Cancer Res Treat.  2019 Jan;51(1):378-390. 10.4143/crt.2018.070.

Plasma Macrophage Migration Inhibitory Factor and CCL3 as Potential Biomarkers for Distinguishing Patients with Nasopharyngeal Carcinoma from High-Risk Individuals Who Have Positive Epstein-Barr Virus Capsid Antigen-Specific IgA

• Xue N
• Lin JH
• Xing S
• Liu D
• Li SB
• Lai YZ
• Wang XP
• Mao MJ
• Zhong Q
• Zeng MS
• Liu WL

• KMID: 2437628
• DOI: http://doi.org/10.4143/crt.2018.070

Abstract

PURPOSE
The purpose of this study was to identify novel plasma biomarkers for distinguishing nasopharyngeal carcinoma (NPC) patients from healthy individuals who have positive Epstein-Barr virus (EBV) viral capsid antigen (VCA-IgA).
MATERIALS AND METHODS
One hundred seventy-four plasma cytokines were analyzed by a Cytokine Array in eight healthy individuals with positive EBV VCA-IgA and eight patients with NPC. Real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were employed to detect the expression levels of macrophage migration inhibitory factor (MIF) and CC chemokine ligand 3 (CCL3) in NPC cell lines and tumor tissues. Plasma MIF and CCL3 were measured by ELISA in 138 NPC patients, 127 EBV VCA-IgA negative (VN) and 100 EBV VCA-IgA positive healthy donors (VP). Plasma EBV VCA-IgA was determined by immunoenzymatic techniques.
RESULTS
Thirty-four of the 174 cytokines varied significantly between the VP and NPC group. Plasma MIF and CCL3 were significantly elevated in NPC patients compared with VN and VP. Combination of MIF and CCL3 could be used for the differential diagnosis of NPC from VN cohort (area under the curve [AUC], 0.913; sensitivity, 90.00%; specificity, 80.30%), and combination of MIF, CCL3, and VCA-IgA could be used for the differential diagnosis of NPC from VP cohort (AUC, 0.920; sensitivity, 90.00%; specificity, 84.00%), from (VN+VP) cohort (AUC, 0.961; sensitivity, 90.00%; specificity, 92.00%). Overexpressions of MIF and CCL3 were observed in NPC plasma, NPC cell lines and NPC tissues.
CONCLUSION
Plasma MIF, CCL3, and VCA-IgA combination significantly improves the diagnostic specificity of NPC in high-risk individuals.

Keyword

Biomarkers; Macrophage migration inhibitory factor; Chemokine CCL3; Nasopharyngeal carcinoma; Diagnosis; Microarray

MeSH Terms

Biomarkers*
Blotting, Western
Capsid*
Cell Line
Chemokine CCL3
Cohort Studies
Cytokines
Diagnosis
Diagnosis, Differential
Enzyme-Linked Immunosorbent Assay
Herpesvirus 4, Human*
Humans
Immunoglobulin A*
Immunohistochemistry
Macrophages*
Plasma*
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Tissue Donors
Biomarkers
Chemokine CCL3
Cytokines
Immunoglobulin A

Figure

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  Fig. 1. Cluster analysis of antibody-based cytokine microarray in eight healthy individuals with positive Epstein-Barr virus viral capsid antigen (S1-S8) and eight patients with nasopharyngeal carcinoma (NPC) (S9-S16). (A) The analysis shows that 34 cytokines divided healthy person from patients with NPC. (B) The fold change of 10 top up-regulated proteins from the list of 34 potential markers. (C) The expression of cytokines in Dodd’s and Qian’s dataset.

• 

  Fig. 2. Expression of macrophage migration inhibitory factor (MIF) in nasopharyngeal carcinoma (NPC) plasma, NPC cell lines, and NPC tumor tissues. (A) Determination by enzyme-linked immunosorbent assay of plasma baseline levels of MIF in 25 viral capsid antigen (VCA-IgA) negative (VN), 15 VCA-IgA positive (VP), and 40 patients of NPC. Inbox bares show median levels for each cytokine of each group of person. The levels of mRNA and protein in the immortalized nasopharyngeal epithelial cell lines (NPEC1, NPEC2, and N5-Tert) and NPC cell lines were determined by real-time polymerase chain reaction (B) and Western blotting (D). Expression level was normalized by β-actin and α-tubulin, respectively. Error bars represent standard deviations (SD) calculated from three parallel experiments. (C) The level of MIF in supernatant was measured by enzyme-linked immunosorbent assay. (E) The normal nasopharyngeal epithelial tissue showed lower or no expression of MIF. Low, medium, and high expression of MIF were showed in the NPC tissues.

• 

  Fig. 3. Expression of CC chemokine ligand 3 (CCL3) in nasopharyngeal carcinoma (NPC) plasma, NPC cell lines, and NPC tumor tissues. (A) Determination by enzyme-linked immunosorbent assay (ELISA) of plasma baseline levels of CCL3 in 25 viral capsid antigen (VCA-IgA) negative (VN), 15 VCA-IgA positive (VP), and 40 patients of NPC. Inbox bares show median levels for each cytokine of each group of person. The levels of mRNA and protein in the immortalized nasopharyngeal epithelial cell lines (NPEC1, NPEC2, and N5-Tert) and NPC cell lines were determined by real-time polymerase chain reaction (B) and Western blotting (D). Expression level was normalized by β-actin and α-tubulin, respectively. Error bars represent standard deviations calculated from three parallel experiments. (C) The level of CCL3 in supernatant was measured by ELISA. (E) The normal nasopharyngeal epithelial tissue showed lower or no expression of CCL3. Low, medium, and high expression of CCL3 were showed in the NPC tissues.

• 

  Fig. 4. The plasma concentration of macrophage migration inhibitory factor (MIF) and CC chemokine ligand 3 (CCL3) in the test cohort. Plasma levels of MIF (A) and CCL3 (B) were measured in viral capsid antigen (VCA-IgA) negative cohort, VCA-IgA positive cohort and NPC patient. Bottom, MIF and CCL3 plasma levels in different groups. p-value was obtained by Kruskal-Wallis test.

• 

  Fig. 5. Diagnosis efficacy of macrophage migration inhibitory factor (MIF), CC chemokine ligand 3 (CCL3), and viral capsid antigen (VCA-IgA) in the diagnosis of nasopharyngeal carcinoma (NPC). (A) Receiver operating characteristic (ROC) curves for diagnosing NPC from VCA-IgA negative (VN) cohort (MIF: area under the curve [AUC], 0.843; CCL3: AUC, 0.874; MIF+CCL3: AUC, 0.913). (B) ROC curves for diagnosing NPC from VCA-IgA positive (VP) cohort (MIF: AUC, 0.732; MIF: AUC, 0.824; VCA-IgA: AUC, 0.736; MIF+CCL3+VCA-IgA: AUC, 0.920). (C) ROC curves for the diagnostic strength to identify NPC from (VN+VP) using MIF, CCL3 and VCA-IgA (MIF: AUC, 0.782; MIF: AUC, 0.852; VCA-IgA: AUC, 0.881; MIF+CCL3+VCA-IgA: AUC, 0.961).

Reference

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