The clinical impact of DNA methylation frequencies of JAK2 negative regulators in patients with essential thrombocythemia.

Suppressors of cytokine signalling (SOCS) and protein tyrosine phosphatase (PTPN) proteins are negative regulators of Janus Kinase 2 (JAK2). They are thought to be involved in the molecular pathogenesis of essential thrombocythaemia (ET) particularly in patients with unmutated JAK2. In this study we compared DNA methylation of SOCS1, SOCS3 and PTPN6 in peripheral blood cells between 39 ET patients (24 JAK2 V617F mutated) and 22 healthy controls by methylation specific PCR (MSP) and analysed the clinical outcome of patients with respect to DNA methylation. In SOCS1, ET patients showed significantly less methylation (P<0.05) than healthy controls, and in SOCS3 and PTPN6 such a tendency was shown. However, there were no significant differences in the methylation frequencies between JAK2 wildtype and mutated ET patients. In addition, no correlation was detected between methylation of SOCS and PTPN and any clinical outcome parameters. Taken together, regarding the genomic regions investigated our data indicate a minor role of methylation of JAK2 negative regulators for the clinical course of ET.

Sequence capture and next generation resequencing of the MHC region highlights potential transplantation determinants in HLA identical haematopoietic stem cell transplantation.

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor-recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.

Clinical significance of HLA-E*0103 homozygosity on survival after allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. METHODS: We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. RESULTS: HLA-E*0103 homozygous patients showed a higher probability of overall survival (P=0.003) and disease-free survival (P=0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (P=0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (P=0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (P=0.01). CONCLUSIONS: We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.

A single nucleotide polymorphism at chromosome 2q21.3 (LCT -13910C>T) associates with clinical outcome after allogeneic hematopoietic stem cell transplantation.

A single nucleotide polymorphism (SNP) responsible for lactase persistence (LCT -13910C>T) changes intestinal microflora. Considering the influence of bacterial microflora on various immune effects, we tested DNA from 111 recipients/donors and analyzed whether this SNP interferes with survival and the incidence of acute graft-versus-host disease (aGVHD) after allogeneic hematopoetic stem cell transplantations (HSCT). Median overall survival (OS) was significantly longer when donors had a CC genotype (not reached after 133 vs 11.1 months, P = .004). Multivariate analysis identified a donor T allele (hazard ratio 2.63, 95% confidence interval 1.29-5.33, P = .008) as independent risk factor for death. Surprisingly, recipient genotypes did not influence outcome and there were no differences regarding aGVHD. Transplantation-related mortality (TRM), relapse and pneumonia were significantly less frequent in patients with CC donors. These findings add to the growing list of non-HLA polymorphisms with impact on outcome after allogeneic HSCT.

Augmentation of DNA vaccine potency through secretory heat shock protein-mediated antigen targeting.

In an effort to enhance the potency of DNA vaccines, we have developed a new strategy to increase antigen presentation by dendritic cells, one that results in markedly improved cytotoxic T-lymphocyte responses, antibody production, and antitumor effects in vivo. Here, we present the rationale and design of a vaccine encoding a secreted antigen-heat shock protein 70 fusion molecule, targeted to the MHC class I cross-presentation pathway of dendritic cells. Using the human papilloma virus 16 E7 protein as a model antigen, we illustrate the preparation of this vaccine and the main experimental procedures used to test such constructs.