Phenotypic characterization of the epithelial and myoepithelial components in canine and feline mammary tumours

In veterinary medicine, the ability to classify mammary tumours based on the molecular profile and also determine whether the immunophenotype of the regional lymph node and/or systemic metastases is equal to that of the primary tumor may be predictive on the estimation of the effectiveness of various cancer treatments that can be scheduled. Therefore, aims, developed as projects, of the past three years have been (1) to define the molecular phenotype of feline mammary carcinomas and their lymph node metastases according to a previous modified algorithm and to demonstrate the concordance or discordance of the molecular profile between the primary tumour and lymph node metastasis, (2) to analyze, in female dogs, the relationship between the primary mammary tumor and its lymph node metastasis based on immunohistochemical molecular characterization in order to develop the most specific prognostic-predictive models and targeted therapeutic options, and (3) to evaluate the molecular trend of cancer from its primary location to systemic metastases in three cats and two dogs with mammary tumors. The studies on mammary tumours, particularly in dogs, have drawn gradually increasing attention not exclusively to the epithelial component, but also to the myoepithelial cells. The lack of complete information on a valid panel of markers for the identification of these cells in the normal and neoplastic mammary gland and lack of investigation of immunohistochemical changes from an epithelial to a mesenchymal phenotype, was the aim of a parallel research. While investigating mammary tumours, it was noticed that only few studies had focused on the expression of CD117. Therefore, it was decided to further deepen the knowledge in order to characterize the immunohistochemical staining of CD117 in normal and neoplastic mammary tissue of the dog, and to correlate CD117 immunohistochemical results with mammary histotype, histological stage (invasiveness), Ki67 index and patient survival time.

Synthese von tumor-assoziierten MUC1-Mucin-Glycopeptid-Vakzinen und deren immunologische Evaluierung

Eine alternative Methode zur Therapie von Tumorerkrankungen bestünde in einer Immuntherapie ausgelöst durch synthetische Antitumor-Vakzine. Ein vielversprechendes Zielmolekül für eine solche Aktivimmunisierung ist das Glycoprotein MUC1, das auf nahezu allen Epithelgeweben exprimiert und auf Tumorgeweben stark überexprimiert wird. Seine extrazelluläre Domäne enthält eine Vielzahl von Tandem-Repeat-Sequenzen der Art: HGVTSAPDTRPAPGSTAPPA mit fünf potentiellen O-Glycosylierungs-Positionen. Da die Form der Glycosylierung des MUC1 in Tumorzellen stark von der auf normalen Zellen abweicht, liegen auf Tumorzellen eine Reihe tumor-assoziierter Saccharidantigene und Peptidepitope vor.rnIn dieser Arbeit wurden tumor-assoziierte Glycopeptidantigene aus der MUC1-Tandem-Repeat-Region hergestellt. Die synthetisierten MUC1-Glycopeptide tragen in verschiedenen Positionen eine Glycosylierung mit den tumor-assoziierten Tn- und STn-Saccharid-Antigenen. Zur Gewinnung von Vakzinen wurden diese Glycopeptid-Antigene über einen Spacer mit immunstimulierenden Komponenten verknüpft. Als Immunstimulanzien wurden ein T-Zell-Epitop aus dem Ovalbumin (OVA323-339) sowie die Carrier-Proteine Rinderserumalbumin (BSA) und Tetanus-Toxoid (TTox) verwendet. rnDie synthetischen MUC1-Glycopeptide wurden durch Immunisierung von Mäusen einer immunologischen Evaluierung unterzogen. Insbesondere die synthetischen MUC1-Glycopeptid-TTox-Vakzine lösen sehr starke Immunantworten aus. Es konnte gezeigt werden, dass die induzierten Antikörper stark an Tumorzellen und auch an Mammakarzinom-Gewebe binden, was für die Entwicklung von Antitumor-Vakzinen als vielversprechend einzustufen ist.

Die Rolle von Chemokinrezeptor CXCR4 und Connective Tissue Growth Factor innerhalb der Tumor-Stroma-Interaktion in der akuten myeloischen Leukämie

Die akute myeloische Leukämie (AML) ist eine heterogene Erkrankung der hämatopoetischen Vorläuferzelle, die durch unkontrollierte Vermehrung und ein reduziertes Differenzierungsverhalten gekennzeichnet ist. Aufgrund von Therapieresistenzen und häufig vorkommenden Rückfällen ist die AML mit einer schlechten Langzeitprognose verbunden. Neue Studienergebnisse zeigen, dass leukämische Zellen einer hierarchischen Ordnung unterliegen, an deren Spitze die leukämische Stammzelle (LSC) steht, welche den Tumor speist und ähnliche Charakteristika besitzt wie die hämatopoetische Stammzelle. Die LSC nutzt den Kontakt zu Zellen der hämatopoetischen Nische des Knochenmarks, um die erste Therapie zu überdauern und Resistenzen zu erwerben. Neue Therapieansätze versuchen diese Interaktion zwischen leukämischen Zellen und supportiv wirkenden Stromazellen anzugreifen. rnrnIn dieser Arbeit sollte die Bedeutung des CXC-Motiv Chemokinrezeptors Typ 4 (CXCR4) und des Connective Tissue Growth Factors (CTGF) innerhalb der AML-Stroma-Interaktion untersucht werden. CXCR4, der in vivo dafür sorgt, dass AML-Zellen in der Nische gehalten und geschützt werden, wurde durch den neuwertigen humanen CXCR4-spezifischen Antikörper BMS-936564/MDX-1338 in AML-Zelllinien und Patientenzellen in Zellkulturversuchen blockiert. Dies induzierte Apoptose sowie Differenzierung und führte in Kokulturversuchen zu einer Aufhebung des Stroma-vermittelten Schutzes gegenüber der Chemotherapie. Für diese Effekte musste teilweise ein sekundärer Antikörper verwendet werden, der die CXCR4-Moleküle miteinander kreuzvernetzt.rnDie Auswertung eines quantitativen Real time PCR (qPCR)-Arrays ergab, dass CTGF in der AML-Zelllinie Molm-14 nach Kontakt zu Stromazellen hochreguliert wird. Diese Hochregulation konnte in insgesamt drei AML-Zelllinien sowie in drei Patientenproben in qPCR- und Western Blot-Versuchen bestätigt werden. Weitere Untersuchungen zeigten, dass diese Hochregulation (i) unabhängig von der Stromazelllinie ist, (ii) den direkten Kontakt zum Stroma benötigt und (iii) auch unter hypoxischen Bedingungen, wie sie innerhalb des Knochenmarks vorherrschen, stattfindet. Der durch Zell-Zell- oder Zell-Matrix-Kontakt gesteuerte Hippo-Signalweg konnte aus folgenden Gründen als möglicher upstream-Regulationsmechanismus identifiziert werden: (i) Dessen zentraler Transkriptions-Kofaktor TAZ wurde in kokultivierten Molm-14-Zellen stabilisiert, (ii) der shRNA-gesteuerte Knockdown von TAZ führte zu einer reduzierten CTGF-Hochregulation, (iii) CTGF wurde in Abhängigkeit von der Zelldichte reguliert, (iv) Cysteine-rich angiogenic inducer 61 (Cyr61), ein weiteres Zielgen von TAZ, wurde in kokultivierten AML-Zellen ebenfalls verstärkt exprimiert. Der Knockdown von CTGF führte in vitro zu einer partiellen Aufhebung der Stroma-vermittelten Resistenz und die Blockierung von CTGF durch den Antikörper FG-3019 wirkte im AML-Mausmodell lebensverlängernd. rn rnDie Rolle von CTGF in der AML ist bisher nicht untersucht. Die vorliegenden Ergebnisse zeigen, dass CTGF ein interessantes Therapieziel in der AML darstellt. Es bedarf weiterer Untersuchungen, um die Bedeutung von CTGF in der Tumor-Stroma-Interaktion näher zu charakterisieren und nachgeschaltete Signalwege zu identifizieren.

VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease

Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.

Incomplete ovariosalpingectomy and subsequent malignant granulosa cell tumor in a female green iguana (Iguana iguana)

Case Description-A 9-year-old spayed female green iguana (Iguana iguana) was evaluated because of a distended coelom and weight loss. History included a single episode of egg binding and subsequent bilateral ovariosalpingectomy.;Clinical Findings-Physical examination revealed a mass within the coelomic cavity. Ultrasonography revealed a large, irregular mass with hypoechoic regions and coelomic effusion. Clinicopathologic derangements included heterophilia, monocytosis, lymphopenia, basophilia, hypocholesterolemia, hypoproteinemia, and hypercalcemia. Results of cytologic evaluation of the mass were suggestive of malignant epithelial neoplasia, but neoplastic cells were not found in the effusion. An ovarian tumor was suspected on the basis of clinical signs, clinicopathologic findings, and results of cytologic evaluation of the mass.;Treatment and Outcome-Surgical exploration revealed a large left ovary, a normal-appearing contralateral ovary, and a mass in the fat body, all of which were removed and submitted for histologic examination. The histologic diagnosis was granulosa cell tumor with metastasis to the fat body. The patient died 11 months after evaluation, and disseminated granulosa cell tumor was confirmed at necropsy; histologic examination at that time also identified systemic mastocytosis.;Clinical Relevance-Granulosa cell tumors are uncommon in reptiles, and this was the first granulosa cell tumor described antemortem cytologically, histologically, and ultrastructurally in an iguana. Findings in this iguana underscored concerns associated with incomplete oophorectomy of iguanas; cytologic and histopathologic findings were similar to those observed in other domestic animals. Oophorectomy should be considered as an alternative to standard ovariosalpingectomy to avoid potential complications in pet reptiles, and use of microsurgical instruments and vascular clips is advised. (J Am Vet Med Assoc 2011239:237-242)

Sarcomatous evolution of oligodendroglioma ("oligosarcoma"): confirmatory report of an uncommon pattern of malignant progression in oligodendroglial tumors

By analogy to gliosarcoma, the neologism "oligosarcoma" is to describe an uncommon form of biphasic central nervous system tumor composed of contiguous neuroepithelial and mesenchymal elements, each of which individually meet the criteria of oligodendroglioma and sarcoma, respectively. By virtue of its distinctive genotype (codeletion 1p/19q), oligodendroglioma is a particularly inviting paradigm to test the assumption that such mixed tumors are clonally derived from a glial primary. We observed this constellation in a 41-year-old male who underwent two resection procedures for a recurring right frontal tumor at five years' interval. On imaging, both lesions were contrast-enhancing, and measured 7 cm × 7 cm × 6.8 cm and 7 cm × 6.5 cm × 4cm, respectively. Following the first operation, temozolomide monotherapy was administered. Whereas initial histology showed conventional anaplastic oligodendroglioma, the recurrence consisted mostly of a fibrosarcoma-like, fascicular neoplasm that was immunoreactive for vimentin, smooth muscle actin, S100 protein, and focally epithelial membrane antigen. In between, a subset of otherwise indistinguishable spindle cells expressed GFAP, and focally merged with residues of oligodendroglioma. Molecular testing for loss of heterozygosity confirmed codeletion of 1p/19q in both the primary tumor and the sarcomatous recurrence. Similarly, generalized immunoreactivity for the mutant R132H form of isocitrate dehydrogenase in both lesions indicated an identical mutation of the IDH1 gene. By the above standards, biologically consistent "oligosarcomas" are felt to be exceedingly rare, and possibly participate of a nosologically heterogeneous group of combined glial/mesenchymal lesions that may also include iatrogenically induced second malignancies as well as true collision tumors.

GLP-2 receptors in human disease: high expression in gastrointestinal stromal tumors and Crohn's disease

Peptide hormones of the glucagon-like peptide (GLP) family play an increasing clinical role, as reported for GLP-1 in diabetes therapy and insulinoma diagnostics. GLP-2, despite its known trophic and anti-inflammatory intestinal actions translated into preliminary clinical studies using the GLP-2 analogue teduglutide for treatment of short bowel syndrome and Crohn's disease, remains poorly characterized in terms of expression of its receptor in tissues of interest. Therefore, the GLP-2 receptor expression was assessed in 237 tumor and 148 non-neoplastic tissue samples with in vitro receptor autoradiography. A GLP-2 receptor expression was present in 68% of gastrointestinal stromal tumors (GIST). Furthermore, GLP-2 receptors were identified in the intestinal myenteric plexus, with significant up-regulation in active Crohn's disease. The GLP-2 receptors in GIST may be used for clinical applications like in vivo targeting with radiolabelled GLP-2 analogues for imaging and therapy. Moreover, the over-expressed GLP-2 receptor in the myenteric plexus may represent the morphological correlate of the clinical target of teduglutide in Crohn's disease.

Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients

Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed monospecific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.

Molecular cloning and characterization of equine thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that plays a central role in T helper 2 (Th2) cell differentiation and allergic inflammation. It is predominantly expressed by epithelial cells, and its expression is increased in patients with atopic dermatitis and asthma. Mice overexpressing TSLP in the skin develop allergic dermatitis and mice overexpressing TSLP in lungs develop asthma-like disease. However, it is not known whether TSLP plays an important role in equine allergies. Therefore, we cloned and sequenced the complete translated region of equine TSLP gene and measured its expression in various tissues. The equine TSLP gene is organized in 4 exons and encodes a protein of 143 amino acids, which has 62% amino acid identity with human TSLP.

Thymic stromal lymphopoietin stimulates the formation of eosinophil extracellular traps

Thymic stromal lymphopoietin (TSLP) that is released by epithelial cells upon certain environmental triggers activates cells of the innate and adaptive immune system resulting in a preferential T helper 2 immune response. By releasing eosinophil extracellular traps (EETs), eosinophils achieve an efficient extracellular bacterial killing. Eosinophil extracellular traps release, however, has been observed in both infectious and noninfectious eosinophilic diseases. Here, we aim to investigate whether eosinophils generate functional EETs as a direct response to TSLP, and further to study the extra- and intracellular mechanisms involved in this process as well as TSLP receptor (TSLPR) expression by eosinophils in vitro and in vivo.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

Papillary glioneuronal tumor

The descriptive term papillary glioneuronal tumor (PGNT) has been repeatedly applied to a morphologic subset of low-grade mixed glial-neuronal neoplasia of juvenile and young adult patients. We report on a 13-year-old boy with PGNT of the left temporal lobe, who presented with headaches and a single generalized seizure. On magnetic resonance imaging, tumor was seen as a large, moderately enhancing paraventricular mass with cyst-mural nodule configuration and slight midline shift. Perifocal edema was virtually absent. Gross total resection could be performed, followed by an uneventful recovery. Histologically, the tumor exhibited similar, if not identical, features as reported previously. These comprised a patterned biphasic mixture of sheets of synaptophysin-expressing small round cells and pseudorosettes of GFAP-positive rudimentary astrocytes along vascular cores. Focally, the latter imprinted a pseudopapillary aspect on this otherwise solid lesion. Both cellular components expressed non-polysialylated neural cell adhesion molecule (NCAM)-L species, and several overlapping areas of synaptophysin and GFAP immunoreactivity were present. The mean MIB-1 labeling index remained below 1%. Signs of anaplasia, in particular mitotic figures, endothelial proliferation, or necrosis were consistently lacking. We perceive PGNT as a clinically and morphologically well-delineated subgroup of extraventricular neurocytic neoplasia, whose paradigmatic presentation may allow for consideration as an entity.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.