Thrombomodulin Has a Significant Impact on Transplant Outcomes after HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Standard Risk Hematologic Malignancies
Authors: Nomoto Haruka , Morishita Eriko , Takami Akiyoshi , Katsu Shiori , Yamaguchi Daisuke , Morishima Yasuo , Onizuka Makoto , Kashiwase Koichi , Fukuda Takahiro , Kodera Yoshihisa , Nakao Shinji , Ohtake Shigeki 
Published: October 2019

Abstract
Background:Endothelial injury is involved in graft-versus-host-disease (GVHD), thrombotic microangiopathy, or sinusoidal obstruction syndrome. Thrombomodulin (THBD) plays a role of anti-coagulation or anti-inflammatory responses, and there are increasing evidences suggesting recombinant human soluble thrombomodulin (rTHBD) is effective on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation. THBD gene has several single nucleotide polymorphisms (SNP) which are associated with the risk of venous thrombotic events or coronary heart disease. In this study, we analyzed the impact of THBD polymorphism A2729C on transplant outcomes in recipients undergoing unrelated HLA-fully-matched T-cell-replete bone marrow transplantation through the Japan Donor Marrow Program and examined allele-specific gene expression of A2729C. Moreover, we investigated the effect of treatment with rTHBD prior to total body irradiation (TBI) on endothelial cells in vitro models.
Methods and Results: The SNP A2729C was analyzed using the TaqMan system (Applied Biosystems), and the results were analyzed using the Allelic Discrimination software program (Applied Biosystems). The THBD polymorphisms were retrospectively analyzed in a cohort of 399 pairs of patients with hematologic malignancies and their unrelated donors. The genotype frequencies of A/A, A/C and C/C were 10%, 35% and 55% in recipients and 51%, 39% and 10% in donors (p=0.41). In standard risk disease group, the recipient C/C genotype was associated with a better overall survival (OS) than the recipient A/A and A/C genotype (p<0.10), while the recipient C/C genotype was not significantly associated with a better transplant-related mortality (TRM) than the recipient A/A and A/C genotype (p=0.11). On the multivariate analysis, the beneficial effects of the recipient C/C genotype remained significant for OS (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.02 to 0.99; p<0.05) and TRM (HR, 4.2x10-5; 95% CI, 1.3x10-5 to 1.4x10-4; p<0.0001). In standard risk disease group, moreover, the donor C/C genotype was associated with a better TRM than the donor A/A and A/C genotype (p<0.10), but was not associated with OS (p=0.80). On the multivariate analysis for TRM, there was the beneficial effect of the donor C/C genotype (HR, 3.6x10-5; 95% CI, 0.30 to 1.6; p<0.0001). The donor/recipient THBD genotype showed no significant effects on the relapse, aGVHD and cGVHD. To further investigate the effect of rs3176123 on transcription of mRNA, we performed Allele-specific transcript quantification on human primary monocytes from 6 healthy donors with heterozygous genotypes of rs3176123. In these cells the mean ratio (C allele vs A allele) was 0.53, which is significantly higher than that of DNA amplicons (ratio 0.50 ; p<0.01). In vitro, human umbilical vein endothelial cells (HUVECs) were pretreated with 1 μg/ml rTHBD for 1 hour, and irradiated with X-rays with single doses of 10 Gy. Total RNA was extracted 3 hours after radiation, and mRNA levels were determined by RT-PCR. Pretreatment with rTHBD significantly inhibited irradiation-induced tissue factor (p<0.03), IL-6 (p<0.02), E-selectin (p<0.03) and ICAM-1 (p<0.05) up-regulation in HUVECs.
Conclusions: These results suggest an association of the recipient THBD genotype with overall survival and TRM after unrelated BMT in the standard risk disease group, and may substantiate that the higher THBD activity by donor with the THBD C allele likely accounts for a reduced risk for TRM in their recipients.These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT.

Disclosures
No relevant conflicts of interest to declare.