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A. Radujkovic et al.
patient and general populations demonstrated that circu- lating ADMA is also an independent risk factor for all- cause mortality.11,12 In view of these findings, the present retrospective study investigated the impact of pre-trans- plant serum ADMA levels on major outcome measures in the setting of allogeneic SCT.
Methods
Patients
Assessment of serum levels of asymmetric dimethyl- arginine and endothelium-related serum factors
Serum levels of ADMA and endothelium-related serum factors were assessed by enzyme-linked immunosorbent assay retrospec- tively in the last serum sample taken before the start of the condi- tioning treatment.
Statistical analysis
Overall survival (OS), progression-free survival (PFS), time to relapse, and NRM (death in the absence of prior relapse) were cal- culated from the date of allogeneic SCT to the appropriate end- point. PFS was defined as the time from transplantation to relapse of the underlying disease or death. Since acute GvHD and its treat- ment are major contributors to post-transplant mortality, OS, PFS and NRM were also assessed after acute GvHD (i.e. from the date of onset of acute GvHD). NRM and recurrence of the underlying malignancy were considered as competing events. For the inci- dence of acute GvHD, the competing events were relapse and death in remission without acute GvHD. The Kaplan-Meier method was used to estimate distributions of survival times. Follow-up times were calculated by the reverse Kaplan-Meier esti- mate.13 Cumulative incidence functions were implemented to account for the competing risks,.
Due to the relatively wide range of supposedly normal ADMA serum levels, pre-transplant ADMA was analyzed as a continuous variable. Since ADMA serum levels did not follow a normal distri- bution and the data were log2-transformed, the prognostic effect of pre-transplant ADMA on OS, PFS, relapse, and NRM as a con- tinuous variable was evaluated using Cox regression models. Cox proportional hazards regression modeling was used for OS and PFS (overall and after the onset of acute GvHD). Relapse and NRM (overall and after the onset of acute GvHD) were analyzed by cause-specific Cox models.
To illustrate the continuous effect of pre-transplant ADMA on the different endpoints, patients were grouped according to ADMA quartiles, and the observation period was restricted to the first year after transplantation or acute GvHD.
To assess the dependency of endothelium-related serum mark- ers [free interleukin (IL)-18, sCD141, and nitrates] on ADMA lev- els, the Jonckheere trend test was applied comparing serum mark- er data in four increasing intervals of pre-transplant ADMA levels determined by the quartiles of the ADMA distribution. The Jonckheere trend test is a rank-based nonparametric test which is used to test for an ordered difference in medians.14
All statistical tests were two-sided. Hazard ratios (HR) were
estimated with 95% confidence intervals (95% CI). P values <0.05 were considered statistically significant.
Details regarding prophylaxis, diagnosis, and treatment of GvHD, the assessment of serum levels of ADMA and endotheli- um-related serum factors, SNP analyses in the inducible nitric oxide synthase gene (INOS) and additional statistical methods are described in the Online Supplement.
Results
Patients’, disease and treatment characteristics and graft-versus-host disease incidence
A total of 938 patients met the eligibility criteria for this study. The patients’, disease and transplant characteristics are given in Table 1. A total of 493 patients died after allo- geneic SCT, of whom 309 died within the first year after transplantation. The cumulative incidences of relapse and NRM at 1 year after transplantation were 23.0% (95% CI: 20.3%-25.7%) and 18.2% (95% CI: 15.8%-20.7%), respectively. The cumulative incidences of acute GvHD
Table 1. Patients’, disease and transplant characteristics.
Adult patients (age ≥18 years) who were allografted between 2002 and 2013 at two centers (Heidelberg, n=518, and Essen, n=420) and who had serum samples available for ADMA meas- urement (collected directly before the start of conditioning chemotherapy prior to allogeneic SCT) were included in this study. Written informed consent to sample and data collection according to the Declaration of Helsinki was obtained from all patients, and the local ethics committees approved the study.
Parameter
Age [in years] at allogeneic SCT, median, (IQR)
Recipient sex, n (%) Female
Male
Disease, n (%) Myeloida Lymphoidb
Disease stage at allogeneic SCTc, n (%) Early
Late/intermediate
Conditioningd regimen, n (%) Reduced intensity Myeloablative
Stem cell source, n (%) Peripheral blood Bone marrow
Donor type, n (%) Unrelated Related
Donor-recipient HLA-matching, n (%) Mismatched
Matched
Antithymocyte globulin treatment, n (%) No
Yes
Donor sex, n (%) Female
Male
ADMA: asymmetric dimethylarginine; SCT: stem cell transplantation; HLA: human leukocyte antigen; IL-18: interleukin 18; IQR, interquartile range; aMyeloid: acute myeloid leukemia, myelodysplastic and myeloproliferative syndromes. bLymphoid: acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma and multi- ple myeloma.cAccording to Gratwohl et al.41 dAccording to Bacigalupo et al.42 eBased on 938 serum samples. fBased on 375 serum samples. gBased on 746 serum samples. hBased on 776 serum samples.
N=938
54 (46-62)
424 (45)
514 (55)
665 (71) 273 (29)
306 (33)
632 (67)
804 (86) 134 (14)
861 (92)
77 (8)
700 (75) 238 (25)
242 (26)
696 (74)
225 (24) 713 (76)
311 (33)
Pre-transplant ADMAe, mM, median (IQR)
Pre-transplant nitrate levelsf, mM, median (IQR)
Pre-transplant free IL-18g, pg/mL, median (IQR) Pre-transplantsolubleCD141h,pg/mL,median(IQR) 4179(3386-5376)
627 (67) 0.73 (0.59-0.97) 22.4 (15.0-35.7) 442 (316-683)
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