Assessment of Anti-Oxidant Markers of Inflammation in Patients with Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a late-occurring inflammatory condition post- allogeneic hematopoietic cell transplantation (HCT), which involves multiple distinct interactions among alloreactive dysregulated T and B cells. Prior studies have shown that autoantibodies to platelet-derived growth factor receptor (PDGFR) induce tyrosine phosphorylation and accumulation of Reactive Oxygen Species (ROS), causing expression of collagen type-1 gene through the Ha-Ras-Erk1/2–ROS signaling pathway. We hypothesize that antioxidant markers of inflammation (8-OHdG, total antioxidant levels, CXCL4 and pH2Ax) will be elevated in patients with scleroderma cGVHD compared to patients who do not develop cGVHD or have non-scleroderma cGVHD. Patients (n=36) were consented on an IRB-approved protocol for a cross-sectional single time point biospecimen collection. Patients with established cGVHD (n=29) or ≥2 years post-HCT without clinical manifestations of cGVHD (n=7) were included. Patients with cGVHD were divided into Scleroderma cGVHD (n=14) and Non-scleroderma cGVHD (n=15). Median age was 49 (range: 21-69) and 56 years (range: 18-72) for patients with scleroderma (group 1) and without scleroderma or no GVHD (group 2), respectively. Median prior lines of therapy was 3.2 (range: 2-5) for patients in group 1. All patients is group 1 and 77% of patients in group 2 received PBCSs as the graft source. Transplant was from a matched unrelated (group 1: 75%, group 2: 73%) or a matched sibling donor (group 1: 25%, group 2: 23%). Tacrolimus/sirolimus was the most commonly used GVHD prophylactic regimen in both groups (group 1: 83% and group 2: 64%). Peripheral blood samples were drawn at a single time point post-HCT, and serum samples were used for ELISA assays for levels of total antioxidants, 8-OHdG, and CXCL4. Flow cytometric analysis was performed to investigate percentage of Tregs and pH2AX+ cells. There were no differences in the total antioxidant levels, CXCL4 and Tregs between groups 1 and 2. Using a cutoff threshold of 10 ng/ml of 8-OHdG, 11 patients (78%) in group 1 and 9 patients (40.9%) in groups 2 had elevated levels of 8-OHdG (p=0.029, Fisher's exact). Percentage of pH2AX expressing cells was lower in group 2. At the cutoff threshold of 37%, only one patient (7%) in group 1 and 13 patients (59%) in group 2 had more than 37% pH2AX-expressing CD45+ cells in their peripheral blood (p=0.002, Fisher's exact). In conclusion, our pilot cross-sectional study showed that scleroderma cGVHD is associated with increased ROS levels (8-OHdG) without elevation of pH2AX cells in peripheral blood T cells. Our results indicate that ROS elevation in scleroderma patients may be a stress response to inflammatory milieu and is independent of DNA damage. Our data justify further prospective cohort studies to define the role of ROS markers in scleroderma cGVHD and to develop novel strategies to treat/prevent cGVHD.