Background:Whether methylation of the microRNA (mir)-124-3 CpG island is of relevance for the clinical course of a solid cancer and whether it shows association with clinicopathology or survival of patients with renal cell cancer (RCC) is not known as yet.Methods:In a cross-sectional study, relative methylation of mir-124-3 was measured in 111 RCC samples and 77 paired normal appearing tissues using quantitative methyl-specific PCR. Results were statistically compared with tumour histology, clinicopathological parameters and disease recurrence.Results:We found tumour-specific hypermethylation of mir-124-3 in samples of RCCs with clear cell histology (ccRCC) compared with paired normal appearing tissues (P<0.0001). Methylation was significantly increased in tumours with state of advanced disease (P<0.0001). Higher relative methylation was associated with worse recurrence-free survival in both univariate (hazard ratio=9.37; P=0.0005) as well as bivariate Cox regression analyses considering age, sex, diameter of tumours and state of advanced disease, metastasis and lymph node metastases as covariates (hazard ratios=5.9–18.2; P-values of 0.0003–0.008).Conclusion:We identified mir-124-3 CpG islands (CGI) methylation as a relevant epigenetic mark for ccRCC thus underlining the need for functional studies of potentially affected signalling pathways in kidney tumour models. Methylation of mir-124-3 is suggested as an independent prognosticator for ccRCC.

ObjectivesWe here investigated whether experimental gingivitis enhances systemic markers of inflammation which are also known as surrogate markers of atherosclerotic plaque development.BackgroundGingivitis is a low-level oral infection induced by bacterial deposits with a high prevalence within Western populations. A potential link between the more severe oral disease periodontitis and cardiovascular disease has already been shown.Methods37 non-smoking young volunteers with no inflammatory disease or any cardiovascular risk factors participated in this single-subject interventional study with an intra-individual control. Intentionally experimental oral inflammation was induced by the interruption of oral hygiene for 21 days, followed by a 21-days resolving phase after reinitiation of oral hygiene. Primary outcome measures at baseline, day 21 and 42 were concentrations of hsCRP, IL-6, and MCP-1, as well as adhesion capacity and oxLDL uptake of isolated blood monocytes.ResultsThe partial cessation of oral hygiene procedures was followed by the significant increase of gingival bleeding (34.0%, P<0.0001). This local inflammation was associated with a systemic increase in hsCRP (0.24 mg/L, P = 0.038), IL-6 (12.52 ng/L, P = 0.0002) and MCP-1 (9.10 ng/l, P = 0.124) in peripheral blood samples between baseline and day 21, which decreased at day 42. Monocytes showed an enhanced adherence to endothelial cells and increased foam cell formation after oxLDL uptake (P<0.050) at day 21 of gingivitis.ConclusionsBacterial-induced gingival low-level inflammation induced a systemic increase in inflammatory markers. Dental hygiene almost completely reversed this experimental inflammatory process, suggesting that appropriate dental prophylaxis may also limit systemic markers of inflammation in subjects with natural gingivitis. International Clinical Trials Register Platform of the World Health Organization, registry number: DRKS00003366, URL: http://apps.who.int/trialsearch/Default.aspx

BackgroundTo investigate the microbial composition of biofilms at inflamed peri-implant and periodontal tissues in the same subject, using 16S rRNA sequencing.MethodsSupra- and submucosal, and supra- and subgingival plaque samples were collected from 7 subjects suffering from diseased peri-implant and periodontal tissues. Bacterial DNA was isolated and 16S rRNA genes were amplified, sequenced and aligned for the identification of bacterial genera.Results43734 chimera-depleted, denoised sequences were identified, corresponding to 1 phylum, 8 classes, 10 orders, 44 families and 150 genera. The most abundant families or genera found in supramucosal or supragingival plaque were Streptoccocaceae, Rothia and Porphyromonas. In submucosal plaque, the most abundant family or genera found were Rothia, Streptococcaceae and Porphyromonas on implants. The most abundant subgingival bacteria on teeth were Prevotella, Streptococcaceae, and TG5. The number of sequences found for the genera Tannerella and Aggregatibacter on implants differed significantly between supra- and submucosal locations before multiple testing. The analyses demonstrated no significant differences between microbiomes on implants and teeth in supra- or submucosal and supra- or subgingival biofilms.ConclusionDiseased peri-implant and periodontal tissues in the same subject share similiar bacterial genera and based on the analysis of taxa on a genus level biofilm compositions may not account for the potentially distinct pathologies at implants or teeth.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6831-14-157) contains supplementary material, which is available to authorized users.

Neurofilament Heavy polypeptid (NEFH) belongs to the group of type IV intermediate filament proteins. DNA methylation of the NEFH promoter and loss of expression have previously been shown to activate the AKT/β-catenin pathway in tumor cells. When identifying hypermethylation of the NEFH CpG island (CGI) in renal cell cancer (RCC) we asked whether methylation could provide clinical or prognostic information for RCC and/or predict therapy response in patients with metastatic RCC (mRCC) undergoing antiangiogenic therapy. Relative methylation of the NEFH CGI was analyzed in 132 RCC samples and 83 paired normal tissues using quantitative methylation-specific PCR. Results were statistically compared with tumor histology, clinicopathological parameters, progression-free survival (PFS) as well as with overall survival (OS) in a subset of 18 mRCC patients following antiangiogenic therapy regimens. The NEFH CGI methylation demonstrated a tumor-specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005). Higher relative methylation was also significantly associated with a poor PFS (HR = 8.6, P < 0.001) independent from the covariates age, gender, diameter of tumors, state of advanced disease, and local and distant metastasis. Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028). We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti-vascular endothelial growth factor-based therapy response.

VEGF-targeted therapy increases both the progression-free (PFS) and overall survival (OS) of patients with metastasized renal cell cancer (mRCC). Identification of molecular phenotypes of RCC could improve risk-stratification and the prediction of the clinical disease course. We investigated whether gene-specific DNA hypermethylation can predict PFS and OS among patients undergoing anti-VEGF-based therapy. Primary tumor tissues from 18 patients receiving targeted therapy were examined retrospectively using quantitative methylation-specific PCR analysis of CST6, LAD1, hsa-miR-124-3, and hsa-miR-9-1 CpG islands. PFS and OS were analyzed for first-line and sequential antiangiogenic therapies using the log rank statistics. Sensitivity and specificity were determined for predicting first-line therapy failure. Hypermethylation of CST6 and LAD1 was associated with both a shortened PFS (log rank p = 0.009 and p = 0.004) and OS (p = 0.011 and p = 0.043). The median PFS observed for the high and low methylation groups of CST6 and LAD1 was 2.0 vs.11.4 months. LAD1 methylation had a specificity of 1.0 (95% CI 0.65–1.0) and a sensitivity of 0.73 (95% CI 0.43–0.90) for the prediction of first-line therapy. CST6 and LAD1 methylation are candidate epigenetic biomarkers showing unprecedented association with PFS and OS as well as specificity for the prediction of the response to therapy. DNA methylation markers should be considered for the prospective evaluation of larger patient cohorts in future studies.

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Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58–81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.

GATA-binding proteins 1 (GATA1) and 2 (GATA2) are zinc-finger transcription factors and belong to the GATA family proteins 1-6. GATA1 interacts with the TP53 tumor suppressor gene, and both GATAs have been shown to be involved in cell growth, apoptosis, and tumorigenesis of several solid tumors. GATA1 and GATA2 expression alterations are associated with poor survival and adverse clinicopathology in prostate and colorectal cancer, while the significance and prognostic value in clear cell renal cell carcinoma (ccRCC) has not been investigated as yet. We investigated relative messenger RNA (mRNA) expression levels of GATA1 and GATA2 in 77 ccRCC and 58 paired adjacent noncancerous renal tissues by quantitative real-time reverse-transcribed PCR. Relative mRNA expression levels were determined using the ΔΔCt method. GATA1 and GATA2 expression levels were significantly decreased in tumor tissues compared with normal tissues (p < 0.001, paired t test). In univariate logistic regression analysis, decreased GATA1 and GATA2 expression levels were associated with advanced tumor disease (p = 0.005 and 0.008), positive distant metastasis (p = 0.03 and 0.001), and lymph node metastasis status (p = 0.011 and 0.038). Reduced expression levels of GATA1 and GATA2 were associated with an increased risk of disease recurrence (p = 0.005 and 0.006; hazard ratio = 0.05 and 0.21). Pairwise bivariate analysis after adjusting for clinicopathological parameters revealed relative mRNA expression of GATA1, but not GATA2, as an independent candidate prognosticator for ccRCC. Our results support that GATA1 and GATA2 are involved in ccRCC tumor biology possibly affecting tumor development and aggressiveness.