Tenascin-C and carcinoma cell invasion in oral and urinary bladder cancer.

Carcinoma invasion is a complex process regulated by genetic and epigenetic factors as well. A relevant supportive condition for cancer cell migration is the reorganization of the extracellular matrix (ECM), which is realized in an orchestrated multicellular manner including carcinoma cells and stromal fibroblasts. An important key player in the process of ECM reorganization is Tenascin-C (Tn-C). The molecule occurs as different isoforms generated by alternative splicing and de novo glycosylation. Large variants of Tn-C are abundantly re-expressed in the invasive front of many carcinoma types. A special role for initiating migration and accompanied epithelial to mesenchymal transition has been suggested. Here, we review the current knowledge concerning the tumor biological importance of Tn-C, the synthesis and alternative splicing during the invasive process in general, and give an overview on the impact of Tn-C in urothelial carcinoma of the urinary bladder (UBC) and oral squamous cell carcinoma (OSCC).

Selective imaging of chronic cardiac rejection using a human antibody specific to the alternatively spliced EDA domain of fibronectin.

BACKGROUND: Chronic cardiac rejection is intimately associated with cardiac allograft vasculopathy and fibrosis, both causing severe complications that cannot be reversed. Thus, there is an urgent need for early diagnosis and for development of therapeutic agents. Chronic rejection is accompanied by the dramatic upregulation of EDA(+) fibronectin (EDA(+) Fn), which is virtually undetectable in the normal healthy adult. METHODS: In this study, we evaluated the potential of the monoclonal antibody F8, specific to that molecule, to selectively accumulate in chronically rejected allografts. RESULTS: A syngeneic immunocompetent heterotopic rat heart transplantation model was used to induce chronic rejection within 70 days. The F8 antibody or an antibody of irrelevant specificity, labeled with the dye DY-682, was administered and near-infrared fluorescence (NIRF) imaging was performed. Targeting performance was assessed by macroscopic organ imaging and fluorescence microscopy. A selective accumulation of the F8 antibody (but not of the negative control antibody) was observed by NIRF imaging in cardiac allografts. The antibody localized to diseased blood vessels as well as to fibrotic regions, where the cognate antigen is abundantly expressed. CONCLUSIONS: This is the first example of antibody-mediated imaging of chronic cardiac rejection. The findings pave the way to immuno-positron emission tomography (immuno-PET) imaging of this clinical condition in patients using the human F8 antibody labeled with a suitable radionuclide (e.g., iodine-124). Furthermore, it would be conceivable to use the F8 antibody as a delivery vehicle to assess experimentally whether a bioactive payload (e.g., drug or cytokine) may be able to reduce disease progression.

Primary and secondary leiomyosarcoma of the oral and perioral region--clinicopathological and immunohistochemical analysis of a rare entity with a review of the literature.

PURPOSE: Leiomyosarcoma (LMS) rarely occurs in the head and neck region. These tumors present with a wide range of clinical features, so the diagnosis is predicated on conventional microscopic findings coupled with immunohistochemical analysis. PATIENTS AND METHODS: Clinical and histologic data of 7 patients with LMS of the head and neck were recorded retrospectively. In addition to routine immunohistochemistry, staining for cell cycle regulator proteins p16 and p21 was performed. RESULTS: Five LMSs (4 intraoral, 1 dermal cheek) occurred primarily in the oral and perioral region. Two LMSs (parietal and sinonasal) were diagnosed as metastases originating from the uterus and pelvis. Treatment of the primary LMSs consisted of radical tumor resection with clear margins. Distant metastases from LMSs were irradiated or excised as palliative treatment. Three of 5 patients (60%) with primarily excised LMS developed recurrence after an average of 7 months, with lung metastases occurring after 17 months. In 1 patient, cervical lymph node metastases were detected after 10 months. Of all patients, 5 died after an average survival period of 2.4 years. The mean survival period of the 5 patients with primary LMS of the head and neck was 3.3 years. All tumors were positive for vimentin and α-smooth muscle actin, with 57% of tumors showing positive nuclear expression of p16 and 71% of p21. Lack of p16 nuclear expression was associated with a shorter mean survival time (1.3 vs 4.3 yr for p16 positivity). CONCLUSION: Lung and cervical lymph node metastases often occur in LMS of the head and neck. Presurgical staging, including gynecologic examination, whole-body computed tomography, and sometimes positron-emission or computed tomography, to rule out LMS metastasis is mandatory. Surgical resection of the tumor should be given top priority. Lack of p16 reactivity may have a prognostic value for LMS because it was related to a trend toward poorer survival.

Paclitaxel enhances therapeutic efficacy of the F8-IL2 immunocytokine to EDA-fibronectin-positive metastatic human melanoma xenografts.

The selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer therapies. In this study, we show that the antibody F8, which recognizes perivascular and stromal EDA-fibronectin (EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn-expressing melanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before the immunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas, enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2-induced natural killer (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support of this likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, this combination reduced both the tumor burden and the number of pulmonary metastatic nodules. The combination did not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumor stroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting the use of immunochemotherapy in the treatment of metastatic melanoma.

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery.

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(®). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.

Expression of Wnt-1, TGF-ß and related cell-cell adhesion components following radiotherapy in salivary glands of patients with manifested radiogenic xerostomia.

BACKGROUND: Radiation-induced xerostomia still represents a common symptom following radiotherapy of head and neck malignancies, which significantly impairs the patient's quality of life. In this cross-sectional study, human salivary glands were investigated to assess the role of Wnt/ß-catenin and TGF-ß pathways in the pathogenic process of radiogenic impairment of salivary function. METHODS: Irradiated human salivary glands were investigated in patients with manifested xerostomia. Alteration of Wnt-1 and cell-cell adhesion was evaluated immunohistologically as well as changes in the expression of TGF-ß were assessed in salivary gland tissue. RESULTS: We assessed two alteration patterns in which Wnt-1 expression represents one change along with up-regulation of ß-catenin and E-cadherin in irradiated but viable acinar cells. Increased expression of tenascin-C was observed in sites of epithelial-mesenchymal interaction and loss of cell-cell adhesion was assessed in translocated epithelial cells in the stroma. CONCLUSION: Increased transdifferentiation and remodeling of acinar structures was associated with decrease of viable acinar structures. The role of Wnt and TGF signaling may provide a potential therapeutic approach to prevent radiation-induced damage to salivary glands during radiotherapy for head and neck cancer.

Comparative analysis of oncofetal fibronectin and tenascin-C expression in right atrial auricular and left ventricular human cardiac tissue from patients with coronary artery disease and aortic valve stenosis.

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17 × AVS; 13 × AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r = 0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r = 0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.

Expression of extra domain A containing fibronectin in chronic cardiac allograft rejection.

BACKGROUND: Cardiac allograft vasculopathy (CAV) and fibrosis are important in chronic cardiac allograft rejection. The aim of our study was to analyze the up-regulation of extra domain A (ED-A) containing fibronectin (ED-A(+) Fn) in cardiac allografts after heterotopic rat heart transplantation using a human recombinant antibody applicable for targeted drug delivery. METHODS: Cardiac allografts were subjected to immunofluorescence double labelling procedures combining a human recombinant small immunoprotein (SIP) format antibody recognizing ED-A(+) Fn (F8) with antibodies recognizing CD31, ASMA or CD45. Protein expression levels of ED-A(+) Fn were measured by quantitative confocal laser scanning microscopy and messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction. RESULTS: A distinct re-expression of ED-A(+) Fn was detectable with the F8 antibody, especially in vessel structures exhibiting CAV and in fibrotic areas. ED-A(+) Fn protein deposition but not messenger RNA expression levels increased with rising rejection grade (p ≤ 0.001). There were clear co-localizations of ED-A(+) Fn and α-smooth muscle actin in vessels and in fibrotic areas. CONCLUSIONS: We could show first that ED-A(+) Fn is expressed in rat cardiac allografts in association with CAV and cardiac fibrosis. The protein is detectable with the human recombinant antibody F8 usable for targeted drug delivery to the side of disease. Second, protein expression levels increase with rising rejection grade. Thus, ED-A(+) Fn might be usable to monitor and target CAV as well as fibrosis after heart transplantation.

Altered expression of cell-cell adhesion molecules ß-catenin/E-cadherin and related Wnt-signaling pathway in sporadic and syndromal keratocystic odontogenic tumors.

Differential diagnosis of the keratocystic odontogenic tumor (KCOT) still represents a challenging problem especially if compared with the dentigerous cyst, which is similar in clinical and radiological course. Histological assessment of this entity may therefore draw crucial attention since various radical procedures are recommended for such lesions in contrast to dentigerous cysts. Since recent reports could prove the involvement of wingless(Wnt)-signaling pathway and ß-catenin in the pathogenesis of many odontogenic and neoplastic lesions indicating impairment of cell-cell adhesion, we investigated the expression of two Wnt-signaling pathways, Wnt-1 and Wnt-10A as well as ß-catenin and E-cadherin along with other related proteins in both lesions. We found a significant down-regulation in the expression of cell adhesion proteins ß-catenin and E-cadherin along with alteration of Wnt-1 and Wnt-10A expression in the epithelium of KCOT. We assessed a specific focal distribution pattern of p63 in the suprabasal cell layer and a significant up-regulation of cyclin D1. Furthermore, laminin α-2 was a characteristic marker labelling only the basement membrane of dentigerous cysts. These results provide a new hypothesis explaining a molecular mechanism to understand initiating and development of KCOTs and an alternative therapeutic approach, especially for syndromal patients, where these multilocal lesions may involve and destroy wide orofacial bony structures.

EGF/TGFß1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332.

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFß1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFß1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and ß1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

The tumour-targeting human L19-IL2 immunocytokine: preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma.

BACKGROUND: L19-IL2, a tumour-targeting immunocytokine composed of the recombinant human antibody fragment L19 (specific to the alternatively-spliced EDB domain of fibronectin, a well characterised marker of tumour neo-vasculature) and of human IL2, has demonstrated strong therapeutic activity in animal cancer models. This phase I/II trial was performed to evaluate safety, tolerability, recommended phase II dose (RD) and early signs of activity of L19-IL2. PATIENTS AND METHODS: Five cohorts of patients with progressive solid tumours (n=21) received an intravenous infusion of L19-IL2 (from 5 to 30 Mio IU IL2 equivalent dose) on days 1, 3 and 5 every 3 weeks. This treatment cycle was repeated up to six times. In the following expansion phase, patients with metastatic renal cell carcinoma (RCC) (n=12) were treated at the RD of L19-IL2. Clinical data and laboratory findings were analysed for safety, tolerability and activity. RESULTS: Preclinical studies in rats and monkeys did not raise any safety concerns. The RD was defined to be 22.5 Mio IU IL2 equivalent. Pharmacokinetics of L19-IL2 was dose proportional over the tested range, with a terminal half-life of 2-3h. Toxicities were manageable and reversible with no treatment-related deaths. We observed stable disease in 17/33 patients (51%) and 15/18 with mRCC (83%) after two cycles. Median progression-free survival of RCC patients in the expansion phase of the study was 8 months (1.5-30.5). CONCLUSIONS: L19-IL2 can be safely and repeatedly administered at the RD of 22.5 Mio IU IL2 equivalent in advanced solid tumours. Preliminary evaluation suggests clinical activity of L19-IL2 in patients with mRCC.

A comparative analysis of oncofetal fibronectin and tenascin-C incorporation in tumour vessels using human recombinant SIP format antibodies.

Tumour angioneogenesis is associated with the reexpression of oncofetal fibronectin (oncFn) and tenascin-C (oncTn-C) splice variants, which may serve as targets for antibody-based pharmacodelivery. Knowledge of the vascular distribution and organization in different tumours is of importance for the understanding of tumour vessel formation and might be crucial for therapy. Therefore, human SIP format antibodies against Fn ED-A, Fn ED-B and Tn-C A and C splice domains were used for immunofluorescence labelling in renal, lung, oral, colon, breast and urinary bladder carcinoma specimens and in a renal carcinoma xenograft. The spatial relation to stroma, vessels and vascular basement membrane (vBM) was analysed including CD31 and laminin alpha4 chain antibodies. Renal cell carcinomas and atypical carcinoid of the lung revealed vessel-restricted oncFn and/or oncTn-C depositions; all other entities showed a variable stroma positivity including vessels. The individual pattern of oncFn/oncTn-C incorporation in the vBM depended on tumour type, vessel size and intratumoural heterogeneity. There was a stratification of the vessel wall showing luminal oncFn and extraluminal oncTn-C depositions. As shown in the xenograft, perivascular oncTn-C is provided by carcinoma cells. In conclusion, tumours differ in the pattern of Fn or Tn-C isoform positivity in the vessel wall, potentially representing a tumour type specific endothelial cell-tumour cell-stromal cell interaction. Carcinoma cells themselves are involved in vascular Tn-C matrix organization. Up to antigen distribution, Fn and Tn-C domain antibodies may serve as vehicles for antiangiogenetic and antifibrotic agents; oncFn/oncTn-C based targeting should be adapted individually.

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies.

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

BACKGROUND: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS: Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS: Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS: Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

The effects of vandetanib on paclitaxel tumor distribution and antitumor activity in a xenograft model of human ovarian carcinoma.

This study was designed to determine the effects of vandetanib, a small-molecule receptor tyrosine kinase inhibitor of vascular endothelial growth factor and epidermal growth factor receptor, on paclitaxel (PTX) tumor distribution and antitumor activity in xenograft models of human ovarian carcinoma. Nude mice bearing A2780-1A9 xenografts received daily (5, 10, or 15 days) doses of vandetanib (50 mg/kg per os), combined with PTX (20 mg/kg intravenously). Morphologic and functional modifications associated with the tumor vasculature (CD31 and alpha-smooth muscle actin staining and Hoechst 33342 perfusion) and PTX concentrations in plasma and tumor tissues were analyzed. Activity was evaluated as inhibition of tumor growth subcutaneously and spreading into the peritoneal cavity. Vandetanib treatment produced no significant change in tumor vessel density, although a reduced number of large vessels, an increased percentage of mature vessels, and diminished tumor perfusion were evident. Pretreatment with vandetanib led to decreased tumor PTX levels within 1 hour of PTX injection, although 24 hours later, tumor PTX levels were comparable with controls. In efficacy studies, the combination of vandetanib plus PTX improved antitumor activity compared with vandetanib or PTX alone, with greater effects being obtained when PTX was administered before vandetanib. The combination of PTX plus vandetanib reduced tumor burden in the peritoneal cavity of mice and significantly increased their survival. Analysis of vascular changes and PTX tumor uptake in vandetanib-treated tumors may help to guide the scheduling of vandetanib plus PTX combinations and may have implications for the design of clinical trials with these drugs.

No incidence of BRAF mutations in salivary gland carcinomas--implications for anti-EGFR therapies.

BRAF is the main effector of KRAS in the RAS-RAF-MAPK axis, a signaling pathway downstream of EGFR. The activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules. Recent studies in metastatic colorectal cancer found that an oncogenic activation of BRAF by a point mutation in exon 15 (V600E) could bypass the EGFR-initiated signaling cascade with the effect that patients bearing the mutant BRAF allele are not likely to benefit from EGFR-targeted therapies. We designed an allele-specific PCR and screened 65 salivary gland carcinoma (SGC) of the main histopathological types for the BRAF V600E mutation. All 65 SGC in this cohort (100%) presented the BRAF wildtype. In a previous study, we found a KRAS wildtype in 98.5% of SGC. These findings imply that SGC rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of EGFR and this pleads in favor of further therapeutic trials with EGFR-targeting monoclonal antibodies.

Metastatic glioblastoma cells use common pathways via blood and lymphatic vessels.

Generally, gliomas do not metastasize. Therefore, larger series are not available to investigate the pathways of tumour spread. Here, we present the case of a young man with a glioblastoma multiforme WHO grade IV and distant metastases in several tissues. The glioblastoma multiforme WHO grade IV of a young male patient recurred within a very short time along the surgical resection pathway within the temporalis muscle. After removal of the tumour bulk, the patient developed a distant intracranial tumour lesion around the contralateral ventricular system and a pulmonary tumour. Later on, the patient underwent an operation on a facial lesion representing a local extracranial glioblastoma recurrence and containing metastases within lymph nodes and lymphatic vessels. Our case report indicated a lymphatic pathway of metastasis, which could be demonstrated by our histopathological analysis. We suggest that altered gene expression stimulated by glioblastoma-environment interaction altered the properties of glioblastoma cells, whether caused by a spontaneous genetic shift or induced by factors provided by the extracranial tissue.

Glioblastoma cells express functional cell membrane receptors activated by daily used medical drugs.

PURPOSE: Calcium ions are highly versatile spacial and temporal intracellular signals of non-excitable cells and have an important impact on nearly every aspect of cellular life controlling cell growth, metabolism, fluid secretion, information processing, transcription, apoptosis, and motility. Neurons and glia respond to stimuli, including neurotransmitters, neuromodulators, and hormones, which increase the intracellular calcium concentration. The function of intracellular calcium in gliomas is unknown. Lots of daily used drugs may act via receptors that can be linked to the intracellular calcium system and therefore could influence glioma biology. METHODS: Glioma cells were loaded with the calcium ion sensitive dye Fura 2-AM. Subsequently, cells were stimulated with 25 different medical drugs for 30 s. The increase of free intracellular calcium ions was measured and calculated by a microscope-camera-computer-unit. RESULTS: Except for the buffer solution HEPES that served as negative control and for the cortisol derivative dexamethasone, all other 24 tested drugs induced a rise of intracellular calcium ions. The cellular calcium responses were classified into seven functional groups. The tested substances activated several types of calcium channels and receptors. CONCLUSIONS: Our study impressively demonstrates that medical drugs are potent inducers of intracellular calcium signals. Totally unexpected, the results show a high amount of functional cellular receptors and channels on glioma cells, which could be responsible for certain biological effects like migration and cell growth. This calcium imaging study proves the usability of the calcium imaging as a screening system for functional receptors on human glioma cells.

KRAS status and epidermal growth factor receptor expression as determinants for anti-EGFR therapies in salivary gland carcinomas.

Salivary gland carcinomas (SGC) are rare cancers with poor prognosis and limited response to conventional chemotherapy. New strategies based on molecular targeted therapy are needed and the EGFR signaling cascade is considered a possible key pathway for therapeutic molecules. We have analyzed 65 SGC of the main histopathological types for the expression of EGFR and and the mutation status of its downstream effector KRAS. EGFR overexpression (+2, +3) has been identified by immunohistochemistry in 75.4%. KRAS mutation analysis was performed by direct genomic sequencing and revealed a KRAS wildtype in 98.5% except of one adenoid cystic carcinoma with a GGT-GAT transition at codon 12 (Gly12Asp). EGFR overexpression and KRAS wildtype are prerequisites for a successful anti-EGFR therapy. The results of this study plead in favor of further therapeutic trials with EGFR-targeting monoclonal antibodies in SGC.

B and C domain containing tenascin-C: urinary markers for invasiveness of urothelial carcinoma of the urinary bladder?

PURPOSE: Surveillance of urothelial carcinoma of the urinary bladder (UBC) patients with respect to tumour recurrence and invasiveness is crucial for therapy and prognosis. Therefore, evaluation of non-invasive methods to monitor tumour progression is of high clinical interest. The study was aimed at investigating urinary concentrations of tenascin-C splicing domains for their value as tumour surveillance markers. METHODS: Urinary concentration of B and C domain containing tenascin-C (Tn-C) was analysed by ELISA technology in 104 UBC patients, 11 patients with cystitis and 15 healthy donors as control. The investigation was supplemented by Tn-C immunohistochemistry and Western blotting. RESULTS: A statistically significant increase in urinary concentrations of both Tn-C B and C domain with tumour progression could be evidenced. A concordant tumour-associated enhanced protein deposition in the carcinoma stroma could be demonstrated by immunohistochemistry in invasive UBC. Western blotting reveals proteolytic fragmentation of urinary Tn-C. CONCLUSIONS: In summary, detection of Tn-C splicing domains in urine is suggested as a marker for the surveillance of UBC recurrence and invasiveness.