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Objective: This phase II, multicenter, single-arm, two-stage study in platinum-resistant, advanced epithelial ovarian or primary peritoneal cancer evaluated the efficacy, safety, and tolerability of weekly single-agent volociximab. Pharmacokinetic/pharmacodynamic (PK/PD) studies were also performed., Methods: Sixteen patients were enrolled in Stage 1. Volociximab was administered at 15mg/kg IV qwk until progression of disease or drug intolerability. Tumor response was assessed every 8weeks. Serum samples for PK or whole blood for the evaluation of circulating tumor cells, endothelial cells, and endothelial progenitor cells were obtained on Days 1, 8, 15, 29, and 50. Ascites from one patient was collected for volociximab concentration analysis. Archived tumor tissue was analyzed by immunohistochemistry (IHC) for α5 integrin expression., Results: Safety data are available on all 16 patients; 14 were evaluable for efficacy. One patient had stable disease at 8weeks. The remaining 13 progressed on treatment. Twelve patients (75%) experienced study-related adverse events (AEs); the most common (≥20%) were headache and fatigue. Three patients experienced possible study-related serious AEs (SAEs): reversible posterior leukoencephalopathy syndrome, pulmonary embolism, and hyponatremia. Peak serum concentrations of volociximab increased 2-3 fold from Day 1 to Day 50. Clinically relevant trough levels were achieved (>150μg/mL). IHC analysis of archived tumor sections showed low-to-moderate expression of α5 integrin on all ovarian cancer tissue evaluated., Conclusion: Despite insufficient clinical activity in this refractory patient population to continue the study, weekly volociximab was well tolerated, and the gained understanding of the mechanism of action of volociximab will inform future development efforts., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer., Experimental Design: Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored., Results: Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo., Conclusions: This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication., (©2010 AACR.)

The safety and efficacy of temporary pericardial pacing wires have been accepted and their use is common after cardiac operations. Complications related to pacing wire removal are unusual but it can be serious and even catastrophic. We report an unusual case of bleeding due the laceration and rent created in the saphenous vein graft wall by the metallic tip of the pacing wire at the time of pacing wire removal.

Objective: To determine whether endometriosis-associated endometrioid cancer (EAOC) is a specific entity compared with endometrioid cancer not associated with endometriosis (OC)., Design: Case-control study., Setting: University hospital research laboratory., Patient(s): Seven patients with endometriosis-associated ovarian cancer EAOC and five patients each with OC, ovarian endometriosis, and benign ovaries., Intervention(s): Ovarian tissue samples were collected from surgical procedures., Main Outcome Measure(s): We hybridized cRNA samples to the Affymetrix HG-U133A microarray chip. Representative genes were validated by real time polymerase chain reaction., Result(s): We identified two main groups of genes: The first group contained the genes SICA2, CCL14, and TDGF1. These genes were equally regulated in endometriosis and EAOC but not in OC and benign ovaries. The second group contained the genes StAR, SPINT1, Keratin 8, FoxM1B, FOLR1, CRABP1, and Claudin 7. They were equally regulated in EAOC and OC but not in ovarian endometriosis and benign ovaries., Conclusion(s): That the first group is composed of the cytokines SICA2 and CCL14 and the growth factor TDGF1 indicates that the regulation of the autoimmune system and of inflammatory cytokines may be very important in the etiology of endometriosis and EAOC. That the second group is composed of genes that play a central role in cell-cell interaction, differentiation, and cell proliferation indicates that they may be important in the development of ovarian cancer in women with endometriosis., (Copyright (c) 2010. Published by Elsevier Inc.)

The biology of ovarian carcinoma differs from that of hematogenously metastasizing tumors because ovarian cancer cells primarily disseminate within the peritoneal cavity and are only superficially invasive. However, since the rapidly proliferating tumors compress visceral organs and are only temporarily chemosensitive, ovarian carcinoma is a deadly disease, with a cure rate of only 30%. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Ovarian carcinoma could originate from any of three potential sites: the surfaces of the ovary, the fallopian tube, or the mesothelium-lined peritoneal cavity. Ovarian cacinoma tumorigenesis then either progresses along a stepwise mutation process from a slow growing borderline tumor to a well-differentiated carcinoma (type I) or involves a genetically unstable high-grade serous carcinoma that metastasizes rapidly (type II). During initial tumorigenesis, ovarian carcinoma cells undergo an epithelial-to-mesenchymal transition, which involves a change in cadherin and integrin expression and up-regulation of proteolytic pathways. Carried by the peritoneal fluid, cancer cell spheroids overcome anoikis and attach preferentially on the abdominal peritoneum or omentum, where the cancer cells revert to their epithelial phenotype. The initial steps of metastasis are regulated by a controlled interaction of adhesion receptors and proteases, and late metastasis is characterized by the oncogene-driven fast growth of tumor nodules on mesothelium covered surfaces, causing ascites, bowel obstruction, and tumor cachexia.

CD95 (also called Fas and APO-1) is a prototypical death receptor that regulates tissue homeostasis mainly in the immune system through the induction of apoptosis. During cancer progression CD95 is frequently downregulated or cells are rendered apoptosis resistant, raising the possibility that loss of CD95 is part of a mechanism for tumour evasion. However, complete loss of CD95 is rarely seen in human cancers and many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Furthermore, cancer patients frequently have elevated levels of the physiological ligand for CD95, CD95L. These data raise the possibility that CD95 could actually promote the growth of tumours through its non-apoptotic activities. Here we show that cancer cells in general, regardless of their CD95 apoptosis sensitivity, depend on constitutive activity of CD95, stimulated by cancer-produced CD95L, for optimal growth. Consistently, loss of CD95 in mouse models of ovarian cancer and liver cancer reduces cancer incidence as well as the size of the tumours. The tumorigenic activity of CD95 is mediated by a pathway involving JNK and Jun. These results demonstrate that CD95 has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity rather than to enhance it should be considered during cancer therapy.

The ovarian surface epithelium (OSE) origin of ovarian cancers has been controversial because these cancers often exhibit Müllerian-like features. One hypothesis is that ovarian neoplasia involves the gain of growth advantages by OSE cells via activation of Müllerian programs. The homeobox gene HOXA10 controls formation of the uterus from the Müllerian ducts, and is not expressed in normal OSE. We previously found that HOXA10 is expressed in ovarian cancers with endometrial-like features, and induces transformed OSE cells to form glandular tumors in mice. In the current study, we found that induction of HOXA10 in OSE cells promotes homophilic cell adhesion and prevents anoikis. HOXA10 expression stimulated interactions of OSE cells with the extracellular matrix proteins vitronectin and fibronectin, and with mesothelial cells of the omentum which is a common attachment site for ovarian cancer cells. HOXA10 also stimulated interactions of OSE cells with omental fibroblasts, and these interactions promoted OSE cell growth. Our findings indicate that aberrant activation of a Müllerian program in OSE cells confers growth advantages by stimulating cellular interactions with the microenvironment.

Deregulated expression of the hepatocyte growth factor (HGF) receptor, c-Met, in cancer contributes to tumor progression and metastasis. The objective of this study was to determine whether blocking c-Met with an orally available c-Met inhibitor, PF-2341066, reduces tumor burden and increases survival in a xenograft model of ovarian cancer metastasis. Treatment of mice injected interperitoneally with SKOV3ip1 cells showed reduced overall tumor burden. Tumor weight and the number of metastases were reduced by 55% (P < .0005) and 62% (P < .0001), respectively. Treatment also increased median survival from 45 to 62 days (P = .0003). In vitro, PF-2341066 reduced HGF-stimulated phosphorylation of c-Met in the tyrosine kinase domain as well as phosphorylation of the downstream signaling effectors, Akt and Erk. It was apparent that inhibition of the pathways was functionally important because HGF-induced branching morphogenesis was also inhibited. In addition, proliferation and adhesion to various extracellular matrices were inhibited by treatment with PF-2341066, and the activity of matrix metalloproteinases was decreased in tumor tissue from treated mice compared with those receiving vehicle. Overall, these data indicate that PF-2341066 effectively reduces tumor burden in an in vivo model of ovarian cancer metastasis and may be a good therapeutic candidate in the treatment of patients with ovarian cancer.

The role of the vitronectin receptor (alpha(v)beta(3)-integrin) as a tumor promoter seems well established, and, consequently, therapies that block this integrin are currently in clinical testing. We undertook the current study to determine whether alpha(v)beta(3)-integrin is an appropriate target in ovarian cancer treatment. Expression of beta(3)-integrin in SKOV3ip1 ovarian cancer cells led to the overexpression of alpha(v)beta(3)-integrin on the cell surface and increased adhesion. However, alpha(v)beta(3)-integrin-overexpressing cells showed impaired invasion, protease expression, and colony formation. These results were recapitulated in xenograft studies: alpha(v)beta(3)-integrin-expressing cells showed increased adhesion to mouse peritoneum, but the overall number of metastatic nodules (105 versus 68 tumors) and tumor weight were significantly lower than those in the parental SKOV3ip1 cells. The alpha(v)beta(3)-integrin-overexpressing cells had a decreased proliferation rate mediated by inhibition of cyclin B1 and induction of phospho-Cdc2 and p53 expression, consistent with a G(2)M cell cycle arrest. Confirming the above results, inhibition of beta(3)-integrin in cultured or primary OvCa cells decreased adhesion but increased invasion and proliferation. Patients with tumors expressing high beta(3)-integrin had significantly better disease-free and overall survival (52 months versus 27 months, P < 0.05). This study shows that alpha(v)beta(3)-integrin expression on tumor cells actually slows tumor progression and acts as a tumor suppressor. Therefore, the vitronectin receptor might not be an appropriate therapeutic target in ovarian cancer.

Recent evidence suggests a close association between extracellular E-cadherin mutation in diffuse-type gastric carcinoma and the acquisition of a migratory phenotype of tumour cells. To characterize the cellular machinery that mediates the gain of motility of tumour cells with mutant E-cadherin, we turned to the small Rho GTPases Rac1 and Rho because they have been implicated in pathological processes including tumour cell migration and invasion. In the present study, we analyse the activity of Rac1 and Rho in relation to E-cadherin harbouring an in-frame deletion of exon 8 and prove for the first time that the mutation reduces the ability of E-cadherin to activate Rac1 and to inhibit Rho. We provide evidence that the lack of Rac1 activation observed in response to mutant E-cadherin influences the downstream signalling of Rac1, as is shown by the decrease in the binding of the Rac1 effector protein IQGAP1 to Rac1-GTP. Moreover, reduced membranous localization of p120-catenin in mutant E-cadherin expressing cells provides an explanation for the lack of negative regulation of Rho by mutant E-cadherin. Further, we show by time-lapse laser scanning microscopy and invasion assay that the enhanced motility and invasion associated with mutant E-cadherin is sensitive to the inhibition of Rac1 and Rho. Together, these findings present evidence that the mutation of E-cadherin influences Rac1 and Rho activation in opposite directions and that Rac1 and Rho are involved in the establishment of the migratory and invasive phenotype of tumour cells that have an E-cadherin mutation.

Although epidemiologic evidence for the ability of combined oral contraception (OC) to reduce the risk of ovarian cancer (OvCa) is convincing, the biological mechanisms underlying this effect are largely unknown. We conducted the present study to determine if OC also influences ovarian carcinogenesis in a genetic mouse model and, if so, to investigate the mechanism underlying the protective effect. LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with ethinyl estradiol plus norethindrone, contraceptive hormones commonly used in combined OC, or norethindrone alone, or a gonadotropin-releasing hormone agonist. The combined OC had a 29% reduction in mean total tumor weight compared with placebo (epithelial tumor weight, -80%). Norethindrone alone reduced mean total tumor weight by 42% (epithelial tumor weight, -46%), and the gonadotropin-releasing hormone agonist increased mean total tumor weight by 71% (epithelial tumor weight, +150%). Large variations in tumor size affected the P values for these changes, which were not statistically significant. Nonetheless, the OC reductions are consistent with the epidemiologic data indicating a protective effect of OC. Matrix metalloproteinase-2 activity was decreased in association with OC, indicating that OC may affect ovarian carcinogenesis by decreasing proteolytic activity, an important early event in the pathogenesis of OvCa. In contrast, OC increased invasion in a K-ras/Pten OvCa cell line established from the mouse tumors, suggesting that OC hormones, particularly estrogen, may have a detrimental effect after the disease process is under way. Our study results support further investigation of OC effects and mechanisms for OvCa prevention.

During early pregnancy, cytotrophoblast cells differentiate into extravillous trophoblast (EVT) cells and invade the uterine spiral arteries. This physiological process is essential for the development of maternal-fetal circulation. Because EVT cells are exposed to a low-oxygen environment during this process, we investigated the role of hypoxia in the mechanism that regulates the invasive behavior of EVT cells. Real-time PCR and immunofluorescent analysis were performed to investigate how hypoxia influences the expression of E-cadherin in villous explants cultures and in trophoblast-derived BeWo cells. We determined that hypoxia induced E-cadherin down-regulation through Snail up-regulation in villous explant cultures. The influence of E-cadherin loss was examined by analyzing the expression of alpha(5)-integrin and phosphorylated focal adhesion kinase (FAK) by Western blot and evaluating trophoblast invasion using a matrigel invasion assay. E-cadherin loss induced the up-regulation of alpha(5)-integrin, which leads to the tyrosine phosphorylation of FAK, resulting in an increase in the invasive activity of EVT cells. An alpha(5)-integrin neutralizing antibody inhibited the invasion of EVT cells by attenuating FAK tyrosine phosphorylation. Immunohistochemical analysis using clinical placental bed biopsies revealed that alpha(5)-integrin was up-regulated and FAK tyrosine phosphorylated (Try(861)) as EVT cells invade the uterine myometrium, whereas E-cadherin expression was down-regulated. These results suggest that alpha(5)-integrin up-regulation induced by E-cadherin loss under hypoxia has a crucial role in regulating the migration of EVT cells. This finding should help us reach a better understanding of the pathogenesis of critical gestational diseases, such as preeclampsia.

The most common site (80%) of ovarian cancer metastasis is the omentum, a large (15 x 10 x 2 cm) peritoneal fold covering the small bowel. Because of the absence of model systems that accurately reproduce the microenvironment of the human omentum, the biological mechanism of early ovarian cancer metastasis is poorly understood. Using a new organotypic 3D culture of the omentum, we show that when cancer cells adhere, matrix-metalloproteinase (MMP)-2 is upregulated and proteolytically activated in these cells. The activated MMP-2 cleaves the matrix proteins fibronectin, vitronectin and collagen I into smaller fragments. The cleaved extra-cellular matrix (ECM) fragments then facilitate and accelerate cancer cell adhesion and invasion by binding to their cognate integrin receptors. In vivo inhibition of MMP-2 before adhesion by using a siRNA or a blocking antibody significantly reduced the number of metastasis and tumor weight in a xenograft mouse model. After metastasis had been established, blocking MMP-2 produced less of an effect. Our data identify tumor-derived proteolytically active MMP-2 as an early regulator of ovarian cancer metastasis.

The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin fixed and paraffin embedded (FFPE) for histopathologic evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody (mAb), designated MET4, from a panel of MET-avid mAbs, based on its specific staining pattern in FFPE preparations. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability of 6.25% in intra-assay and interassay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histologic subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-MET on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 mAb accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.

Objective: To determine if race impacts the survival of patients with epithelial ovarian cancer in a large academic medical center., Methods: Demographic and clinical-pathologic information from patients treated at the University of Chicago from 1992-2007 were analyzed. Continuous variables were analyzed with t tests and categorical variables with chi square tests. Survival curves were evaluated using Kaplan-Meier methods and Cox proportional hazard models were constructed for both overall and disease free survival., Results: 209 women with epithelial ovarian cancer were included in the study, 163 (78%) white and 46 (22%) African American. The baseline demographic characteristics and clinico-pathologic factors such as disease stage, grading, CA-125 levels, rates of optimal debulking (<1 cm residual tumor), platinum sensitivity and American Society of Anesthesiologists score (ASA) were similar between the groups. The median overall survival for African American women was similar: 37.2 months (95% Confidence Interval (CI): 22.5, 52.9) while it was 34.1 months (95% CI: 27.4, 42.6) for white women., Conclusions: There is no evidence of a racial disparity in either treatment or survival for ovarian cancer patients treated at a large academic center. Given that large epidemiologic studies suggested a difference in survival between African American and white women, other sources of disparities must be sought.

Personalized medicine and targeted therapy is the best hope for patients with cancer. C-Met-HGF/SF inhibition may be an effective cancer treatment for ductal carcinoma of the breast as it is expected to be an important signalling target in a large number of malignancies.

Cancer progression has similarities with the process of epithelial-to-mesenchymal transition (EMT) found during embryonic development, during which cells down-regulate E-cadherin and up-regulate Vimentin expression. By evaluating the expression of 207 microRNAs (miRNAs) in the 60 cell lines of the drug screening panel maintained by the Nation Cancer Institute, we identified the miR-200 miRNA family as an extraordinary marker for cells that express E-cadherin but lack expression of Vimentin. These findings were extended to primary ovarian cancer specimens. miR-200 was found to directly target the mRNA of the E-cadherin transcriptional repressors ZEB1 (TCF8/deltaEF1) and ZEB2 (SMAD-interacting protein 1 [SIP1]/ZFXH1B). Ectopic expression of miR-200 caused up-regulation of E-cadherin in cancer cell lines and reduced their motility. Conversely, inhibition of miR-200 reduced E-cadherin expression, increased expression of Vimentin, and induced EMT. Our data identify miR-200 as a powerful marker and determining factor of the epithelial phenotype of cancer cells.

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, alpha5beta1 and alphaVbeta3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in beta1-integrin mRNA and beta1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both beta1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to beta1-integrin. These results suggest that thrombin induces expression and association of beta1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.

The microRNA let-7 regulates late embryonic development by suppressing expression of a number of genes such as c-myc and RAS as well as the embryonic gene high mobility group, A2 (HMGA2). We now demonstrate that HMGA2 is more efficiently targeted by let-7 than RAS. Its expression inversely correlates with the expression of let-7 in the NCI60 cells lines, and the expression of RAS does not change when amounts of let-7 that efficiently silence expression of HMGA2 are introduced into tumor cells. We did not find a difference in the expression of HMGA2 between primary ovarian cancer samples and matching metastases, suggesting that the expression of HMGA2 represents an early event during cancer progression. The late repression of HMGA2 by let-7 during embryonic development, and the early reexpression of HMGA2 during cancer development, is in line with the hypothesis that cancer development represents a case of reverse embryogenesis.

The omentum is a major site of ovarian cancer metastasis. Our goal was to establish a three-dimensional (3D) model of the key components of the omental microenvironment (mesothelial cells, fibroblasts and extracellular matrices) to study ovarian cancer cell adhesion and invasion. The 3D model comprised of primary human fibroblasts extracted from normal human omentum, mixed with ECM and covered by a layer of primary human mesothelial cells, also from normal human omentum. After addition of ovarian cancer cells, the histological appearance of the 3D culture mimicked microscopic metastases to the omentum from patients with ovarian cancer. When ovarian cancer cells, SKOV3ip.1 and HeyA8, were applied to the 3D omental culture, 60% and 68% of all cells attached, respectively, but only 18% and 25% were able to invade. Ovarian cancer cells preferentially adhered to and invaded collagen I, followed by binding to collagen IV, fibronectin, vitronectin, laminin 10 and 1. Omental mesothelial cells significantly inhibited ovarian cancer cell adhesion and invasion, while omental fibroblasts induced adhesion and invasion. This effect is clearly mediated by direct cell-cell contact, since conditioned medium from mesothelial cells or fibroblasts has a minimal, or no, effect on ovarian cancer cell adhesion and invasion. In summary, we have established a 3D model to study the early steps of ovarian cancer metastasis to the human omentum, and found that omental mesothelial cells inhibit, while omental fibroblasts and the ECM enhance, the attachment and invasion of ovarian cancer cells.

The early phases of carcinogenesis resemble embryonic development, often involving the reexpression of embryonic mesenchymal genes. The NCI60 panel of human tumor cell lines can genetically be subdivided into two superclusters (SCs) that correspond to CD95 Type I and II cells. SC1 cells are characterized by a mesenchymal and SC2 cells by an epithelial gene signature, suggesting that SC1 cells represent less differentiated, advanced stages of cancer. miRNAs are small 20- to 22-nucleotide-long noncoding RNAs that inhibit gene expression at the posttranscriptional level. By performing miRNA expression analysis on 10 Type I and 10 Type II cells, we have determined that SC1 cells express low and SC2 cells high levels of the miRNA let-7, respectively, suggesting that let-7 is a marker for less advanced cancers. Expression of the let-7 target high-mobility group A2 (HMGA2), an early embryonic gene, but not of classical epithelial or mesenchymal markers such as E-cadherin or vimentin, inversely correlated with let-7 expression in SC1 and SC2 cells. Using ovarian cancer as a model, we demonstrate that expression of let-7 and HMGA2 is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. These data identify loss of let-7 expression as a marker for less differentiated cancer.

Aims: Tyrosine kinase receptors Her2/neu and c-Met play an important role in breast cancer development and progression. Our aim was to determine the expression of c-Met, its ligand hepatocyte growth factor/scatter factor (HGF/SF) and Her2/neu in ductal carcinoma in situ (DCIS) lesions of the breast (n = 39) by two different immunocytochemical techniques, classical immunohistochemistry and immunofluorescence, and to correlate their expression levels with histopathological and clinical characteristics., Methods and Results: Both methods revealed similar c-Met staining patterns in both the in situ component and the adjacent normal tissue (P < 0.001). However, an imbalance in c-Met expression between tumour and surrounding normal tissue was correlated with high-grade DCIS (Van Nuys Grade 3). No correlation existed between Her2/neu and c-Met expression. High HGF/SF immunoreactivity was observed in 43.6% of the cases, yet the adjacent cellular stroma revealed only low levels of HGF/SF. No correlation existed between c-Met, Her2/neu or HGF/SF expression and clinicopathological factors., Conclusion: An imbalance in c-Met expression between tumour and surrounding normal tissue is associated with an aggressive DCIS phenotype. Moreover, c-Met and HGF/SF may contribute to tumour development by different means than those controlled by Her2/neu.

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.

Objective: Uterine sarcomas are a heterogeneous group of tumors with a propensity for metastasis and resistance to conventional therapy. Recent success in the treatment of other solid tumors with the targeted tyrosine kinase inhibitor imatinib mesylate offers new avenues for investigation. The primary target of imatinib is c-kit, but the drug also inhibits PDGFR-alpha and PDGFR-beta. Given the lack of identified molecular targets in endometrial stromal sarcomas, leiomyosarcomas, and carcinosarcomas, the purpose of this study was to determine the protein expression of c-kit, PDGFR-alpha, and PDGFR-beta in these tumors as a preliminary step to determining their susceptibility to directed therapy. A secondary goal was to identify specific gene mutations that might be associated with activation of these proteins in uterine sarcomas., Methods: Archived tissue from 42 cases of uterine sarcomas was stained for c-kit, PDGFR-alpha, and PDGFR-beta using immunohistochemistry. Laser-capture microdissected samples of uterine carcinosarcomas, or homogeneous areas of leiomyosarcomas or endometrial stromal sarcomas, were subjected to genetic analysis of PDGFR-alpha exons 12 and 18., Results: The majority (38/42, 90%) of uterine sarcomas lacked c-kit expression and 90% (38/42) demonstrated negative or weak staining for PDGFR-beta. In contrast, 70% (30/42) of cases had strong staining for PDGFR-alpha in the tumor but not in normal myometrium or endometrium. Sequencing results revealed no mutations in exons 12 or 18 of PDGFR-alpha., Conclusion: c-kit and PDGFR-beta are unlikely to represent primary treatment targets in uterine sarcomas. The strong expression of PDGFR-alpha in uterine sarcoma specimens suggests a role for this receptor in tumor development, although its potential as a therapeutic target requires further investigation.

The hepatocyte growth factor receptor c-Met is a receptor tyrosine kinase that plays an important role in tumor growth by activating mitogenic signaling pathways. The goal of this study was to evaluate the role of c-Met in the biology of ovarian cancer and to determine its potential as a therapeutic target. c-Met protein expression was detected by immunohistochemistry in 138 advanced-stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Fifteen of 138 (11%) tissues had c-Met overexpression. Median survival for patients with high c-Met levels was 17 months versus 32 months (P = 0.001) for patients with low c-Met expression. Infection of SKOV-3ip1 cells with an adenovirus expressing a small interfering RNA (siRNA) against c-Met efficiently inhibited c-Met protein and mRNA expression as well as extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. It also inhibited adhesion to different extracellular matrix components, human primary mesothelial cells, and full-thickness human peritoneum and, in vivo, to mouse peritoneum. This was paralleled by a significant reduction in alpha(5) and beta(1) integrin protein and mRNA expression as well as a reduction of urokinase and matrix metalloproteinase (MMP)-2/MMP-9 activity. In SKOV-3ip1 ovarian cancer xenografts, i.p. treatment with the c-Met siRNA significantly reduced tumor burden, ascites formation, protease activity, and the number of peritoneal implants but not tumor size or angiogenesis. These results suggest that c-Met overexpression is a prognostic factor in ovarian cancer and that targeting c-Met in vivo inhibits peritoneal dissemination and invasion through an alpha(5)beta(1) integrin-dependent mechanism. Therefore, c-Met should be explored further as a therapeutic target in ovarian cancer.

A subset of tyrosine kinases are activated by mutations which contribute to the malignant transformation, growth, and metastasis of human cancers. Mutations change the expression, conformation and/or stability of tyrosine kinases, often leading to constitutive activation of the signaling pathways the kinases regulate. Given that tyrosine kinases are key members of signaling cascades, mutations have multiple effects on various cellular proteins. This review will focus on four kinases (EGFR, c-Met, c-Kit, and PI3-kinase) known to be mutated in human cancer. It will discuss the effects that these mutations have on the biology of tumors, and how our understanding of the structure and function of kinases and their mutations is currently being used to design targeted treatments.

Purpose: To identify dosimetric parameters associated with acute hematologic toxicity (HT) and chemotherapy delivery in cervical cancer patients undergoing concurrent chemotherapy and intensity-modulated pelvic radiotherapy., Methods and Materials: We analyzed 37 cervical cancer patients receiving concurrent cisplatin (40 mg/m(2)/wk) and intensity-modulated pelvic radiotherapy. Pelvic bone marrow (BM) was contoured for each patient and divided into three subsites: lumbosacral spine, ilium, and lower pelvis. The volume of each region receiving 10, 20, 30, and > or =40 Gy (V(10), V(20), V(30), and V(40), respectively) was calculated. HT was graded according to the Radiation Therapy Oncology Group system. Multivariate regression models were used to test associations between dosimetric parameters and HT and chemotherapy delivery., Results: Increased pelvic BM V(10) (BM-V(10)) was associated with an increased Grade 2 or worse leukopenia and neutropenia (odds ratio [OR], 2.09; 95% confidence interval [CI], 1.24-3.53; p = 0.006; and OR, 1.41; 95% CI, 1.02-1.94; p = 0.037, respectively). Patients with BM-V(10) > or =90% had higher rates of Grade 2 or worse leukopenia and neutropenia than did patients with BM-V(10) <90% (11.1% vs. 73.7%, p < 0.01; and 5.6% vs. 31.6%, p = 0.09) and were more likely to have chemotherapy held on univariate (16.7% vs. 47.4%, p = 0.08) and multivariate (OR, 32.2; 95% CI, 1.67-622; p = 0.02) analysis. No associations between HT and V(30) and V(40) were observed. Dosimetric parameters involving the lumbosacral spine and lower pelvis had stronger associations with HT than did those involving the ilium., Conclusion: The volume of pelvic BM receiving low-dose radiation is associated with HT and chemotherapy delivery in cervical cancer patients undergoing concurrent chemoradiotherapy.

Twenty yeast strains have recently been isolated in pure cultures from natural and industrial sources and identified based mainly on physiological properties. The majority of the strains (15) are alcohologenic belonging to the genus Saccharomyces and comprise two brewer's (beer) yeast strains (S. carlsbergensis= S. uvarum A and B), two baker's yeast strains (S. cerevisiae CA and CP), one spirit yeast strain (S. cerevisiae CF) and ten wine yeast strains (S. cerevisiae var. ellipsoideus = S. ellipsoideus 1, 3, 4, 6, 8 and 9; S. oviformis 2, 5 and 7; and S. uvarum 10). The other 5 yeast strains belong to different species: Kloeckera apiculate, Candida mycoderma (Mycoderma vini), Pichia membranaefaciens, Rhodotorula glutinis and Torulopsis holmii, respectively.

The aim of this study was to investigate the familial clustering of nasopharyngeal carcinoma (NPC) in a non-endemic geographical region on the basis of two case reports and a review of the literature. Following an upper respiratory infection, NPC (WHO type III) was detected in a 57-year-old female (Case 1) who presented with nasal symptoms and a year later in her 36-year-old son (Case 2) who presented with enlarged lymph nodes. After a full diagnostic work-up, cT2a cN0 cM0 (stage IIA; Case 1) and cT2a cN2 cM0 (stage III; Case 2) disease were identified, and telecobalt irradiation was administered to both patients. The mother achieved complete remission and has been disease-free during a 14-year follow-up period. After initial complete remission, the son experienced regional (cervical) and base of the skull relapses within 2 years, which were treated unsuccessfully by means of radical neck dissection, a second course of radiotherapy and chemotherapy. Epstein-Barr virus (EBV) was detected in pathology sections from both patients. The authors review 20 additional well-documented cases of familial clustering of NPC in non-endemic geographical regions from the English language literature. This clinical entity typically has WHO type III histology; it may occur following an upper respiratory tract infection, and EBV-related serological titers were elevated in all 20 investigated cases. No consequent promoting factors were identified. The present two cases and the review of the literature strongly suggest that familial clustering of NPC in non-endemic geographical areas may be related to EBV infections. The difference in outcome of our two cases may be explained by the fact that the disease in Case 2 was diagnosed 1 year later than that in Case 1 and hence at a more advanced stage.

Aim: To report the role of liver angiography in the staging of medullary thyroid cancer (MTC) patients., Material and Methods: Sixty MTC patients with persistent or recurrent hypercalcitonemia (n=49), a characteristic general symptom (diarrhea, n=4) or a normal basal calcitonin level without general symptoms (n=7) were investigated by dynamic liver CT, MRI and angiography between 06/1998 and 06/2002., Results: Dual-phase CT and MRI investigations identified hepatic metastases with relatively low frequency (8/58 on MRI, and 7/60 on CT). Angiography indicated liver involvement in 54/60 cases. The hepatic metastases were typically multiple, hypervascular, small foci (only 13 foci measured >/=10 mm). With one exception significant disease progression was not observed over 5 years of follow-up., Conclusions: Liver angiography is a powerful tool to reveal hepatic metastases in MTC patients. Frequent, inoperable liver metastases in hypercalcitoninemic MTC patients demonstrate that secondary lymph node dissection is an inefficient technique for restoration of a normal calcitonin level.

Receptor tyrosine kinases play an important role in malignant transformation of epithelial cells by activating signal transduction pathways important for proliferation, invasion and metastasis. In a pilot study (n = 40), we evaluated expression of the c-Met and Her2/neu receptor tyrosine kinases and the c-Met ligand hepatocyte growth factor/scatter factor (HGF/SF) in primary breast cancers and their lymph node metastases using both conventional immunohistochemistry and confocal immunofluorescence. Neither c-Met and HGF/SF nor Her2/neu expression correlated with established prognostic factors such as age, lymph node involvement, estrogen receptor (ER), progesterone receptor (PR), tumor size, or grade. Both staining methods confirmed a significant correlation between c-Met overexpression and a high risk of disease progression. Furthermore, among tumors with c-Met overexpression, only 50% also overexpress Her2/neu, thus identifying a subset of patients with aggressive disease in addition to Her2/neu. Median disease-free survival in patients with c-Met overexpressing tumors was 8 months compared to 53 months when c-Met expression was low (p = 0.037; RR = 3.0). This significant impact of c-Met on tumor aggressiveness independent of Her2/neu was also confirmed by multivariate analysis. In conclusion, the role of c-Met expression as a prognostic variable and consequently as an interesting target for novel therapeutic approaches deserves further analysis in a larger cohort of patients.

A novel splice variant of Rac1, designated Rac1b, is expressed in human breast and colon carcinoma cells. Rac1b contains an additional 19 amino-acid insert immediately behind the switch II domain, a region important for Rac1 interaction with regulators and effectors. Recent studies showed that Rac1b exhibited the biochemical properties of a constitutively activated GTPase, yet it showed impaired interaction with downstream effectors, suggesting that Rac1b may be defective in biological activity. Whether Rac1b is a biologically active protein was not addressed. Therefore, we evaluated the biochemical, signaling and growth-promoting properties of authentic Rac1b. Similar to previous observations, we found that Rac1b showed enhanced intrinsic guanine nucleotide exchange activity, impaired intrinsic GTPase activity, and failed to interact with RhoGDI. Surprisingly, we found that Rac1b, like the constitutively-activated and transforming Rac1(Q61L) mutant, promoted growth transformation of NIH3T3 cells. Rac1b-expressing cells also showed a loss of density-dependent and anchorage-dependent growth. Surprisingly, unlike activated Rac1(61L), Rac1b did not show enhanced activation of the nuclear factor kappaB (NF-kappaB) transcription factor or stimulate cyclin D1 expression, the signaling activities that best correlate with Rac1 transforming activity. However, Rac1b did promote activation of the AKT serine/threonine kinase. Therefore, we suggest that Rac1b selectively activates a subset of Rac1 downstream signaling pathways to facilitate cellular transformation.

Ras-related C3 toxin substrate 1 (Rac1) is a small Rho-GTPase with important functions in fundamental cellular processes such as cytoskeleton rearrangements, signal transduction, cell cycle progression and malignant transformation. Using Rac1 primer, we identified a 5.5-kb DNA sequence on chromosome 4 (Chr. 4) in the human genome, containing the intronless protein coding sequence of Rac1. Sequence analysis revealed features of a processed pseudogene, which we named psi1Rac1, that could be detected by Southern blot and polymerase chain reaction (PCR) on genomic DNA. A psi1Rac1 pseudogene transcript was not detected by reverse transcription-polymerase chain reaction (RT-PCR), nor had the psi1Rac1 promoter any transcriptional activity. In addition, three other intronless pseudogenes of Rac1 on chromosomes 4, 13 and X were identified (psi1Rac1-psi4Rac1) sharing an 86-96% sequence similarity with Rac1. Neither RT-PCR with pseudogene specific restriction enzymes, nor the sequencing of 130 cDNA clones from benign and malignant breast tissue and cell lines, detected the transcription of any of the Rac1 pseudogenes (psi2Rac1-psi4Rac1). Existence of Rac1 pseudogenes should be taken into consideration when analyzing genomic alterations of the human Rac1 gene.

Positron emission tomographic (PET) investigations were performed to obtain in vivo information on symptomless radiation-induced pathological changes in the human spinal cord. PET investigations were carried out prior to radiotherapy and during the regular follow-up in an early hypopharyngeal cancer patient (the spinal cord was irradiated with a biologically effective dose of 80 Gy2), with [18F]fluorodeoxyglucose (FDG), [11C]methionine and [15O]butanol as tracers; radiosensitivity and electroneuronographic (ENG) studies were also performed. A very low background FDG accumulation (mean standardized uptake values, i.e. SUV: 0.84) was observed in the spinal cord before the initiation of radiotherapy. An increased FDG uptake was measured 2 months after the completion of radiotherapy (mean SUV: 1.69), followed by a fall-off, as measured 7 months later (mean SUV: 1.21). By 44 months after completion of irradiation, the FDG accumulation in the irradiated segments of the spinal cord had decreased to a level very close to the initial value (mean SUV: 1.11). The simultaneous [15O]butanol uptake results demonstrated a set of perfusion changes similar to those observed in connection with the FDG accumulation. The patient exhibited an extremely low [11C]methionine uptake within the irradiated and the nonirradiated spinal cord during the clinical course. She has not had any neurological symptoms, and the results of central ENG measurements before radiotherapy and 2 months following its completion proved normal. Radiobiological investigations did not reveal unequivocal signs of an increased radiosensitivity. A transitory increased spinal cord FDG uptake following radiotherapy may be related to the posttherapeutic mild inflammatory and regenerative processes. The normal [11C]methionine accumulation observed is strong evidence against intensive cell proliferation. The high degree of normalization of the temporarily increased FDG uptake of the irradiated spinal cord segments by 44 months is in good agreement with the results of monkey studies, which demonstrated a nearly complete recovery from radiation-induced spinal cord injury.

Purpose: To investigate the pathophysiology of the radiation-induced, chronic Lhermitte's sign (LS) on the basis of long-standing case histories with partial functional recovery., Patients and Methods: As radiotherapy in two nasopharyngeal cancer patients, a biologically effective dose (BED) of 103.8 Gy(2) (case 1) and 94.8 Gy(2) (case 2) was delivered to the cervical spinal cord. Neurologic signs relating to the irradiated spinal cord segments developed after 2 months (case 1) and 5 years (case 2), with radiation-induced damage equivalent to grade 3 (case 1) and grade 2 (case 2) toxicity (Common Toxicity Criteria, Version 2.0). The clinical status improved to grade 2 (case 1) and grade 1 (case 2). Positron emission tomography (PET) and fibroblast clonogen assay were applied 25 and 7 years postirradiation, respectively, to characterize this rare clinical picture., Results: PET demonstrated increased [(18)F]fluorodeoxyglucose (FDG) accumulation and [(15)O]butanol perfusion, but negligible [(11)C]methionine uptake in the irradiated spinal cord segments in both patients. In clonogenic assays, fibroblasts from case 1 displayed much higher radiation sensitivity than in healthy controls, while in case 2 the fibroblasts sensitivity was normal., Conclusions: These data suggests a close direct relationship between regional perfusion and metabolism of the spinal cord, similarly as in the brain. The postirradiation recovery may be related to energy-demanding conduction, explaining the increased metabolism and perfusion. The increased radiosensitivity and higher spinal cord BED may have contributed to the more severe sequelae in case 1.

Purpose: To study the efficacy of reirradiation as salvage treatment in patients with locally recurrent nasopharyngeal carcinoma., Patients and Methods: Between 1993 and 2000, 20 consecutive patients (twelve males and eight females) with nasopharyngeal cancer, previously irradiated in different Hungarian institutions, were reirradiated for biopsy-proven locally recurrent tumor. Histologically, 85% of the patients had WHO type III, 5% type II, and 10% type I disease. Stages I-IV (AJCC 1997 staging system) were assigned to five (25%), seven (35%), five (25%), and three (15%) patients, respectively; none of them had distant metastases, and only eight (40%) displayed regional dissemination. The median time period between termination of primary treatment and local recurrence was 30 (range, 10-204) months. Brachytherapy was the method most frequently used: in ten cases alone (especially for rT1 tumors), and in eight cases in combination with external beam therapy. Two patients with locally advanced disease underwent external beam therapy only. The median dose in the event of brachytherapy alone was 20 Gy (4 x 5 Gy or 5 x 4 Gy, range, 16-36 Gy), and the dose range for exclusive external irradiation was 30-40 Gy. In cases of combined irradiation, a median 20-Gy brachytherapy (range, 16-40 Gy) was associated with 30-40 Gy of external irradiation. Radiotherapy was supplemented by neck dissection (six patients), nasopharyngectomy (one patient), or chemotherapy (eleven patients)., Results: 16 patients were reirradiated once, three twice, and one patient three times, with a median equivalent dose for tumor effect of 36 Gy (mean, 44 Gy; range, 19-117 Gy; the estimated alpha/beta-ratio was 10 Gy). The median equivalent dose of reirradiation for late effect on normal tissue (with an estimated 70% delivery of the tumor dose) amounted to 30 Gy (mean, 37 Gy; range, 13-101 Gy, estimated alpha/beta-ratio 3 Gy). After a median follow-up of 37 (range, 12-72) months, the overall survival was 60% (12/20). Seven of the twelve surviving patients are currently tumor-free. After primary irradiation, xerostomy occurred in all patients as an unavoidable side effect of treatment. Following reirradiation, a severe (grade 3 or higher) late toxicity (CTC criteria, version 2) has been observed in two tumor-free patients (10%) so far (necrosis of soft palate and paresis of glossopharyngeal nerve)., Conclusion: Retreatment of nasopharyngeal carcinoma with radiotherapy (preferably a combined modality), can result in longterm local control and survival in a substantial proportion of patients, at the price of an acceptable morbidity.

Mucosal melanomas comprise about 1% of all malignant melanomas and exhibit far more aggressive behaviour than that of skin melanomas: they are more inclined to metastatize into regional and distant sites or recur locally, regionally or in distant locations, resulting in a high rate of cause-specific death. Mucosal melanomas in the head and neck region account for half of all mucosal melanomas, occurring mainly in the upper respiratory tract, oral cavity and pharynx. They appear with equal gender distribution and with a peak incidence in the age range 60-80 years. In consequence of their hidden location, they are usually diagnosed in a locoregionally advanced clinical stage, with a rate of 5-48% of regional and 4-14% of distant dissemination. The typical therapeutic approach is surgery, postoperative irradiation and systemic therapy. Local control with either surgery or radiotherapy is frequently (60- 70%) achieved, but the rates of local, regional and distant recurrences are high (50-90%, 20-60% and 30-70%, respectively). The reported 5-year actual survival rates are poor (17-48%), which is attributed mainly to a haematogenous dissemination. These characteristics demonstrate that identification of the precursor lesions and more effective local and systemic approaches are needed to improve the therapeutic results.

Radiation myelopathy is a rare, but extremely serious side-effect of radiotherapy. Recovery from radiation-induced motor sequelae is rare, whereas, the regeneration of sensory losses is relatively frequent. Among the sensory radiogenic injuries of the spinal cord, Lhermitte's sign (LS) is most frequent. This review describes the clinical picture and diagnostic imaging signs of radiogenic LS. There have been only a few studies on large patient groups with radiogenic LS, demonstrating a rate of occurrence of 3.6-13%, relating mainly to mantle irradiation or the radiotherapy of head and neck tumors. These cases typically manifest themselves 3 months following radiotherapy and gradually disappear within 6 months. Only 3 LS cases have been described in the English literature with extraordinarily severe symptoms lasting for more than 1 year. MRI, a sensitive tool in the detection of demyelination, failed to reveal any pathological sign accompanying radiogenic LS. However, positron emission tomography demonstrated increased [18F]fluorodeoxyglucose accumulation and [15O]butanol perfusion, but a negligible [11C]methionine uptake in the irradiated spinal cord segments in patients with long-standing LS. These imaging data are suggestive of a close direct relationship between the regional perfusion and metabolism of the spinal cord, very much like the situation in the brain. We postulate that an altered, energy-demanding conduction along the demyelinated axons of patients with chronic radiogenic LS may explain the increased metabolism and perfusion.

Objective: Authors analysed the prevalence of depressive symptomatology among the patients of a day time rehabilitation department for mobility disorders., Methods: Beck's Depression Inventory was used as a screening instrument. 57 subjects completed the inventory both at admission and discharge., Results: 33 subjects of 57 showed depressive symptoms at admission and 13 persons at discharge. The number of patients with mild depressive symptomatology decreased from 24 to 10, with medium level depression from 5 to 2 and with severe depression from 4 to 1. None of them have taken antidepressive drugs., Conclusions: These results confirm the high importance of the proper psychological conduction of patients with depressive symptoms.

Endothelial migration on extracellular matrix is regulated by integrins and proteolysis. Previous studies showed that beta(3)-integrins regulate expression of the urokinase-type plasminogen activator receptor (uPAR) through outside-in signalling involving the cytoplasmic domain. Here we show that overexpression of the integrin-binding protein beta(3)-endonexin decreased uPAR promoter (-398 base-pair fragment) activity that is constitutively active in endothelial cells. Mutation of the NF-kappaB promoter binding site (-45 bp) impaired the ability of beta(3)-endonexin to downregulate uPAR promoter activity. Immunoprecipitation studies showed that beta(3)-endonexin interacts directly with the p50/p65 transactivation complex and thereby inhibits binding of kappaB oligonucleotides to the p50/p65 complex. Moreover, binding of beta(3)-endonexin to p50 was inhibited in the presence of kappaB but not mutated kappaB oligonucleotides, suggesting a sterical competition between beta(3)-endonexin and kappaB DNA for the p50/p65 complex. We therefore propose that beta(3)-endonexin acts as regulator of uPAR expression in beta(3)-integrin-mediated endothelial cell migration through direct interaction with p50/p65. Since NF-kappaB regulates the expression of matrix degrading enzymes, the present results define a role of beta(3)-endonexin in regulating beta(3)-integrin-mediated adhesion and pericellular proteolysis.

Patients and Methods: In the period between 1993 and 2000, 20 patients with nasopharyngael cancer were re-irradiated for locally recurrent carcinomas. The median duration between primary treatment and recurrence was 30 months. Brachytherapy was the method most frequently used in 10 cases alone, and in 8 cases in combination with external beam therapy. 2 patients underwent only external beam therapy. The external irradiation was performed with CT/MRI-based treatment planning. Brachytherapy involved a high dose rate afterloading method. The cumulative dose of re-irradiation was 20-60 Gy., Results: After a median follow-up of 37 months the overall survival and the local control were 60% (12/20) and 58% (7/12), respectively. 7 of the 12 surviving patients are currently tumour-free. After primary irradiation xerostomy occurred in all patients as an unavoidable side-effect of the treatment. A severe (grade 3 or higher according to the Common Toxicity Criteria version 2.0) late-side effect has so far been observed in 2 cases (10%). Authors' results lie in the medial range of the data in the international literature, though the rate of radiogen side-effects in the patient group is low. Both results are assumed to be a consequence of the re-irradiation dose being lower than the value considered optimum in the recent literature., Conclusions: Re-irradiation of locally recurrent nasopharyngeal cancers is an efficient treatment modality, which should be used as a combination of external beam therapy and brachytherapy. The optimum cumulative dose is about 50-60 Gy. This dose results in a 5 years survival rate of about 40% with an acceptable (30%) risk. The results of re-irradiation may be improved, if PET is used to determine the extent of the surviving tumour tissue. This can help in the choice of the proper treatment modality.

Objective: To investigate the pathomechanism of the rare radiogenic lower motor neurone disease (LMND) on the basis of a case history involving a partial functional recovery., Patient: A 31-year-old seminoma patient received postoperative para-aortic and para-iliac telecobalt irradiation with a biologically effective dose of 88 Gy(2) (44 Gy in 2 Gy fractions/day, with an estimated alpha/beta of 2 Gy) delivered to the spinal cord following a single cycle of chemotherapy. LMND developed 4 months after the completion of radiotherapy. The patient exhibited flaccid paraparesis of the lower extremities (without sensory or vegetative signs), followed by a worsening after further chemotherapy, due to pulmonary metastatization. A gradual spontaneous functional improvement commenced and led several years later to a stabilized state involving moderately severe symptoms., Methods: In the 15th year of the clinical course, magnetic resonance imaging (MRI) and positron emission tomography (PET) with [(18)F]fluorodeoxyglucose (FDG) and [(11)C] methionine were conducted. Four lines of experiments (clonogenic assay using fibroblasts isolated from a skin biopsy sample of the patient, comet assay, micronucleus assay, and the testing of chromosome aberrations after in vitro irradiation of peripheral blood samples) were performed in a search for an increased individual radiosensitivity., Results: MRI investigations failed to reveal any pathological change. PET demonstrated an increased FDG accumulation, but a negligible [(11)C] methionine uptake in the irradiated spinal cord segments. The radiobiological investigations did not indicate any sign of an increased individual radiosensitivity., Conclusions: We suggest that the observed partial functional recovery and stabilization of the symptoms of radiogenic LMND may be explained by the higher than normal density of sodium channels expressed along the demyelinated axons of the restored conduction. The increased energy demands of this type of conduction are proved by a higher metabolic rate (increased FDG uptake) of the irradiated spinal cord segments without a substantial regenerative process (lack of detectable protein synthesis).

The authors have reviewed the financial considerations of oncological FDG PET examinations by the guidelines of the Health Care Financing Administration (USA). By critical assessment of large number of clinical investigations, the cost-effectiveness of FDG PET scans has been confirmed in the following cases: differential diagnosis of solitary pulmonary nodule, diagnosis, staging and restaging of non-small cell lung cancer, colorectal cancer, malignant lymphomas, melanoma malignum, esophageal neoplasms and cancers of the head and neck. The role of this method in breast cancer is currently under intensive investigation. Due to the correct staging, PET examinations in these indications enable the clinicians to choose the optimal treatment ensuring the maximum probability of recovery and being cost-effective as unnecessary medical interventions become avoidable.

Following the failure of conventional diagnostic procedures, whole-body FDG-PET investigations were carried out in 42 metastatic cancer patients to localize occult primary carcinomas. During the clinical follow-up, the presence of malignant tumor was ruled out in 3 cases, and 2 patients originally believed to have carcinoma were confirmed to be suffering from a malignant hematological disease. These false diagnoses were associated with the use of imaging methods only (2 cases) or cytology only (1 case), lack of double, independent pathological investigations (2 cases) or immunophenotyping (2 cases) and the occurrence of an unrecognized rare tumor in a hospital with a small patient turnover (1 case). The discovered 11 occult primaries (4 lung, 3 breast, 2 hypopharynx and 1 base of the tongue carcinomas and 1 non-Hodgkin lymphoma) led to a 28% diagnostic efficacy of PET (11/39 malignant pathological reports). This efficacy is doubled (60%) if PET reveals < or = 5 malignant lesions and the locations of the pathological foci are tumor-specific. We suggest PET investigations in the search for occult primaries following a controlled pathological diagnosis and the failure of conventional diagnostic procedures.

Whole-body FDG PET examinations in 10 cases of epipharyngeal tumour (8 males, 2 females, mean age: 48 years) have been performed since January 1999. The PET examinations were aimed at the accurate staging, follow-up of the patients after the treatment, identification of recurrencies and localization of the unknown primary tumor. Functional imaging resulted in "upstaging" in 3 patients as compared to staging by the conventional diagnostic tools. Four additional patients with hyperplastic epipharyngeal tissue were investigated for occult primary cancer after negative results of multiple excisions, resulting in one case of primary epipharyngeal cancer. Correct staging, early detection of recurrencies, localization of occult primary tumor and the better post-therapeutic assessment of epipharyngeal masses all facilitate a more reasonable therapeutic approach, which may improve survival results.

The authors have reviewed the financial considerations of oncological FDG PET examinations by the guidelines of the Health Care Financing Administration (USA). By critical assessment of large number of clinical investigations,the cost-effectiveness of FDG PET scans has been confirmed in the following cases: differential diagnosis of solitary pulmonary nodule, diagnosis,staging and restaging of non-small cell lung cancer, colorectal cancer, malignant lymphomas, melanoma malignum, esophageal neoplasms and cancers of the head and neck. The role of this method in breast cancer is currently under intensive investigation. Due to the correct staging, PET examinations in these indications enable the clinicians to choose the optimal treatment ensuring the maximum probability of recovery and being cost-effective as unnecessary medical interventions become avoidable.