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Abstract: We determined circulating T helper, T regulatory and ICOS+T regulatory as well as DC cell counts in 29 patients with cerebral gliomas. Samples from patients with gliomas vs. healthy controls and from patients with glioblastomas vs. patients with glioma WHO grades I–III contained significantly (p<0.05) decreased numbers of total as well as mature, i.e. myeloid and plasmacytoid DCs. Patients with glioblastomas demonstrated significantly lower values of CD4+ as well as an increased fraction of ICOS+T regulatory/CD4+ cells. Higher CD4+ cell counts (≥225cells/μl, median) were associated with improved survival in glioblastomas. [Copyright &y& Elsevier]

Objectives: To identify an appropriate reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies. Methods: Serum from patients with prostate cancer ( n = 24), bladder cancer ( n = 24), renal cell carcinoma ( n = 24) and control subjects ( n = 48) was spiked with cel-miR-39, and then ribonucleic acid was isolated. Quantitative real-time polymerase chain reaction was used to determine the levels of candidate reference genes ( RNU1-4, RNU6-2, SNORD43, SNORD44, SNORD48, S NORA74A, miR-let-7a-1, miR-106a). Reference gene stability was determined using the NormFinder, geNorm and comparative delta-Ct algorithm. The effect of normalization was tested with miR-21 as the target gene, as this was previously suggested to be upregulated in cancer patients' serum. Results: Recovery of cel-miR-39 (mean 11.6%, range 1-56%) was similar in control subjects and cancer patients. SNORD44 and SNORD74A levels were around the detection limit of the assay and were thus omitted. All remaining candidates showed satisfying stability; SNORD43 was the most stable reference gene using all three algorithms. A combination of two genes ( SNORD43, RNU1-4) increases the stability somewhat. The level of miR-21 was similar in cancer patients and healthy controls, irrespective of the normalization strategy. Conclusions: SNORD43 is a suitable reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies. Our study questions the suitability of miR-21 as a biomarker for uro-oncological patients. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Cell-free circulating mitochondrial DNA (mtDNA) has been proposed as universal diagnostic and prognostic biomarker in cancer patients. Patients and methods: Cell-free DNA was isolated from 1 ml serum from patients with bladder cancer (BCA, n = 84), renal cell carcinoma (RCC, n = 33), and prostate cancer (CaP, n = 23), and compared with healthy individuals (n = 79). Quantitative real-time PCR was used to analyze the levels of a 79 bp (mtDNA-79), and 220 bp (mtDNA-220) fragment of the mitochondrial specific 16S-RNA. The mitochondrial DNA integrity (mtDNA-integrity) was defined as ratio of mtDNA-220 to mtDNA-79 fragments. Results: In healthy controls, mtDNA-79 levels were increased in male volunteers; mtDNA-230 levels and mtDNA-integrity were correlated with age. Neither mtDNA levels nor mtDNA-integrity were correlated with age or gender in cancer patients. Circulating mtDNA-79 (median 8.75 × 106 vs. 0.43 × 106 copies/ml) and mtDNA-230 (8.11 × 106 vs. 0.27 × 106 copies/ml) levels were significantly increased in cancer patients and allowed sensitive (84%) and specific (97%) discrimination from healthy controls. mtDNA levels were unequally distributed among the different cancer entities (mtDNA-79: BCA 9.54 × 106 vs. RCC 6.69 × 106 vs. CaP 4.48 × 106 copies/ml; mtDNA-230: BCA 9.78 × 106 vs. RCC 6.74 × 106 vs. CaP 1.94 × 106 copies/ml). The mtDNA-integrity was increased in RCC and BCA patients compared to control subjects and CaP patients. Serum mtDNA-integrity was correlated with pathological stage in RCC and with tumor grade in BCA patients. Conclusion: Circulating mtDNA levels are associated with gender and age in healthy individuals, but not in cancer patients. Quantification of circulating mtDNA may help identify patients with urologic malignancies. [Copyright &y& Elsevier]

Global histone modification patterns have been shown to be a predictive factor of recurrence in various cancers. We analyzed global histone-3-lysine-27 (H3K27) methylation in prostate cancer (PCA) tissues. H3K27 mono-, di-, and tri-methylation patterns were different in nonmalignant prostate tissue, localized PCA, metastatic PCA, and castration-resistant PCA. H3K27 mono-methylation was correlated with pT-stage, capsular penetration, seminal vesicle infiltration, and Gleason score in localized PCA and may therefore indicate adverse prognosis. [ABSTRACT FROM AUTHOR]

What's known on the subject? and What does the study add? Epigenetic alterations are important during carcinogenesis, and earlier studies suggested that global histone modification levels are predictive for patients' outcome in various tumor entities. We demonstrate that H3K27me1 and H3K27me3 are markers of progression-free survival in patients with renal cell carcinoma. OBJECTIVE • To evaluate if histone H3 lysine 27 (H3K27) methylation plays a role in renal cell carcinoma (RCC) tissue and whether its expression is a predictor of cancer recurrence in RCC. MATERIALS AND METHODS • A tissue microarray (TMA) with 193 RCC specimens (comprising 142 clear-cell, 31 papillary, 10 chromophobe and 10 sarcomatoid RCC), 10 oncocytoma tissue specimens and a TMA with 30 benign renal tissue samples were stained with antibodies against H3K27-monomethyl (H3K27me1), H3K27-dimethyl (H3K27me2) and H3K27-trimethyl (H3K27me3). • Sections were scored according to staining intensity and the proportion of epithelial cells showing nuclear staining. • H3K27 methylation levels were correlated with established clinical-pathological variables (tumour-node-metastasis [TNM] stage, Fuhrman grade) and progression-free/cancer-specific survival. RESULTS • H3K27me1/-me2/-me3 staining was significantly more intense in papillary RCC then in clear-cell RCC. • H3K27me3 levels were higher in oncocytoma than in RCC. • H3K27me1/-me2/-me3 methylation levels were inversely correlated with Fuhrman grading and pT-stage. • Global H3K27me1/-me2/-me3 methylation levels were always higher in benign renal tissue than in RCC with tumour relapse (H3K27me1 P < 0.001, H3K27me2 P= 0.032, H3K27me3 P= 0.004). • Progression-free survival was shorter in patients with lower levels of H3K27me1 and H3K27me3 in the univariate analysis. The newly created H3K27me score (combining the staining levels of the single modifications) was a significant and independent predictor of RCC progression-free survival. CONCLUSION • The present study on H3K27-methylation supports the hypothesis that global histone modifications are potential markers of cancer prognosis in RCC. One reason could be that decreased H3K27 indicates transcriptional activation and therefore predicts cancer activation. [ABSTRACT FROM AUTHOR]

Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Epigenetic alterations play an essential role during carcinogenesis. While DNA methylation has been extensively studied in bladder cancer, the relevance of histone modifications remains to be clarified. Earlier studies suggested that global histone modification levels are predictive for patients' outcome in various tumour entities (e.g. prostate, lung, breast and kidney cancer). The possibility to determine global histone modification levels easily and inexpensively using immunohistochemistry increases a potential routine use in the future. Our aim was therefore to investigate the global levels of histone H3K4 and H4K20 mono-, di- and trimethylation. For this purpose we prepared tissue microarrays with non-muscle-invasive bladder cancer (NMIBC), muscle-invasive bladder cancer (MIBC), bladder cancer metastases (METS) and normal urothelium (NU) tissue to compare global H3K4 and H4K20 methylation in these tissues, as well to assess the prognostic value of histone modifications. We show that global histone modification levels (H3K4me1, H3K4me3, H4K20me1, H4K20me2, H4K20me3) are lower in bladder cancer than in NU tissue. Furthermore, there was a decrease of histone modification levels (H3K4me1, H4K20me1, H4K20me2, H4K20me3) from NU over NMIBC and MIBCto METS. Histone modifications are correlated to advanced pathological stage in NMIBC and MIBC. Furthermore, H4K20me3 appeared to be a significant and independent prognostic predictor of bladder cancer-specific survival in patients with MIBC undergoing radical cystectomy. Our findings therefore provide a rationale for further investigation of histone modifications and their manipulation in bladder cancer. OBJECTIVE • To determine the role of global histone methylation as a prognostic parameter in patients with bladder cancer. PATIENTS AND METHODS • We used a tissue microarray with samples from patients with non-muscle-invasive bladder cancer (NMIBC; n= 161), muscle-invasive bladder cancer (MIBC, n= 127), normal urothelium (NU; n= 31) and bladder cancer metastases (METS; n= 31) to determine global histone methylation (me) levels at histone H3 lysine 4 (H3K4) and H4K20. RESULTS • Global histone modification levels (H3K4me1, H3K4me3, H4K20me1, H4K20me2, and H4K20me3) were lower in bladder cancersamples than in NU tissue • Global levels of H3K4me1, H4K20me1, H4K20me2 and H4K20me3 were decreasing from NU over NMIBC and MIBC to METS. • H4K20me1 levels were increased in patients with NMIBC with advanced pTstage and less differentiated bladder cancer. • In patients with MIBC, pTstage was negatively correlated with H3K4me1, H4K20me1 and H4K20me2 levels. • H4K20me3 levels were significantly correlated in a univariate and multivariate model with bladder cancer-specific mortality after radical cystectomy in patients with MIBC. CONCLUSION • Global histone methylation levels may help to identify patients with bladder cancerwith poor prognosis after radical cystectomy. [ABSTRACT FROM AUTHOR]

Abstract: Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria. [Copyright &y& Elsevier]

Abstract: The presence of small amounts of circulating DNA in plasma was demonstrated 60 years ago. Since then, cell-free DNA has been tested for quantity, fragmentation pattern, and tumor-specific sequences in patients with various malignancies. Recent studies have shown that cell-free DNA levels are distinctly increased in most patients with prostate cancer (PCA) and that the DNA fragmentation pattern is different from healthy individuals and patients with benign prostate disease. The origin of this circulating DNA remains largely unknown, but it is established that a small fraction of the DNA is derived from the tumor itself, and genetic (allelic imbalances) and epigenetic (DNA methylation) alterations are regularly detected in patients with PCA. The detection of increased DNA levels and tumor-specific DNA sequences may provide diagnostic and prognostic information. The recent findings in the emerging field of cell-free DNA will be discussed. [Copyright &y& Elsevier]

Scars arise in the late phase of wound healing and are characterized by fibroplasia. Previous controversial studies have discussed the regenerative wound healing capacity of Murphy Roths Large (MRL) mice. The aim of this study was to investigate the mechanisms of improved wound healing in a skin transplantation model. Skin grafts from MRL and haplotypically identical B10.BR mice were cross-transplanted. At day 10, B10.BR and MRL grafts on B10.BR recipients deposited collagen and showed severe apoptosis. Grafts of MRL recipients were not affected by such alterations and showed an enhanced healing progress. They were characterized by higher partial pressure of tissue oxygen, increased microcirculation, exceptionally intense neovascularization, and a blunted inflammatory response. This phenotype was accompanied by increased vascular endothelial growth factor expression, augmented by enhanced signal transducer and activator of transcription 3 (STAT3) phosphorylation. These effects were combined with a decreased STAT1 expression and phosphorylation. STAT1 pattern variation was associated with decreased Smad7 levels. Furthermore, MRL recipients showed improved stem cell recruitment to the wound area. The basic accelerated wound healing mechanism in MRL mice found in this skin transplantation model is improved engraftment; this is based on enhanced neovascularization and reduced inflammation. These effects are most likely due to higher vascular endothelial growth factor levels and changes in the STAT/Smad signal pathway, which may enhance transforming growth factor-β signaling, reducing proinflammatory responses. [ABSTRACT FROM AUTHOR]

OBJECTIVE To analyse the diagnostic and prognostic value of cell-free mitochondrial (mt)DNA in patients with testicular cancer, as increased levels of cell-free circulating mtDNA have been reported in patients with cancer. PATIENTS, SUBJECTS AND METHODS In all, 74 patients with testicular cancer (seminoma in 39, nonseminoma in 35) and 35 healthy individuals were included in the study. Circulating DNA was isolated from 1 mL of serum. A quantitative real-time polymerase chain reaction was used to analyse the levels of a 79-bp (mtDNA-79) and 220 bp (mtDNA-220) fragment of the mitochondrial specific 16S-RNA. The mtDNA integrity was expressed as the ratio of mtDNA-220 to mtDNA-79. RESULTS mtDNA-79 and mtDNA-220 levels were significantly ( P < 0.001) greater in patients with testicular cancer than in healthy individuals. The mtDNA integrity was similar in patients and healthy controls ( P = 0.435). Receiver operator characteristic curve analysis showed that cell-free mtDNA (mtDNA-79) levels distinguished, with a sensitivity of 59.5% and a specificity of 94.3%, between patients and healthy individuals (area under curve, 0.787). Also, mtDNA-79 levels could be used to distinguish between patients (31) with conventional markers (α-fetoprotein, human chorionic gonadotrophin, placental alkaline phosphatase and lactate dehydrogenase) within normal ranges and healthy individuals, with a sensitivity of 64.5% and specificity of 91.4% (area under curve 0.797). Cell-free mtDNA levels were not correlated with any clinicopathological variable (pT stage, lymph node invasion, vascular invasion, clinical stage, International Germ Cell Cancer Collaborative Group classification, tumour markers; all P > 0.05). CONCLUSION Cell-free mtDNA levels are greater in patients with testicular cancer and might provide valuable information for managing patients with testicular anomalies, especially those with normal levels of established tumour markers. [ABSTRACT FROM AUTHOR]

OBJECTIVE To investigate the role of circulating mitochondrial DNA (mtDNA) in patients with localized prostate cancer, as recent reports show that patients with advanced cancer have increased levels of mtDNA. PATIENTS AND METHODS DNA was isolated from the serum of 100 patients with prostate cancer and 18 with benign prostate hyperplasia (BPH). A quantitative real-time polymerase chain reaction was used to amplify 79 bp and 230 bp fragments of the mitochondrial 16s-RNA gene, the short fragment representing total mtDNA, including mtDNA truncated by apoptosis, and the long fragment representing mostly mtDNA from other cell death entities. mtDNA integrity was defined as the ratio of long to short mtDNA fragments. RESULTS The short and long mtDNA levels, and mtDNA integrity, were similar in patients with BPH and cancer ( P = 0.940, 0.211 and 0.441, respectively), and were not correlated with clinical or pathological variables, e.g. age, prostate-specific antigen (PSA) level, cT stage, pT stage, seminal vesicle infiltration, lymph node invasion, or Gleason score ( P = 0.075 to 0.961). However, patients with high levels of short mtDNA (>75th percentile) had a greater risk of PSA progression and this variable was the strongest predictor of PSA recurrence in a multivariate Cox analysis ( P = 0.023; hazard ratio 0.31; 95% confidence interval 0.113–0.851). CONCLUSION Circulating mtDNA levels did not distinguish between patients with prostate cancer or BPH. However, there was a significant increase in short mtDNA fragments in patients with early PSA recurrence after radical prostatectomy. [ABSTRACT FROM AUTHOR]

Objectives: CpG island hypermethylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. We investigated its role at multiple gene sites during prostate carcinogenesis. Methods: A quantitative, methylation-specific polymerase chain reaction was used to analyze the hypermethylation patterns at nine gene loci (Annexin2, APC, EDNRB, GSTP1, PTGS2, MDR1, RARbeta, Reprimo, and TIG1) in 80 patients with prostate cancer (PCa) and 26 patients with benign prostatic hyperplasia (BPH). Results: Hypermethylation was more frequent in PCa than in BPH tissues (EDNRB, 100% versus 88%; TIG1, 96% versus 12%; RARbeta, 95% versus 35%; GSTP1, 93% versus 15%; APC, 80% versus 50%; MDR1, 80% versus 31%; PTGS2, 68% versus 15%; Reprimo, 59% versus 19%; and Annexin2, 4% versus 0%). TIG1 and GSTP1 hypermethylation distinguished between PCa and BPH with a specificity of greater than 85% and sensitivity of greater than 93%. Hypermethylation at a single gene locus did not correlate with any clinicopathologic variables. In contrast, hypermethylation at two genes (eg, APC and TIG1, APC and GSTP1, APC and PTGS2, APC or MDR, GSTP1 or PTGS2) correlated significantly with the pathologic stage and/or Gleason score (P = 0.033 to 0.045). Hypermethylation at APC and Reprimo, as well as DNA hypermethylation at more than five genes, correlated significantly with the rate of prostate-specific antigen recurrence after radical prostatectomy (P = 0.0078 and P = 0.0074, respectively). Conclusions: Our results have confirmed that the hypermethylation patterns are helpful in the diagnosis and prognosis of PCa. Increases in CpG island hypermethylation at multiple gene sites occur during PCa progression and indicate early biochemical recurrence after radical prostatectomy. [Copyright &y& Elsevier]

Background: Little is known about the local accumulation and function of immune cells in peritoneal fluid after elective surgery of the upper and lower gastrointestinal tract. Our study was designed to investigate whether systemic immune cell response mirrors the local response. We focused on the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α and on monocytes, natural killer (NK) cells, and T cells that play an important role in eliciting the innate and adaptive immune response. Methods: Blood samples were taken prospectively from 25 patients 24 h before surgery, as well as 24 h and 48 h afterward. Abdominal drainage fluids were collected intraoperatively 1 h after the abdomen was opened and 24 h and 48 h postoperatively. Apart from the white blood cells, intracellular T-helper-cell (TH1/TH2) cytokine production (interferon-γ, IL-2, IL-4, IL-13) and HLA-DR on monocytes were measured by four-color flow cytometry, IL-6, and TNF-α with the fast immunoluminescence method. Results: Cells of the innate immune system (NK cells, monocytes, NK-T cells, CD5+ B cells) rapidly decreased in abdominal fluids (P &#60; 0.05: +24 h; +48 h) after surgery, which was paralleled by a concomitant decline in peripheral blood. The percentage of abdominal interferon-γ, IL-2, IL-4, and IL-13-producing TH cells increased in a way that distinctly counteracted the decrease of the natural immune cells. HLA-DR expression on monocytes in peripheral blood declined significantly (P &#60; 0.05: +24 h; +48 h). In contrast, monocytes in abdominal fluids had high HLA-DR expression. Furthermore, abdominal fluids contained significantly higher concentrations of TNF-α (P &#60; 0.05: +24 h; +48 h) and IL-6 (P &#60; 0.05: +24 h) compared with peripheral blood. Conclusions: Specific immune cell recruitment and cytokine production play an important role in post-trauma events. Measuring distinct local immune cell repertoires and cytokines provides answers as to how the different phases of postoperative immune events proceed. The evaluation of the local response may provide additional criteria for the evaluation of operative trauma. This knowledge may be helpful in detecting postoperative pathological aberrancies. [Copyright &y& Elsevier]

Objectives: To analyze circulating microRNAs (miRNA) in serum as non-invasive biomarker in patients with localized prostate cancer (PCA), benign prostate hyperplasia (BPH) and healthy individuals (HI). Methods: Total RNA was isolated from serum samples and the circulating levels of different RNA species (miRNA, miR-16; small nuclear RNA, RNU1A-1; messenger RNA, HPRT1), as well as of 4 oncogenic miRNAs (miR-26a, miR-32, miR-195, miR-let7i), were determined using a quantitative real-time polymerase chain reaction. We also evaluated miRNA levels in a second cohort of 10 PCA patients in cancer/nonmalignant tissue, and pre- and post-prostatectomy serum samples. Results: The levels of miR-16 and RNU1A-1were reliably measured, whereas HPRT1 levels were often below the detection limit of our assay. Circulating oncogenic miRNA levels were different, and especially the miR-26a level allowed sensitive (89%) discrimination of PCA and BPH patients at a moderate specificity (56%; area under the curve [AUC]: 0.703); the analysis of oncogenic miRNAs in combination increased the diagnostic accuracy (sensitivity: 78.4%; specificity: 66.7%; AUC: 0.758). Despite the low number of patients limiting the statistical power of the study, we observed correlations with clinical-pathologic parameters: miR-16, miR-195, and miR-26a were significantly correlated with surgical margin positivity; miR-195 and miR-let7i were significantly correlated with the Gleason score. Tissue miRNA levels were correlated with preprostatectomy miRNA levels in serum, and serum miRNA decreased after prostatectomy, thereby indicating tumor-associated release of miRNA. Conclusions: Tumor-associated miRNAs in serum allow noninvasive discrimination of PCA and BPH. [ABSTRACT FROM AUTHOR]

Gene silencing by promoter DNA hypermethylation is common in PCA. The role of HXKy methylation as an up-/downstream or parallel silencing mechanism is not yet clear. We analyzed HXKy methylation at gene sites that are aberrantly DNA-methylated in PCA (i.e. GSTP1, RARβ, APC and PTGS2). Cancerous (PC3, DU145, LNCaP) and benign prostate hyperplasia (BPH1) cell lines were studied using chromatin immunoprecipitation and methylation specific PCR. Gene expression was analyzed using quantitative RT-PCR. In PCA, histone modifications characteristic of gene repression (di-/tri-methylated H3K9, H3K27, H4K20) were predominantly observed at the studied gene sites and paralleled aberrant DNA hypermethylation. In BPH, no aberrant DNA hypermethylation and mostly histone modifications supporting active gene expression (H3K4, H3K79 di-/tri methylation) were seen at the respective gene sites. Mono-methylated H3K4 and H3K9 modifications that have moderate effects on gene expression were frequent in both PCA and BPH. PC3 cells grown in the presence of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (aza) resulted in DNA demethylation and moderate gene reexpression whereas repressive HXKy di-methylation increased 1.5-3 fold and tri-methylation was mostly unchanged. Removal of aza reverses gene expression. These findings suggest that HXKy methylation is upstream to DNA hypermethylation in PCA. [ABSTRACT FROM AUTHOR]

Cancer patients have increased levels of serum DNA fragments (SDF) compared to most patients with non-malignant disease and healthy individuals. In PCA, DNA hypermethylation is also frequently observed (>80%) at promoter CpG islands of GSTP1, PTGS2 and TIG1. Therefore we analysed hypermethylation of these genes in SDF from patients with PCA (n=168) and benign prostate hyperplasia (BPH; n=42) and controls (n=11) for information about the cellular origin of DNA. SDF concentrations and the hypermethylated fractions were analysed using a methylation-sensitive restriction enzyme based real-time PCR. SDF levels were significantly increased in PCA vs BPH vs controls (65 vs 20 vs 7 ng/mL; p<0.001). Hypermethylation of GSTP1, TIG1 or PTGS2 was more frequently detected in PCA than in BPH patients and controls (overall 47% vs 8% vs 0%; p<0.001), but did not exceed 10% of total DNA. SDF levels distinguished PCA from BPH patients with a sensitivity of 83% and specificity of 69%. The diagnostic significance of SDF was greater than that of hypermethylation data. In conjunction with SDF, hypermethylation numbers did not contribute any further information. Both SDF levels and the hypermethylation status were not correlated to advanced tumor stage or grade (p>0.05). In summary SDF is a promising diagnostic marker in PCA. It does not primarily originate from tumor cells but from apoptotic and necrotic cells surrounding the tumor. [ABSTRACT FROM AUTHOR]