Preemptive endoluminal vacuum therapy with the VACStent-A pilot study to reduce anastomotic leakage after Ivor Lewis hybrid esophagectomy.

Introduction: Endoscopic treatment by vacuum therapy (EVT) or covered stents has emerged as an improved treatment option for upper gastrointestinal wall defects and is regarded as an improved treatment option for anastomotic leakage (AL) after esophagectomy. However, endoluminal EVT devices may lead to obstruction of the GI tract; and a high rate of migration and missing functional drainage has been shown for covered stents. The recently developed VACStent, a combination of a fully covered stent within a polyurethane sponge cylinder may overcome these issues allowing EVT while stent passage is still open. Initial clinical applications have demonstrated efficacy, practicability and safety in the treatment of esophageal leaks (AL). Methods: In this pilot study, 9 patients with high-risk anastomosis after neoadjuvant therapy undergoing hybrid esophagectomy received the VACStent in a preemptive setting for the assessment of the reduction of the AL rate, postoperative morbidity and mortality. Results: Technical success of the application of the VACStent® was achieved in all interventions. One patient experienced anastomotic leakage 10 days after esophagectomy and was successfully treated with two consecutive VACStents and a VAC Sponge. In summary, mortality in-hospital was 0% and anastomotic healing was uneventful without septic episodes. No severe device-related adverse events (SADE) nor significant local bleeding or erosion could be observed. Oral intake of liquids or food was documented in all patients. The device handling was regarded uncomplicated. Discussion: The preemptive application of the VACStent offers a promising new option for improved clinical treatment avoiding of critical situations in hybrid esophagectomy, which should be validated in a large clinical study.

Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1ß via a Caspase-1-Independent Mechanism.

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.

Multiple mechanisms mediate resistance to sorafenib in urothelial cancer.

Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor ß, PDGFR-α/-ß; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.

Concomitant downregulation of the imprinted genes DLK1 and MEG3 at 14q32.2 by epigenetic mechanisms in urothelial carcinoma.

BACKGROUND: The two oppositely imprinted and expressed genes, DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified. RESULTS: We observed that both DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences. CONCLUSIONS: Our data support the idea that the main cause of the prevalent downregulation of DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.

Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers.

OBJECTIVE: To determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat. MATERIALS AND METHODS: Expression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21(CIP1), thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs. RESULTS: Prominent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed. CONCLUSIONS: In urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation.