[Status of the availability and use of next generation sequencing (NGS) in bladder cancer-a questionnaire within the uropathology working group]. / Status der Verfügbarkeit und Anwendung von "next generation sequencing" (NGS) beim Harnblasenkarzinom ­ eine Umfrage in der Arbeitsgemeinschaft Uropathologie.

BACKGROUND: Technical advancement and availability of high-throughput analysis has advanced molecular subtyping of most cancers. Thus, new possibilities for precision oncology have emerged. AIM: Therefore, we aimed to collect data regarding availability and use of next generation sequencing (NGS) for urothelial cancer within the uropathology working group of the German Society of Pathology. METHODS: We collected data by questionnaires and additionally asked for sequencing results of bladder cancers in the participating institutions. RESULTS: A total of 13 university-affiliated institutes of pathology took part in the survey. All university institutes offer NGS-based molecular panel diagnostics and provide panels covering between 15 and 170 genes. Altogether, only 20 bladder cancers were sequenced in routine diagnostics and for 10 cancers potential targeted treatment options were available. DISCUSSION: So far, despite availability of NGS diagnostics at university institutes of pathology, only few bladder cancer samples have been sequenced. Based on current data from the molecular subtyping of bladder cancers, we recommend a step-by-step protocol with basic immunohistochemistry analysis and subsequent subtype-dependent analyses, e.g., alterations of the fibroblast growth factor receptors (FGFR) or comprehensive gene panel analyses.

AnnexinA5-pHrodo: a new molecular probe for measuring efferocytosis.

Efferocytosis, the clearing of dead or dying cells from living tissues, is a highly programmed, vital process to maintain the healthy functioning of every organism. Disorders of efferocytosis have been linked to several chronic diseases including atherosclerosis and auto-immune diseases. To date several different assays to determine phagocytosis, using microscopy or FACS analysis with labelled targets, have been developed. However, many of these are unable to differentiate between cells that have truly been phagocytosed and those still present on the surface of the macrophages hindering exact assessment of efferocytotic capacity. We herein describe AnxA5-pHrodo and its negative control M1234-pHrodo as new molecular probes to measure in vitro as well as ex-vivo efferocytotic capacity.

Microwave-Assisted Cross-Polarization of Nuclear Spin Ensembles from Optically Pumped Nitrogen-Vacancy Centers in Diamond.

The ability to optically initialize the electronic spin of the nitrogen-vacancy (NV) center in diamond has long been considered a valuable resource to enhance the polarization of neighboring nuclei, but efficient polarization transfer to spin species outside the diamond crystal has proven challenging. Here we demonstrate variable-magnetic-field, microwave-enabled cross-polarization from the NV electronic spin to protons in a model viscous fluid in contact with the diamond surface. Further, slight changes in the cross-relaxation rate as a function of the wait time between successive repetitions of the transfer protocol suggest slower molecular dynamics near the diamond surface compared to that in bulk. This observation is consistent with present models of the microscopic structure of a fluid and can be exploited to estimate the diffusion coefficient near a solid-liquid interface, of importance in colloid science.

Glucose-dependent insulinotropic peptide secretion is induced by inflammatory stimuli in an interleukin-1-dependent manner in mice.

Recently, glucagon-like peptide-1 (GLP-1) levels have been found to be increased in response to inflammatory stimuli, leading to insulin secretion and prevention of hyperglycaemia during endotoxemia in mice. In the present study, we assess the relevance of the other incretin hormone, glucose-dependent insulinotropic peptide (GIP), as a regulator of glucose metabolism under inflammatory conditions. We found that lipopolysaccharide (LPS) increased GIP secretion in a time- and dose-dependent manner in C57BL/6J mice. To elucidate the underlying mechanisms, mice were injected with inflammatory cytokines known to be released by LPS. Circulating GIP levels significantly increased in response to interleukin (IL)-1ß but not IL-6 or tumour necrosis factor (TNF)-α administration. Using respective knockout mice we found that LPS-mediated GIP secretion was selectively dependent on IL-1 signalling. To evaluate the functional relevance of inflammatory GIP secretion we pretreated mice with the GIP-receptor antagonist (Pro3)GIP. This blunted LPS-induced TNF-α and IL-6 secretion but did not affect LPS-induced insulin secretion or blood glucose-lowering. In conclusion, GIP provides a novel link between the immune system and the gut, with proinflammatory-immune modulatory function but minor glucose regulatory relevance in the context of acute endotoxemia.

[Molecular classification of bladder cancer. Possible similarities to breast cancer]. / Molekulare Klassifikation des Harnblasenkarzinoms. Mögliche Ähnlichkeit zum Mammakarzinom.

Therapeutic decisions for breast cancer are increasingly becoming based on subtype-specific gene expression tests. For bladder cancer very similar subtypes have been identified by genome-wide mRNA analysis, which as for breast cancer differ with respect to the prognosis and response to therapy on the basis of their hormone dependency. At the DNA level, however, the type of mutations and their frequencies within the subtypes are strikingly different between bladder and breast cancers. It will be interesting to see whether possible driver mutations can serve as therapeutic targets in both indications. In contrast, the apparent hormone dependency of a substantial number of bladder carcinomas suggests that hormonal and anti-hormonal treatment can be valid therapy options similar to breast cancer. Moreover, gender-specific differences with respect to the incidence and aggressiveness of male compared to female bladder cancers can be explained by hormonal effects. Together with forthcoming immunomodulatory therapies these multiple therapy options raise and give new hope to efficiently combat this aggressive disease.

ITCH modulates SIRT6 and SREBP2 to influence lipid metabolism and atherosclerosis in ApoE null mice.

Atherosclerosis is a chronic inflammatory disease characterized by the infiltration of pro-inflammatory macrophages into a lipid-laden plaque. ITCH is an E3 ubiquitin ligase that has been shown to polarize macrophages to an anti-inflammatory phenotype. We therefore investigated the effect of ITCH deficiency on the development of atherosclerosis. ApoE-/-ITCH-/- mice fed a western diet for 12 weeks showed increased circulating M2 macrophages together with a reduction in plaque formation. Bone marrow transplantation recreated the haemopoietic phenotype of increased circulating M2 macrophages but failed to affect plaque development. Intriguingly, the loss of ITCH lead to a reduction in circulating cholesterol levels through interference with nuclear SREBP2 clearance. This resulted in increased LDL reuptake through upregulation of LDL receptor expression. Furthermore, ApoE-/-ITCH-/- mice exhibit reduced hepatic steatosis, increased mitochondrial oxidative capacity and an increased reliance on fatty acids as energy source. We found that ITCH ubiquitinates SIRT6, leading to its breakdown, and thus promoting hepatic lipid infiltration through reduced fatty acid oxidation. The E3 Ubiquitin Ligase ITCH modulates lipid metabolism impacting on atherosclerosis progression independently from effects on myeloid cells polarization through control of SIRT6 and SREBP2 ubiquitination. Thus, modulation of ITCH may provide a target for the treatment of hypercholesterolemia and hyperlipidemia.

[Personalized urooncology based on molecular uropathology: part 1: what is diagnostic routine?]. / Personalisierte Uroonkologie auf Grundlage einer molekularen Uropathologie : Teil 1: Was ist diagnostischer Alltag?

The approval of new therapeutic procedures for the three main malignancies of the urogenital tract in recent years has generated a need for personalization of urooncology. As a consequence the diagnostic procedures are no longer limited to histology and immunohistochemistry but also include the analysis of genetic alterations (mutations and chromosomal aberrations).

[Bladder cancer in focus: update 2012 of the German Bladder Cancer Association]. / Das Harnblasenkarzinom im Fokus : Update 2012 des Deutschen Forschungsverbundes Blasenkarzinom e. V.

The German Bladder Cancer Association (DFBK) invited its members to the 3rd annual meeting 2012 in Hannover 4 years after the official founding. The meeting was directed to discuss the progress of ongoing and newly initiated projects and collaborations. In this article we will introduce current research activities and collaborations of the DFBK and would like to invite interested researchers to join this national interdisciplinary research association. The aim of the DFBK is to initiate interdisciplinary collaboration and to support scientific discussions among its members. For further information please visit our website at www.forschungsverbund-blasenkarzinom.de.

Optical detection of a single rare-earth ion in a crystal.

Rare-earth-doped laser materials show strong prospects for quantum information storage and processing, as well as for biological imaging, due to their high-Q 4f↔4f optical transitions. However, the inability to optically detect single rare-earth dopants has prevented these materials from reaching their full potential. Here we detect a single photostable Pr(3+) ion in yttrium aluminium garnet nanocrystals with high contrast photon antibunching by using optical upconversion of the excited state population of the 4f↔4f optical transition into ultraviolet fluorescence. We also demonstrate on-demand creation of Pr(3+) ions in a bulk yttrium aluminium garnet crystal by patterned ion implantation. Finally, we show generation of local nanophotonic structures and cell death due to photochemical effects caused by upconverted ultraviolet fluorescence of praseodymium-doped yttrium aluminium garnet in the surrounding environment. Our study demonstrates versatile use of rare-earth atomic-size ultraviolet emitters for nanoengineering and biotechnological applications.

KRAS mutation in metastatic pancreatic ductal adenocarcinoma: results of a multicenter phase II study evaluating efficacy of cetuximab plus gemcitabine/oxaliplatin (GEMOXCET) in first-line therapy.

BACKGROUND: Genetic alterations within the epidermal growth factor receptor (EGFR) pathway, including KRAS mutations, have been demonstrated to be associated with response to EGFR inhibitors like cetuximab in colorectal cancers. Mutations in the KRAS gene have been found in 70-90% of pancreatic cancers. Unfortunately, the addition of cetuximab to chemotherapy did not increase response or survival in patients with advanced pancreatic cancer in phase II and phase III studies. The aim of this study was to evaluate the relationship between KRAS mutations and response or survival in patients with metastatic pancreatic cancer treated with cetuximab plus chemotherapy. METHODS: Within a multicenter phase II trial, 64 patients with metastatic pancreatic cancer were treated with cetuximab in combination with gemcitabine and oxaliplatin until disease progression. Analyses of the EGFR pathway, including KRAS mutations, could be performed in 25 patients. Analyses were carried out following microdissection of the tumor. RESULTS: Fourteen (56%) of the 25 patients examined harbored a point mutation in codon 12 of the KRAS gene. No differences between the groups were noted in median progression-free survival (104 days in KRAS wild-type patients vs. 118 days in patients with KRAS mutations). Overall survival was longer in wild-type patients compared to patients with KRAS mutations (263 vs. 162 days), but the difference did not reach statistical significance. A further analysis of our clinical phase II trial showed that the presence of a rash was significantly correlated with overall survival. CONCLUSIONS: KRAS mutation in codon 12 may be associated with reduced survival compared to KRAS wild type. The role of KRAS mutations for cetuximab therapy in pancreatic cancer warrants further investigation in larger trials to exclude an epiphenomenon. Furthermore, the development of a rash is indicative of clinical benefit.

[Bladder cancer at an early age in father and son]. / Harnblasenkarzinom in jungen Jahren bei Vater und Sohn.

Bladder cancer may be caused by external factors like tobacco smoking, but may also be familial. We report on a father and son who developed this tumour at the ages of 45 and 35. Testing various genetic markers including the mismatch repair proteins MLH1, MSH2 and MSH6, whose loss is associated with a higher risk for hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), did not point to a familial disease. Thus the heavy smoking habits of the two patients must be considered as causal.

[Bladder cancer update: what was new at the 2010 annual congress of the German Association of Urology in Düsseldorf?]. / Blasenkarzinom - Update : Was gab es neues auf dem Jahreskongress 2010 der Deutschen Gesellschaft für Urologie in Düsseldorf?

One of the principal objects of the scientific research network "German Bladder Cancer Network" is to consolidate research activities on bladder cancer. An overview about directions of current projects on this research topic was given at the annual meeting of the German Association of Urology in Düsseldorf from September 22 to 25 September 2010. As representatives of the"German Bladder Cancer Network" we summarize and comment on some of the most interesting projects on bladder cancer presented at this meeting. A special focus will be on current developments in the field of uropathology and on different aspects in preclinical research on bladder cancer.

[EMMPRIN (CD147). A prognostic and potentially therapeutic marker in urothelial cancer]. / EMMPRIN (CD147). Ein prognostischer und potenzieller therapeutischer Marker im Urothelkarzinom.

In 50% of all cases, bladder cancer patients develop tumor progression despite modern surgical methods such as radical cystectomy. A solution to the problem might be the identification and understanding of molecular biomarkers which could result in the development of advanced methods with better preventive, diagnostic, and therapeutic potential. One suitable approach is the identification of a bladder cancer-specific molecular marker in order to enhance patients' outcome. We and others have identified EMMPRIN as a prognostic biomarker in a variety of tumor diseases. EMMPRIN (CD147, extracellular matrix metalloproteinase inducer) is a cell surface protein that is expressed among other cell types, in particular in tumor cells. Since its first description in 1982 it is established that overexpression of EMMPRIN correlates with tumor progression and patient outcome. EMMPRIN expression levels can be used as an independent prognostic factor for survival. Recently, EMMPRIN has been defined as a potential target for tumor therapy in preclinical studies.

[Symposium of the German Bladder Cancer Association]. / Symposium des Deutschen Forschungsverbundes Blasenkarzinom e.V.

One year after the official founding of the German Bladder Cancer Association the interdisciplinary association organized its first symposium on bladder cancer at the meeting center"Schloss Mickeln" in Düsseldorf in June 2009. The focus of the symposium was on the initiation and implementation of primary objectives of the association. Members from national and international research groups presented and discussed current projects. A period of 12 months has been required to establish a national tissue bank for bladder cancer speciem which will be started in January 2010. The meeting not only stirred new ideas but also promoted new collaborations and served as an innovative platform to discuss novel questions and methodological approaches to study bladder cancer. Additionally, the website www.forschungsverbund-blasenkarzinom.de will provide further information about the German Bladder Cancer Association.

[EMMPRIN (CD147). A new key protein during tumor progression in bladder cancer]. / EMMPRIN (CD147). Ein neues Schlüsselprotein in der Tumorprogression des Harnblasenkarzinoms.

EMMPRIN (CD147) is a cell surface protein that is highly expressed on tumor cells. Elevated EMMPRIN levels have been detected in a variety of malignant tumors and have been associated with tumor progression in experimental and clinical conditions. Recent studies have shown that EMMPRIN is an independent prognostic factor for overall survival in bladder cancer patients. In a multicenter phase II trial, antibodies against EMMPRIN were shown to be successful in hepatocellular cancer therapy. We are characterizing the functional importance of EMMPRIN in bladder cancer in order to evaluate this protein as a new target molecule for therapy.

[Precancerous lesions of the urothelium. From Feulgen staining to single cell CGH]. / Präkanzerosen des Urothels. Von der Feulgen-Färbung bis zur Einzelzell-CGH.

Feulgen staining represents a staining method to quantitatively document the DNA content of a nucleus. Thus it is an excellent and straightforward method to reflect the irregular increase in DNA content of a malignant cell as a sign of genetic instability. Genetic instability of the tumour cell is the key feature of the 2004 WHO classification of bladder tumours, in which flat and papillary neoplasia are grouped into low- and high-grade lesions. "High grade" represents the tumor with genetic instability and consequently a higher likelihood of progression. Concomitant distinct genetic aberrations other than the numeric ones are increasingly identified as discriminators and help group the entities. The current status of genetic investigations, especially those in precancerous lesions, will be outlined in this review in the context of morphology (histology and cytology) as well as clinical situation.

[Plasmacytoid carcinoma. Five case reports of a rare variant of urothelial carcinoma]. / Das plasmazytoide Urothelkarzinom. 5 Fallberichte einer seltenen Variante des konventionellen Urothelkarzinoms.

Plasmacytoid carcinoma is a rare variant of urothelial carcinoma and is characterised by distinct histopathological and clinical characteristics. The incidence varies between 2.7% and 3.1% of all muscle-invasive urothelial carcinoma. It is an aggressive, high-grade tumor with poor prognosis. Negative E-cadherin expression seems to be important for exact diagnosis. Systemic chemotherapy of plasmacytoid carcinoma could lead to prolonged patient survival.

[Promoter methylation and microsatellite mutation reveals the clonal relationship of multiple urothelial carcinomas with mutator phenotype]. / Promotor-hypermethylierung und Mikrosatelliten-Instabilität in multifokalen Urothelkarzinomen mit Mutator-Phänotyp.

AIMS: The clonality of multiple urothelial carcinomas (UC) is subject to debate and affects treatment. Evidence derived from X-chromosome mosaicism and patterns of molecular alterations supports both a mono- and polyclonal relationship. In contrast to most UC, tumours with the mutator phenotype have frequent mutations in repetitive sequences (MSI) and promoter methylation. The aim of this study was to investigate the clonality of multifocal UC with MSI. METHODS: We have screened 400 UC for MSI and found it to occur in 1% of bladder and 15% of upper tract UC. Of these, 9 patients, whose tumours had MSI, developed or presented with multiple UC. A total of 32 UC (occurring over 0-6 years, 2-12 TCC per patient), 2 cases of CIS and 9 normal urothelial samples were screened for MSI at 17 loci and aberrant promoter methylation at 7 genes. RESULTS: In 8 of 9 patients, the pattern of microsatellite mutation and promoter methylation suggested that the multiple tumours had a clonal origin. Patterns of aberrant methylation in multiple tumours were more similar than microsatellite mutations, suggesting an earlier carcinogenic timing. MSI and promoter methylation were present in macroscopically normal urothelium from these patients. CONCLUSIONS: Aberrant promoter methylation occurs before microsatellite alteration in UC with mutator phenotype. The majority of recurrent UC with MSI are monoclonal in origin and macroscopically normal urothelium harbours multiple molecular abnormalities. Thus, at the time of apparently successful treatment, there is molecular evidence of residual tumour that subsequently develops into recurrent disease.