Aberrant Expression of Syndecan-1 in Cervical Cancers.

Syndecan-1, is a transmembrane heparan/chondroitin sulfate proteoglycan necessary for cell-cell and cell-matrix interactions. Its decreased level on the cell surface correlates with poor prognosis in several tumor types. Aberrant stromal localization of syndecan-1 is also considered an unfavorable prognostic factor in various human malignancies. In the presented work the question was addressed if changes in syndecan-1 expression are related to the prognosis of cervical cancer. Immunohistochemistry for syndecan-1 extracellular domain was performed on surgical specimens of primary cervical cancer. To follow the communication between tumor cells and stromal fibroblasts, their mono-and co-cultures were studied, detecting the expression of syndecan-1, smooth muscle actin, vimentin, and desmin. Immunohistochemistry of tumorous specimens revealed that while cell surface syndecan-1 expression was reduced on cancer cells, it appeared on the surface of tumor-associated fibroblasts. Until year 7, the cohort with high cell surface syndecan-1 expression had significantly longer survival. No difference in the same time-period could be detected when stromal syndecan-1 expression was analyzed. In vitro analysis revealed, that tumor cells can induce syndecan-1 expression on fibroblast, and fibroblasts showed that fibroblast-like cells are built by two cell types: (a) syndecan-1 positive, cytokeratin negative real fibroblasts, and (b) syndecan-1 and cytokeratin positive epithelial-mesenchymal transformed tumor cells. Syndecan-1 on the surface of cancer cells appears to be a positive prognostic marker. Although syndecan-1 positive fibroblasts promote tumor cell proliferation in vitro, we failed to detect their cancer promoting effect in vivo.

Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma.

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as alphav-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

The presence of human papillomavirus 16 in neural structures and vascular endothelial cells.

Human papillomavirus (HPV) is known as a strictly epitheliotropic pathogen. Our results raised the possibility that HPV 16 is present in neural cells and in the vascular endothelium. By in situ hybridization, we have detected HPV 16 E6 ORF sequence in small blood vessels and peripheral nerves adjacent to oral and cervical cancers. The same structures have clearly shown immunohistochemical reactivity for the E6 oncoprotein. These results were verified by PCR applied to E6 and L1 ORFs following microscopic laser dissection of the immunohistochemically positive nerves and vessels. These observations suggest that HPV 16 DNA and protein are present in neurons and endothelial cells in the vicinity of HPV-associated tumors. The HPV 16 genome presumably exists in a non-replicating form in the neurons and constitutively produces high levels of E6 and E7 proteins with an unknown neuropathological outcome.

Prognostic significance of high-risk HPV status in advanced cervical cancers and pelvic lymph nodes.

OBJECTIVE: In this study, we investigated the presence of high-risk (HR) HPV types most prevalent in the Hungarian population in surgically removed cervical cancers and pelvic lymph nodes. The aim of our work was to determine the prognostic significance of HPV status in the lymph nodes draining the tumor. METHODS: Primary tumor specimens from 150 patients and 900 lymph node samples (six per case) were studied. Fifty-six/150 were early (FIGO IA-IB) stage, while 94/150 were advanced (FIGO IIA-IIIB) stage cancers. Beside histopathological evaluation, DNA extracted from the tissue samples was subjected to nested PCR to detect characteristic type-specific sequences of HPVs 16, 18 and 33. Moreover, clinicopathological data were collected for an average 48-month postoperative follow-up period for the purposes of statistical analysis. RESULTS: The presence of HR-HPV types in the lymph nodes shows no correlation with disease-free survival, whereas the presence of lymph node metastases significantly decreases life expectancy (P = 0.002). Lymph nodes with metastases more frequently carry HR-HPV than nodes with no evidence of tumorous infiltration (65% versus 36%, P < 0.001); however, a high number of metastases surrounding HR-HPV-positive tumors were found negative for the viruses (42/120). CONCLUSIONS: HR-HPV status of pelvic lymph nodes draining cervical cancers has no noticeable influence on the life expectancy of the patients. HR-HPV-positive tumor cells do not necessarily have a selective advantage in forming metastases. Presumably, a number of alterations in cellular genes rather than the presence of papillomavirus DNA may have a decisive role in the progression of cervical cancers.

Stromal syndecan-1 expression is an adverse prognostic factor in oral carcinomas.

Syndecan-1, a transmembrane proteoglycan, may exert anti-proliferative effects and promote cell growth by binding various growth factors. Malignant epithelial cells often down-regulate their own syndecan-1 production, and are capable of inducing an aberrant syndecan-1 expression in stromal fibroid cells. Decreased tumor cell syndecan-1 levels in human head and neck squamous cell carcinomas indicate poor prognosis, however, no correlation between stromal syndecan-1 expression and clinical parameters has previously been established. By means of immunohistochemistry, we observed a decrease in tumor cell syndecan-1 reactivity in 33/39 oral carcinoma cases, the degree of which, however, correlated only weakly with the clinical outcome (p = 0.097). Conversely, stromal syndecan-1 positivity proved to be a significant risk factor of recurrence (Cox analysis, p = 0.03) and tumor-specific death (p = 0.023) within a 24-month period after operation. Taken together, stromal expression of syndecan-1 is a reliable factor of adverse prognosis in oral carcinomas.

The expression of five different claudins in invasive breast carcinomas: comparison of pT1pN1 and pT1pN0 tumors.

The evaluation of the role of claudins (CLDNs) in breast carcinogenesis has recently begun. We investigated the expression of CLDNs 1, 2, 3, 4, and 7 in pT1pN0 and pT1pN1 invasive ductal breast carcinomas. Tissue arrays of 30-30 pT1pN0 and pT1pN1 invasive ductal breast carcinomas of different grades were constructed, and the expression of CLDN 1, 2, 3, 4, and 7 proteins was analyzed using standard and immunofluorescent immunohistochemistry. The results were evaluated by light and confocal microscopy. Regarding CLDN 1, 4, and 7 expressions, differences were noted between normal and tumor cells and also between tumors of different grades, while no remarkable differences were noted between pT1pN0 and pT1pN1 tumors. CLDNs 1 and 7 were found to be downregulated in tumor cells compared to the normal epithelium, while CLDN 4 expression was decreased in grade 1 tumors. CLDN 7 protein was abundant in normal epithelia, and the staining decreased in grade 3 tumors. There were no differences between normal and neoplastic cells regarding CLDN 2 and 3 expressions. As a preliminary result, our observations suggest that the analyzed CLDNs do not promote tumor metastasis. On the basis of our findings, it seems that CLDN 1, CLDN 4, and CLDN 7 may rather have an important role in tumorigenesis or in cell-to-cell adhesion.

Spatial distribution of keratan sulfate in the rabbit cornea following photorefractive keratectomy.

PURPOSE: To examine the keratan sulfate content of the stroma and to assess its correlation with the healing process (epithelialization and keratocyte density) after photorefractive keratectomy (PRK). METHODS: Using an Aesculap Meditec MEL 70(G-scan) excimer laser, -6.0 diopters (6.0-mm diameter, 82 microm photoablation depth), PRK was carried out on the right eye of 32 New Zealand pigmented rabbits. After enucleation (at days 1, 4, 7, 14, and 28 and months 2, 3, and 7; sub-groups of 4 animals), fluorescent immunohistochemistry was performed on sections from the central comea using monoclonal mouse anti-keratan-sulfate antibody, immunohistochemistry with proliferative cell nuclear antigen antibody, and hematoxylin-eosin histology. The left, untreated eyes served as controls. Cellular morphology and spatial distribution of keratan sulfate were recorded, stromal thickness measured, and keratocyte density calculated. RESULTS: Keratan sulfate was found on the surface of migrating epithelial cells in the early stage (from days 1 to 7). In the stroma, three phases were noted. (Phase 1) Day 1 to 14, intense granular fluorescence appeared in the anterior stroma with hypocellularity. (Phase 2) Month 1 to 2, newly synthesized lamellar keratan sulfate restored the repopulating anterior stroma. Endothelial cells became keratan sulfate positive, while in the posterior stroma, lamellar-form keratan sulfate increased from week 1 and peaked at month 1 (100% increase). (Phase 3) Month 2 to 7, remodeling and deposition of keratan sulfate was noted, which was produced in phase 2. CONCLUSIONS: Keratan sulfate was found in the epithelium, stroma, and endothelium. By controlling the interlamellar spacing, keratan sulfate plays a role in postoperative edema, remodeling of the corneal stroma, and simultaneous regulation of inflammation after PRK.

Effect of induction chemotherapy on changes of laminin and syndecan expression in oral squamous cell carcinomas: a prospective, randomized, clinicopathologic and immunohistochemical study.

Sixty patients with tumors of the floor of the mouth or of the tongue (T2N0-1-2M0) were randomized into three treatment groups. The first two groups participated in low-dose inductive chemotherapy, surgery, and then radiotherapy, whereas the third control group underwent only surgery and radiotherapy. In all three groups, studies were made of the stage, grade, sex, localization, extents of expression of the pretreatment laminin and syndecan-1 and the cancer specific survival rate, and the correlations among these. The response to neoadjuvant chemotherapy was assessed by means of a method that we developed, involving measurement of the degree of histologic regression observed in response to chemotherapy. Immunohistochemical methods were applied to investigate the changes in degree of expression of laminin and syndecan-1 in response to the medication and their correlations with the survival. As concerns the overall tumorfree survival rate, a significant difference was not found between the two chemotherapeutic groups. However, there was a significant difference between the survival indices of those who participated in cytostatic treatment (70%) and the control group (40%). In the clinical and immunohistochemical examinations, the initial laminin and syndecan-1 levels obtained from biopsy samples could be used as prognostic factors. Our model measuring the extent of histologic regression clearly demonstrated that the survival indices of the patients who responded to the neoadjuvant cytostatic treatment with adequate tissue regression were better than those of the patients who responded to the treatment to only a decreased extent or not at all. The changes in the expressions of laminin and syndecan-1 in response to cystostatic treatment proved to be important predictive factors. The increase or stagnation of these clearly forecast a good prognosis, whereas their decrease was a definite indication of poor prognosis.