Imaging the Nonlinear Plasmoemission Dynamics of Electrons from Strong Plasmonic Fields.

We use subcycle time-resolved photoemission microscopy to unambiguously distinguish optically triggered electron emission (photoemission) from effects caused purely by the plasmonic field (termed "plasmoemission"). We find from time-resolved imaging that nonlinear plasmoemission is dominated by the transverse plasmon field component by utilizing a transient standing wave from two counter-propagating plasmon pulses of opposite transverse spin. From plasmonic foci on flat metal surfaces, we observe highly nonlinear plasmoemission up to the fifth power of intensity and quantized energy transfer, which reflects the quantum-mechanical nature of surface plasmons. Our work constitutes the basis for novel plasmonic devices such as nanometer-confined ultrafast electron sources as well as applications in time-resolved electron microscopy.

Exome sequencing characterizes the somatic mutation spectrum of early serrated lesions in a patient with serrated polyposis syndrome (SPS).

BACKGROUND: Serrated or Hyperplastic Polyposis Syndrome (SPS, HPS) is a yet poorly defined colorectal cancer (CRC) predisposition characterised by the occurrence of multiple and/or large serrated polyps throughout the colon. A serrated polyp-CRC sequence (serrated pathway) of CRC formation has been postulated, however, to date only few molecular signatures of serrated neoplasia (BRAF, KRAS, RNF43 mutations, CpG Island Methylation, MSI) have been described in a subset of SPS patients and neither the etiology of the syndrome nor the distinct genetic alterations during tumorigenesis have been identified. METHODS: To identify somatic point mutations in potential novel candidate genes of SPS-associated lesions and the involved pathways we performed exome sequencing of eleven early serrated polyps obtained from a 41 year-old female patient with clinically confirmed SPS. For data filtering and analysis, standard pipelines were used. Somatic mutations were identified by comparison with leukocyte DNA and were validated by Sanger sequencing. RESULTS: The BRAF p.V600E or KRAS p.G12D mutation was identified in six polyps (~50%) and not found in polyps from the distal colon. In addition, we found seven unique rare somatic alterations of seven different genes in four serrated tumours, all of which are missense variants. The variant in ABI3BP and CATSPERB are predicted to be deleterious. No established cancer gene or candidate genes related to serrated tumorigenesis were affected. CONCLUSIONS: Somatic mutations seem to be rare events in early hyperplastic and serrated lesions of SPS patients. Neither frequently affected genes nor enrichment of specific pathways were observed. Thus, other alterations such as non-coding variants or epigenetic changes might be the major driving force of tumour progression in SPS.

Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4.

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

Oral CPSI-2364 treatment prevents postoperative ileus in swine without impairment of anastomotic healing.

BACKGROUND: Postoperative ileus (POI) is an iatrogenic complication of abdominal surgery, mediated by a severe inflammation of the muscularis externa (ME). We demonstrated that orally applicated CPSI-2364 prevents POI in rodents by blockade of p38 MAPK pathway and abrogation of NO production in macrophages. In the present experimental swine study we compared the effect of orally and intravenously administered CPSI-2364 on POI and examined CPSI-2364 effect on anastomotic healing. METHODS: CPSI-2364 was administered preoperatively via oral or intravenous route. POI was induced by intestinal manipulation of the small bowel. ME specimens were examined by quantitative PCR for CCL2 chemokine gene expression and myeloperoxidase activity. Functional analyzes included measurement of ileal smooth-muscle ex vivo contractility, in vivo intestinal and colonic transit. Furthermore, anastomotic healing of a rectorectostomy after CPSI-2364 treatment was assessed by perianastomotic hydroxyproline concentration, a histochemically evaluated healing score and anastomotic bursting pressure (ABP). RESULTS: CPSI-2364 abolished inflammation of the ME and improved postoperative smooth muscle contractility and intestinal transit independently of its application route. Hydroxyproline concentration and ABP measurement revealed no wound healing disturbances after oral or intravenous CPSI-2364 treatment whereas histological scoring demonstrated delayed anastomotic healing after intravenous treatment. CONCLUSION: CPSI-2364 effectively prevents POI in swine independently of its application route. Impairment of anastomotic healing could be observed after intravenous but not oral preoperative CPSI-2364 treatment. Subsumed, an oral preoperative administration of CPSI-2364 appears to be a safe and efficient strategy for prophylaxis of POI.

Histone methylation defines an epigenetic entity in penile squamous cell carcinoma.

PURPOSE: Earlier studies indicate that epigenetics contribute to the pathogenesis of penile squamous cell carcinoma. Histone methylation patterns are frequently altered during carcinogenesis. Therefore, we investigated the methylation pattern of the histones H3K4, H3K9 and H3K27 in penile carcinoma and normal tissue. MATERIALS AND METHODS: A tissue microarray was constructed with 65 penile carcinomas, 6 metastatic lesions and 30 control tissues. Global histone methylation was assessed using immunohistochemistry. RESULTS: Global levels of H3K4me1, H3K9me1, H3K9me2, H3K27me2 and H3K27me3 were decreased, whereas H3K9me3 was increased in penile carcinoma. Histone methylation levels defined an epigenetic entity that allowed accurate differentiation of cancer and normal samples. We observed no correlation of histone methylation levels with clinicopathological parameters or patient outcome. CONCLUSIONS: The description of a definite epigenetic entity in penile carcinoma provides a rationale for testing epigenetic agents in patients with metastatic disease.

Hepatocarcinogenesis in non-cirrhotic liver is associated with a reduced number of clonal hepatocellular patches in non-tumorous liver parenchyma.

We investigated circumscribed cell proliferations in healthy livers in comparison to non-cirrhotic livers bearing hepatocellular carcinoma. Using histochemical staining for cytochrome c oxidase, the fourth complex of the respiratory chain, we visualized patch-forming descendents of regeneratively active liver cells. The clonal nature of these patches was verified by laser-capture microdissection and Sanger sequencing of the enzyme's core subunits in patches carrying marker mutations on the mtDNA. We demonstrate a highly significant increase of the patch size and also a highly significant increase in the number of patches carrying marker mutations between hepatocellular carcinoma-free and -bearing livers. Thus, the carcinoma-bearing livers accumulated more genetic damage on mtDNA than the control group. Furthermore, for the first time, we present evidence in hepatocellular carcinoma-bearing non-cirrhotic livers of a significantly reduced pool of regeneratively active liver cells that are genetically and functionally altered. The analogy to ageing-related changes is suggestive of premature ageing of stem cells in non-cirrhotic hepatocellular carcinoma-bearing liver as an early step to hepatocarcinogenesis.

Classifying prostate cancer malignancy by quantitative histomorphometry.

PURPOSE: Prostate cancer is routinely graded according to the Gleason grading scheme. This scheme is predominantly based on the textural appearance of aberrant glandular structures. Gleason grade is difficult to standardize and often leads to discussion due to interrater and intrarater disagreement. Thus, we investigated whether digital image based automated quantitative histomorphometry could be used to achieve a more standardized, reproducible classification outcome. MATERIALS AND METHODS: In a proof of principle study we developed a method to evaluate digitized histological images of single prostate cancer regions in hematoxylin and eosin stained sections. Preprocessed color images were subjected to color deconvolution, followed by the binarization of obtained hematoxylin related image channels. Highlighted neoplastic epithelial gland related objects were morphometrically assessed by a classifier based on 2 calculated quantitative and objective geometric measures, that is inverse solidity and inverse compactness. The procedure was then applied to the prostate cancer probes of 125 patients. Each probe was independently classified for Gleason grade 3, 4 or 5 by an experienced pathologist blinded to image analysis outcome. RESULTS: Together inverse compactness and inverse solidity were adequate discriminatory features for a powerful classifier that distinguished Gleason grade 3 from grade 4/5 histology. The classifier was robust on sensitivity analysis. CONCLUSIONS: Results suggest that quantitative and interpretable measures can be obtained from image based analysis, permitting algorithmic differentiation of prostate Gleason grades. The method must be validated in a large independent series of specimens.

Global histone H3K27 methylation levels are different in localized and metastatic prostate cancer.

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.

Global histone H3 lysine 27 (H3K27) methylation levels and their prognostic relevance in 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.

Four-and-a-half LIM domain protein 2 is a novel regulator of sphingosine 1-phosphate receptor 1 in CCL19-induced dendritic cell migration.

We identified the four-and-a-half LIM domain protein 2 (FHL2) as a novel regulator of CCL19-induced dendritic cell (DC) migration. Initiation of migration is a hallmark of DC function and plays a central role in the induction and regulation of immune responses. In vivo, DCs continuously acquire Ag in the periphery and migrate to draining lymph nodes, under the influence of local environmental chemotactic factors like CCL19/21 or sphingosine 1-phosphate (S1P). We investigated the role of S1P- and RhoA-regulated FHL2 in this process. We found reduced nuclear localization of FHL2 in mature bone marrow-derived DCs (BMDCs), compared with immature BMDCs, following stimulation with CCL19. Furthermore, in vitro-generated murine FHL2(-/-) BMDCs displayed a significantly increased migratory speed, directionality, and migratory persistence toward the chemokine CCL19 compared with wild-type BMDCs. Moreover, in vivo, FHL2(-/-) BMDCs showed increased migration toward lymphoid organs. FHL2(-/-) BMDCs increased the expression of S1PR1, which was associated with greater Rac activation. An S1PR1 antagonist and knock-down of S1PR1 abrogated the increased migratory speed of FHL2(-/-) BMDCs. Our results identify FHL2 as an important novel regulator of DC migration via regulation of their sensitivity toward environmental migratory cues like S1P and CCL19.

Alterations of global histone H4K20 methylation during prostate carcinogenesis.

BACKGROUND: Global histone modifications have been implicated in the progression of various tumour entities. Our study was designed to assess global methylation levels of histone 4 lysine 20 (H4K20me1-3) at different stages of prostate cancer (PCA) carcinogenesis. METHODS: Global H4K20 methylation levels were evaluated using a tissue microarray in patients with clinically localized PCA (n = 113), non-malignant prostate disease (n = 27), metastatic hormone-naive PCA (mPCA, n = 30) and castration-resistant PCA (CRPC, n = 34). Immunohistochemistry was performed to assess global levels of H4K20 methylation levels. RESULTS: Similar proportions of the normal, PCA, and mPCA prostate tissues showed strong H4K20me3 staining. CRPC tissue analysis showed the weakest immunostaining levels of H4K20me1 and H4K20me2, compared to other prostate tissues. H4K20me2 methylation levels indicated significant differences in examined tissues except for normal prostate versus PCA tissue. H4K20me1 differentiates CRPC from other prostate tissues. H4K20me1 was significantly correlated with lymph node metastases, and H4K20me2 showed a significant correlation with the Gleason score. However, H4K20 methylation levels failed to predict PSA recurrence after radical prostatectomy. CONCLUSIONS: H4K20 methylation levels constitute valuable markers for the dynamic process of prostate cancer carcinogenesis.

Macroscopic and histopathologic findings in a laparoschisis model in fetal sheep: comparisons with gastroschisis in human fetuses and implications for prenatal interventions.

PURPOSE: In order to further define the challenges, minimally invasive fetal surgery will have to overcome human fetuses with gastroschisis. The purpose of this study was to compare macroscopic and histopathologic characteristics of experimental laparoschisis in sheep fetuses with actual cases of gastroschisis from a postmortem series of human fetuses. METHODS: Employing fetoscopy, we created a laparoschisis model in eight fetal sheep between 74 and 92 days of gestation (median 86.5 days). Twenty to 31 days after fetoscopic creation of fetal laparoschisis, six surviving fetuses were harvested for macroscopic and histopathologic assessments of the experimental lesion. These findings were compared to those of ten human fetuses with gastroschisis after termination of pregnancy. RESULTS: In the six sheep fetuses, both macroscopic and histopathologic intestinal changes achieved by this animal model resembled those of the human fetuses with gastroschisis. The surface of the intestine, liver and stomach exposed to the amniotic fluid was covered by a thick pseudocapsule made up of reactive fibroblasts and a dense capillary network. Parts of the capsule showed a foreign body-type reaction. CONCLUSIONS: Macroscopic and histopathological findings in a new minimally invasive laparoschisis model in sheep resemble those found in human fetuses with gastroschisis. The new model seems therefore suitable for assessing the potential of prenatal minimally invasive fetoscopic interventions in this condition.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome.

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

FHL2 expression in peritumoural fibroblasts correlates with lymphatic metastasis in sporadic but not in HNPCC-associated colon cancer.

Four and a half LIM domain protein-2 (FHL2) is a component of the focal adhesion structures and has been suggested to have an important role in cancer progression. This study analyses the role of FHL2 in peritumoural fibroblasts of sporadic and hereditary non-polyposis colorectal cancer (HNPCC). Tissue specimens of 48 sporadic and 49 hereditary colon cancers, respectively, were stained immunohistochemically for FHL2, transforming growth factor (TGF)-ß1 ligand and α-SMA. Myofibroblasts at the tumour invasion front co-expressed α-SMA and FHL2. Sporadic colon cancer but not HNPCC cases showed a correlation between TGF-ß1 expression of the invading tumour cells and FHL2 staining of peritumoural myofibroblasts. Overexpression of FHL2 in peritumoural myofibroblasts correlated to lymphatic metastasis in sporadic colon cancer but not in HNPCC. In cultured mouse fibroblasts, TGF-ß1 treatment induced myofibroblast differentiation, stimulated FHL2 protein expression and elevated number of migratory cells in transwell motility assays, suggesting that FHL2 is regulated downstream of TGF-ß. Physical contact of colon cancer cells and myofibroblasts via FHL2-positive focal adhesions was detected in human colon carcinoma tissue and in co-culture assays using sporadic as well as HNPCC-derived tumour cell lines. Our data provide strong evidence for an important role of FHL2 in the progression of colon cancers. Tumour-secreted TGF-ß1 stimulates FHL2 protein expression in peritumoural fibroblasts, probably facilitating the invasion of tumour glands into the surrounding tissue by enhanced myofibroblast migration and tight connection of fibroblasts to tumour cells via focal adhesions. These findings are absent in HNPCC-associated colon cancers in vivo and may contribute to a less invasive and more protruding tumour margin of microsatellite instable carcinomas.

Regulatory BCL2 promoter polymorphism (-938C>A) is associated with adverse outcome in patients with prostate carcinoma.

Molecular markers predictive of prostate cancer prognosis are urgently needed. Overexpression of the antiapoptotic protein, Bcl-2, has repeatedly been shown to be associated with adverse outcome in this malignancy. We hypothesized that a regulatory BCL2 -938C>A promoter polymorphism, which significantly affects promoter activity and Bcl-2 expression in different malignancies, may influence survival. Reporter assays and electrophoretic mobility shift assays reveled that the -938C>A BCL2 promoter polymorphism significantly affects promoter activity and transcription factor binding in prostate cancer cells. Significantly higher BCL2 mRNA expression was observed in primary prostate carcinomas derived from patients with the AA, compared to CC, genotype. Survival analysis showed that the -938AA genotype was an independent, unfavorable prognostic factor for relapse-free survival in a primary cohort of 142 patients and in an independent replication cohort of 148 patients, with hazard ratios (HR) of 4.4 (95% CI, 1.3-15.1; p = 0.018) and 4.6 (95% CI, 1.5-14.2; p = 0.009). Furthermore, the -938AA genotype was independently associated with worse overall survival in the replication series, with a HR of 10.9 (95% CI, 1.2-99.3; p = 0.034). We conclude that the BCL2 -938C>A polymorphism is an independent predictor of relapse-free and overall survival in patients with prostate cancer. The BCL2 -938C>A polymorphism should be evaluated prospectively and may also have promise in assisting optimal patient choice for treatment with BCL2-targeted drugs already in evaluation for prostate cancer treatment.

DNA hypermethylation in papillary renal cell carcinoma.

OBJECTIVE: To investigate the pattern of DNA CpG island hypermethylation in papillary renal cell carcinoma (pRCC). MATERIAL AND METHODS: DNA from pRCC (n= 32) and adjacent normal tissue (n= 15) was isolated. A quantitative methylation-specific PCR was performed to analyse the methylation pattern at APC (actin beta), CDH1 (E-cadherin), GSTP1 (glutathione S-transferase pi 1), RASSF1A (Ras association domain family member 1A) and TIMP3 (TIMP metallopeptidase inhibitor 3); a sequence of ACTB without CpG was used to normalize for DNA input and to calculate the relative amount of methylated DNA (normalized index of methylation, NIM). RESULTS: RASSF1A hypermethylation was observed in most pRCC and normal samples (100 vs 94.4%), but the median NIM was significantly higher in pRCC samples (2.11 vs 0.61; P < 0.001). RASSF1A hypermethylation allowed discrimination of pRCC and normal tissue with a sensitivity of 87.5% and a specificity of 73.3% as determined via receiver operator characteristic analysis (area under curve = 0.814). Hypermethylation at APC (3.0 vs 6.7%), CDH1 (15.6 vs 0%), GSTP1 (21.9 vs 6.7%) and TIMP3 (6.3 vs 0%) was infrequent in pRCC and normal tissue. CDH1 was significantly correlated with pathological stage (P= 0.015), and patients with methylated CDH1 methylation showed a trend towards shorter recurrence-free survival (log-rank P= 0.057). The number of methylated gene sites was correlated with pathological stage (P= 0.007) and lymph node metastasis (P= 0.008). CONCLUSIONS: DNA hypermethylation at RASSF1A is common in pRCC tissue irrespective of the histological subtype, but also frequently seen at lower levels in normal adjacent tissue. Aberrant hypermethylation could be a prognostic marker for pRCC.

Colonic anastomotic healing in the context of altered macrophage function and endotoxemia.

PURPOSE: Prevention of perioperative activation of intestinal muscularis macrophages is a promising intervention to avoid post-traumatic gastrointestinal tract dysfunction. However, impaired macrophage function could have deleterious consequences on anastomotic healing, especially in complications aggravating the healing process itself, such as infectious problems either as preexisting local inflammation or infection (e.g., complicated diverticulitis) or endotoxemia due to early postoperative infections (e.g., pneumonia). Aim of this study was to investigate colonic anastomotic healing in macrophage-depleted mice in the presence of endotoxemia. METHODS: Colonic anastomoses were performed, and mice were randomized into six groups (wild type; wild type with endotoxemia; pharmacological depletion of macrophages; pharmacological depletion with endotoxemia; genetically conditioned within the gut muscularis macrophage-deficient osteopetrotic mice; osteopetrotic mice with endotoxemia). Anastomotic tissues were removed 2, 5, and 10 days after surgery and used for functional, histological, biochemical, and molecular investigations. RESULTS: After pharmacological pretreatment, an almost complete depletion of macrophages was found in the muscularis up to 24 h postoperatively. Bursting pressure was significantly lower than 10 days after anastomotic procedure in osteopetrotic mice during endotoxemia, in marked contrast to transient pharmacologically macrophage-depleted mice. Pharmacological depletion during endotoxemia did not affect hydroxyproline concentration. Finally, in osteopetrotic mice during endotoxemia, collagen-3 expression was significantly lower compared to controls. CONCLUSIONS: In our current model, we demonstrate that perioperative pharmacological macrophage depletion and inactivation transiently diminishes muscularis macrophages and does not affect intestinal anastomotic healing in the presence of endotoxemia. However, a long-lasting macrophage absence or dysfunction impairs anastomotic healing and could be a risk factor for postoperative anastomotic leakage.

Prognostic relevance of global histone H3 lysine 4 (H3K4) methylation in renal cell carcinoma.

Epigenetic alterations play an important role in carcinogenesis. Recent studies suggested that global histone modifications are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone H3 lysine 4 mono-methyl (H3K4me1), -di-methyl (H3K4me2) and -trimethyl (H3K4me3) patterns in renal cell carcinoma (RCC) using a tissue microarray with 193 RCC (including 142 clear cell, 31 papillary, 10 chromophobe and 10 sarcomatoid RCC) and 10 oncocytoma specimens: H3K4me3 staining was more intense in papillary RCC, whereas H3K4me1 and H3K4me2 were similar in the diverse RCC subtypes. H3K4me2 and H3K4me3 levels were increased in oncocytoma. H3K4me1-3 levels were inversely correlated with Fuhrman grading, pT stage, lymph node involvement and distant metastasis. Progression-free survival and cancer-specific survival were shorter in patients with low levels of H3K4me1-3 in the univariate analysis, but we did not observe a significant correlation of a single modification in a multivariate model, which also included the established prognostic parameters TNM-stage and Fuhrman grade. In comparison, the H3K4me score, which combined staining levels of the H3K4 modifications, was an independent predictor of RCC progression-free survival. Our study on H3K4 methylation supports the concept of global histone modifications as potential cancer prognosis markers.

Global histone acetylation levels: prognostic relevance in patients with renal cell carcinoma.

Epigenetic alterations play an important role in carcinogenesis. Recent studies have suggested that global histone modifications are predictors of cancer recurrence in various tumor entities. Global histone acetylation levels (histone H3 lysine 9 acetylation [H3K9Ac], histone H3 lysine 18 acetylation [H3K18Ac], total histone H3 acetylation [H3Ac] and total histone H4 acetylation [H4Ac]) were determined in patients with renal cell carcinoma (RCC) using immunohistochemistry in a tissue micro array with 193 RCC and 10 oncocytoma specimens. The histone acetylation pattern was not different among the diverse histological subtypes of RCC or oncocytoma samples. The H3Ac levels were inversely correlated with pT-stage (P = 0.005), distant metastasis (P = 0.036), Fuhrman grading (P = 0.001) and RCC progression (P = 0.029, hazard ratio 0.87). H4Ac deacetylation was correlated with pT-stage (P = 0.011) and grading (P = 0.029). H3K18Ac levels were an independent predictor of cancer-progression following surgery for localized RCC in the univariate (P = 0.001, hazard ratio 0.78) and multivariate (P = 0.005, hazard ratio 0.82) analysis. In conclusion, our study supports the concept of global histone modification levels as a universal cancer prognosis marker, and provides evidence for the use of histone deacetylases inhibitors as future drugs in the therapy of RCC.

Global levels of histone modifications predict prostate cancer recurrence.

PURPOSE: Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It was reported that global histone modification patterns are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone lysine (H(x)K(y)) and histone acetyl (H(x)Ac) modifications in prostate tissue. MATERIALS AND METHODS: A tissue microarray with 113 prostate cancer (PCA), 23 non-malignant prostate tissues was stained with antibodies against H3K4 mono-(H3K4me1), di-(H3K4me2), tri-(H3K4me3) methylation, H3K9me1, H3K9me2, H3K9me3, H3 and H4 pan-acetylation (H3Ac, H4Ac). We also analyzed H3K4 methylation in patients with advanced PCA (hormone-refractory PCA-HRPC, n = 34; hormone-dependent PCA, n = 30). Sections were scored according the staining intensity and the proportion of epithelial cells showing nuclear staining. RESULTS: H3K4me1, H3K9me2, H3K9me3, H3Ac, and H4Ac were significantly reduced in PCA compared to non-malignant prostate tissue. H3Ac and H3K9me2 levels allowed discrimination of PCA and non-malignant prostate tissue highly specifically (>91%) and sensitively (>78%) as determined via ROC analyses (AUC >0.91). Histone lysine methylation and histone acetylation marks were correlated with clinical-pathological parameters (i.e., digital rectal examination, preoperative PSA, pT-stage, lymph node metastasis, Gleason score). In addition, H3K4me1 was a significant predictor of PSA recurrence following radical prostatectomy. H3K4me1, H3K4me2, and H3K4me3 levels were significantly increased in HRPC. CONCLUSIONS: Global histone modification levels may help to identify patients with adverse prognosis, and represent a target for the future therapy of PCA.