Aleksandar Radujkovic

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

PURPOSE Steroid-refractory graft-versus-host disease (GVHD) is a major and often fatal complication after allogeneic stem-cell transplantation (alloSCT). Although the pathophysiology of steroid refractoriness is not fully understood, evidence is accumulating that endothelial cell stress is involved, and endothelial thrombomodulin (THBD) plays a role in this process. Here we assess whether single-nucleotide polymorphisms (SNPs) within the THBD gene predict outcome after alloSCT. PATIENTS AND METHODS Seven SNPs within the THBD gene were studied (rs1962, rs1042579, rs1042580, rs3176123, rs3176124, rs3176126, and rs3176134) in a training cohort of 306 patients. The relevant genotypes were then validated in an independent cohort (n = 321). RESULTS In the training cohort, an increased risk of nonrelapse mortality (NRM) was associated with three of seven SNPs tested: rs1962, rs1042579 (in linkage disequilibrium with rs3176123), and rs1042580. When patients were divided into risk groups (one v no high-risk SNP), a strong correlation with NRM was observed (hazard ratio [HR], 2.31; 95% CI, 1.36 to 3.95; P = .002). More specifically, NRM was predicted by THBD SNPs in patients who later developed GVHD (HR, 3.03; 95% CI, 1.61 to 5.68; P < .001) but not in patients without GVHD. In contrast, THBD SNPs did not predict incidence of acute GVHD. Multivariable analyses adjusting for clinical variables confirmed the independent effect of THBD SNPs on NRM. All findings could be reproduced in the validation cohort. CONCLUSION THBD SNPs predict mortality of manifest GVHD but not the risk of acquiring GVHD, supporting the hypothesis that endothelial vulnerability contributes to GVHD refractoriness.

Rituximab maintenance therapy after autologous stem cell transplantation prolongs progression-free survival in patients with mantle cell lymphoma

Rituximab maintenance therapy after autologous stem cell transplantation prolongs progression-free survival in patients with mantle cell lymphoma

Normal-Tissue Radioprotection by Overexpression of the Copper-Zinc and Manganese Superoxide Dismutase Genes

Background and Purpose:Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation.Material and Methods:HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1–6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined.Results:After transduction, 90.0% ± 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 ± 12.6, CuZnSOD: 95.1 ± 17.1, MnSOD: 108.5 ± 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06–1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07–1.43; p = 0.01).Conclusion:Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.Hintergrund und Ziel:Der Ansatz, Normalgewebszellen gegen bestrahlungsinduzierte Schäden zu schützen, kann möglicherweise die therapeutische Breite strahlentherapeutischer Ansätze erhöhen. Ein potentieller Ansatz wäre die gentherapeutische Überexpression der Kupfer-Zink-(CuZnSOD) oder Mangan-Superoxiddismutase (MnSOD) mit rekombinanten Adeno-assoziierten viralen (rAAV2) Vektoren. Das Ziel dieser Studie war die Bestimmung der bestrahlungsmodulierenden Effekte der SOD-Gene auf humane primäre Lungenfibroblasten (HPLF).Material und Methodik:HPLF wurden mit rAAV2-Vektoren transduziert, die die cDNA für CuZnSOD, MnSOD oder ein Kontrollgen enthielten. Die Zellen wurden mit 1–6 Gy bestrahlt und deren Gentransfereffizienz, Apoptoserate, Proteinexpression/-aktivität und Strahlensensitivität mit einem Koloniebildungsassay bestimmt.Ergebnisse:Nach Transduktion exprimierten 90,0% ± 6,4% der Zellen das entsprechende Transgen. Für beide SOD konnte mittels Immunhistochemie sowie SOD-Aktivitätsassay eine signifikante Überexpression (fünffach; Kontrolle: 21,1 ± 12,6, CuZnSOD: 95,1 ± 17,1, MnSOD: 108,5 ± 36,0 U SOD/mg Gesamtprotein) gezeigt werden. Diese Überexpression führte zu einer signifikanten Radioprotektion der HPLF im Vergleich zu den Kontrollen (Überlebensfraktion-[SF-]Quotient SOD/Kontrolle > 1): CuZnSOD: 1,18-fach (95%-Konfidenzintervall [CI]: 1,06–1,32; p = 0,005), MnSOD: 1,23-fach (95%-CI: 1,07–1,43; p = 0,01).Schlussfolgerung:Im Vergleich zu den Kontrollen führte die Überexpression von CuZnSOD oder MnSOD in HPLF zu einem erhöhten klonogenen Überleben nach Bestrahlung. Da in vorherigen Arbeiten eine mangelnde Radioprotektion nach Überexpression der SOD-Gene in Tumorzellen beobachtet werden konnte, sind rAAV2-Vektoren erfolgversprechend für die Übertragung von radioprotektiven Genen in Normalgewebszellen.

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

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.

Pretransplant metabolic distress predicts relapse of acute myeloid leukemia after allogeneic stem cell transplantation

Background The impact of nutritional status on outcome of allogeneic stem cell transplantation (alloSCT) is controversial. This study investigates the influence of pretransplant weight loss and serologic indicators of nutritional homeostasis on relapse and death of acute myeloid leukemia (AML) after alloSCT. Methods Pretransplant weight loss along with serum levels of total serum protein (TSP), albumin, C-reactive protein, and leptin were collected retrospectively in a training cohort (n = 149) and correlated with clinical outcome. Metabolic risk groups were defined and tested in an independent validation cohort (n = 167). Results We identified pretransplant weight loss and TSP as strong independent predictors of relapse and death. Patients in the metabolic high-risk group (low TSP and weight loss) had an increased risk for relapse (P = 0.0002) and death (P = 0.002), but a similar risk for acute graft-versus-host disease. Weight loss coincided with reduced pretransplant serum leptin levels. The adverse influence of weight loss and high metabolic risk on relapse and overall survival could be confirmed in the validation cohort and similarly in patients with less than or more than 5% blasts before alloSCT. Multivariate analysis of both cohorts revealed a hazard ratio for relapse of 7.78 (2.59–23.36, P = 0.0003) in the metabolic high risk group. Conclusion Altered nutritional homeostasis before alloSCT correlates with recurrence of AML after transplantation. Studies addressing pretransplant nutritional interventions to reduce AML relapse rates are warranted.

Transplant-associated thrombotic microangiopathy is an endothelial complication associated with refractoriness of acute GvHD

There is increasing evidence that endothelial dysfunction is involved in refractoriness of acute GvHD (aGvHD). Here we investigated the hypothesis that another endothelial complication, transplant-associated thrombotic microangiopathy (TMA), contributes to the pathogenesis of aGvHD refractoriness. TMA was retrospectively assessed in 771 patients after allogeneic stem cell transplantation (alloSCT). Incidences of TMA and refractory aGvHD were correlated with biomarkers of endothelial damage obtained before alloSCT for patients receiving or not receiving statin-based endothelial prophylaxis (SEP). Diagnostic criteria for TMA and refractory aGvHD were met by 41 (5.3%) and 76 (10%) patients, respectively. TMA was overrepresented in patients with refractory aGvHD (45.0 vs 2.3% in all other patients, P<0.001). TMA independently increased mortality. Elevated pretransplant suppressor of tumorigenicity-2 and nitrates along with high-risk variants of the thrombomodulin gene were associated with increased risk of TMA. In contrast, SEP abolished the unfavorable outcome predicted by pretransplant biomarkers on TMA risk. Patients on SEP had a significantly lower risk of TMA (P=0.001) and refractory aGvHD (P=0.055) in a multivariate multistate model. Our data provide evidence that TMA contributes to the pathogenesis of aGvHD refractoriness. Patients with an increased TMA risk can be identified pretransplant and may benefit from pharmacological endothelium protection.

Expression of CDKN1C in the bone marrow of patients with myelodysplastic syndrome and secondary acute myeloid leukemia is associated with poor survival after conventional chemotherapy

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34+ cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin‐dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p < 0.001). In multivariable analysis, higher CDKN1C expression was associated with worse OS (p = 0.02). On the BM level, a total of 84 (38%) patients showed CDKN1C protein expression before start of treatment. Patient, disease and treatment characteristics did not differ between CDKN1C‐positive and ‐negative patients. Positive CDKN1C BM status was associated with shorter OS in multivariable analysis (HR 1.54, p = 0.04). There was an interaction between CDKN1C BM status and subsequent treatment with negative impact on OS being most pronounced in patients receiving conventional cytotoxic chemotherapy (n = 83, 2‐year OS 30% versus 58%, p = 0.002). In conclusion, low‐proliferative phenotype and CDKN1C expression were associated with shorter OS. CDKN1C protein expression in the BM of newly diagnosed, treatment‐naïve MDS and secondary AML patients was identified as a prognostic factor for poor survival in patients treated with antiproliferative chemotherapy.

Azacitidine and low-dose cytarabine in palliative patients with acute myeloid leukemia and high bone marrow blast counts – a retrospective single-center experience

We retrospectively analyzed and compared the efficacy and toxicity of azacitidine (AZA) and low‐dose cytarabine (LD‐Ara‐C) in 65 palliative patients with acute myeloid leukemia (AML) showing high bone marrow blast counts (≥30%) before start of treatment. Twenty‐seven and 38 patients received AZA and LD‐Ara‐C, respectively. The median patient age was 71 yr. Patient and disease characteristics did not differ between the treatment groups, except for BM blast counts, and peripheral leukocyte and blast counts which were significantly higher in the LD‐Ara‐C group. AZA and LD‐Ara‐C were first‐line treatment in 12 (44%) and 17 patients (45%), respectively. Response and hematologic improvement rates were low and similar in both treatment groups. In both treatment groups, most common non‐hematologic toxicities included febrile neutropenia, pneumonia, and bleedings without significant differences regarding frequencies. Estimated 1‐yr survival rates were 15% (95% CI 8–22) and 13% (95% CI 7–19) in the AZA and LD‐Ara‐C groups, respectively, without statistically significant difference. In multivariate analysis (n = 65), previous treatment (HR 2.27, 95% CI 1.00–5.22, P = 0.05) and adverse cytogenetics (HR 2.50, 95% CI 1.20–5.22, P = 0.02) were independent predictors of poor survival. In our center and within the limitations of a retrospective study, both treatment regimens showed similar but limited efficacy in palliative patients with AML and high BM blast counts.

Haploidentical transplant in patients with myelodysplastic syndrome

The only curative treatment in patients with intermediate or high-risk myelodysplastic syndrome (MDS) is allogeneic hematopoietic stem cell transplantation (HSCT), which usually results in a long-term, disease-free survival rate of between 30% and 50%, depending on the disease risk and the type of donor. In patients without an HLA-matched sibling donor, a family haploidentical donor is an alternative option. The present study reports the European Group for Blood and Marrow Transplantation activity for haploidentical transplantation in MDS patients. A total of 228 patients transplanted from a mismatched HLA-related donor between 2007 and 2014 were studied. The median age at transplant was 56 years. Eighty-four (37%) patients had MDS transformed into acute myeloid leukemia at the time of transplant. Ex vivo T-cell depletion was used in 34 patients. One hundred ninety-four patients received a T-cell replete transplant and 102 patients received posttransplant cyclophosphamide (PT-CY) as graft-versus-host disease (GVHD) prophylaxis. The cumulative incidences of acute and chronic GVHD in PT-CY vs other patients were 25% vs 37% and 37% vs 24%, respectively. The cumulative incidence of nonrelapse mortality was 55% in patients who did not receive PT-CY (no PT-CY) and 41% in patients who did receive PT-CY. Three-year overall survival was 28% in no PT-CY patients and 38% in PT-CY patients. In multivariable analysis, the main risk factors were the intensity of the conditioning regimen and the use of PT-CY. In conclusion, the outcomes of MDS patients who received an haploidentical transplant are close to the results other transplantations from HLA-mismatched donors with approximately one-third of patients alive and free of disease 3 years after transplant, and the use of PT-CY may improve their outcomes.

Visceral leishmaniasis in a patient with relapsed multiple myeloma receiving high-dose melphalan and autologous stem cell transplant

Visceral leishmaniasis in a patient with relapsed multiple myeloma receiving high-dose melphalan and autologous stem cell transplant Aleksandar Radujkovic, Michael Hundemer, Christoph Eisenbach, Thomas Luft, Roland Penzel, Hartmut Goldschmidt, Anthony D. Ho & Frauke Bellos a Department of Internal Medicine V b Department of Gastroenterology and Infectious Diseases, University of Heidelberg, Heidelberg, Germany c Department of General Pathology, Institute of Pathology, Heidelberg, Germany d MLL Munich Leukemia Laboratory, Munich, Germany Published online: 01 Jun 2015.