A novel predictive approach for GVHD after allogeneic SCT based on clinical variables and cytokine gene polymorphisms.

Despite considerable advances in our understanding of the pathophysiology of graft-versus-host disease (GVHD), its prediction remains unresolved and depends mainly on clinical data. The aim of this study is to build a predictive model based on clinical variables and cytokine gene polymorphism for predicting acute GVHD (aGVHD) and chronic GVHD (cGVHD) from the analysis of a large cohort of HLA-identical sibling donor allogeneic stem cell transplant (allo-SCT) patients. A total of 25 SNPs in 12 cytokine genes were evaluated in 509 patients. Data were analyzed using a linear regression model and the least absolute shrinkage and selection operator (LASSO). The statistical model was constructed by randomly selecting 85% of cases (training set), and the predictive ability was confirmed based on the remaining 15% of cases (test set). Models including clinical and genetic variables (CG-M) predicted severe aGVHD significantly better than models including only clinical variables (C-M) or only genetic variables (G-M). For grades 3-4 aGVHD, the correct classification rates (CCR1) were: 100% for CG-M, 88% for G-M, and 50% for C-M. On the other hand, CG-M and G-M predicted extensive cGVHD better than C-M (CCR1: 80% vs. 66.7%, respectively). A risk score was calculated based on LASSO multivariate analyses. It was able to correctly stratify patients who developed grades 3-4 aGVHD (P < .001) and extensive cGVHD (P < .001). The novel predictive models proposed here improve the prediction of severe GVHD after allo-SCT. This approach could facilitate personalized risk-adapted clinical management of patients undergoing allo-SCT.

Bone marrow VEGFC expression is associated with multilineage dysplasia and several prognostic markers in adult acute myeloid leukemia, but not with survival.

Vascular endothelial growth factor C (VEGFC) stimulates leukemia cell proliferation and survival, and promotes angiogenesis. We studied VEGFC expression in bone marrow samples from 353 adult acute myeloid leukemia (AML) patients and its relationship with several clinical, cytogenetic, and molecular variables. We also studied the expression of 84 genes involved in VEGF signaling in 24 patients. We found that VEGFC expression was higher in AML patients with myelodysplasia-related changes (AML-MRC) than in patients with non-AML-MRC. We also found an association between VEGFC expression and the patient cytogenetic risk group, with those with a worse prognosis having higher VEGFC expression levels. No correlation was observed between VEGFC expression and survival or complete remission. VEGFC expression strongly correlated with expression of the VEGF receptors FLT1, KDR, and NRP1. Thus, in this series, VEGFC expression was increased in AML-MRC and in subgroups with a poorer prognosis, but has no impact on survival.

Association of RBP4 genetic variants with childhood obesity and cardiovascular risk factors.

BACKGROUND: Recent data suggest that retinol-binding protein 4 (RBP4) gene variants could be associated with a risk of obesity and its co-morbidities, such as metabolic syndrome, which increases the risk of developing type 2 diabetes mellitus and cardiovascular disease. OBJECTIVES: The present study examined the potential association of RBP4 single nucleotide polymorphisms (SNPs) with childhood obesity and its metabolic complications. METHODS: Four RBP4 SNPs, rs3758538 (3944A>C), rs3758539 (4406G>A), rs12265684 (12177G>C) and rs34571439 (14684T>G), were genotyped in a population of 180 Spanish Caucasian children (97 obese and 83 normal-weight children). Association of RBP4 SNPs with obesity, metabolic risk factors (blood pressure, triglycerides, high-density lipoprotein cholesterol, insulin resistance) and markers of vascular inflammation, such as high-sensitive C-reactive protein (hs-CRP), was tested. RESULTS: We found SNP rs3758538 to be associated with obesity (p = 0.007). Specifically, each copy of the minor allele C was associated with an increased risk of obesity, by more than twofold, in respect of being homozygous for the major allele A (odds ratio = 2.4; 95% confidence interval = 1.2-4.8). The rs3758538 and rs34571439 RBP4 SNPs correlated with plasma RBP4 levels. The SNPs rs12265684 and rs34571439 correlated with plasma triglyceride levels. The rs34571439 was also associated to hs-CRP levels. Marginal association of RBP4 SNPs with plasma high-density lipoprotein levels (rs34571439), blood pressure (rs12265684) and insulin resistance (rs3758539) was also observed. CONCLUSIONS: These findings suggest that childhood obesity may be associated with variations in RBP4 gene. The presence of selective SNPs in the RBP4 gene may account for metabolic complications.

Risk factors for non-melanoma skin cancer in patients with essential thrombocythemia and polycythemia vera.

OBJECTIVES: Population-based studies have reported an increased incidence of skin cancer in patients with essential thrombocythemia (ET) and polycythemia vera (PV). We have examined the risk factors for non-melanoma skin cancer (NMSC) in patients diagnosed with ET or PV during 1973-2012. METHODS: A case-control study was performed to compare the clinical and treatment-related data of 51 ET/PV patients who had NMSC with that of 401 patients who did not. We also evaluated whether polymorphisms in 12 genes involved in DNA integrity predisposed to NMSC. RESULTS: By multivariate logistic regression analysis, risk factors for NMSC were older age (OR: 1.7, 95% CI: 1.3-2.1, P < 0.001), male sex (OR: 2.1, 95% CI: 1.1-3.8, P = 0.023), higher cumulated hydroxycarbamide dose (OR: 1.3, 95% CI: 1.1-1.7, P = 0.017), and busulphan exposure (OR: 3.2, 95% CI: 1.05-10.0, P = 0.041). On the time-to-event prognostic model, factors independently associated with increased cumulative incidence of NMSC were age (5% increased risk per year; P < 0.001), male sex (91% increased risk; P = 0.022), and hydroxycarbamide exposure (22% increased risk; P = 0.065). No susceptibility gene variant was identified. CONCLUSIONS: These findings suggest that the risk to develop NMSC in ET/PV patients results from the combined effect of common risk factors (age, male sex) together with cytoreductive treatment.

The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation.

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08-0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients.

BCL2 gene polymorphisms and splicing variants in chronic myeloid leukemia.

Recent data suggest that constitutional genetic variation in the antiapoptotic BCL2 gene could be associated with the susceptibility to develop chronic myeloid leukemia (CML) and the clinical outcome in several hematological malignancies. The present study examines whether BCL2 single nucleotide polymorphisms (SNPs) predispose to CML or may potentially influence the disease characteristics at diagnosis. Notably, no association was observed between the four candidate BCL2 SNPs and the risk of developing CML. Instead, the 4777C>A (rs2279115) and the 5735A>G (rs1801018) SNPs were significantly associated with the disease risk profile as determined by the Sokal score. We found that such polymorphisms correlated with the expression of BCL2 alternative splicing transcripts (BCL2-α, BCL2-ß) in healthy donors, but not in CML patients, although the relative levels of BCL2 mRNA splicing variants were shown to change during the clinical course of CML. Our findings suggest that BCL2 polymorphisms could influence the clinical features of CML patients at diagnosis. However, the pathogenic mechanisms involved in such association remain to be ascertained.

A polymorphism in the TYMP gene is associated with the outcome of HLA-identical sibling allogeneic stem cell transplantation.

Thymidine phosphorylase (TYMP), an enzyme involved in nucleotide synthesis, has been implicated in critical biological processes such as DNA replication, protection against mutations, and tissue repair. In this work, we retrospectively evaluated the influence of a polymorphism in the TYMP gene (rs112723255; G/A) upon the outcome of 448 patients subjected to allogeneic stem cell transplantation (allo-SCT) from an human leukocyte antigen (HLA)-identical sibling donor. The TYMP genotype of patients correlated with overall survival-carriers of the minor allele (A) being at an increased risk of dying after transplantation (hazard ratio, HR = 1.9; P = 0.004). This effect was mostly due to differences in transplant toxicity-related mortality (HR = 2.5; P = 0.029). In addition, the TYMP genotype of donors was associated with the risk of chronic graft-versus-host disease (GVHD)-carriers of the minor allele being at an increased risk of developing this complication ([HR] = 1.7; P = 0.039). The impact of such polymorphism on the risk of chronic GVHD is limited to patients transplanted in early stage disease (HR = 2.2; P = 0.019). The combination of a donor harboring the minor allele with a patient homozygous for the major allele was associated with the highest risk of chronic GVHD (HR = 2.8; P = 0.008). These findings provide the first evidence of the significant impact of the TYMP genotype upon the clinical outcome of patients treated with HLA-identical sibling allo-SCT.

A polymorphism in the XPD gene predisposes to leukemic transformation and new nonmyeloid malignancies in essential thrombocythemia and polycythemia vera.

Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have an increased incidence of acute myeloid leukemia and new nonhematologic malignancies compared with the general population. However, information on the factors determining the risk for such complications is limited. In the present study, we investigated whether constitutional genetic variations in DNA repair predispose to leukemic transformation and new nonmyeloid neoplasias in patients with ET and PV. Case-control studies for predisposition to both types of malignancies were nested in a cohort of 422 subjects diagnosed with ET or PV during the period 1973-2010 in several institutions in Spain. A total of 64 incidence cases of leukemia and 50 cases of primary nonmyeloid cancers were accrued. At conditional regression analysis, the Gln/Gln genotype in the XPD codon 751 showed the strongest association with both leukemic transformation (odds ratio [OR] = 4.9; 95% confidence interval [95% CI], 2.0-12) and development of nonmyeloid malignancies (OR = 4.2; 95% CI, 1.5-12). Additional predictive factors were exposure to cytoreductive agents for leukemic transformation (OR = 3.5; 95% CI, 2.0-6.2) and age for nonmyeloid malignancies (OR = 2.0; 95% CI, 1.4-2.8). These findings provide further evidence about the contribution of inherited genetic variations to the pathogenesis and clinical course of myeloproliferative neoplasms.

Donor CTLA-4 genotype influences clinical outcome after T cell-depleted allogeneic hematopoietic stem cell transplantation from HLA-identical sibling donors.

CTLA-4 (cytotoxic T-lymphocyte antigen-4) plays a pivotal role in inhibiting T cell activation through competitive interaction with B7 molecules and interruption of costimulatory signals mediated by CD28. Polymorphisms on the CTLA-4 gene have been previously associated with autoimmune diseases, predisposition to leukemic relapse, and with graft-versus-host disease (GVHD) or relapse after allogeneic transplant. As CTLA-4 is expressed on T-lymphocytes, the aim of this study was to determine whether the donor CTLA-4 CT60 genotype also influences clinical outcome even after T cell depletion with CD34-positive selection. We studied 136 patient-donor pairs. Overall survival (OS) was worse for those patients who received grafts from a donor with the CT60 AA genotype rather than from a donor with the AG or GG genotype (35.6% vs 49.4%; P = .043). This association was confirmed through multivariate analysis, which identified the donor CT60 genotype as an independent risk factor for OS (P = .008; hazard ratio [HR]: 2.24, 95% confidence interval [CI]: 1.23-4.08). The donor CT60 AA genotype was also associated with lower disease-free survival, this being related to an increased risk of relapse (P = .001; HR: 3.41, 95% CI: 1.67-6.96) and a trend toward higher transplant-related mortality. These associations were stronger when considering only patients in the early stage of disease. Our results suggest that graft-versus-leukemia (GVL) activity after T cell depletion is conditioned by the donor CTLA-4 genotype.

Functional polymorphisms in SOCS1 and PTPN22 genes correlate with the response to imatinib treatment in newly diagnosed chronic-phase chronic myeloid leukemia.

The function of the natural modulators of BCR-ABL-induced signaling pathways could influence the results to imatinib treatment. We assessed the association between single nucleotide polymorphisms (SNPs) on genes of the phosphatase family and the suppressors of cytokine signaling and the response to imatinib in 105 patients newly diagnosed with chronic-phase CML. SNPs in SOCS1 (rs243327) and PTPN22 (rs2476601) genes correlated with the risk of primary resistance to imatinib. A high-risk Sokal score, the T allele in PTPN22 SNP, and each copy of the C allele in SOCS1 SNP were adverse prognostic factors for failure-free survival (FFS). Based on such parameters, three risk groups were identified, with the 5-year FFS for each group being 95%, 75%, and 50%, respectively (P<0.001). A simple predictive model including Sokal score and genotype of SOCS1 and PTPN22 SNPs may be useful in the selection of the initial treatment in CML.

An XRCC1 polymorphism is associated with the outcome of patients with lymphoma undergoing autologous stem cell transplant.

High-dose chemotherapy supported by autologous stem cell transplant (ASCT) remains the treatment of choice for patients with lymphoma failing first-line chemotherapy. Recent evidence suggests a relationship between the genetic variations in genes involved in DNA repair and the outcome of patients with a number of malignancies. In this work, we retrospectively evaluated the influence of an XRCC1 polymorphism (rs25487) on the treatment results in a series of 73 patients with lymphoma subjected to ASCT. The factors correlated to overall survival were the disease status at transplant and XRCC1 genotype. Carriers of a mutant A allele had a two-fold higher risk of death than those with the wild-type genotype. In addition, patients harboring one or two copies of the A allele (GA/AA) were 4.5-fold more likely to develop therapy-related acute myeloid (t-AML). Thus, the cumulative probability of t-AML at 10 years was 37 ±â€Š13% in patients with the mutant A allele as compared to 8.5 ±â€Š6% in the remaining cases (p = 0.04). Our findings suggest that genetic variation in the DNA repair gene XRCC1 may play a role in the results of transplant in patients with lymphoma.

Correlation between genetic polymorphisms of the hOCT1 and MDR1 genes and the response to imatinib in patients newly diagnosed with chronic-phase chronic myeloid leukemia.

The association between seven polymorphisms in the genes hOCT1 and MDR1, encoding for imatinib transporter proteins, and the response to imatinib 400mg/daily was investigated in 65 patients newly diagnosed with chronic-phase chronic myeloid leukemia. The AA genotype at the rs6935207 hOCT1 polymorphic locus was not detected in patients with inadequate response to imatinib. The CC genotype at the rs1045642 (C3435T) MDR1 locus was associated with primary failure, whereas a T allele at the rs2032582 (G2677T/A) MDR1 locus seemed to protect from primary failure. Beside, the MDR1 haplotype 1236T-2677G-3435C was more frequently found in patients primarily resistant to imatinib.

XPC genetic polymorphisms correlate with the response to imatinib treatment in patients with chronic phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is driven by the BCR-ABL protein, which promotes the proliferation and viability of the leukemic cells. Moreover, BCR-ABL induces genomic instability that can contribute to the emergence of resistant clones to the ABL kinase inhibitors. It is currently unknown whether the inherited individual capability to repair DNA damage could affect the treatment results. To address this, a comprehensive analysis of single nucleotide polymorphisms (SNPs) on the nucleotide excision repair (NER) genes (ERCC2-ERCC8, RPA1-RPA3, LIG1, RAD23B, XPA, XPC) was performed in 92 chronic phase CML patients treated with imatinib upfront. ERCC5 and XPC SNPs correlated with the response to imatinib. Haplotype analysis of XPC showed that the wild-type haplotype (499C-939A) was associated with a better response to imatinib. Moreover, the 5-year failure free survival for CA carriers was significantly better than that of the non-CA carriers (98% vs. 73%; P = 0.02). In the multivariate logistic model with genetic data and clinical covariates, the hemoglobin (Hb) level and the XPC haplotype were independently associated with the treatment response, with patients having a Hb < or =11 g/dl (Odds ratio [OR] = 5.0, 95% confidence interval [CI] = 1.5-16.1) or a non-CA XPC haplotype (OR = 4.1, 95% CI = 1.6-10.6) being at higher risk of suboptimal response/treatment failure. Our findings suggest that genetic polymorphisms in the NER pathway may influence the results to imatinib treatment in CML.

Influence of DNA damage and repair upon the risk of treatment related leukemia.

Therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) are malignancies occurring after exposure to chemotherapy and/or radiotherapy. Several studies have addressed cumulative dose, dose intensity and exposure to specific agents of preceding cytotoxic therapy in relation to the risk of developing such leukemia. Since only a small percentage of patients exposed to cytotoxic therapy develop t-MDS/AML, it has been suggested that some genetic predisposition may be involved, specifically associated to polymorphisms in certain genes involved in chemotherapy/radiotherapy response - fundamentally genes intervening in drug detoxification and DNA synthesis and repair. A review is made of the genetic studies related to t-MDS/AML predisposition, focusing on the mechanistic findings of how specific chemotherapeutic drug exposure produces DNA damage and induces the chromosomal abnormalities characteristic of t-MDS/AML, the molecular pathways involved in repairing such drug induced damage, and the way in which they influence t-MDS/AML genesis. Specific issues are (a) the interaction of topoisomerase II inhibitors, alkylators and antimetabolite drugs with DNA repair mechanisms and their impact on t-MDS/AML leukemogenicity and (b) the influence of DNA polymorphisms in genes involved in DNA repair, drug metabolization and nucleotide synthesis, paying special attention to the relevance of folate metabolism. Finally, we discuss some aspects relating to study design that are most suitable for characterizing associations between drug exposure and genotypes related to t-MDS/AML risk - stressing the importance of the inclusion of chemotherapy-exposed control groups.

Targeted oligonucleotide delivery in human lymphoma cell lines using a polyethyleneimine based immunopolyplex.

The efficacy of antisense gene therapy depends on efficient delivery of oligonucleotides into targeted cells. Although polyethyleneimine based polyplexes have been reported as good transfection reagents, they are inefficient in lymphoid cell transfection. We report the construction of an immunopolyplex, a targeted nonviral vector based on a polyplex backbone and its application for oligonucleotide transfer on human lymphoma cell lines. The salient characteristic of immunopolyplex lies in the possibility of easily replacing the targeting element (antibody), leaving the polyplex backbone intact. Furthermore, a study was made of the influence of endocytosis inhibitors on immunopolyplex activity. The capacity of the immunopolyplex for oligonucleotide transfer was studied in vitro using FITC-labeled oligonucleotides as fluorescent reporters, an anti-CD3 antibody as targeting element, and a CD3-positive cell line (Jurkat) as a target cell line, in the absence and presence of endocytosis inhibitors. A CD3-negative Jurkat-derived mutant cell line (J.RT3-T3.5) was used as control. A nine-fold increase in fluorescence in the CD3-positive above that in the CD3-negative cell line was observed, indicating that oligonucleotide transfer is mainly specific. Low fluorescence values were obtained in the presence of endocytosis inhibitor or with untargeted polyplexes. We conclude that the immunopolyplex is a good nonviral vector for specific oligonucleotide delivery. Abolition of immunopolyplex activity in the presence of endocytosis inhibitor suggests that targeted oligonucleotide transfer occurs through an endocytic pathway.

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines.

BACKGROUND: Specific and efficient delivery of genes into targeted cells is a priority objective in non-viral gene therapy. Polyethyleneimine-based polyplexes have been reported to be good non-viral transfection reagents. However, polyplex-mediated DNA delivery occurs through a non-specific mechanism. This article reports the construction of an immunopolyplex, a targeted non-viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines. METHODS: Targeting elements (biotin-labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19-), Granta 519 (CD3-/ CD19+), and J.RT3-T3.5 (CD3-/CD19-)] using the EGFP gene as a reporter gene and anti-CD3 and anti-CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry. RESULTS: A significant selectivity of gene delivery was observed, since the anti-CD3 immunopolyplex worked only in Jurkat cells while the anti-CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3- cell mixture with anti-CD3 immunopolyplexes showed up to 16-fold more transfection in CD3+ than in CD3- cells. Several non-specific transfection reagents showed poor or no transfection activity. CONCLUSION: It is concluded that immunopolyplex is a good non-viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) - the streptavidin-polyplex backbone remaining intact - thereby conferring high versatility.