Cytokines associated with organ manifestations in chronic graft-versus-host disease

Prior studies have identified cytokines associated with the general syndrome of chronic graft-versus-host disease (cGVHD). The goal of this cross-sectional study was to see if these biomarkers were associated with specific organ manifestations. Patients with cGVHD (N=529) from Chronic GVHD Consortium studies with available clinical data and plasma samples had the following predefined cytokines measured by Luminex: Th1 (IFNa, IFNg, TNFa, IL-2, IL-1b, IL-12, IL-12/IL-23p40, IL-18), Th2 (IL-4, IL-10, IL-13), Th17/Tc17 (IL-6, IL-17A, IL-21, IL-23), B cell (BAFF), and monocyte factors (M-CSF, MCP-1, GM-CSF). Chemokines (IL-8, CXCL9, CXCL10), ST2 and REG3a were also measured. Of these, we tested IL-12p70, IL-12/IL-23p40, IL-18, IL-6, IL-21, BAFF, M-CSF, MCP-1, GM-CSF, IL-8, CXCL9, CXCL10, ST2, and REG3a for association with GVHD clinical features. The other cytokines were not tested further due to low levels. At time of blood draw, median age was 53 years (IQR 40, 62), median time from HCT to draw was 11.8 months (mos) (IQR 7.2, 22) and median time from cGVHD diagnosis to draw 0.9 (IQR 0.2-9.2) mos. 63% were within 3 mos of cGVHD diagnosis and 39% were female. Donors were 50% HLA-matched unrelated, 32% matched related and the rest umbilical cord blood, haploidentical or mismatched unrelated donors. 90% received peripheral blood grafts. Methotrexate and calcineurin inhibitor acute GVHD (aGVHD) prophylaxis was used for 53%. Half had prior grade II-IV aGVHD. 246 (47%) had moderate and 189 (36%) had severe cGVHD with the following organ involvement: skin (67%), mouth (60%), eye (50%), joint (36%), GI (31%), lung (24%), and liver (18%). Correlations of cytokine levels with organ involvement were tested using logistic regression (involved vs. not involved) for liver and lung cGVHD due to low incidence and prior grade II-IV aGVHD, and ordinal logistic regression (severity of organ involvement vs. not involved) for skin mouth, eye, joint, GI, and overall cGVHD severity, with p≤0.05 considered significant after Benjamini-Hochberg adjustment. After adjustment for batch effects, 8 cytokines were associated with GVHD characteristics in univariate analysis and are presented as (OR, 95% confidence intervals): Increased IL-18 (Th1 cytokine) was associated with liver cGVHD (1.66, 95% CI 1.33-2.09) and more severe overall cGVHD (1.28, 1.09-1.50). Increased IL-6 (Th17/Tc17 cytokine) was associated with skin (1.48, 95% confidence interval, 1.27-1.74), joint (1.33, 1.12-1.57) and lung cGVHD (1.38, 1.14-1.68) and more severe overall cGVHD severity (1.37, 1.16-1.62). Elevated IL-12/IL-23p40 was negatively associated with lung cGVHD (OR 0.75, 95% CI 0.61-0.91) and this was not seen with IL-12p70. Increased BAFF (B cell cytokine) was associated with skin (1.25, 1.07-1.46) and liver cGVHD (1.36, 1.10-1.70). Increased MCP-1 (monocyte cytokine) was associated with lung cGVHD (1.38, 1.12-1.71) but less mouth involvement (0.79, 0.67-0.93). Increased IL-8 (chemokine) was associated with liver (1.66, 1.35-2.05) and GI cGVHD (1.36, 1.14-1.62). Increased CXCL9 and CXCL10 (chemokines) were associated, respectively, with skin cGVHD (1.29, 1.10-1.50 and 1.33, 1.14-1.55), mouth (1.39, 1.18-1.64 and 1.36, 1.16-1.61) and liver cGVHD (1.41, 1.13-1.76 and 1.45, 1.15-1.84). Increased REG3a was associated with GI cGVHD (1.75, 1.45-2.13) but negatively associated with joint cGVHD (0.67, 0.56-0.81). Grade II-IV aGVHD was not associated with any of the cytokines. No associations were identified for IL-12p70, IL-21, M-CSF, GM-CSF, ST2, or eye cGVHD. In conclusion, in this large cohort of patients with cGVHD, we identified T cell, B cell and monocyte cytokines and chemokines associated with specific organ manifestations and overall severity of cGVHD. Testing of additional samples is ongoing. Future analyses will include multivariate adjustment for other clinical characteristics and testing for biomarkers predictive of response to treatment.