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Background: Data on routine systemic treatment of patients with ovarian cancer are currently available only to a limited degree. The alkylating agent treosulfan is approved in oral (p.o.) and intravenous (i.v.) form for the treatment of ovarian carcinoma. The present non-interventional study analyzed the clinical use of treosulfan in Germany, evaluating the mode of application, toxicity, and response and survival rate., Patients and Methods: Two hundred and forty-eight ovarian cancer patients in 57 Centers, who received treosulfan mainly either i.v. (5,000-8,000 mg/m(2) d1, q21d or q28d) or p.o. (400-600 mg/m(2) d1-14 or 21, q28d) for at least one therapy cycle were evaluable and were included in the study., Results: With a median age of 70 years (range=36-92 years), predominantly elderly patients received treosulfan treatment. Most participants presented serous histology (131, 52.8%) and advanced-stage FIGO III (122, 49%) or IV (55, 22%) disease. Median ECOG status was 1 (range=0-2), whereas cardiac co-morbidity was common (31%). Treosulfan was usually administered as second- (26%), third- (21%) or fourth-line (17%) therapy. Two hundred and one patients received i.v. and 47 p.o., Treatment: The most common reason for dose modifications was due to hematological toxicity (46%). The main reason for a therapy discontinuation was progressive disease (38.5%). Response was observed in 25.8% of participants, disease stabilization in 28.6 % and progress in 45.6%. The median progression-free and overall survival was 196 and 405 days, respectively., Conclusion: In predominantly elderly and heavily pre-treated patients with recurrent ovarian cancer, treosulfan featured a clinical relevant efficacy and well-manageable, mostly hematological, toxicity, which resulted in a positive therapeutic index., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

TGF-beta is one of the most effective suppressors of cell proliferation and acts as a potent inducer of matrix formation by promoting the neosynthesis of the extracellular matrix and inhibiting the synthesis of matrix degrading enzymes. In this study we analysed the expression of TGF-beta 1 protein and mRNA in normal laryngeal mucosa and in invasive laryngeal carcinomas, to find out any differences in the amount of expressed TGF-beta 1 and to identify the cells which are actively involved in the synthesis of TGF-beta 1. In addition, we analysed the biological effect of TGF-beta 1 on the rate of tumor cell proliferation and the amount of neosynthesis of the extracellular matrix. The study comprised a series of 24 laryngeal squamous cell carcinomas (SCC) of different degree of tumor cell differentiation and 10 cases of normal laryngeal mucosa, which was immunhistochemically analyzed using antibodies against TGF-beta 1, the cell proliferation antigen Ki-67 (Mib-1 antibody) and major interstitial collagen types (III, V, VI) and tenascin. The TGF-beta 1-mRNA was identified by non-radioactive in-situ hybridization using a 360bp cDNA-clone for TGF-beta 1. All immunostainings and the in-situ hybridization were quantitatively evaluated by morphometric analysis and subjected to a statistical evaluation. In the normal laryngeal mucosa we found TGF-beta 1 protein mainly in the suprabasal epithelial cell layer and in some stroma cells below the epithelium. The mRNA of TGF-beta 1 was located in the basal cell layer of the normal mucosa as well as in the stroma cells next to the squamous cell epithelial. All cases of invasive carcinoma analysed showed a positive cytoplasmatic staining for TGF-beta 1 in the tumor cells as well as in stroma cells with a significantly more intense staining in the tumor cells than in the stroma cells. The in-situ hybridisation for TGF-beta 1-mRNA provided also positively stained tumor as well as stroma cells. Again, the amount of positively labeled tumor cells outnumbered the amount of stained stroma cells. In addition, we found a dramatic increase of the proliferation index in the invasive laryngeal carcinomas which was correlated with the amount of TGF-beta 1 expression. The collagens III, V and VI as well as tenascin were found strongly in the invasive carcinomas when compared to the normal mucosa, with statistically positive correlation. In conclusion, we found that invasive tumor growth of the larynx is associated with an increase of the TGF-beta 1 protein and mRNA expression. The synthesis of TGF-beta 1 is mainly performed by the tumor cells, but also to a lesser extent by the stroma cells. We provide evidence, that the TGF-beta 1 is biologically active on the neofomation of extracellular matrix components. In contrast the lack of a suppressive effect on the proliferative capacity of the tumor cells, indicates a "dissociation" of the TGF-beta effect in SCCs. A possible reason for this dissociation may lie in a partial dysfunction of the TGF-beta receptors of the tumor cells or in defects of the intracellular signaling pathways.

The key event of invasive growth of malignant epithelial tumors is the dissolution of the peritumoral basement, membrane (BM). Accordingly, numerous immunohistochemical studies have shown that particularly in breast carcinomas there is an almost complete loss of the BM, even in well-differentiated carcinomas. In order to find out the significance of tumor-associated BM-degradation we localized major matrix metalloproteinases (MMPs) as the most important proteolytic enzymes for connective tissue dissolution. As a prerequisite, we had to identify antibodies reacting specifically on paraffin-embedded tissue material. Extensive pretesting of MMP-2, -3 and -9 antibodies of various sources provided evidence that only a small proportion of the antibodies analyzed showed a specific, positive staining result, as most of the commercial antibodies did not react on the paraffin material or revealed non-specific staining results. Using the specifically reacting antibodies, we analyzed material from 65 cases of invasive ductal breast cancer by immunohistochemistry for the localization of MMP-2, -3 and -9 and by the non-radioactive in-situ hybridization technique for the localization of the MMP-3-mRNA. The specificity of the in-situ hybridization was analyzed using the sense control. We observed a distinct positive immunoreaction for MMP-2, -3 and -9 over both invasive, as well as non-invasive tumor cells, without apparent differences in the staining intensity. Remarkably, there was a significant staining of tumor cell complexes undergoing lymphangiotic dissemination. In addition to this tumor cell staining pattern, a positive immunoreaction, although to reduced proportion, was observed over peritumoral fibroblastic and endothelial stroma cells. Normal breast tissue also revealed a positive immunostaining of epithelial and stromal cells. Using in-situ hybridization, we observed mRNA expression for MMP-3 both in tumor and stroma cells, comparable to the protein data. Normal breast epithelia reacted weakly positive for MMP-3-mRNA. Our data indicate that there is a major active expression of important MMPs in invasive breast carcinomas as the possible cause for the matrix dissolution. These MMPs are synthesized both by tumor and peritumoral stroma cells which may interact with each other. However, the de-novo synthesis and the amount of immunoreactive enzyme protein does not seem to be significantly enhanced in invasive versus noninvasive tumor areas or normal breast epithelia, indicating that other mechanisms, such as enzyme activation and/or differences in the levels of proteinase inhibitors may be biologically essential factors.

In the present study we compared the localization of major basement membrane (BM) components and their mRNAs between invasive carcinomas of the breast (adenocarcinomas) and larynx carcinomas (squamous cell carcinomas, SCC), in order to determine the extent of BM production and deposition in malignant tumors of biologically different behaviour. Thus, breast carcinomas usually show a rapid locoregional/systemic spread, while the laryngeal SCCs normally show a more locally restricted growth pattern. While normal mammary glands and laryngeal mucosa revealed an intact epithelial BM as evidenced by a continuous linear staining for collagen IV, laminin-1, heparan sulfate proteoglycan (perlecan) and fibronectin-as well as collagen VII in the larynx mucosa-, this continuous staining was lost in the invasive carcinomas, however, affecting the two tumor types differently. In the breast carcinomas, a complete loss was seen even in well differentiated tumors affecting the various BM components similarly, while in the SCCs well differentiated carcinomas had retained significantly more BM material than poorly differentiated ones. In the SCCs, an "early" loss of collagen VII contrasted with a "later" loss of collagen IV, laminin, perlecan and fibronectin the extent of which was, however, associated with a decreasing degree of differentiation. In contrast to the protein findings, by use of the in-situ hybridization we observed a significant expression of mRNA for collagen IV, perlecan and fibronectin. The resulting pattern was comparable between both tumor types and not significantly related to the tumor cell differentiation. Both tumor cells and stroma cells were positively labelled with a more extensive labelling of the stroma cells. Our observations indicate a similar upregulation of the mRNAs for BM-components in breast and larynx carcinomas, but significant differences in the BM-protein deposition so that either major differences in presumed BM-proteolysis or further translational defects are suggested. Furthermore, it can be speculated that the far lesser amount of BM-material in the breast carcinomas may be linked to the more aggressive metastatic spread of those tumors, particularly when compared to the SCCs.

In the present study we used immunohistochemistry and in-situ hybridization for the localization of major basement membrane (BM) components and their mRNA, respectively, in order to determine the extent of BM production and deposition in normal mammary tissue as well as in invasive mamma carcinomas. While normal mammary tissue showed an intact epithelial BM, as evidenced by a continuous linear staining for collagen i.v., laminin, heparan sulfate proteoglycan (perlecan) and fibronectin, this staining was widely lost in the invasive carcinomas. Non-invasive intraductal areas of the carcinomas (carcinoma-in-situ) revealed focal fragmentation and duplication of the epithelial BM. Using in-situ hybridization, we observed only focally positive mRNA-expression for collagen i.v.-, perlecan- and fibronectin-mRNA in normal glands, while mRNA-signals were significantly enhanced in one case of fibroadenoma and particularly in invasive and non-invasive carcinomas, regardless of the degree of tumor cell differentiation. In these instances both tumor and stroma cells were positively labelled. In addition, we could demonstrate a significant increase in the level of TGF-beta 1-mRNA--as the most active cytokine for the induction of matrix component production--by carcinoma cells and to lesser extent by stroma cells. The discrepancy between significantly enhanced mRNA-synthesis and loss in protein deposition points either to an upregulated activity of matrix degrading proteinases (matrix-metalloproteinases) or a posttranslational block of protein synthesis or both.