Tumor markers: from laboratory to clinical utility.
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Antigens, Neoplasm↗
Biomedical subjects
Manfred Schmitt
Publications and source records attributed to Manfred Schmitt.
Human kallikrein 8, a novel biomarker for ovarian carcinoma.
Human kallikrein 8 (hK8; neuropsin) is a serine protease and new member of the hK family. The aim of this study was to examine if hK8 may serve as a novel cancer biomarker. An hk8-ELISA, developed in-house, was used to study the distribution of hK8 in various biological fluids and tissue extracts from healthy individuals and ovarian cancer patients of different stages of the disease (International Federation of Obstetrics and Gynecology II-IV). For ovarian cancer patients, very high levels in ascites fluid were observed (<or=1000 microg/liter; n = 85 samples). Elevated serum levels were seen in 24 of 40 (62%) of ovarian cancer patients. Higher ascites fluid hK8 concentration was associated with better ovarian cancer progression-free survival (P = 0.02). In both serum and ascites fluid, there is a significant correlation between hK8 and CA125 concentration (r = 0.51 and 0.58, respectively). The serum concentration of hK8 was an indicator of progression on regression on longitudinal monitoring of an ovarian cancer patient. These data suggest that hK8 protein is detectable in ovarian cancer tissue extracts, serum, and ascites fluid, indicating that it may serve as a new ovarian cancer marker.
Ascitic Fluid↗
Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer.
PURPOSE: The independent clinical relevance of invasion factors urokinase-type plasminogen activator (uPA)/PAI-1 and HER2 status was evaluated in lymph node-negative breast cancer patients (N = 118) without adjuvant systemic therapy after long-term follow-up of more than 10 years (median, 126 months). PATIENTS AND METHODS: Levels of uPA and its inhibitor PAI-1 were prospectively measured by enzyme-linked immunosorbent assay in primary tumor tissue extracts. HER2 gene amplification (HER2_AMP) was evaluated by fluorescence in situ hybridization (FISH; Ventana Medical Systems HER-2/neu probe; Tucson, AZ), and HER2 protein overexpression (HER2_EXP) was evaluated by immunohistochemistry (IHC; Oncogene Science antibody Ab-3; Cambridge, MA) on parallel-cut formalin-fixed paraffin-embedded tissue sections. RESULTS: uPA/PAI-1 was high (either one or both factors were high) in 44% of the tumors. HER2_AMP was detected by FISH in 33% of the patients, and HER2_EXP was found by IHC in 44% of the patients. In a multivariate analysis of established and tumor-biologic prognostic factors, uPA/PAI-1 was the only independent prognostic factor for disease-free survival ([DFS]; P <.001; relative risk [RR], 8.3; 95% confidence interval [CI], 3.4 to 20.4). Although HER2_AMP and HER2_EXP did not reach significance for DFS, they were significant for overall survival (OS), even in multivariate analysis (HER2_AMP: P =.004; RR, 3.7; 95% CI, 1.5 to 9.2; HER2_EXP: P =.009; RR, 3.4; 95% CI, 1.4 to 8.7). CONCLUSION: After long-term follow-up, uPA/PAI-1 levels in primary tumor tissue reliably and strongly indicate an aggressive course of disease in lymph node-negative breast cancer independent of HER2 status. The particular prognostic effect of HER2 status on OS may reflect its ability to predict resistance to systemic therapy.
Adult↗
Detailed analysis of five mutations in dihydropyrimidine dehydrogenase detected in cancer patients with 5-fluorouracil-related side effects.
Complete or partial loss of dihydropyrimidine dehydrogenase (DPD or DYPD) function has been described in cancer patients experiencing severe side effects upon administration of the fluoropyrimidine anticancer drug 5-fluorouracil (5-FU). To investigate a genetic predisposition for 5-FU intolerance due to inherited DPD defects, we established a mutation detection assay based on denaturing HPLC. Analyzing four individuals with symptoms of 5-FU-related toxicity, we detected six distinct sequence variants in the dihydropyrimidine dehydrogenase gene (DPYD): one novel mutation, c.775A>G (K259E); four known missense mutations, c.85T>C (C29R), c.496A>G (M166V), c.1601G>A (S534N), c.1627A>G (I543V); and one silent mutation c.1896T>C affecting the codon for F632. One cancer patient possessing a total of four gene mutations resulting in four amino acid substitutions (C29R, M166V, S534N, I543V) displayed significantly reduced DPD activity. The rare combination of the highly conserved mutation sites M166V and S534N was additionally found in one of the other patients. DPD enzyme activity was low, but yet within normal range. The K259E mutation did not provoke a decrease in DPD function in a heterozygous individual. Based on the protein structure of crystalline pig DPD and the deduced homology models, we have additionally investigated the amino acid positions in their three-dimensional network which correspond to the five missense mutations discovered in the patients.
Antimetabolites, Antineoplastic↗
The urokinase receptor (uPAR, CD87) as a target for tumor therapy: uPA-silica particles (SP-uPA) as a new tool for assessing synthetic peptides to interfere with uPA/uPA-receptor interaction.
Many different processes in the physiology and pathophysiology of human beings are regulated protein/protein interactions such as receptor/ligand interactions. A more detailed knowledge of the nature of receptor/ligand binding sites and mechanisms of interaction is necessary as well in order to understand the process of cancer spread and metastasis. For instance, the cell surface receptor uPAR (CD87) and its ligand, the serine protease urokinase-type plasminogen activator (uPA), facilitate tumor invasion and metastasis in solid malignant tumors. Besides its proteolytic function in activating the zymogen plasminogen into the serine protease plasmin, binding of uPA to tumor cell-associated uPAR initiates various cell responses such as tumor cell migration, adhesion, proliferation, and differentiation. Hence, the tumor-associated uPA/uPAR system is considered a potential target for cancer therapy. Here we briefly describe a new technology using micro-silica particles coated with uPA (yields SP-uPA) and reaction of SP-uPA with recombinant soluble uPAR (suPAR) to test the competitive antagonistic potential of synthetic uPA peptides by flow cytofluorometry (FACS). We discuss the data obtained with the SP-uPA system from two different points of view: (1) The enhanced potential of improved uPA-derived synthetic peptides compared to previously described peptides, and (2) comparison of the new technique to other test systems currently used to identify uPA/uPAR or other protein/protein interactions.
Animals↗
Inhibition of the tumor-associated urokinase-type plasminogen activation system: effects of high-level synthesis of soluble urokinase receptor in ovarian and breast cancer cells in vitro and in vivo.
Tumor cell invasion and metastasis depend on the coordinated and temporal expression of proteolytic enzymes to degrade the surrounding extracellular matrix and of adhesion molecules to remodel cell-cell and/or cell-matrix attachments. The tumor cell-associated urokinase-type plasminogen activator system, consisting of the serine protease uPA, its substrate plasminogen, its membrane-bound receptor uPAR, as well as its inhibitors PAI-1 and PAI-2, plays an important role in these pericellular processes. Especially, association of the proteolytic activity of uPA with the cell surface via interaction with uPAR significantly increases the invasive capacity of tumor cells. Consequently, various approaches have been pursued to interfere with the expression or activity of uPA and/or uPAR, including antisense strategies and the development of active-site inhibitors of uPA or inhibitors of uPA/uPAR interaction. In this review, we focus on the results obtained in vitro and in vivo with tumor cells producing high levels of a recombinant soluble form of uPAR, which efficiently inhibits uPA binding to cell surface-associated uPAR and, by this, acts as a scavenger for uPA.
Animals↗
Minimal residual disease in breast cancer and gynecological malignancies: phenotype and clinical relevance.
In breast cancer, about 35% of patients without any clinical signs of overt distant metastases already have disseminated tumor cells in bone marrow aspirates at the time of primary therapy. A significant prognostic impact of these disseminated tumor cells has been shown by many international studies: patients with tumor cells in their bone marrow have a significantly worse prognosis than those without them. Even in malignancies where the skeletal system is not a preferred location for distant metastasis, such as ovarian cancer, early presence of minimal residual disease (MRD) is correlated with poor patient outcome. Thus, besides analysis of the primary tumor, detection of MRD can be used for assessment of patient prognosis and for prediction or monitoring of response to systemic therapy. Disseminated tumor cells are also the targets for novel tumor biological therapy approaches such as specific antibody-based therapies against target cell-surface antigens such as HER2, Ep-CAM (17-1A), and uPA-R. In breast cancer, a first antibody-based tumor therapy against HER2 (Herceptin) has already been approved for clinical use in recurrent disease. However, patient selection for such tumor biological therapies becomes rather difficult due to phenotype changes, which may manifest themselves as differences between primary lesion and disseminated tumor cells. Therefore, not only identification of disseminated tumor cells but even more so their characterization at the protein and gene levels have become increasingly important. In conclusion, characterization of tumor biological properties of disseminated tumor cells allows identification of patients with breast cancer or gynecological malignancies at risk for relapse who are likely to benefit from systemic treatment and/or novel tumor biological therapy approaches.
Breast Neoplasms↗
Advanced statistical methods for the definition of new staging models.
Adequate staging procedures are the prerequisite for individualized therapy concepts in cancer, particularly in the adjuvant setting. Molecular staging markers tend to characterize specific, fundamental disease processes to a greater extent than conventional staging markers. At the biological level, the course of the disease will almost certainly involve interactions between multiple underlying processes. Since new therapeutic strategies tend to target specific processes as well, their impact will also involve interactions. Hence, assessment of the prognostic impact of new markers and their utilization for prediction of response to therapy will require increasingly sophisticated statistical tools that are capable of detecting and modeling complicated interactions. Because they are designed to model arbitrary interactions, neural networks offer a promising approach to improved staging. However, the typical clinical data environment poses severe challenges to high-performance survival modeling using neural nets, particularly the key problem of maintaining good generalization. Nonetheless, it turns out that by using newly developed methods to minimize unnecessary complexity in the neural network representation of disease course, it is possible to obtain models with high predictive performance. This performance has been validated on both simulated and real patient data sets. There are important applications for design of studies involving targeted therapy concepts and for identification of the improvement in decision support resulting from new staging markers. In this article, advantages of advanced statistical methods such as neural networks for definition of new staging models will be illustrated using breast cancer as an example.
Biomarkers, Tumor↗
Pooled analysis of prognostic impact of uPA and PAI-1 in breast cancer patients.
In this report we present an extension of the pooled analysis of the prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I in breast cancer patients. We analyzed a different endpoint, metastasis-free survival (MFS). We checked the consistency of the estimates for uPA and PAI-1 for relapse-free survival (RFS) and MFS exploring possible sources of heterogeneity. Nodal status, the most important prognostic factor for breast cancer, introduced heterogeneity in the uPA/PAI-1 survival analyses, reflecting the interaction between nodal status and uPA/PAI-1. The estimates for uPA and PAI-1 were found to be consistent, even when a different transformation of their values was used. The heterogeneity of the separate data sets decreased if the levels of uPA and PAI-1 were ranked, data sets were pooled, and the analyses corrected for the base model that included all traditional prognostic factors, and stratified by data set. We conclude that uPA and PAI-1 are ready to be used in the clinic to help classify breast cancer patients into high and low risk groups.
Breast Neoplasms↗
Synergistic person x situation interaction in distributive justice behavior.
A person x situation interaction is synergistic when a personality trait amplifies the effect of a situational factor. The present study tested how individuals' justice attitudes and situational factors jointly affect the allocation of financial burdens. Six insurance cases were described to 80 participants. Economic status of client (high, low) and responsibility of client for damage of the insured (high, low) were manipulated between subjects. Participants suggested a percentage of the total costs that they considered a fair contribution by the client. In accordance with the synergistic model, justice attitude (person factor) and responsibility for damage (situation factor) interacted and explained 5% of the variance of the dependent variable. With increasing negativity of attitude toward equality, the effect of responsibility was larger. Several cognitive mechanisms, such as motivated perception, selective attention, and the availability of attitude congruent situation schemas, that may account for synergistic interactions in justice behavior and in other domains were discussed.
Attitude↗
Ovarian cancer cell proliferation and motility is induced by engagement of integrin alpha(v)beta3/Vitronectin interaction.
During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin alpha(v)beta3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized alpha(v)beta3 directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of alpha(v)beta3 and was accompanied by rapid and transient tyrosine-phosphorylation of p44(erk-1)/p42(erk-2) mitogen-activated protein kinase. Moreover, overexpression of alpha(v)beta3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon alpha(v)beta3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-alpha (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of human ovarian cancer cells.
Cell Adhesion↗
Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems.
Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.
Cell Line, Tumor↗
Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems.
Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.
Animals↗
Molecular and functional interdependence of the urokinase-type plasminogen activator system with integrins.
The serine protease urokinase-type plasminogen activator (uPA), its inhibitor PAI-1, and its cellular receptor uPA-R (CD87) are of crucial importance during cellular invasion and migration, required for a variety of physio- and pathophysiological processes. It has become increasingly evident in recent years that the uPA/uPA-R-system has far more functional properties than plasminogen activation alone. This is reflected by its involvement in cellular events such as proliferation, adhesion, migration, and chemotaxis. Since uPA-R lacks a transmembrane domain and thus on its own is not capable of transmitting signals into cells, association and functional cooperation with other signaling molecules/receptors is needed. In this respect, one group of adhesion and signaling receptors, the integrins, have been identified which constitute, together with the uPA/uPA-R-system, an interdependent biological network by which the uPA/uPA-R-system broadly affects integrin functions and vice versa. Moreover, there is a growing body of evidence that cellular uPA, uPA-R, and PAI-1 expression is under control of specific ECM/integrin interactions and also that integrins are regulated by components of the uPA/uPA-R-system. By this multifaceted crosstalk, cells may modulate their proteolytic, adhesive, and migratory activities and monitor ECM integrity in their microenvironment.
Animals↗
Clinical relevance of matrix metalloproteinase-13 determined with a new highly specific and sensitive ELISA in ascitic fluid of advanced ovarian carcinoma patients.
Matrix metalloproteinases (MMPs) are involved in many physiological and pathophysiological processes, including tumor cell invasion and metastasis. For one member of this family, MMP-13 (collagenase-3), a new, highly specific ELISA with a sensitivity of 0.5 ng MMP-13/ml was established. The protein levels of MMP-13 in ascitic fluids of 30 patients with advanced ovarian cancer FIGO stage III (n = 19) and IV (n = 11) were measured with this ELISA. Using a cut-off value of 0.5 ng MMP-13/mg total protein, two patient subpopulations with short (median 16 months) and long (median 36 months) overall survival were identified. Together with other prognostic markers, determination of MMP-13 in ascitic fluid may help to identify patients at risk for early death and help to individualize adjuvant therapy.
Adult↗
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR): development of antagonists of uPA/uPAR interaction and their effects in vitro and in vivo.
In cancer, increased levels of the tumor-associated serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are linked to tumor progression, metastasis, and shortened survival in patients afflicted with this disease. Strong clinical and experimental evidence has accumulated that the cell surface interaction of uPA with uPAR facilitates extravasation and intravasation of cancer cells by regulating local proteolysis and attachment of the cells to components of the extracellular matrix. Moreover, the uPA/uPAR system is also implicated in proliferation of some tumor cells and migration of tumor and endothelial cells. Thus, metastasis formation is facilitated via tumor cell spread through the blood circulation system and neovascularization at the metastatic site. This multifunctional potential has rendered the uPA/uPAR system an attractive novel target for anti-metastatic therapy. Consequently, inhibitors of the uPA/uPAR interaction have been and are currently developed for suppression of tumor growth and angiogenesis. In addition to antibodies and recombinant uPA- or uPAR-derived proteins, various linear and cyclic peptides as well as small molecules have been designed and synthesized which potently interfere with the uPA/uPAR interaction, leading to reduced tumor progression in experimental animals. Such compounds affecting the uPA/uPAR system represent novel tumor biology-based therapeutic agents, thereby opening new ways for patient optimized and individualized cancer therapy.
Animals↗
Theme Issue: update on the role of the fibrinolysis/plasminogen activation system in a cellular context.
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Animals↗
Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer.
The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.
Adult↗