Frequency of interleukin 6 polymorphisms
The IL6-174 polymorphism was examined in 300 recipients and 295 donors. The IL6-597 polymorphism was analyzed in 299 recipients and 296 donors.
153 patients (47.8%) had the GC genotype of the IL6-174 SNP, 85 patients (26.6%) the GG genotype and 62 patients (19.4%) the CC genotype. In 20 cases, genotype was not available.144 donors (45.0%) were heterozygous for the IL6-174 GC genotype. 96 donors (30.0%) were homozygous for the GG genotype and 55 donors (17.2%) for the CC genotype. In 25 cases, DNA was not available for genotyping.
We found the IL6-597 GA, GG and AA genotypes in 149 recipients (46.6%), 91 recipients (28.4%) and 59 recipients (18.4%), respectively. We observed the GA genotype of the IL6-597 in 147 donors (45.9%), the GG genotype in 96 donors (30.0%) and the AA genotype in 53 donors (16.6%). In 21 recipients and 24 donors, DNA was not available for genotyping.
An overview of the genotypes’ frequencies is presented in Table 2.
The distribution of genotypes was similar to those reported in the literature (Ambruzova et al. 2009; Mullighan et al. 2004; Gao et al. 2014).
The comparison of the distribution of the SNPs revealed a strong linkage disequilibrium between IL6-174 GG/ IL6-597 GG, IL6-174 GC/IL6-597 GA and IL6-174 CC/ IL6-597 AA in recipients and donors. Only in 10 recipients (3.1%) and 8 donors (2.5%), the above-mentioned genotypes were not linked. This result is in line with the findings of Muller-Steinhardt et al. (2004).
Frequency of graft-versus-host disease
We observed acute GVHD in 145 patients (45.3%). 88 patients (27.5%) had a moderate to severe acute GVHD (grade II to IV) and 31 patients (9.7%) had a severe acute GVHD (grade III to IV). The median time to onset of acute GVHD was 28 days.
Chronic GVHD occurred in 52 patients which led to a chronic GVHD incidence of 16.3%. 37 patients (11.6%) developed an extended form of chronic GVHD and 15 patients (4.7%) a limited form of chronic GVHD. The median number of days between allogeneic HSCT and onset of chronic GVHD was 164 days.
Genetic associations with acute graft-versus-host disease
To investigate a possible association between recipients’ and donors’ IL6-174 and IL6-597 SNPs and the occurrence of acute GVHD, we compared the proportion of genotypes between recipients with clinically significant acute GVHD (grade II to IV) and severe acute GVHD (grade III to IV). We observed a significantly increased incidence of moderate to severe acute GVHD (grade II to IV) in recipients with IL6-174 GG genotype compared to those with IL6-174 GC/CC genotype (GG vs. GC/CC; P=0.024; Figure 1). Furthermore, patients with IL6-597 GG genotype developed grade II to IV acute GVHD more frequently than individuals with the GA or AA genotype (GG vs. GA vs. AA; P=0.013; Figure 2). Severe acute GVHD (grade III to IV) was significantly more frequent in patients with IL6-597 GG genotype than in those with IL6-597 GA/AA genotype (GG vs. GA/AA; P=0.042). Analysis of the influence of donor SNPs on the occurrence of acute GVHD did not reveal any significant results. In summary, for both IL6 polymorphisms a recipient GG genotype was associated with an increased risk of acute GVHD. The donor genotype had no influence on the occurrence of acute GVHD.
Genetic associations with chronic graft-versus-host disease
The cytokine gene polymorphisms showed an association with the occurrence of chronic GVHD. Recipients with the IL6-174 GG genotype developed chronic GVHD more frequently than individuals with the C allele (GG vs. GC/CC; P=0.049; Figure 3). The chronic GVHD was present in 19 out of 85 patients (22.4%) with the GG genotype compared to 29 out of 215 patients (13.5%) with the GC/CC genotype. Similarly, we observed a significant association with an increased risk of chronic GVHD in recipients with IL6-597 GG genotype compared with patients with the other genotypes (GG vs. GA vs. AA; P=0.043; GG vs. GA/AA; P= 0.012; Figure 4). A total of 49 in the group of recipients’ IL6-597 SNP developed chronic GVHD: 22 of 91 patients (24.2%) with the GG genotype, 8 of 59 patients (13.6%) with the AA genotype and 19 of 149 patients (12.8%) with the GA genotype. In our study, the genotype of donors did not affect the incidence of chronic GVHD.
In addition to the occurrence of acute GVHD and chronic GVHD, we investigated the influence of the IL6-174 and IL6-597 polymorphisms on OS, EFS, RI and TRM. The two SNPs had no significant impact on OS of the patients. We found no association between the polymorphisms and the EFS. There was no relationship between RI or TRM and the IL6-174 and IL6-597 SNPs.
Multivariate analysis was performed to examine the significance of clinical factors potentially influencing the occurrence of acute GVHD and chronic GVHD. To evaluate whether IL6-174 and IL6-597 SNPs are independent prognostic factors, we included following possible confounding variables: age at time of transplantation, donor-recipient-gender match, source of stem cells, and HLA compatibility.
In comparison with the above-mentioned variables, the recipients’ IL6-174 polymorphism remained to be an independent significant risk factor for the occurrence of acute GVHD (P=0.030) and chronic GVHD (P=0.045). The results of recipients’ IL6-174 polymorphism and acute GVHD and chronic GVHD are presented in Table 3. Furthermore, the IL6-597 SNP of the recipients turned out to be an independent significant prognostic factor for acute GVHD (P=0.007) and chronic GVHD (P=0.015). The results of recipients’ IL6-597 polymorphism and acute GVHD and chronic GVHD are shown in Table 4.
In addition to these findings, age at time of transplantation was a significant risk factor for the occurrence of chronic GVHD (P=0.003). This is in line with the reported observation that older patient age is associated with an increased development of chronic GVHD (Lee et al. 2003; MacDonald et al. 2017).