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

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nThe purpose of the present retrospective study was to investigate whether the predictive value of pre-transplant levels of parameters representing endothelial cell function, such as angiopoietin-2, could be further refined by measuring pre-transplant nitrate levels, and to analyze whether negative influences on clinical outcome could be attenuated by (potentially endothelium-protective) concomitant statin administration. Methods Eligibility of patientsPatients were eligible for this study if they had undergone allogeneic SCT at our institution between June 2002 and December 2011 and their blood samples were available for nitrate measurement (collected directly before the start of conditioning chemotherapy prior to allogeneic SCT). Written informed consent to sample and data collection in accordance with to the declaration of Helsinki was obtained from all eligible patients as well as normal donors (n=49) and sample and data collection was approved by the responsible Institutional Review Board. Graft-versus-host disease prophylaxis, treatment, and supportive careGVHD prophylaxis was performed as previously described 2,3 and remained unchanged over the whole study period. GVHD was clinically and histologically diagnosed and graded using standard criteria.24 Steroid-refractory GVHD was defined as histologically confirmed disease not responding to standard prednisone therapy (2x1 mg/kg body weight, for intestinal GVHD combined with mycophenolate mofetil 2x1 g/day) and requiring second-line salvage immunosuppressive therapy which was generally pentostatin 25 (see Online Supplementary Methods). Enzyme-linked immunosorbent assays, multiplex analyses, nitrate assay and western blotsSerum levels of angiopoietin-2 were quantified by multiplex protein array technology in 327 patients as reported before.3 TNF levels were measured in 417 patients by enzyme-linked immunosorbent assay (BD Biosciences Pharmingen, San Diego, CA, USA). Serum nitrates (n=417 samples) were determined using the Griess Reagent System (see Online Supplementary Methods).The following monoclonal antibodies were used for the western blots: rabbit anti-TNF-α (Cell Signaling Technology through New England Biolabs, Frankfurt, Germany), mouse anti-iNOS (2D2-B2) (R&D Systems, Wiesbaden-Nordenstadt, Germany) and mouse anti-β-actin (AC-15) (Abcam, Cambridge, UK). Corresponding secondary antibodies were purchased from SigmaAldrich (Deisenhofen, Germany). Statistical analysisCategorical and continuous variables of the patients' characteristics were compared using the Fisher exact test and the MannWhitney test, respectively. Cumulative incidences of refractory GVHD were compared using the log-rank test. The choice of cutoffs for nitrates and angiopoietin-2 is explained in the Online Supplementary Methods.Non-relapse mortality (NRM) was calculated as the time from allogeneic SCT to death in the absence of relapse, considering recurrence of the underlying malignancy as a competing event. the NRM rates of differ...

Single-Nucleotide Polymorphisms Within the Thrombomodulin Gene (THBD) Predict Mortality in Patients With Graft-Versus-Host Disease

“…Because pretransplantation ANG2 and serum nitrates have been shown to predict refractoriness of acute GVHD episodes, 6 we also tested if these serum markers correlated with high-risk THBD genotypes. As summarized in Table 1, high-and low-risk THBD genotypes had similar pretransplantation serum levels of ANG2 and nitrates.…”

“…Serum markers were measured before conditioning therapy: Sera for measuring nitrates, soluble thrombomodulin, and ANG2 were available for 240 patients. 6 The validation cohort consisted of patients who underwent allografting at the Charité, Campus Benjamin Franklin (Berlin, Germany), between August 1995 and December 2011 and had DNA available for testing for the three significant SNPs (rs1962, rs1042579, and rs1042580). Written informed consent according to the Declaration of Helsinki was obtained for all eligible patients, and our local ethics committee approved the sample and data collection.…”

“…5 This hypothesis was further substantiated by the observation that markers of altered endothelial homeostasis, such as serum nitrates and angiopoietin-2 (ANG2), even when measured pretransplantation, were associated with an increased risk of refractoriness in those patients who developed GVHD. 6 Although soluble THBD levels were strongly associated with refractory courses of GVHD after alloSCT, pretransplantation THBD levels did not predict outcome. 3,5 An explanation for this could be that single-nucleotide polymorphisms (SNPs) within the THBD gene might contribute to a locally abnormal reactivity of intestinal endothelial cells to immunologic challenge during GVHD.…”

Thrombomodulin alleviates murine GVHD in association with an increase in the proportion of regulatory T cells in the spleen

“…Recent studies suggested that vascular endothelial vulnerability and endothelial dysfunction are involved in the pathogenesis of GVHD. [11][12][13] Elevated pretransplant serum levels of angiopoietin-2, a hormone mediating vascular vulnerability, and nitrate are noted in steroidrefractory GVHD patients. 11,12 A marker of endothelial damage including soluble TM is elevated in patients with steroid-refractory GVHD.…”

“…[11][12][13] Elevated pretransplant serum levels of angiopoietin-2, a hormone mediating vascular vulnerability, and nitrate are noted in steroidrefractory GVHD patients. 11,12 A marker of endothelial damage including soluble TM is elevated in patients with steroid-refractory GVHD. 13 These observations encouraged us to hypothesize that the prophylactic use of rTM with cytoprotective as well as antiinflammatory activity would decrease the incidence of GVHD in transplant patients.…”