Alexander Schütz

Expression and Regulation of CD97 in Colorectal Carcinoma Cell Lines and Tumor Tissues

The expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-beta, which inhibits proliferation in transforming growth factor-beta-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion.

CD97, but not its closely related EGF-TM7 family member EMR2, is expressed on gastric, pancreatic, and esophageal carcinomas

CD97 expression is related closely to the dedifferentiation and tumor stage in thyroid carcinomas. We systematically examined the role of CD97 and its closest relative, EMR2, in normal and malignant gastric, esophageal, and pancreatic tissue. The normal tissues were EMR2-, whereas CD97 was expressed slightly in the parietal cells of gastric mucosa and in exocrine pancreatic cells. Interestingly, intralobular and interlobular pancreatic ducts were CD97+. All tumors were EMR2-. CD97 was expressed by 44 of 50 gastric, 14 of 18 pancreatic, and 10 of 13 esophageal carcinomas. Of the 44 gastric cancers, 27 showed disseminated or scattered tumor cells at the invasion front with stronger CD97 expression than tumor cells located in solid tumor formations. There was no correlation between CD97 levels in the tumors or soluble CD97 in the serum samples and the clinicopathologic features of the patients. Taken together, significant numbers of gastric, esophageal, and pancreatic carcinomas are CD97+, whereas its homolog, EMR2, does not have any role in such tumors.

Expression profiles of p53, p63, and p73 in benign salivary gland tumors.

Abstract. The tumor-suppressor protein p53 has recently been shown to belong to a family that includes two structurally related proteins, p63 and p73. In contrast to p53, p63 and p73 play an essential role in epithelial development, stem cell identity and cellular differentiation. Salivary gland tumors carry a wide spectrum of histopathological forms, which may share a common single-cell origin from the epithelial progenitor basal duct cells and have a different tendency of malignant progression. This study was performed to examine the expression of p53, p63, and p73 in benign salivary gland tumors. Expression and mutation of p53, p73, and p63 were examined by direct DNA sequencing, reverse transcription PCR using isoform-specific primers, and by immunohistochemistry in normal parotid tissue (n=10), and various tumors of the salivary gland (42 pleomorphic adenomas, 12 myoepitheliomas, 8 basal cell adenomas, 5 oncocytomas, 5 canalicular adenomas, and 20 adenolymphomas). In normal parotid tissue the expression of p63 and p73 was restricted to few basal and myoepithelial cells. Ductal luminal and acinus cells were completely negative for the expression of all three family members. In contrast, in salivary gland tumors, strong nuclear staining for p63 and p73 was observed. Myoepithelial and basaloid cells and the basal epithelial layer of adenolyphomas and oncocytomas were positive for p63 and also, to a lesser extent, to p73. Mutations of p53 were detected in 4 of 42 (10%) pleomorphic adenomas, in 3 of 12 (25%) myoepitheliomas, and in 1 of 8 (13%) basal cell adenomas but not in other tumors. We failed to detect specific mutations of p63 and p73. Using isoform-specific PCR, we found that all isoforms of p63 were expressed in normal parotid tissue whereas the pleomorphic adenomas, myoepitehliomas, and basal cell adenomas dominantly expressed the transactivation-incompetent truncated isoforms. Our data indicate that p63 and p73 are upregulated in salivary gland tumors and may serve as a marker of epithelial and myoepithelial progenitor cells in salivary glands. The prevalence of p53 mutations and the observation of the expression of ΔNp63 isoforms only in pleomorphic adenomas, myoepitheliomas, and also basal cell adenomas may reflect their possible malignant potential.

Expression of ADAM15 in lung carcinomas

ADAM15, a member of the ADAM (a disintegrin and metalloprotease) family, is a membrane protein containing both protease and adhesion domains and may, thus, be involved in tumor invasion and metastasis. The aim of this study was to analyze the expression of ADAM15 and its potential ligand, integrin αvβ3 (CD51/CD61), in lung carcinoma cell lines and tissues. Most small cell lung carcinomas (SCLCs) and non-SCLC cell lines were ADAM15, αv and β3 integrin mRNA positive. Half of the cell lines expressed ADAM15, and three expressed the αvβ3 heterodimer at the cell surface as shown using flow cytometry. Paraffin sections of pulmonary epithelial tumors, including SCLCs (n=26), squamous cell cancer (SCCs, n=27) and adenocarcinomas (ACs, n=17) were stained with antibodies to the ectosolic and cytosolic domain of ADAM15 and αvβ3 integrin complex. The results were scored (0–12, according to Remmele’s score). Normal epithelial cells of the lung were negative or slightly positive for ADAM15 (score<2). The score was always significantly higher for tumor cells. ACs showed the strongest staining (tumor center; ADAM15ecto; mean±SEM; 5.47±1.04), whereas SCLCs only showed weak ADAM15 expression (2.67±0.42; SCCs: 3.62±0.62). Frequently, significantly stronger ADAM15 expression has been shown in tumor cells located at the front of invasion compared with those within solid formations. Overall analysis of all tumor specimens and each tumor type revealed no significant correlation between tumor stage or degree of differentiation and ADAM15 ectosolic or cytosolic domain expression in tumor cells. Both molecules are often co-localized in the same tumor cells in ADAM15- and αvβ3 integrin-positive carcinomas. In summary, lung carcinoma cell lines and tissues were frequently ADAM15 positive.

Projection of non-cholinergic basal forebrain neurons ensheathed with perineuronal nets to rat mesocortex

The existence of non-cholinergic (GABAergic) components in the septo-hippocampal system but also in basal forebrain projections terminating in the olfactory bulb and certain cortical areas has been documented by several authors using retrograde and anterograde tracing techniques. On the other hand, the basal forebrain also contains a high number of mainly parvalbumin-positive neurons ensheathed by a lattice-like matrix of polyanionic proteoglycans forming so-called perineuronal nets of as yet unknown function. By a combination of retrograde tracing using Fluoro-Gold injection into mesocortical areas of rats and staining of perineuronal nets by Wisteria floribunda agglutinin (WFA) the present study describes the projection pattern and distribution of non-cholinergic projection neurons characterized by perineuronal nets in the anterior parts of the basal forebrain complex (medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus). After tracer injection into the cingulate cortex labelled net-associated neurons were distributed within the rostrocaudal extension of the basal forebrain complex but were predominantly found in the horizontal limb of the diagonal band of Broca. Retrograde labelling of neurons with perineuronal nets after tracer injection into the retrosplenial cortex was more pronounced in the medial septum. Choline acetyltransferase-immunoreactive (ChAT-ir) projection neurons were in no case associated with perineuronal nets. The results demonstrate that a large portion of the non-cholinergic projection neurons of the basal forebrain are endowed with a specialized microenvironment of proteoglycans and form a strong input system of mesocortical components of the limbic system.

Alterations of the INK4a-ARF gene locus in pleomorphic adenoma of the parotid gland

Pleomorphic adenomas of the parotid gland are benign tumours composed of epithelial and mesenchymal cells. The INK4a‐ARF (CDKN2A) locus on chromosome 9p21 encodes two tumour suppressor proteins, p16INK4a and p14ARF, which act as upstream regulators of the Rb‐CDK4 and p53 pathways. To study the contribution of each pathway in pleomorphic adenomas, this study analysed alterations of p14ARF, p16INK4a, p53, and pRb in these tumours. After microdissecting the different histological components, 42 pleomorphic adenomas of the parotid gland were analysed for INK4a‐ARF inactivation by DNA sequence analysis, methylation‐specific PCR (MSP), restriction enzyme‐related polymerase chain reaction (RE‐PCR), mRNA expression, microsatellite analysis, and immunohistochemistry. In addition, microdeletion of p14ARF and p16INK4a were assessed by differential PCR. The status of p53 and Rb was examined by direct sequencing and immunohistochemistry. Using microdissection, it was possible to examine the tumour components, i.e. epithelial, mesenchymal, and transitional, separately after immunohistochemical identification. Methylation of p14ARF was found in 1/42 cases and alterations of p16INK4a occurred in 12/42 of pleomorphic adenomas, which correlated with loss of mRNA transcription. Microdeletions or specific mutations of either exon were not detected. Methylation was detected exclusively in the epithelial and transitional components and not within the mesenchymal part of the tumour. p53 mutations were detected in 4/42 adenomas, also occurring solely in the epithelial components of the tumours. pRb was detected immunohistochemically in 40/42 adenomas. In normal, corresponding parotid tissue, p14ARF, p16INK4a, p53, and pRb alterations were not observed. The observation that alterations of p14ARF and p16INK4a, and also p53 mutations, occurred exclusively in the epithelial and transitional components of pleomorphic adenoma supports the theory that these areas are prone to malignant transformation to carcinoma in adenoma. Copyright

Diversity of CD97 in smooth muscle cells.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97stalk). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97EGF) did not bind to normal SMC. Vascular SMC, which was also CD97EGF-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97stalk. CD97stalk-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97stalk. A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97stalk and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97stalk and CD97EGF. As expected, CD97EGF-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.

Lung Adenocarcinomas and Lung Cancer Cell Lines Show Association of MMP-1 Expression With STAT3 Activation

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in the majority of lung cancer. This study aims at defining connections between STAT3 function and the malignant properties of non–small cell lung carcinoma (NSCLC) cells. To address possible mechanisms by which STAT3 influences invasiveness, the expression of matrix metalloproteinase-1 (MMP-1) was analyzed and correlated with the STAT3 activity status. Studies on both surgical biopsies and on lung cancer cell lines revealed a coincidence of STAT3 activation and strong expression of MMP-1. MMP-1 and tyrosine-phosphorylated activated STAT3 were found co-localized in cancer tissues, most pronounced in tumor fronts, and in particular in adenocarcinomas. STAT3 activity was constitutive, although to different degrees, in the lung cancer cell lines investigated. Three cell lines (BEN, KNS62, and A549) were identified in which STAT3 activitation was inducible by Interleukin-6 (IL-6). In A549 cells, STAT3 activity enhanced the level of MMP-1 mRNA and stimulated transcription from the MMP-1 promoter in IL-6–stimulated A549 cells. STAT3 specificity of this effect was confirmed by STAT3 knockdown through RNA interference. Our results link aberrant activity of STAT3 in lung cancer cells to malignant tumor progression through up-regulation of expression of invasiveness-associated MMPs.

Evaluation of the invasion front pattern of squamous cell cervical carcinoma by measuring classical and discrete compactness

The invasion front pattern of squamous cell carcinoma (SCC) is a conspicuous histological phenomenon, which is assessed without precise criteria. The current study was performed to introduce the classical (C(C)) and discrete compactness (C(D)) as new morphometric parameters for quantification of this pattern. A retrospective analysis of 76 surgically treated patients with cervical carcinoma was conducted and the pattern of invasion was qualitatively classified as closed, finger-like or diffuse, respectively, by two pathologists. After digitization of the histological slides with a field of view of 10.4 mm x 8.3mm, tumor areas were labeled and C(C) and C(D) were computed based on the drawings (binary images). Additionally, intraindividual variation of compactness was evaluated for 12 selected tumors. The qualitative pattern assessment by the pathologists was moderately reproducible with an interobserver agreement of 72% and a kappa coefficient of 0.44. The values of C(C) and C(D) referring to the invasion front patterns assigned by both pathologists were significantly different between the three classified groups (p< or =0.01 and p< or =0.0001), so that, both theoretically and in practice, compactness regards the same morphological feature. In due consideration of the analysis of the area under the ROC (receiver operating characteristic) curves and the variation coefficient of different tumor regions, C(D) is more suitable for practical use than C(C). Tumors with a microscopic invasion into the parametria and with lymph-vascular space invasion were found to have a lower value of C(D), which indicates a more diffuse pattern of invasion (p=0.028 and p=0.033). We conclude that the discrete compactness C(D) is a new and reproducible parameter for a computer assisted quantification of the invasion front pattern and, thus, defines a further phenotypic feature of SCC of the uterine cervix.

Diagnosis of aspergilloma in a pleural cavity (persistent pneumothorax) using classic imaging methods

The diagnosis of pulmonary aspergillosis is based on serum‐analysis, as well as histological and microbiological analysis of bronchial lavage and transbronchial biopsies. When Aspergillus develops within a preformed cavity, however, these tests are likely to be negative. In this situation, classic imaging techniques such as chest X‐ray and high resolution‐computed tomography (HR‐CT) can be of great diagnostic use. We here describe the case of a 62‐year‐old woman with a history of breast cancer and subsequent ablation of the left breast and radiotherapy. The case demonstrates an example of a pleuropulmonary aspergilloma, in which sero‐ and micro‐biological detection failed. Thorax HR‐CT exhibited the cavity, a small persistent pneumothorax, partially filled by an oval density. This density clearly dislocated according to gravity following a positional change of the patient from supine to prone. The density thus revealed mobility which was typical of aspergilloma. Following excision, this diagnosis was confirmed. A density within a cavity may be differentiated by its mobility from differential diagnoses such as lung cancer which would not be expected to exhibit mobility.