Genetic Risk of Severe Chronic Graft-Versus-Host Disease Defined By Host-Derived CXCR3 Ligands
Authors: Dai Hao , Rachakonda Sivaramakrishna P. , Penack Olaf , Radujkovic Aleksandar , Müller-Tidow Carsten , Blau Olga , Blau Igor-Wolfgang , Kumar Rajiv , Dreger Peter , Luft Thomas 
Published: October 2019

Abstract
Introduction
Severe chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and mortality in long-term survivors after allogeneic stem cell transplantation (alloSCT). The CXCR3 signalling pathway may be implicated in cGVHD pathophysiology, since CXCR3 and its ligands (CXCL4, CXCL9, CXCL10 and CXCL11) are involved in attracting activated Th1 cells into inflamed tissues. To better understand the role of the CXCR3 axis in cGVHD, we measured serum levels of CXCR3 ligands in allograft recipients pre-transplant and on day 28 post-transplant, and correlated them to the single nucleotide polymorphisms (SNPs) in recipient CXCR3/CXCR3L genes in the context of severe cGVHD.
Patients and methods:
287 patients who were allografted at Heidelberg University Hospital, survived more than 6 months after alloSCT, and did not receive statin-based endothelial prophylaxis (SEP) constituted the no-SEP training cohort, whereas 401 patients who received SEP constituted the SEP cohort. DNA for genotyping was available for 545 patients (no-SEP 242, SEP 303) and sera for measuring CXCL9, CXCL10 and CXCL11 by ELISA were collected at pre-transplant for 405 patients (no-SEP 109, SEP 296) and at day +28 for 494 patients (no-SEP 152, SEP 342). The no-SEP validation cohort consisted of 202 patients who had been allografted at Berlin Charité and survived more than 6 months. CGVHD was diagnosed and graded using the National Institutes of Health's 2005 consensus criteria.
Eighteen SNPs (7 in CXCL9-11, 7 in CXCL4 and 4 in CXCR3 loci) were selected and analyzed for association with severe cGVHD, treating non-relapse death and relapse without severe cGVHD as competing events. Associations of SNPs with serum chemokine levels were studied by Mann-Whitney U-test. Hazard ratios (HR) with 95% confidence interval (CI) were estimated using (cause-specific) Cox regression. Covariates considered in multivariate analysis were age, diagnosis, donor type, sex of donor and recipient and usage of ATG.
Results:
Overall, 50 of 287 patients (17.4%) in the no-SEP training cohort, 53 of 401 patients (13.2%) in the SEP cohort and 48 of 202 patients (23.8%) in the no-SEP validation cohort developed at least one episode of severe cGVHD. In the no-SEP training cohort, higher serum CXCL9 levels at day +28 were significantly associated with a higher risk of severe cGVHD in univariate analysis (HR 1.38 for every log2-fold change, 95% CI 1.10-1.75, P=0.01; Figure 1a). No significant association was found for serum CXCL10 and CXCL11 pre- or post-transplant with severe cGVHD.
The rs884304 SNP in CXCL9-11 locus showed a significant association with severe cGVHD; patients with AA/AG genotypes carried a HR of 2.32 (95%CI 1.21-4.46, P=0.01) compared to patients with GG genotypes. In addition, 3 other SNPs (rs3733236 and rs4282209 in CXCL9-11, rs655328 in CXCL4 loci) were selected based on the effect on severe cGVHD (P <0.10) to calculate a combined genetic risk score. Patients with any low-risk genotypes (rs884304GG, rs3733236AA/AG, rs4282209AA/AG and rs655328TT) were classified as the low-risk. All others were considered as high-risk. Taken together, high-risk patients were found in 21.4% (52/242) of the no-SEP training cohort, 22.4% (68/303) of the SEP cohort and 19.8% (40/202) of the no-SEP validation cohort. Patients in the high-risk group had significantly higher serum CXCL9 levels at day +28 (Figure 1b) and a significantly higher risk of severe cGVHD (Figure 1c) on both univariate (HR 2.68, 95%CI 1.45-4.95, P=0.001) and multivariate analyses (HR 2.49, 95%CI 1.33-4.66, P=0.004). The effect of the combined score was confirmed in the no-SEP validation cohort (HR 3.02, 95%CI 1.60-5.72, P=0.001).
In contrast, in the SEP cohort the adverse effect of high risk genotypes was not observed (HR 1.30, 95% CI 0.60-2.79, P=0.50). In addition, SEP reduced day +28 CXCL9 levels in patients with high-risk genotype but not in low-risk patients.
Conclusion:
In the absence of SEP, the risk of severe cGVHD could be predicted both by a genetic score of 4 SNPs in recipient CXCR3L genes and by serum CXCL9 levels at day +28. The genetic score influenced serum CXCL9 levels at day +28. Our results suggest that in high-risk patients, host-derived CXCR3 ligands are upregulated early after alloSCT and may promote the development of severe cGVHD. Endothelial prophylaxis may reduce the risk of severe cGVHD by regulating serum CXCL9 levels and, thus, warrants further study.

Disclosures
No relevant conflicts of interest to declare.