Martin Wurm

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.

Aims Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. Material and methods EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term ‘EpCAM overexpression’ was reserved for tissues showing a total immunostaining score >4. Results EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. Conclusion EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.

Bilirubin Inhibits Tumor Cell Growth via Activation of ERK

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.

Survival Signaling by C-RAF: Mitochondrial Reactive Oxygen Species and Ca2+ Are Critical Targets

ABSTRACT Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca2+. Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca2+ levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca2+, which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca2+ overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca2+ homeostasis.

Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer

Epithelial cell adhesion molecule (EpCAM) is a widely used immunohistochemical marker for epithelial human malignancies. Antibodies to target EpCAM are usually directed against its ectodomain (EpEX), but do not detect the intracellular domain (EpICD). The aim of this study was to compare membranous EpEX versus EpICD expression by immunohistochemistry.

Pharmacodiagnostic Value of the HER Family in Head and Neck Squamous Cell Carcinoma

Two protooncogene products, EGFR (Her-1, c-erbB-1) and HER2 (Her-2/neu, c-erbB-2), have been reported to be frequently overexpressed in head and neck squamous cell carcinoma (HNSCC). In order to identify patients who may benefit from targeted cancer treatment for these two molecules, we determined the expression status of EGFR and HER2 in 129 HNSCC tumor specimens. Two pharmacodiagnostic kits (EGFR pharmDx™ and HercepTest™) were used to identify HNSCC tumors that overexpress EGFR or HER2. Overexpression of EGFR was detected in 42.6% of the tumor specimens, while HER2 was only rarely expressed (overexpression was observed in just 3.1% of all cases). Given the necessity of new therapeutic modalities for patients suffering from HNSCC, treatment EGFR signaling inhibitors appears to be warranted, whereas therapeutic intervention with HER2 inhibitors seems to be inappropriate in this tumor type.

Introduction of a Novel Prototype Bioartificial Liver Support System Utilizing Small Human Hepatocytes in Rotary Culture

The aim of this study was to establish a stand-alone, perfused, rotary cell culture system using small human hepatocytes (SH) for bioartificial liver (BAL) support. SH were grown on cytodex 3 microcarriers (beads) to a maximum density of 1.2 +/- 0.3 x 10(7) cells per mL within 12 days. Size of aggregates formed by up to 15 beads was regulated by rotation speed. Cell function was proven by treatment with ammonia and galactose, and metabolism was analyzed. Treatment strategy was comprised of two phases, namely growth phase and treatment phase. Cells were grown for 6 days and subsequently incubated with ammonia or galactose for 2 days, followed by a 2-day regeneration period and another 2-day treatment phase. Consumption of glucose, release of lactate dehydrogenase, formation of lactate, and production of urea and albumin were determined regularly. Mean galactose consumption was 50 microg per 106 cells per hour, ammonia-induced urea formation was 3.6 microg per 106 cells per hour, and albumin production was 110 ng per 106 cells per hour. All metabolic parameters followed a logarithmic trend and were found to be very stable in the second half of the culture period when cells were treated with ammonia or galactose. Dissolved oxygen (%DO), pH, and temperature were monitored in-stream at intervals of 7 min, and the values were logged. Viability and morphology of cells were monitored via confocal microscopy. Viability was around 95% in controls and 90% during treatment. Promising results were obtained in support of our ongoing efforts to establish a fully autonomous BAL support device utilizing SH as a bridge to transplantation.

Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process.1 Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established. Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused “rotary transport system for islets” termed “ROTi.” Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating “standard” shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated “standard” shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

Impairment of mitochondrial respiratory function as an early biomarker of apoptosis induced by growth factor removal

Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40% inhibition. A slight increase in respiration following addition of cytochrome c, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.

Book Reviews / Erratum

The updated fourth edition of the Color Atlas of ENT Diagnosis is an illustrated first introduction examination, diagnosis and therapy of the most frequent ear, nose and throat diseases. The more than 500 good-to-excellent color pictures are of great help to the beginner to become familiar with the conditions typically seen in the ENT field. The text is divided into five main sections: examination; ear; nose and face; pharynx and larynx; head and neck. As the stated purpose of this small pocket book is not to be a textbook but a pictorial survey, the majority of the book is reserved for the excellent pictures. Although most of the color photographs are self-explanatory every illustration is accompanied by a short descriptive text. Moreover, small paragraphs introduce special issues like laryngectomy or audiologic examination. I suggest a reference list for additional reading. All in all this small pocket atlas will be of great help to medical students, ENT trainees and general practitioners seeking a quick reference during daily practice. Elmar Oestreicher, Munich Maria Sanna, Hiroshi Sunose, Fernando Mancini, Alessandra Russo, Abdelkader Taibeh