Yesilda Balavarca

KIR genes and KIR ligands affect occurrence of acute GVHD after unrelated, 12/12 HLA matched, hematopoietic stem cell transplantation

Interactions of polymorphic killer Ig-like receptor (KIR) receptors with KIR ligands have been shown to modify the outcome of hematopoietic SCT (HSCT). The association of these genetic factors with different transplantation endpoints, however, varies substantially, depending on clinical and study setup variables. We aimed to assess whether KIR ligands, KIR genes and KIR haplotypes are associated with HSCT outcome of 124 patients with various hematological malignancies, transplanted with 12/12 HLA matched grafts from unrelated donors. For this purpose, patient and donor KIR gene and KIR ligand polymorphisms were determined and correlated with clinical data in simple and multiple models. We found that a missing HLA-C2 ligand for donor inhibitory KIR2DL1 was significantly associated with an increased risk of acute GVHD (aGVHD) (II–IV) (hazard ratio (HR)=2.23, 95% confidence interval (95% CI): 1.21–4.10, P=0.010), as were the AA KIR haplotypes in patients and donors in HLA-C1CX (HR=2.37, 95% CI: 1.16–4.84, P=0.018) and in HLA-Bw4− (HR=3.20, 95% CI: 1.35–7.60, P=0.008) patients. On the contrary, transplantation of HLA-C1C2 patients with KIR2DS2 positive grafts were associated with a decreased risk of aGVHD (II–IV) (HR=0.24, 95% CI: 0.07–0.85, P=0.027). Thus, our single center study provides evidence for the modification of aGVHD risk by KIRs and their ligands.

The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

The MHC class I chain‐related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)‐cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA‐129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft‐versus‐host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA‐129Val/Val genotype carriers was improved when treated with anti‐thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA‐129Met isoform was characterized by stronger NKG2D signaling, triggering more NK‐cell cytotoxicity and interferon‐γ release, and faster co‐stimulation of CD8+ T cells. The MICA‐129Met variant also induced a faster and stronger down‐regulation of NKG2D on NK and CD8+ T cells than the MICA‐129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA‐129Met variants appeared to reduce the severity of aGVHD.

Impact of HLA-DPB1 allelic and single amino acid mismatches on HSCT

The interpretation of the role of HLA‐DPB1 in unrelated haematopoietic stem cell transplantation (HSCT) is subject to discussion. We have investigated the role of HLA‐DPB1 allele matching in HSCT outcomes in 161 recipients who were HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1‐matched with their unrelated donors at the allelic level (10/10). In addition, we analysed the association of polymorphic amino acid mismatches of DPB1 molecule with HSCT end‐points, and a previously published permissiveness concept. HLA‐DPB1 allele mismatches were significantly associated with an increased incidence of acute graft‐versus‐host disease (aGvHD) and worse overall survival (OS). The mismatch at amino acid position 69 significantly increased the risk for transplant‐related mortality (TRM). Risk factors for aGvHD also included mismatches at positions 8, 9, 35, 76 and 84. This is to our knowledge, the first report of an in vivo effect of single amino acid mismatches on HSCT outcomes. In this study, grouping of allelic mismatches into permissive and non‐permissive categories and their association with transplantation end‐points was relevant for TRM but not for other clinical end‐points.

Association of HLA-E polymorphism with the outcome of hematopoietic stem-cell transplantation with unrelated donors.

HLA-E is a nonclassical human leukocyte antigen (HLA) class I gene with two antithetical products HLAE*0101 and HLA-E*0103. They are found on most tissues (1), HLA-E*0103 being expressed at considerably higher levels than HLA-E*0101. These differences depend on the affinity for available peptides and on the stability of refolded complexes (2). We correlated the HLA-E polymorphism with the outcome of hematopoietic stem-cell transplantation (HSCT) with unrelated donors. The median follow-up time of 124 patients transplanted at the University Hospital in Vienna between September 1995 and December 2005 was 80 months (range 35–158 months). Clinical endpoints were acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD), transplant-related mortality (TRM), relapse, and overall survival. Finally, 121 patients were evaluated for aGvHD and 102 for cGvHD. All patient donor pairs were matched for HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 alleles at four digit resolution. Patient and donor characteristics used in the statistical models are summarized in Table 1. Donor selection criteria, assessment of aGvHD and cGvHD, and supportive care during HSCT have been previously described (3). Statistical analyses comprised simple and multiple Cox regression models and competing risk analyses (4). HLA-E typing was performed by sequencing based typing of axon 3. The distribution of HLA-E alleles was similar in patients (HLA-E*0101: n 45, 37%; HLA-E*0101, *0103: n 53, 43%; HLA-E*0103: n 24, 20%) and donors (HLA-E*0101: n 38, 31%; HLA-E*0101, *0103: n 56, 46%; HLAE*0103: n 28, 23%). Two patients and two donors could not be typed due to missing DNA samples. Simple and competing risk analyses did not show evidence of an association of either of the analyzed genetic factors with clinical endpoints. Multiple regression analyses, however, showed associations with aGvHD (II–IV) (cumulative incidence at 80 days 41%, 95% confidence interval[CI]30–49), overall cGvHD (cumulative incidence at 3 years 39%, 95% CI 28–48), relapse (cumulative incidence at 3 years, 39% 95% CI 29– 48), and TRM (cumulative incidence at 180 days 17%, 95% CI 10–23). As to GvHD, HLA-E*0103, *0103 in donors was associated with a decreased risk of aGvHD (II–IV) (hazards ratio [HR] 0.39, 95% CI 0.16 – 0.99, P 0.047), whereas HLA-E*0101, *0103 in donors was associated with a decreased risk of overall cGvHD (HR 0.36, 95% CI 0.14 – 0.90, P 0.030). Regarding relapse, HLA-E*0103 alleles were associated with a higher risk (HR 2.24, 95% CI 1.03– 4.88, P 0.042). Concerning TRM, HLA-E*0103, *0103 in donors was a risk factor (HR 3.94, 95% CI 1.03– 15.02, P 0.045), whereas the presence of HLA-E*0101 alleles was protective (HR 0.32, 95% CI 0.11– 0.94, P 0.037). In summary, the presence of HLA-E*0103 in donors is associated with decreased risks of aGvHD (II–IV) and cGvHD and an increased risk of relapse. Regarding early posttransplant effects, HLA-E*0103 is associated with an increased risk of TRM. In addition to the genetic factors, multiple analyses showed significantly increasedriskofTRMinpatientswithaGvHD (I–IV), in patients at high-risk disease stage at transplantation, and in patients with donors older than 27 years (data not shown). Tamouza et al. (5) were the first to describe an association of HLA-E polymorphism with TRM in HSCT with unrelated donors. In their study, however, they found an association of HLA-E*0101, *0101 with increased TRM. This discrepancy might be due to differences between study populations (Tamouza et al. included also pediatric patients) and clinical protocols used. Especially, patients with bone marrow failure syndromes were more frequent in the Tamouza et al. study (27% vs. 0.8% in our patients). Patients suffering from bone marrow failure syndromes have high mortality rates after unrelated donor HSCT (6). The source of stem cells represents another difference (7). Although in our study, 56.5% of patients received peripheral blood stem cells, all patients in the study of Tamouza et al. had been transplanted with bone marrow-derived cells, known to prolong the time to hematologic and immunologic reconstitution and, therefore, prolong susceptibility to infections. Even though HLA-E*0103 allele cell surface expression exceeds that of HLAE*0101 (8), a similar correlation in the effectiveness of viral or bacterial antigen presentation has not been shown. Moreover, an impaired efficiency of HLA-E*0103 alleles in minor histocompatibility antigen presentation and T-lymphocyte stimulation hasbeenpostulated(9). Inefficientpresentation of minor histocompatibility antigens by HLA-E*0103 would be consistent with less aGvHD, an increased relapse rate, and increased TRM due to infections, as observed in our patients. Although our donor selection was based on a 12 of 12 allelic match, 47% of the pairs were mismatched for HLA-E alleles indicating different haplotypes. Therefore, it is possible that HLA-E serves as a surrogate marker for adjacent polymorphic loci that confer the effects we have observed. Katarina Ludajic Agathe Rosenmayr Ingrid Faé Gottfried F. Fischer Division of Blood Group Serology Medical University of Vienna Vienna, Austria

Impact of genomic risk factors on outcome after hematopoietic stem cell transplantation for patients with chronic myeloid leukemia

Background Non-HLA gene polymorphisms have been shown to influence outcome after allogeneic hematopoietic stem cell transplantation. Results were derived from heterogeneous, small populations and their value remains a matter of debate. Design and Methods In this study, we assessed the effect of single nucleotide polymorphisms in genes for interleukin 1 receptor antagonist (IL1RN), interleukin 4 (IL4), interleukin 6 (IL6), interleukin 10 (IL10), interferon (IFNG), tumor necrosis factor (TNF) and the cell surface receptors tumor necrosis factor receptor II (TNFRSFIB), vitamin D receptor (VDR) and estrogen receptor alpha (ESR1) in a homogeneous cohort of 228 HLA identical sibling transplants for chronic myeloid leukemia. Three good predictors of overall survival, identified via statistical methods including Cox regression analysis, were investigated for their effects on transplant-related mortality and relapse. Predictive power was assessed after integration into the established European Group for Blood and Marrow Transplantation (EBMT) risk score. Results Absence of patient TNFRSFIB 196R, absence of donor IL10 ATA/ACC and presence of donor IL1RN allele 2 genotypes were associated with increased transplantation-related mortality and decreased survival. Application of prediction error and concordance index statistics gave evidence that integration improved the EBMT risk score. Conclusions Non-HLA genotypes were associated with survival after allogeneic hematopoietic stem cell transplantation. When three genetic polymorphisms were added into the EBMT risk model they improved the goodness of fit. Non-HLA genotyping could, therefore, be used to improve donor selection algorithms and risk assessment prior to allogeneic hematopoietic stem cell transplantation.

Minor ABO-Mismatches are Risk Factors for Acute Graft-versus-Host Disease in Hematopoietic Stem Cell Transplant Patients

We investigated the impact of ABO and Rhesus (Rh) blood group matching on the outcome of hematopoietic stem cell transplantation (HSCT) of 154 patients matched at 10/10 HLA loci with unrelated donors. ABO and Rh, as potential risk factors, were modeled with the clinical outcome--acute and chronic graft-versus-host disease (aGVHD, cGVHD), relapse, treatment-related mortality (TRM), and overall survival (OS)--by simple, multiple, and competing risk analyses. We found that minor ABO-mismatches represent a significant risk factor for aGVHD (II-IV) with an estimated risk increase of almost 3-fold (hazard ratio [HR]=2.92, 95% confidence interval [CI]: 1.43-5.95, P=.003), and even 4-fold for aGVHD (III-IV) (HR=4.24, 95% CI: 1.70-10.56, P=.002), but not for other transplant endpoints. No significant association of the Rh matching status with any of the HSCT endpoints was seen. These results suggest that ABO minor mismatches may play a role in aGvHD pathophysiology, possibly by providing the setting for T cell activation and antibody mediated damage. To decrease the risk of aGVHD, ABO matching should be considered in HSCT.

Correlation of Hsp70-1 and Hsp70-2 Gene Expression With the Degree of Graft-Versus-Host Reaction in a Rat Skin Explant Model

Background. Graft-versus-host disease (GVHD) is the most serious complication after allogeneic hematopoietic stem-cell transplantation. A human skin explant assay has been used to predict the risk of GVHD in patients by histological grading of graft-versus-host reactions (GVHR). New molecular markers of GVHR might help to further increase the predictive value of the assay. Methods. A rat skin explant assay has been developed to further aid in identifying potential novel molecular markers. Results. In inbred rat strains GVHR were observed in skin explants co-cultured with allogeneic lymphocytes stimulated against minor or major histocompatibility antigens. The histological signs of GVHR were similar to those observed in human skin explant assays and acute GVHD lesions occurring in rats after experimental bone marrow transplantation. Heat shock protein (HSP) 70 has been shown to be expressed during GVHR. We therefore investigated the expression of the three major histocompatibility complex (MHC)-linked HSP70 genes in rat skin explants. The two major stress-inducible genes Hsp70-1 and Hsp70-2 were found to be upregulated in the allogeneic rat skin explant assays. The increase in mRNA correlated with the GVHR grade (I–IV). Interestingly, the expression of the third MHC-linked Hsp70 gene Hsp70-3 was not found to be augmented during GVHR. Conclusion. The observed induction of the MHC-encoded Hsp70-1 and Hsp70-2 genes might serve as new markers of GVHR and as potentially novel diagnostic tools for GVHD.

Predicting survival using clinical risk scores and non-HLA immunogenetics

Previous studies of non-histocompatibility leukocyte antigen (HLA) gene single-nucleotide polymorphisms (SNPs) on subgroups of patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) revealed an association with transplant outcome. This study further evaluated the association of non-HLA polymorphisms with overall survival in a cohort of 762 HSCT patients using data on 26 polymorphisms in 16 non-HLA genes. When viewed in addition to an already established clinical risk score (EBMT-score), three polymorphisms: rs8177374 in the gene for MyD88-adapter-like (MAL; P=0.026), rs9340799 in the oestrogen receptor gene (ESR; P=0.003) and rs1800795 in interleukin-6 (IL-6; P=0.007) were found to be associated with reduced overall survival, whereas the haplo-genotype (ACC/ACC) in IL-10 was protective (P=0.02). The addition of these non-HLA polymorphisms in a Cox regression model alongside the EBMT-score improved discrimination between risk groups and increased the level of prediction compared with the EBMT-score alone (gain in prediction capability for EBMT-genetic-score 10.8%). Results also demonstrated how changes in clinical practice through time have altered the effects of non-HLA analysis. The study illustrates the significance of non-HLA genotyping prior to HSCT and the importance of further investigation into non-HLA gene polymorphisms in risk prediction.

Tests for candidate-gene interaction for longitudinal quantitative traits measured in a large cohort.

For the Framingham Heart Study (FHS) and simulated FHS (FHSsim) data, we tested for gene-gene interaction in quantitative traits employing a longitudinal nonparametric association test (LNPT) and, for comparison, a survival analysis. We report results for the Offspring Cohort by LNPT analysis and on all longitudinal cohorts by survival analysis with cohort effect adjustment. We verified that type I errors were not inflated. We compared the power of both methods to detect in FHSsim data two sets of gene pairs that interact for the trait coronary artery calcification. In FHS, we tested eight gene pairs from a list of candidate genes for interaction effects on body mass index. Both methods found evidence for pairwise non-additive effects of mutations in the genes FTO, PON1, and PFKP on body mass index.

Impact of genomic risk factors on survival after haematopoietic stem cell transplantation for patients with acute leukaemia.

The EBMT risk score is an established tool successfully used in the prognosis of survival post‐HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non‐HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non‐HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat‐shock protein 70‐hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non‐HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival.