Posttransplant lymphoproliferative disorder (PTLD) is a relatively uncommon, but frequently devastating complication of solid organ transplantation. Consequences of PTLD include both high mortality as well as high rates of allograft loss (1). The incidence of PTLD varies with the type of solid organ transplanted (2). The highest incidence is seen in hepatic, cardiac, and intestinal transplantation. There has been a considerable number of reports documenting the association of PTLD with Epstein-Barr virus (EBV) infection, and increased risk of developing PTLD with reactivation of EBV and especially primary EBV infection posttransplant (2). Thus, the incidence of PTLD in EBV seronegative recipients ranges from 23% to 50%, as compared with 0.7% to 1.9% for seropositive recipients (2, 3).
Immunosuppression may be the trigger for PTLD as well. The role of newer immunosuppressive agents in the development of PTLD has been the subject of many studies (3, 4). Of particular interest are the various anti-T lymphocyte antibodies, especially the monoclonal OKT3 antibodies, and tacrolimus (3, 5). Despite similar amounts of immunosuppression only a small portion of patients develop PTLD. The identification of factors predisposing to PTLD development is important for the understanding and management of those patients.
Cytokines are soluble proteins or glycoproteins produced mostly, but not exclusively by leukocytes. They act as immunomodulators and chemical communicators between cells. The clinical outcome of many infectious, autoimmune, or malignant diseases appears to be influenced by the overall balance of production profiles of proinflammatory and anti-inflammatory cytokines (6). Different cytokines have been shown to be involved in the regulation of key pathways of B cell proliferation as well as immunity to intracellular pathogens and malignant cells (7). While transforming growth factor (TGF)-beta and interleukin (IL)-10 modulate B cell activation and/or proliferation (7, 8), interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha contribute to viral defense by their proinflammatory activities (7, 9, 10). Genetic variations including single nucleotide polymorphisms have been identified in a number of cytokine genes (6). Regulation of cytokine levels has been shown to be under genetic control through genetic polymorphisms in their coding and promoter sequences (6).
One of the most important characteristics of PTLD is the time of onset after transplantation. The behavior and features of early onset (<1 year posttransplant) and late-occurring PTLD (development more than 1 year after transplantation) are often distinct (2) and therefore may have different risk factors.
In order to enhance the understanding of the etiology of PTLD and identify potential markers of disease susceptibility, we embarked on a study of genetic polymorphisms in cytokines that may be involved in the pathogenesis of late onset PTLD. Here, we report on the results of IL-10, TNF-alpha, IFN-gamma, and TGF-beta1 gene polymorphisms in a case-control study among solid transplant organ recipients.
MATERIALS AND METHODS
From 1,765 solid transplant patients consecutively admitted or referred from other German transplant units to the Transplant Outpatient Units of the University Hospital Charité, Campus Virchow, Berlin, 446 patients were enrolled in the study. There were 38 solid organ transplant patients with EBV-associated PTLD and 408 transplant controls. For PTLD group, enrollment criteria include: age greater than 18 years, recipient of a solid organ, alive at the time of the interview, no previous diagnosis of cancer, EBV-associated B-cell lymphoma, PTLD onset later than 1 year after transplantation. Inclusion criteria for the control group were: age greater than 18 years, stable graft function, no EBV reactivation, no hematological abnormalities. The patients of the control group were ethnically, gender, age and transplanted organ matched with patients of PTLD. The study was approved by the Ethical Committee of the University Hospital Charite, Berlin. All patients gave an informed consent after being advised of the nature of the study.
Within the PTLD group, the median patient and transplant age was 44.4±8.4 years, and 7±3.8 years respectively (Table 1). A total of 66% were male and 34% were female. Respectively, kidney allograft, heart, and liver transplants were performed in 66%, 13%, and 21% of PTLD patients, respectively.
All patients presented with lymphoma and an elevated EBV load. The diagnosis of lymphomas was made based on histological findings and according to World Health Organization classification. All lymphomas were of B cell origin, 35 patients had non-Hodgkin lymphoma (NHL), and the remaining three patients presented with Hodgkin lymphoma. The most important clinical findings including histological characteristics, disease stage, therapy, and response to the therapy are demonstrated in Table 2.
Control group consisted of 408 unrelated, ethnically matched individuals. The subjects showed no signs of EBV-associated PTLD as determined by the absence of enhanced EBV load and hematological abnormalities. The control patients showed stable graft function. The median patient and transplant age of the control group was 46.8±12.1 and 6.7±4.7 years, respectively. A total of 57% were male and 43% were female. Respectively, kidney allograft, heart, and liver transplants were performed in 70%, 11%, and 19% of the control group, respectively.
All patients were under triple (cyclosporine or tacrolimus+prednisone and antithymocyte globulin or anti-IL-2R-antibody as induction therapy) or quadruple (triple+ mycophenolate mofetil) immunosuppressive regimen.
There were no significant differences regarding the age, type of transplant, sex, immunosuppressive regimen, and the transplant age between the study and the control patients.
DNA was obtained from frozen cells or 10 mL of citrated peripheral blood using a commercially available DNA extraction kit (QIAamp Blood Kit, Qiagen, Germany) according to manufacture instructions. For quality control of results, the samples were run in duplicate. Duplicate results with the concordance rates of 100% were used for statistical analysis.
Analysis of TNF-α, TGF-β, IL-10, and IFN-γ Gene Polymorphism
The polymerase chain reaction-sequence specific primers was performed using commercially available cytokine genotyping primer pack (One Lambda, Canoga Park, CA) according to manufacture instructions. Briefly, 50-100 ng of isolated DNA in the volume of 1 μL were added to the master mix consisted of 7 μL D-Mix, 0.05 μL Tag polymerase (5 units/μL), and 2 μL of appropriate primer. Cycling was performed at 96°C for 130 s, 63°C for 60 s, followed by 9 cycles at 96°C for 10 s, 63°C for 60 s, 20 cycles at 96°C for 10 s, 59°C for 50 s, and 72°C for 30 s. The amplified products were monitored by electrophoresis on a 2% agarose gel containing ethidium bromide (0.5 μg/mL), visualized with ultraviolet light on a transilluminator and photographed.
The occurrence of a genotype remains constant unless matings are nonrandom, inappropriate, or mutations accumulate. Therefore, the frequency of genotypes and the frequency of alleles are said to be at “genetic equilibrium.” Genetic equilibrium is a basic principle of population genetics. We estimated frequencies of genotypes from the observed data. Hardy-Weinberg equilibrium was assessed for the genotype distribution using the equation (p2+2pq+q2=1), where p is defined as the frequency of the allele A and q as the frequency of the allele B for a genotype controlled by a pair of alleles (A and B). Contingency 2×2 tables were constructed and the Yate’s corrected chi-square test was run to determine statistical significance of differences in genotype frequency for the cytokine single nucleotide polymorphisms (SNPs) in control and study patients. P values of <0.05 were considered statistically significant. Odds ratios (OR) with confidence intervals (CI) were also calculated for significant associations. For this purpose, the online interactive statistical calculation page (two-way contingency table analysis) was used. Post-hoc comparisons were made using multivariate multiway between-groups analysis of variance (MANOVA) in order to ensure that factors such as posttransplant time period, type of native disease, or type of donor is not a confounding variable and that the genotype related association is an independent risk factor.
Genotype Distribution Fulfilled Hardy-Weinberg Criteria
There was a significant difference in the genotype distribution between controls and patients with PTLD for the −1082IL-10 and the TGF-β1 (codon 25) SNPs (Table 3). The frequency of −1082IL-10 GG genotype coding for the high phenotypic cytokine production was found significantly higher in the control group compared to PTLD patients (odds ratio [OR]=0.5, 95% confidence interval [CI]: 0.25-1.0, P=0.044). The TGF-β1 (codon 25) CC genotype indicative of low cytokine production was detected more frequently in PTLD patients than in controls (OR=0.34, 95% CI: 0.17-0.69, P=0.0022). We failed to observe significant differences in the −308TNF-α, TGF-β1 (codon 10), and +874IFN-γ genotype distribution (Table 3).
No significant associations were found between any of the cytokine genotypes and the course of disease. Analyses of genotype distributions with respect to the stage or aggressiveness of the disease did not show any significant association. There was also no correlation between cytokine polymorphisms and the therapeutic response.
To assess that IL-10 and TGF-β1 (codon 25) SNPs constitute independent risk factors for PTLD development, MANOVA were performed. −1082IL-10 as well as TGF-β1 (codon 25) SNPs together with transplant age, patient age, sex, type of the organ, type of native disease were subjected to these analyses. The analyses confirmed the role of −1082IL-10 and TGF-β1 (codon 25) SNPs as independent risk factors for EBV-associated posttransplant lymphoma. No other factors contributing to the risk of PTLD development could be identified.
Here, we report one of the first data on association between late-onset EBV-associated PTLD and SNPs of certain key immunologic cytokine genes. Due to its dramatic impact on the patients’ outcome, PTLD, despite its relatively low incidence, is an important complication after solid organ transplantation. With poor survival statistics and limited treatment options, the management of PTLD has focused largely on the identification of risk factors to prevent the disease.
During the last decade we learned that EBV plays a major role in the pathogenesis of the PTLD (9, 11). Other variable such as immunosuppression regimen, type of transplanted organ, viral coinfection, age of the recipient, race, and time since transplantation (12, 13) are also reported to be a risk factors for PTLD development. The highest observed rates of lymphoma were among transplanted patients in the first 12 months, so called early-onset of PTLD. The majority of cases occurred in young patients (age <25 years), is associated with an active EBV infection. The proliferating B cells are polyclonal, and PTLD can be often cured by the withdrawal of the immunosuppression. The behavior and characteristics of late-occurring PTLD (development more than 1 year after transplantation) are often distinct (14). The patients are usually older, lymphomas are monomorph, and the reduction or withdrawal of the immunosuppression does not bring any benefit. In our study we focused on the evaluation of the genetic risk factors for the development of the late-onset EBV-associated PTLD. Given the established pathophysiologic role of particular pro-inflammatory and anti-inflammatory cytokines in the immune defense, and provided that individual cytokine SNPs can influence cytokine expression, we analyzed the −1082IL-10, −308TNF, TGF-β1 (codon 10, 25), and +874IFN-γ gene polymorphisms in a cohort of solid organ recipients with and without EBV-associated late-onset PTLD.
We found a significant correlation of −1082IL-10 and TGF-β1 (codon 25) genotypes with EBV-associated PTLD. The present study provides support for the role of genetic factors as determinants for late-onset PTLD susceptibility in solid organ transplanted patients. To the authors’ knowledge, this is the first description of the association mentioned.
TGF-β1 is a pleiotropic cytokine that attracts much attention in hematological and connective tissue diseases because of its effects on immune cells and connective tissue metabolism (15). It is known that the production of TGF-β is under genetic control through polymorphisms in its coding sequence and TGF-β1 (codon 25) GG genotype is associated with the increased expression of the protein (16). In recent years, TGF-β has been increasingly recognized as a potent tumor suppressor in several epithelial and other cell types (16). To date, TGF-β is known for its ability to act as both a tumor suppressor and inhibitor of B cell proliferation (16, 17).
The tumor suppressive effect is especially pronounced during the early benign stage (18). Recently demonstrated lower TGF-β1 plasma levels in patients with EBV-associated nasopharyngeal carcinoma (NPC) compared to the healthy controls suggest a protective role of this cytokine for NPC development (18). The TGF-β (codon 25) GG genotype is known to be associated with the increased expression of the protein (16).
Further, TGF-β inhibits B cell proliferation, induces apoptosis of immature or resting B cells, and blocks B cell activation (15).
Findings of our study demonstrating TGF-β1 (codon 25) GG genotype as a protective factor for PTLD development are in line with these data. Given the potential for TGF-β1 for the inhibition of uncontrolled proliferation of B cells in transplant patients with EBV infection, it is tempting to speculate that lack of inhibitory biological activities results in insufficient control of pathological proliferation of B cells contributing to the development of EBV-associated lymphoma.
Further, we observed a significant increase in the IL-10 (−1082) GG genotype frequency in control group compared to patients with lymphoma suggesting a protective role of this genotype to PTLD. IL-10 is an important cytokine that exerts an anti-inflammatory activity by inhibiting Th1 type responses and monocyte function. There is also substantial evidence supporting the role of IL-10 in lymphomagenesis including EBV-associated PTLD (19).
An in vitro study using peripheral blood mononuclear cells has suggested that the IL-10 (−1082) GG genotype is associated with higher cytokine production than other genotypes (20). Keeping this observation in mind and considering lymphomagenetic properties of IL-10, our established correlation between the IL-10 (−1082) GG genotype and protective ability to PTLD appears difficult to explain. However, it is known that IL-10 enhances antibody responses, thus increasing humoral immunity (21). We may speculate therefore, that high levels of IL-10 lead to the enhancement of immunity against EBV, preventing EBV reactivation, a causative factor of PTLD. In addition, IL-10 strongly controls IL-6 secretion by macrophages and lymphocytes. IL-6 is an important survival and proliferation factor of B cells. High levels of IL-10 might limit the B-cell supporting IL-6 secretion. Another possible explanation for the negative correlation between −1082IL-10-GG genotype and PTLD could be a linkage between IL-10 GG and TGF beta GG polymorphisms. However, this linkage was excluded by the appropriate statistical tests.
Our results are in line with two other recent case control studies performed on a large cohort of general population with and without non-Hodgkin lymphoma. These studies showed a correlation of IL-10 promoter genotype coding for a low IL-10 production and susceptibility to aggressive forms of lymphoma (7, 22).
An important limitation of this study is the moderate patient number in the PTLD group. Since the incidence of the PTLD and especially late-onset EBV-associated PTLD is relatively low, it was not possible to enrol a large number of PTLD patients in the study. To improve the statistical power, we increased the number of patients in the control group and enrolled 408 matched solid organ recipients in our case-control study. To minimize/exclude the impact of other variables on the statistical analyses, patients of the control group were matched with the PTLD group regarding organ, age, transplant age, and regimen of immunosuppression. MANOVA could confirm that these factors are not confounding variables and that the genotype related association is an independent risk factor. Further, patients with severe chronic diseases such as any kinds of chronic active infections, inflammatory diseases, cancer, or hematological abnormalities were excluded from the study.
None of the controls showed episodes of EBV reactivation during the posttransplant follow up compared to 100% EBV reactivation in patients with PTLD. We were not able to demonstrate any association between −308TNF-α, TGF-β1 codon 10, and +874IFN-γ SNPs and PTLD.
The findings of our study do not, therefore, support previous observations (23) that showed an association between a low IFN-γ polymorphisms and PTLD in pediatric liver transplant recipients. Differences may in part be due to study population and geographical location. The number of PTLD patients analyzed in the previous study was smaller (six PTLD cases) than in the present study and the cases were all pediatric liver transplant patients.
In summary, the present study provides support for the role of genetic variants in two key cytokines of the Th2 pathway as risk factors for PTLD development in transplant patients. Hence, the assessment of respective cytokine gene SNPs may potentially allow identification of patients at risk for EBV-associated PTLD and appropriate adjustment of preemptive treatments in these patients.
Taken together, our findings suggest that a shift in the Th1/Th2 balance caused by genetic factors can play an important role in the pathogenesis of the late onset EBV-associated PTLD.
The authors gratefully acknowledge Dr. I. Furgel for his assistance in statistical analysis. The authors also thank Zachary Nearman for English revision.
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