Molekularbiologische Charakterisierung eines CEA424/SV 40 large T-Antigen – induzierten Magentumors im transgenen Mausmodell

HINTERGRUND: Krebserkrankungen sind nach kardiovaskulären Erkrankungen die zweithäufigste Krankheitsgruppe überhaupt in der Medizin. Nach dem aktuellen Stand der Forschung entstehen Tumoren durch Fehlregulation von Proliferation und Differenzierung einzelner Zellen. Diese Vorgänge können in Tiermodellen nachgebildet werden. Eine besonders hohe Aussagekraft haben dabei Tiermodelle, bei denen durch Verwendung eines hybriden Onkogens spontan ein Tumor entsteht. Ein hybrides Onkogen ist aus einem organspezifischen Promotor und einem bekannten Onkogen zusammengesetzt und führt zur Tumorentwicklung in einem bestimmten Organ. Ein Beispiel hierfür sind CEA424/SV 40 large T-Antigen – transgene Mäuse, sie entwickeln reproduzierbar einen Tumor im Antrum des Magens. ZIELSETZUNG: An diesem Tiermodell sollten die Faktoren der organselektiven Entwicklung des Tumors aufgeklärt werden. Hypothesen hierzu waren einerseits, dass dies durch die Integration des Transgens verursacht ist, andererseits, dass im betroffenen Organ spezifische Stimulatoren wirken. Zudem sollte auf der Grundlage von Genexpressionsdaten der Phänotyp der Tumorzellen identifiziert werden. METHODEN: Zunächst wurde mit Walking-PCR die Integrationsstelle identifiziert. Darüber hinaus wurde mit RT-PCR gezielt die Expression von Genen untersucht, welche an der Regulation von Proliferation und Differenzierung des Magens beteiligt sind. Mikroarray-Analysen und ihre computergestützte Auswertung dienten zudem zur Erstellung von Genexpressionsprofilen für verschiedene Zeitpunkte der Tumorentwicklung. ERGEBNISSE: Das Transgen wurde auf dem Chromosom 4 der transgenen Mäuse lokalisiert. Ein Einfluss der Integrationsstelle auf die organspezifische Tumorentstehung konnte dabei nicht gesehen werden. Die Genexpressionsanalyse ergab, dass im Verlauf der Tumorentwicklung die Wnt-Signalkaskade hochreguliert ist. Dieser Signalweg ist als wichtiger Stimulator der Proliferation vor allem in Stammzellmilieus des Gastrointestinaltraktes bekannt. Weiterhin zeigte sich für die Differenzierungsfaktoren Ihh, Notch1 und Pdx1 sowie für das Hormon Gastrin mit fortschreitender Tumorentwicklung eine verminderte Expression, was auf einen Verlust bestimmter Differenzierungswege im Tumorgewebe schließen lässt. In den Genexpressionsprofilen zeigte sich ein neuroendokriner Phänotyp der Tumorzellen

Two-Photon Microscopy Allows Imaging and Characterization of Cochlear Microvasculature In Vivo

Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanateor Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of 90.0 +/- 22.7 mu m. Imaging yielded a mean contrast-to-noise ratio (CNR) of 3.3 +/- 1.7. Mean diameter in vivo was 16.5 +/- 6.0 mu m for arterioles and 8.0 +/- 2.4 mu m for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with 12.2 +/- 1.6 mu m and 6.6 +/- 1.0 mu m, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P < 0.001 and P = 0.022, two-way ANOVA). Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research

Histaminergic H3-Heteroreceptors as a Potential Mediator of Betahistine-Induced Increase in Cochlear Blood Flow

OBJECTIVE Betahistine is a histamine-like drug that is considered beneficial in Ménière's disease by increasing cochlear blood flow. Acting as an agonist at the histamine H1-receptor and as an inverse agonist at the H3-receptor, these receptors as well as the adrenergic \textgreeka2-receptor were investigated for betahistine effects on cochlear blood flow. MATERIALS AND METHODS A total of 54 Dunkin-Hartley guinea pigs were randomly assigned to one of nine groups treated with a selection of H1-, H3- or \textgreeka2-selective agonists and antagonists together with betahistine. Cochlear blood flow and mean arterial pressure were recorded for 3 min before and 15 min after infusion. RESULTS Blockage of the H3- or \textgreeka2-receptors caused a suppression of betahistine-mediated typical changes in cochlear blood flow or blood pressure. Activation of H3-receptors caused a drop in cochlear blood flow and blood pressure. H1-receptors showed no involvement in betahistine-mediated changes of cochlear blood flow. CONCLUSION Betahistine most likely affects cochlear blood flow through histaminergic H3-heteroreceptors

Fingolimod (FTY-720) is Capable of Reversing Tumor Necrosis Factor Induced Decreases in Cochlear Blood Flow

Hypothesis: The potential of Fingolimod (FTY-720), a sphingosine-1-phosphate analogue, to revoke the changes in cochlear blood flow induced by tumor necrosis factor (TNF) was investigated. Background: Impairment of cochlear blood flow has often been considered as the common final pathway of various inner ear pathologies. TNF, an ubiquitous cytokine, plays a major role in these pathologies, reducing cochlear blood flow via sphingosine-1-phosphate-signaling. Methods: Fifteen Dunkin-Hartley guinea pigs were randomly assigned to one of three groups (placebo/placebo, TNF/placebo, TNF/FTY-720). Cochlear microcirculation was quantified over 60 minutes by in vivo fluorescence microscopy before and after topical application of placebo or TNF (5 ng/ml) and after subsequent application of placebo or FTY-720 (200 mu g/ml). Results: Treatment with TNF led to a significant decrease of cochlear blood flow. Following this, application of placebo caused no significant changes while application of FTY-720 caused a significant rise in cochlear blood flow. Conclusions: FTY-720 is capable of reversing changes in cochlear blood flow induced by application of TNF. This makes FTY-720 a valid candidate for potential treatment of numerous inner ear pathologies

Role of cancer stem cell markers ALDH1, BCL11B, BMI-1, and CD44 in the prognosis of advanced HNSCC

PURPOSE Cancer stem cells (CSCs) are held accountable for the progress of head and neck squamous cell carcinoma (HNSCC). In the presented study, the authors evaluated the prognostic value of CSC markers in two particular HNSCC cohorts. METHODS This two cohort study consisted of 85~patients with advanced stage HNSCC, treated with primary radio(chemo)therapy (pRCT), and 95~patients with HNSCC, treated with surgery and partially adjuvant radio(chemo)therapy. Overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were assessed. Samples were assessed for the expression of different molecular stem cell markers (ALDH1, BCL11B, BMI\hbox-1, and CD44). RESULTS In the pRCT cohort, none of the baseline patient and tumor features exhibited a~statistically significant relation with survival in either the cohort or the human papillomavirus (HPV)-stratified subcohorts. High expression of BMI\hbox-1 significantly decreased OS and DFS, while high expression of CD44 decreased all modes of survival. Multivariate analysis showed significant prognostic influence for all tested CSC markers, with high BMI\hbox-1 and CD44 decreasing survival (BMI-1: OS, DFS, DSS; CD44: OS, DFS) and high ALDH1 and BCL11B showing a~beneficial effect on survival (ALDH1: OS, DFS; BCL11B: OS, DSS). In the surgical cohort, classical prognosticators such as HPV status, R1 resection, and nodal status in HPV-negative HNSCC played a~significant role, but the tested CSC markers showed no significant effect on prognosis. CONCLUSION Although validation in independent cohorts is still needed, testing for CSC markers in patients with advanced or late stage HNSCC might be beneficial, especially if many comorbidities exist or disease is irresectable. The findings might guide the development and earlier use of targeted therapies in the future

Benefit of postoperative radiotherapy for early tumors with single ipsilateral lymph node metastasis

Objectives/Hypothesis Indication for postoperative radiotherapy in patients with locally circumscribed tumors (pT1–pT2) and a single ipsilateral lymph node metastasis (pN1) is debatable. The aim of this study was to evaluate the oncological long‐term outcome of patients with pT1‐pT2 pN1 squamous cell carcinoma (SCC) of the oral cavity, the oropharynx, and the hypopharynx without extracapsular spread (ECS) after a margin‐negative surgical resection, who either received or did not receive postoperative (chemo)radiotherapy. Study Design Retrospective case series. Methods The oncological outcome of patients with pT1‐pT2 pN1 SCC without ECS was evaluated retrospectively. All patients underwent primary tumor resection that included transoral laser microsurgery and neck dissection at an academic tertiary referral center. Results Of 65 identified patients treated between 1986 and 2015 (18 oral cavity, 30 oropharynx, 17 hypopharynx), 21 (32%) received postoperative radiotherapy, and 44 (68%) were treated by surgery alone. The group of patients receiving postoperative treatment showed a significantly superior 5‐year disease‐specific (94.4% vs. 73.2%, P = .029) and recurrence‐free survival (85.2% vs. 43.2%, P = .002), as well as a higher local control rate (90.2% vs. 64.9%, P = .042). The overall survival was 71.4% vs. 62.6% (P = .53). The mean follow‐up was 80.7 months. Conclusions Patients with locally circumscribed carcinomas and a single ipsilateral ECS‐negative lymph node metastasis seem to benefit from postoperative radiotherapy. Level of Evidence 4 Laryngoscope, 130:E530–E538, 202

Prognostic impact of additional HPV diagnostics in 102 patients with p16-stratified advanced oropharyngeal squamous cell carcinoma

Purpose!#!p16 overexpression was considered as surrogate marker to identify human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCCs).!##!Methods!#!102 patients with advanced stage OPSCCs treated primarily by transoral lasermicrosurgery were included. Prognostic associations of p16- and HPV-status were analyzed separately and combined.!##!Results!#!In contrast to p16, the HPV-status resulted in no significant survival discrepancies (5-year overall survival (OS) HPV-positive 64.9%, HPV-negative 78.7%). Combining both markers, p16-positive (p16-positive/HPV-positive, p16-positive/HPV-negative) and p16-negative/HPV-negative groups demonstrated comparable high survival (OS 78.1% vs. 85.6% vs. 73.6%). Lowest survival was observed for patients with p16-negative/HPV-positive OPSCCs (OS 40.8%). Never smoking patients with p16-positive OPSCCs demonstrated the highest survival, whereas within former/current smokers with p16-positive and p16-negative disease it was comparable low (OS 90.0% vs. 63.0% vs. 57.4%).!##!Conclusions!#!p16- and HPV-status should not be considered as equivalent markers for a better prognosis. Furthermore, they should not generally predominate patient associated factors like smoking

Cancer stem cell markers in adenocarcinoma of the salivary glands - reliable prognostic markers?

PURPOSE Adenocarcinoma of the salivary glands is of low incidence and a broad range of histopathological subtypes. Cancer stem cell markers (CSC) might serve as novel prognostic parameters. To date, only a few studies examined the expression of CSC in adenocarcinoma of the salivary glands with diverging results. To further investigate the reliability in terms of prognostic value, a histopathological analysis of CSCs on a cohort of patients with adenocarcinomas of the major salivary glands was performed. METHODS Tumor samples of 40 consecutive patients with adenocarcinoma of the major salivary gland treated with curative intend at one tertiary center were stained with the CSCs ALDH1, BMI-1, CD44, Nanog, and SOX2. Expression of these markers was correlated with clinicopathological parameters and survival estimates. RESULTS Correlation of high expression of ALDH1 with higher grading (p < 0.001) and high expression of CD44 with the localization of the neoplasm (p = 0.05), larger tumor size (p = 0.006), positive pN-category (p = 0.023), and advanced UICC stage (p = 0.002) was found. Furthermore, high expression of SOX2 correlated with a negative perineural invasion (p = 0.02). No significant correlation of any investigated marker with survival estimates was observed. CONCLUSION In conclusion, our study did not find a significant correlation of the investigated CSCs with survival estimates in adenocarcinoma of the major salivary glands. Recapitulating the results of our study in conjunction with data in the literature, the CSCs ALDH1, BMI-1, CD44, Nanog, and SOX2 do not seem to serve as reliable prognostic parameters in the treatment of adenocarcinoma of the salivary glands