High pre-transplant serum nitrate levels predict risk of acute steroid-refractory graft-versus-host disease in the absence of statin therapy

Abstract

Steroid-refractory graft-versus-host disease is a life-threatening complication after allogeneic stem cell transplantation. Evidence is accumulating that steroid-refractory graft-versus-host disease is associated with endothelial distress. Endothelial cell homeostasis is regulated by nitric oxide, and serum nitrates are derived from nitric oxide synthase activity or dietary sources. In this retrospective study based on 417 patients allografted at our institution we investigated whether quantification of serum nitrates could predict steroid-refractory graft-versus-host disease. Elevated pre-transplant levels of serum nitrates (>26.5 μM) predicted steroid-refractory graft-versus-host disease (P=0.026) and non-relapse mortality (P=0.028), particularly in combination with high pre-transplant angiopoietin-2 levels (P=0.0007 and P=0.021, respectively). Multivariate analyses confirmed serum nitrates as independent predictors of steroid-refractory graft-versus-host disease and non-relapse mortality. Differences in serum nitrate levels did not correlate with serum levels of tumor necrosis factor or C-reactive protein or expression of inducible nitric oxide synthase in blood cells. Patients with high pre-transplant nitrate levels had significantly reduced rates of refractory graft-versus-host disease (P=0.031) when pravastatin was taken. In summary, patients at high risk of developing steroid-refractory graft-versus-host disease could be identified prior to transplantation by serum markers linked to endothelial cell function. Retrospectively, statin medication was associated with a reduced incidence of refractory graft-versus-host disease in this endothelial high-risk cohort.

Figure 1.

(A)Serum nitrate levels measured prior to allogeneic SCT predict steroid-refractory GVHD and NRM. Patients with high pre-transplant serum nitrate levels (>26.5 μM, dashed line, n=169) had a higher incidence of steroid-refractory GVHD (P=0.026; HR 2.5, 95% CI 1.1–6.2) and a higher NRM rate (P=0.028; HR 1.96, 95% CI 1.08–3.56) than patients with low serum nitrate levels (=26.5 μM, solid line, n=248). Patients who received statins during and after transplantation were excluded from all analyses. (B) Statin intake reduces incidence of steroid-refractory GVHD in patients with high nitrate levels. Statin intake (dashed line) during and after transplantation (n=159) did not reduce the incidence of steroid-refractory GVHD among patients with low pre-transplant serum nitrate levels (=26.5 μM, n=248, P=0.28; HR 1.66, 95% CI 0.66–4.17). In contrast, patients with high pre-transplant serum nitrate levels (n=169, >26.5 μM) did benefit from statin intake and had a significantly lower incidence of steroid-refractory GVHD (P=0.031, HR 0.20, 95% CI 0.05–0.86).

Figure 2.

Serum nitrate levels and angiopoietin-2 (ANG) levels measured prior to allogeneic SCT cooperate to predict steroid-refractory GVHD and NRM: Patients (n=325) were divided into three risk groups: (i) angiopoietin-2 >1,000 pg/mL and nitrates >26.5 μM, n=67; (ii) either angiopoietin-2 >1,000 pg/mL or nitrates >26.5 μM, n=158; and (iii) neither of these risk factors, n=32. Patients with both risk factors (angiopoietin-2 >1,000 pg/mL and nitrates > 26.5 μM) had a significantly higher incidence of steroid-refractory GVHD (P=0.0007; HR 3.9, 95% CI 1.78–8.65) as well as NRM (P=0.021; HR 2.05, 95% CI 1.11–2.78) as compared with the remaining groups.

Figure 3.

Endothelial risk parameters indicate transplant-related mortality only in patients who develop GVHD. Patients were divided into three risk groups: (i) angiopoietin-2 (ANG) >1,000 pg/mL and nitrates >26.5 μM; (ii) either angiopoietin-2 >1,000 pg/mL or nitrates >26.5 μM; and (iii) neither of these risk factors. Patients with both risk factors (angiopoietin-2 >1,000 pg/mL and nitrates >26.5 μM) only had an increased incidence of NRM if they developed GVHD (P=0.0006; HR 3.73, 95% CI 1.75–8.33) but not if they never suffered from GVHD.