Prognostic significance of Gremlin1 (GREM1) promoter CpG island hypermethylation in clear cell renal cell carcinoma.

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P = 0.02), stage (P = 0.013), grade (P = 0.04), tumor (P = 0.001), and endothelial cell (P = 0.0001) proliferation and decreased mean vessel density (P = 0.001) in a hospital-based ccRCC series (n = 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] = 2.35, 95% confidence interval [CI]:1.29 to 4.28), but not in multivariate analysis (HR = 0.88, 95% CI: 0.45 to 1.74). In a population-based validation series (n = 185), GREM1 region iii methylation was associated with increased Fuhrman grade (P = 0.03) and decreased overall survival (P = 0.001) in univariate and multivariate analysis (HR = 2.32, 95% CI: 1.52 to 3.53 and HR = 2.27, 95% CI: 1.44 to 3.59, respectively). The strong correlation between GREM1 region iii promoter methylation and increased malignancy and its correlation with active angiogenesis indicates a role for GREM1 in ccRCC carcinogenesis and tumor angiogenesis.

VHL and HIF signalling in renal cell carcinogenesis.

Hypoxia-inducible factor (HIF) plays an important role in renal tumourigenesis. In the majority of clear cell RCC (ccRCC), the most frequent and highly vascularized RCC subtype, HIF is constitutively activated by inactivation of the von Hippel-Lindau gene. Of the HIF subunits, HIF-2alpha appears to be more oncogenic than HIF-1alpha, in that HIF-2alpha activates pro-tumourigenic target genes. In addition, recent studies indicate that HIF-1alpha, more than HIF-2alpha, can undergo proteasomal degradation in VHL - /- RCC cells. A more detailed understanding of the molecular basis of hypoxia and angiogenesis in renal carcinogenesis has set the stage for the development of targeted therapies, inhibiting multiple HIF-related pathways, such as the phosphatidylinositol 3-kinase-AKT-mTOR, RAS/RAF/MAP, and VEGF signalling routes. However, despite the positive results of these targeting agents in progression-free survival, clinical resistance remains an issue. Recent pre-clinical studies have suggested new targeting approaches such as inhibition of HIF-driven key metabolic enzymes and have introduced new HIF targeting agents, such as histone deacetylase inhibitors, with successful anti-neoplastic effects. In this review, we discuss existing and novel findings about RCC carcinogenesis, with subsequent clinical implications.

Genetics and epigenetics of renal cell cancer.

Renal cell carcinoma (RCC) is not a single disease, but comprises a group of tumors of renal epithelial origin, each with a different histology, displaying a different clinical course and caused by different genetic alterations. Since cure rates are inversely associated with stage and response to the available treatment regimes is limited to a subgroup of patients, diagnostic methods facilitating early detection and new therapeutic modalities are necessary. Increased knowledge of the underlying pathophysiology of RCC has resulted in the identification of genetic alterations involved in renal cell cancer carcinogenesis. Promising agents to target these pathways, especially the angiogenesis pathway, are being developed, some of which are already standard of care. In addition to genetics, knowledge on epigenetics in the process of renal tumorigenesis has been significantly increased in the last decades. Epigenetics will play an increasing role in the development of new therapeutic modalities and may deliver new prognostic and early diagnostic markers. In this review we discuss the background of RCC and the clinical applications of RCC genetics and epigenetics.

A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients.

Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC.Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2'-deoxycytidine-treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan-Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used.Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02-13.00 and HR, 7.54; 95% CI, 2.68-21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02-6.40).Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006-18. ©2016 AACR.

Promoter Methylation of CDO1 Identifies Clear-Cell Renal Cell Cancer Patients with Poor Survival Outcome.

PURPOSE: In this era of molecular diagnostics, prediction of clear-cell renal cell cancer (ccRCC) survival requires optimization, as current prognostic markers fail to determine individual patient outcome. Epigenetic events are promising molecular markers. Promoter CpG island methylation of cysteine dioxygenase type 1 (CDO1), which was identified as prognostic marker for breast cancer, is studied as a potential marker for ccRCC survival. EXPERIMENTAL DESIGN: We collected primary tissues of 365 ccRCC cases identified within the prospective Netherlands Cohort Study (NLCS). In this population-based series, CDO1 promoter methylation was observed in 124 of 324 (38.3%) patients with successful methylation-specific PCR analysis. Kaplan-Meier curves and Wilcoxon tests were used to evaluate 10-year ccRCC-specific survival. Cox regression analysis was used to obtain crude and multivariate HRs and 95% confidence intervals (CI). The relative prognostic value of multivariate models with and without CDO1 promoter methylation was compared using likelihood-ratio tests. RESULTS: Patients with CDO1 promoter methylation have a significantly poorer survival than those without (Wilcoxon P = 0.006). Differences in survival were independent of other prognostic factors, including age and sex (HR, 1.66; 95% CI, 1.12-2.45) and TNM stage, tumor size, and Fuhrman grade (HR, 1.89; 95% CI, 1.25-2.85). Multivariate models performed better with than without CDO1 promoter methylation status (likelihood-ratio P = 0.003). Survival curves were validated in an independent series of 280 ccRCC cases from The Cancer Genome Atlas (TCGA; Wilcoxon P < 0.001). CONCLUSIONS: CDO1 promoter methylation may not substitute common prognostic makers to predict ccRCC survival, but offers additional, relevant prognostic information, indicating that it might be a novel molecular marker to determine ccRCC prognosis.

A comparative study of conventional versus new, magnesium-poor Vacutainer® Sodium Citrate blood collection tubes for determination of prothrombin time and INR.

INTRODUCTION: Conventional Vacutainer® Sodium Citrate blood collection tubes contain a relatively high concentration of contaminating magnesium ions, which may result in shortening of the prothrombin time (PT) and the International Normalized Ratio (INR). Recently the manufacturer of Vacutainer® Sodium Citrate tubes introduced new tubes with a magnesium-poor stopper. The magnesium concentration in the new low-Mg tubes is significantly lower than that in the conventional plastic tubes. The purpose of the present study was to compare PT and INR determined in specimens drawn with the new tubes to those drawn with the conventional tubes. MATERIALS AND METHODS: Venous blood specimens were collected from 22 healthy persons and 65 patients treated with vitamin K-antagonists using conventional Vacutainer® Sodium Citrate tubes and new, low-Mg Vacutainer® tubes. PT and INR were determined with four thromboplastin reagents, i.e., three brands of recombinant human tissue factor and one brand of combined rabbit brain reagent. Magnesium concentrations were determined in the citrate plasmas with a colorimetric method. RESULTS: The differences in PT, INR and International Sensitivity Index (ISI) between the two tubes were significant when using three recombinant human thromboplastin reagents, but were not significant when using the rabbit thromboplastin. The PT and INR differences between the tubes correlated with the magnesium concentration differences (P<0.001). The INR bias between the four reagents was greater for specimens drawn with conventional tubes than the INR bias for specimens drawn with the new tubes. CONCLUSION: Agreement of INR between reagents is improved by using magnesium-poor tubes.

Analysis of promoter CpG island hypermethylation in cancer: location, location, location!

The genetic and epigenetic alterations that underlie cancer pathogenesis are rapidly being identified. This provides novel insights in tumor biology as well as in potential cancer biomarkers. The somatic mutations in cancer genes that have been implemented in clinical practice are well defined and very specific. For epigenetic alterations, and more specifically aberrant methylation of promoter CpG islands, evidence is emerging that these markers could be used for the early detection of cancer as well as prediction of prognosis and response to therapy. However, the exact location of biologically and clinically relevant hypermethylation has not been identified for the majority of methylation markers. The most widely used approaches to analyze DNA methylation are based on primer- and probe-based assays that provide information for a limited number of CpG dinucleotides and thus for only part of the information available in a given CpG island. Validation of the current data and implementation of hypermethylation markers in clinical practice require a more comprehensive and critical evaluation of DNA methylation and limitations of the techniques currently used in methylation marker research. Here, we discuss the emerging evidence on the importance of the location of CpG dinucleotide hypermethylation in relation to gene expression and associations with clinicopathologic characteristics in cancer.

Different angiogenic potential in low and high grade sporadic clear cell renal cell carcinoma is not related to alterations in the von Hippel-Lindau gene.

BACKGROUND: von Hippel-Lindau (VHL) inactivation is common in sporadic clear cell renal cell carcinomas (ccRCC). pVHL is part of the ubiquitin ligase complex that targets the alpha subunits of hypoxia-inducible transcription factor (HIF) for degradation under well-oxygenated conditions. In the absence of wild-type pVHL, as observed in VHL patients and most sporadic ccRCCs, constitutive upregulation of HIF results in transcriptional activation of angiogenesis-related genes, such as VEGF. Differences in angiogenic activity within the group of ccRCCs were reported and strong genotype-phenotype correlations were found in patients with VHL disease, raising a question about the importance of VHL inactivation status in angiogenic behaviour and tumour progression. METHODS: To address this question, we investigated the influence of VHL mutation (direct sequencing)/hypermethylation (methylation-specific PCR) on angiogenesis/tumour parameters (immunohistochemistry) in 150 patients with sporadic ccRCC. RESULTS: We found no significant association between VHL mutation or methylation and angiogenesis/tumour parameters. CONCLUSION: These data indicate that tumour progression and angiogenesis are not directly influenced by VHL alterations and that additional genetic/epigenetic events should be considered to explain the diverse angiogenic and proliferative behaviour during tumour progression.

Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.

The maturation state of (monocyte-derived) dendritic cells (DCs) determines the type of T-cell response. Currently, several maturation cocktails are used in clinical trials, most commonly a cocktail of TNF-alpha, PGE2, IL-1beta, and IL-6. The authors studied DC phenotype and functional ability to stimulate TH1 responses after maturation with different cocktails employing clinically approved agents. DCs were stimulated with the microbial agent Ribomunyl combined with IFN-gamma and various inflammatory cytokine cocktails: TNF-alpha/IL-1beta/IFN-gamma and TNF-alpha/PGE2 combined with monocyte-conditioned medium (MCM) or IL-1beta/IL-6. Regardless of the maturation cocktail used, all DCs possessed the characteristic phenotype of mature, migratory DCs (high expression of CD40, CD80, CD83, CD86, CCR7, MHC class I and MHC class II). Ribomunyl/IFN-gamma matured DCs produced high IL-12p70 levels, whereas other maturation stimuli did not. Even more striking, restimulation of Ribomunyl IFN-gamma mDCs with CD40-activating antibody reactivated IL-12p70 production. No IL-12p70 could be detected when DCs were stimulated with TNF-alpha/PGE2 combined with MCM or IL-1beta/IL-6, presumably by suppression by PGE2. Restimulation of these DCs with CD40-activating antibody failed to activate IL-12p70 production. Moreover, low levels of IL-10 were observed, possibly indicating inhibition of TH1-cell responses. Indeed, T cells stimulated with these DCs produced high levels of type 2 cytokine IL-5 and outgrowth of CD4CD25 T cells. This study shows that DC maturation with cytokine cocktails different from those most commonly used could be beneficial for immunotherapy trials in cancer patients.