Gene expression in patients with no steroid * treatment

In order to eliminate the effects of steroid treatment on the gene regulation in the patient biopsies, we separated the patients that were not treated with steroids or those who were treated with a dosage of ≤ 20 mg/kg steroids (n=69), from those who underwent steroid treatment. This cut off value for the two groups was pre-defined by clinicians. Within the group of patients with no steroid (or low) treatment, we studied the changes in gene expression patterns based on the different variables.

* Patients that were treated with a 20 mg/kg or less steroid dosage and patients that were not treated with steroids.

Genes t-value p-value ( < 0.05)

IL33 -2.400 0.018

p-value (<0.15)

CXCL16 1.647 0.120

CXCL8 -1.522 0.130

LGALS7 1.542 0.125

CD226

(DNAM-1) -1.762 0.081

HCLS1 -1.840 0.068

3.5.3.1 Effect of clinical GI GvHD grade on gene expression patterns of the selected genes in patients with no steroid treatment

In table 3.8, we compared the gene expression during clinical GI aGvHD in patients with no or less steroids. Several genes were significantly increased based on the clinical scores in patients with aGvHD. Genes that were most strongly regulated were the chemokine receptors, CCR5, CCR4, CCR1 and CXCR3. In addition, CXCR4 and the chemokines CCL4, CCL5 and CXCL8 were significantly increased. The Th1 and Th2 cytokines IL2 and IL6 respectively were also increased.

Other regulated genes included genes associated with activation and regulation of Tregs, IL2RA and TGFB1, genes associated with activation of B and T cells, PSTPIP1, PIK3AP1, PTPN7, HCLS1, FCGR3 and FCER1G and genes regulated by IFN-γ, LST1 and MSR1.

Considering most of these genes are not regulated based on the histological scores, it is perceivable that these genes are important for the clinical symptoms in patients such as skin rash, weight loss etc., most likely due to inflammation, and a heightened immune response post transplant. Activation of alloreactive T cells and infiltration of T cells in the GI tissue, as a result of inflammation, explains the heightened clinical symptoms. Most of the regulated genes were also regulated in patients with steroids (Table 3.4), suggesting that the administration of steroids had no or little effect on the regulation of these genes. The main difference between the two groups was in the regulation of MSR1, the macrophage scavenger receptor 1. It could be that infiltration of macrophages in the GI tissue, in addition to other immune cells results in the clinical aGvHD symptoms in the patients.

Table 3.8: Genes regulated in clinically scored GI aGvHD (grade 1-4) biopsies versus no aGvHD (grade 0) in patients with no steroids*. Genes significantly regulated (p < 0.05) based on histological aGvHD in human GI biopsies. Additionally, genes that show a trend of significance ( p < 0.15; n.s.) are listed. t-values represent the size of the relative difference to variation of the sample data; t > 0 shows upregulation and t < 0 shows downregulation. ^*Patient group consisting of patients who were treated with a 20 mg/kg or less steroid dosage and those who were not treated

3.5.3.2 Effect of histological aGvHD score on gene expression patterns of the selected genes in patients with no steroid treatment

3.5.3.2.1 Histological aGvHD grades 2-4 versus no or low aGvHD (grades 0-1)

Table 3.9: Genes regulated in severe histological GI aGvHD (grade 2-4) versus no or low aGvHD (grade 0-1) in patients with no steroids*. Genes significantly regulated (p < 0.05) based on histological aGvHD in human GI biopsies. Additionally, genes that show a trend of significance ( p < 0.15; n.s.) are listed. t-values represent the size of the relative difference to variation of the sample data; t > 0 shows upregulation and t < 0 shows downregulation.

*Patients group consisting of patients who were treated with a 20 mg/kg or less steroid dosage and those who were not treated with steroids.

Since the administration of steroids in patients, as part of treatment post-transplant has a significant effect on the gene expression, we studied the regulation of our selected genes in patients that were not given steroids. In patient biopsies with histological grade 2-4, we observed a significant upregulation of the Th2 cytokine, IL4 and the chemokines and chemokine receptor, CX3CR1 and CXCL16 respectively. The chemokines CCL4 and CXCL8, and PSTPIP1 were decreased in patients with severe aGvHD. Moreover, LILRA5, CXCR4 and CXCL9 were decreased in patients with severe aGvHD but did not reach statistical significance (Table 3.9). Interestingly, these genes were regulated in the same way even in the presence of steroids, suggesting that the presence of steroids did not have a significant effect on these genes. On the other hand, the genes ENPP1 and the NKG2D ligand, ULBP3 were significantly regulated in the presence of steroids (Table 3.5).

No significant changes in the expression of genes were observed in this subgroup analysis between patients with histological aGvHD (grade 1-4) and patients without aGvHD (grade 0).

Genes t-value p-value ( < 0.05)

CX3CR1 3.173 0.0018

CXCL8 -2.988 0.003

CCL4 -2.638 0.009

CXCL16 2.159 0.032

IL4 2.058 0.041

PSTPIP1 -1.973 0.05

p-value (<0.15)

LILRA5 -1.914 0.057

CXCR4 -1.612 0.109

CXCL9 -1.516 0.138

3.5.3.3 Effect of gene regulation on transplant related mortality (TRM) in HSCT patients with no steroid treatment

Table 3.10: Effect of gene regulation on TRM in patients with no steroids. Genes significantly regulated (p < 0.05) based on histological aGvHD in human GI biopsies.

Additionally, genes that show a trend of significance ( p < 0.15; n.s.) are listed. t-values represent the size of the relative difference to variation of the sample data; t > 0 shows upregulation and t < 0 shows downregulation. ^* Patients group consisting of patients who were treated with a 20 mg/kg or less steroid dosage and those who were not treated with steroids.

The presence of steroids had a significant effect on regulation of genes associated with TRM.

Several genes were regulated differently in patients that died due to transplant when no steroids were administered. The DNAM-1 ligand, PVRL2 was higher in patients that died due to transplant compared to patients that were still alive or those that died due to other causes. Moreover, several genes associated with innate immune responses, such as C1QTNF7, LGALS7 and HTRA1 were also increased. The expression of several genes that were associated with clinical aGvHD symptoms in patients (Table 3.8), were also correlated with death due to transplant, such as HTRA1, CXCL8, TGFB1, IL2RA and PSTPIP1 (Table 3.10).