Ceruloplasmin is a potential biomarker for aGvHD following allogeneic hematopoietic stem cell transplantation

Abstract

Acute graft-versus-host-disease (aGvHD) is the major cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, diagnostic biomarkers for aGvHD have been shown to play important roles in evaluating disease status and mortality risk after allo-HSCT. To identify plasma biomarkers for aGvHD with high sensitivity and specificity, a quantitative proteomic approach using 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) was employed to screen differentially expressed proteins in peripheral blood before and after the onset of aGvHD. Four target proteins, ceruloplasmin (CP), myeloperoxidase (MPO), complement factor H (CFH), and alpha-1-acid glycoprotein (AGP), were chosen for preliminary validation with enzyme linked immunosorbent assay (ELISA) in 20 paired samples at both the time of diagnosis of aGvHD and the time of complete response. The most promising candidate, ceruloplasmin, was further validated at fixed time points after allo-HSCT and during aGvHD. The plasma ceruloplasmin levels were significantly increased during the period of aGvHD onset and were markedly decreased as aGvHD resolved. The plasma ceruloplasmin levels at different time points post-transplant in the aGvHD (+) group were significantly higher than those in the aGvHD (-) group (p<0.001). The elevation of ceruloplasmin level in patients with active aGvHD was independent of infection status. Patients whose ceruloplasmin levels were elevated above 670 μg/ml at 7, 14 and 21 days after allo-HSCT had a remarkably increased probability of subsequently developing aGvHD. In conclusion, our results suggest that plasma ceruloplasmin is a potential plasma biomarker of aGvHD, and it also has prognostic value for risk-adapted prophylaxis during the consecutive time points monitored in the first month after allo-HSCT.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Preliminary validation of four candidate proteins.

Four candidate proteins underwent preliminary validation were tested by ELISA in paired samples from 20 patients at active aGvHD onset and after complete response(CR). (A) Ceruloplasmin (CP) (Diagnosis Vs. CR p

Figure 2. Kinetics of ceruloplasmin following allo-HSCT.

(A) comparison of 16 aGvHD+infection- and 15 aGvHD-infection- patients. (B) comparison of 11 aGvHD-infection+ and 15 aGvHD-infection- patients. (C) comparison of 16 aGvHD+infection- and 11 aGvHD-infection+ patients. (D) comparison of 21 aGvHD+infection+ and 11 aGvHD-infection+ patients.

Figure 3. Comparison of ceruloplasmin levels in infection and aGvHD.

Infection+aGvHD- (n = 18; systemic n = 11; symptomatic n = 7) and aGvHD+infection- (n = 37; systemic n = 16; symptomatic n = 21) were compared at diagnosis of complications as well as recovery.

Figure 4. ROC analysis of ceruloplasmin levels at aGvHD diagnosis.

(ROC) curve analysis of patients with aGvHD (n = 72) and without aGvHD (n = 26). At a cutoff value of 780 μg/ml, the corresponding sensitivity was 0.905, the specificity was 0.8, the area under the ROC was 0.902 (95% CI 0.825, 0.979; P

Figure 5. Ceruloplasmin levels during aGvHD recovery.

Comparison of ceruloplasmin level in patients with aGvHD (n = 72) at diagnosis, none remission (NR), complete response (CR).

Figure 6. Ceruloplasmin levels in different types of aGvHD.

Comparison of ceruloplasmin level in patients with different aGvHD involved organs: Skin (n = 40), Gastrointestinal (GI, n = 11) and Mixed (Skin & GI, n = 21).

Figure 7. The cumulative incidence of aGvHD at multiple time points.

Comparison of cumulative incidence of aGvHD in two groups of patients (CP> 670 μg/ml or