Bouke G. Hepkema

Variable EBV DNA Load Distributions and Heterogeneous EBV mRNA Expression Patterns in the Circulation of Solid Organ versus Stem Cell Transplant Recipients

Epstein-Barr virus (EBV) driven post-transplant lymphoproliferative disease (PTLD) is a heterogeneous and potentially life-threatening condition. Early identification of aberrant EBV activity may prevent progression to B-cell lymphoma. We measured EBV DNA load and RNA profiles in plasma and cellular blood compartments of stem cell transplant (SCT; n = 5), solid organ transplant recipients (SOT; n = 15), and SOT having chronic elevated EBV-DNA load (n = 12). In SCT, EBV DNA was heterogeneously distributed, either in plasma or leukocytes or both. In SOT, EBV DNA load was always cell associated, predominantly in B cells, but occasionally in T cells (CD4 and CD8) or monocytes. All SCT with cell-associated EBV DNA showed BARTs and EBNA1 expression, while LMP1 and LMP2 mRNA was found in 1 and 3 cases, respectively. In SOT, expression of BARTs was detected in all leukocyte samples. LMP2 and EBNA1 mRNA was found in 5/15 and 2/15, respectively, but LMP1 mRNA in only 1, coinciding with severe PTLD and high EBV DNA. Conclusion: EBV DNA is differently distributed between white cells and plasma in SOT versus SCT. EBV RNA profiling in blood is feasible and may have added value for understanding pathogenic virus activity in patients with elevated EBV-DNA.

Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53‐SLP) vaccine, twenty patients with recurrent elevation of CA‐125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53‐SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no ≥ grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN‐γ producing p53‐specific T‐cell responses were induced in all patients who received all 4 immunizations as measured by IFN‐γ ELISPOT. An IFN‐γ secretion assay showed that vaccine‐induced p53‐specific T‐cells were CD4+, produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53‐specific response. P53‐specific T‐cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53‐specific T‐cells. As best clinical response, stable disease evaluated by CA‐125 levels and CT‐scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine‐induced immunity. This study shows that the p53‐SLP vaccine is safe, well tolerated and induces p53‐specific T‐cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper‐1 polarization and clinical efficacy.

Epstein-Barr virus-DNA load monitoring late after lung transplantation: A surrogate marker of the degree of immunosuppression and a safe guide to reduce immunosuppression

Background. Posttransplant lymphoproliferative disease (PTLD) is a serious complication after lung transplantation and its relation with Epstein-Barr virus (EBV) is well recognized. It has been postulated that preemptive reduction of immunosuppression guided by EBV-DNA load may lead to a significantly lower incidence of PTLD, because of the reconstitution of T-cell control. In this report, we describe the feasibility of this approach in terms of safety with regard to the risk of acute as well as chronic allograft rejection in 75 lung transplant recipients transplanted between 1990 and 2001 and followed for this study from June 1, 2001 until January 1, 2006. Methods. From all patients visiting our outpatient clinic, EBV-DNA load was measured at least twice a year during the study period. In patients with positive results, measurements were repeated every two to four weeks. EBV reactivation was defined as two consecutive EBV-DNA load measurements with a rising trend; with the last measurement exceeding 10.000 copies/mL under stable immunosuppression. In such case, immunosuppression was reduced. Results. EBV reactivation was observed in 26/75 patients (35%). One (1.5%) of these patients developed PTLD during the study period. Acute rejection, acceleration of chronic allograft rejection, or worse survival were not observed after reduction of immunosuppression. Conclusions. Preemptive reduction of immunosuppression after lung transplantation guided by EBV-DNA load appears to be a safe approach for the prevention of PTLD in lung transplant recipients late after transplantation.

Long-term outcome of lung transplantation is predicted by the number of HLA-DR mismatches

Background. The importance of HLA mismatch in determining long-term outcome in lung transplantation remains largely uncertain. Methods. A retrospective analysis of 102 consecutive primary lung transplants was performed to identify risk factors for poor long-term outcome after lung transplantation defined as graft survival and bronchiolitis obliterans syndrome (BOS) stage I and II. Variables included were patient characteristics (age, sex, prior diagnosis), the number of HLA mismatches between donor and recipient, cold ischemic time, cytomegalovirus serologic concordance, number of acute rejections, and time to first rejection. Variables carrying significance in a univariate analysis were subjected to a proportional hazard regression analysis. Results. In the multivariate analysis, an increased number of acute rejections correlated positively with decreased graft survival (risk ratio [RR]=1.25; 95% confidence interval [CI], 1.05–1.5;P =0.011), development of BOS stage I (RR=1.36/episode; 95% CI, 1.16–1.58;P <0.001), and BOS stage II (RR=1.42/episode; 95% CI, 1.2–1.67;P <0.001). An increased time to rejection correlated positively with reduced graft survival (RR=1.03/day; 95% CI, 1.01–1.06;P =0.02), and BOS stage I and II (both RR=1.04/day; 95% CI, 1.01–1.07;P <0.005). Compared with 2 HLA-DR mismatches, 0 or 1 mismatch was associated with improved graft survival (RR=0.43; 95% CI, 0.19–0.98;P =0.045) and protected against development of BOS stage I (RR=0.47; 95% CI, 0.23–0.98;P =0.044) and BOS stage II (RR=0.35; 95% CI, 0.15–0.83;P =0.017). Conclusions. HLA-DR mismatching appears to be a risk factor for the development of BOS and graft loss. Improved outcome after lung transplantation might be achieved with prospective matching for HLA-DR. Alternatively, the amount and type of immunosuppressive drugs may be guided by the degree of HLA-DR (mis)matching.

Lectin complement pathway gene profile of donor and recipient determine the risk of bacterial infections after orthotopic liver transplantation

Infectious complications after orthotopic liver transplantation (OLT) are a major clinical problem. The lectin pathway of complement activation is liver‐derived and a crucial effector of the innate immune defense against pathogens. Polymorphisms in lectin pathway genes determine their functional activity. We assessed the relationship between these polymorphic genes and clinically significant bacterial infections, i.e., sepsis, pneumonia, and intra‐abdominal infection, and mortality within the first year after OLT, in relation to major risk factors in two cohorts from different transplant centers. Single‐nucleotide polymorphisms in the mannose‐binding lectin gene (MBL2), the ficolin‐2 gene (FCN2), and the MBL‐associated serine protease gene (MASP2) of recipients and donors were determined. Recipients receiving a donor liver in the principal cohort with polymorphisms in all three components i.e., MBL2 (XA/O; O/O), FCN2+6359T, and MASP2+371A, had a cumulative risk of an infection of 75% as compared to 18% with wild‐type donor livers (P = 0.002), an observation confirmed in the second cohort (P = 0.04). In addition, a genetic (mis)match between donor and recipient conferred a two‐fold higher infection risk for each separate gene. Multivariate Cox analysis revealed a stepwise increase in infection risk with the lectin pathway gene profile of the donor (hazard ratio = 4.52; P = 8.1 × 10−6) and the donor‐recipient (mis)match genotype (hazard ratio = 6.41; P = 1.9 × 10−7), independent from the other risk factors sex and antibiotic prophylaxis (hazard ratio > 1.7 and P < 0.02). Moreover, patients with a lectin pathway gene polymorphism and infection had a six‐fold higher mortality (P = 0.9 × 10−8), of which 80% was infection‐related. Conclusion: Donor and recipient gene polymorphisms in the lectin complement pathway are major determinants of the risk of clinically significant bacterial infection and mortality after OLT. (HEPATOLOGY 2010;)

Hla antigens and post renal transplant lymphoproliferative disease : HLA-B matching is critical

Although several risk factors for posttransplant lymphoproliferative disease (PTLD) after solid organ transplantation have been identified, the immunosuppressive regimen probably as most important one, their exact pathogenic role and relevance is still unclear. In hematopoietic stem cell transplantation, HLA mismatching also is a risk factor. We analyzed factors possibly associated with development of PTLD in patients receiving a kidney transplant at our hospital between 1985 and 2002. PTLD was observed in 20 out of 1,013 patients (2.0%). Mismatches at the HLA-B locus, but not at the HLA-A or HLA-DR loci, and anti T-cell antibody therapy were both independently associated with development of PTLD. Hazard ratios increased from 1.4 (0.5–4.1) with one mismatch to 5.1 (1.4–19.0) in case of two HLA-B mismatches. Decreased surveillance by T-cells with dual specificity for Epstein-Barr virus (EBV) as well as for allo HLA antigens on the allograft might facilitate clonal expansion of B-cells latently infected with EBV.

Association of human leukocyte antigen haplotypes with posttransplant lymphoproliferative disease after solid organ transplantation

Background. Posttransplant lymphoproliferative disease (PTLD) after solid organ transplantation (SOT) is commonly characterized by Epstein-Barr virus (EBV)-driven proliferation of recipient B cells due to impaired immune surveillance in the context of immunosuppression. Because EBV-specific T-cell responses are focused on the level of EBV antigen and epitope choice depending on the individual human leukocyte antigen (HLA) alleles, we hypothesized that certain HLA alleles or a distinct HLA haplotype may influence the risk of development of PTLD after SOT. Methods. A multicenter case-control study was performed comparing a group of 155 recipients after SOT with development of PTLD with a group of 1996 recipients after SOT without development of PTLD. Alleles, genotypes, and three locus haplotypes were compared of SOT recipients with and without PTLD. Results. The bivariate analysis showed that carrying HLA-A03 was negatively associated (odds ratio [OR] 0.61, confidence interval [CI] 0.40–0.92, P<0.02) whereas carrying of HLA-B18 (OR 1.79, CI 1.18–2.73, P<0.006) and HLA-B21 (OR 2.08, CI 1.14–3.77, P<0.02) were positively associated with PTLD after SOT. HLA-DR analysis demonstrated a significant negative association between the expression of HLA-DR7 (OR 0.46, CI 0.28–0.78, P<0.004) and PTLD. Three locus haplotype analysis underlined the relevance of a dominant protective effect of HLA-DR7 expression concerning the risk of PTLD development. Conclusions. Our data suggest an influence of HLA variants on the risk of the development of PTLD. We hypothesize that HLA genes or non-HLA genes within the HLA loci confer a risk modification for the individual patient.

HLA-DR4, DR13(6) and the ancestral haplotype A1B8DR3 are associated with ANCA-associated vasculitis and Wegener's granulomatosis

OBJECTIVES As the HLA system is involved in recognition of self and non-self, an association with the development of ANCA-associated vasculitis (AAV) seems probable. In this study, the relation between HLA antigens and AAV and its severity were investigated. METHODS Consecutive patients diagnosed with AAV at our centre, who were followed for at least 2 years, were included. The frequency of HLA antigens of AAV and WG patients was compared with 5872 healthy blood donors from the same region and with 4000 healthy Dutch controls originating from a Eurotransplant database. RESULTS From 304 AAV patients, sufficient data were available. We found DR13(6) to be less prevalent and both DR4 and the ancestral haplotype A1B8DR3 more prevalent in patients with AAV compared with controls, particularly in patients with WG. In addition, DR1 was less prevalent in patients with WG in comparison with controls. Further, DR8 was more prevalent in patients with CSS compared with other forms of vasculitis and controls. There were no associations between HLA antigens and disease characteristics or course of AAV or WG. CONCLUSIONS AAV is associated with increased prevalence of DR4 and the ancestral haplotype A1B8DR3 and with decreased prevalence of DR13(6), particularly in patients with WG. In patients with WG, prevalence of DR1 was decreased, whereas in patients with CSS DR8 was increased. No associations between HLA antigens and disease characteristics or course of AAV were found.

Heme Oxygenase-1 Genotype of the Donor Is Associated With Graft Survival After Liver Transplantation

Heme oxygenase‐1 (HO‐1) has been suggested as a cytoprotective gene during liver transplantation. Inducibility of HO‐1 is modulated by a (GT)n polymorphism and a single nucleotide polymorphism (SNP) A(‐413)T in the promoter. Both a short (GT)n allele and the A‐allele have been associated with increased HO‐1 promoter activity. In 308 liver transplantations, we assessed donor HO‐1 genotype and correlated this with outcome variables. For (GT)n genotype, livers were divided into two classes: short alleles (<25 repeats; class S) and long alleles (≥25 repeats; class L). In a subset, hepatic messenger ribonucleic acid (mRNA) expression was correlated with genotypes. Graft survival at 1 year was significantly better for A‐allele genotype compared to TT‐genotype (84% vs. 63%, p = 0.004). Graft loss due to primary dysfunction (PDF) occurred more frequently in TT‐genotype compared to A‐receivers (p = 0.03). Recipients of a liver with TT‐genotype had significantly higher serum transaminases after transplantation and hepatic HO‐1 mRNA levels were significantly lower compared to the A‐allele livers (p = 0.03). No differences were found for any outcome variable between class S and LL‐variant of the (GT)n polymorphism. Haplotype analysis confirmed dominance of the A(‐413)T SNP over the (GT)n polymorphism. In conclusion, HO‐1 genotype is associated with outcome after liver transplantation. These findings suggest that HO‐1 mediates graft survival after liver transplantation.

HLA associations in classical Hodgkin lymphoma: EBV status matters.

The pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients using a PCR-based sequence-specific oligonucleotide probe (SSOP) hybridization approach. The allele frequencies were compared to HLA typings of more than 6,000 controls. The age of the cHL patients varied between 13 and 81 years with a median of 35 years. Nodular sclerosis subtype was the most common subtype (87%) and EBV was detected in 25% of the cHL patients. HLA-B5 was significantly increased and HLA-DR7 significantly decreased in the total cHL patient population as compared to controls. Two class II associations were observed to be specific for the EBV− cHL population with an increase of HLA-DR2 and HLA-DR5. Allele frequencies of HLA-A1, HLA-B37 and HLA-DR10 were significantly increased in the EBV+ cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in Caucasians. The allele frequency of HLA-A2 was significantly decreased in the EBV+ cHL population. Analysis of haplotypes with a frequency of >1% revealed a significant increase of HLA-A2-B7-DR2 in EBV− cHL as compared to controls. SSOP association analysis revealed significant differences between EBV+ and EBV− cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV+ cHL. Furthermore several new protective and predisposing HLA class I and II associations for the EBV+, the EBV− and the entire cHL population were identified.