (TMA) and individual tissue sections. An in-house TMA block was constructed using the FFPE blocks and included 6 controls (6 GSD cases) and 14 GBC cases (2 early stage and 12 advanced stage). Each TMA block consisted of tissue cores of 2 mm diameter and 4 µm sections were cut from the TMA block for carrying out IHC. Individual tissue sections (FFPE) of 33 GBC (11 early stage and 22 advanced stage) and 17 controls (10 GSD and 7 XGC cases) were also for IHC analysis. IHC results showed positive expression for NT5E in 53.84% of early and 50% of advanced stage GBC cases while MME showed positive expression in 23.07% of early and 23.52% of advanced stage GBC cases. (B) The statistical analysis showed a significant difference between cases and controls for NT5E and MME for both early stage and advanced stage GBC. The scale bar is shown as red line. IHC scoring is shown in “Methodology” section “Immunohistochemistry analysis”.