International consensus guidelines for scoring the histopathological growth patterns of liver metastasis.

BACKGROUND: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs. METHODS: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined. RESULTS: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006). CONCLUSIONS: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

Microvessel density and endothelial cell proliferation levels in colorectal liver metastases from patients given neo-adjuvant cytotoxic chemotherapy and bevacizumab.

The treatment of patients with colorectal liver metastasis has improved significantly and first line therapy is often combined chemotherapy and bevacizumab, although it is unknown who responds to this regimen. Colorectal liver metastases grow in different histological growth patterns showing differences in angiogenesis. To identify possible response markers, histological markers of angiogenesis were assessed. Patients who underwent resection of colorectal liver metastasis at Rigshospitalet, Copenhagen, Denmark from 2007 to 2011 were included (n = 254) including untreated and patients treated with chemotherapy or chemotherapy plus bevacizumab. The resected liver metastases were characterised with respect to growth pattern, endothelial and tumour cell proliferation as well as microvessel density and tumour regression. Tumour regression grade of liver metastases differed significantly between untreated/chemotherapy treated patients in comparison to chemotherapy plus bevacizumab treated patients (both p < 0.0001). Microvessel density was decreased in liver metastases from patients treated with bevacizumab in comparison to those from untreated/chemotherapy-treated patients (p = 0.006/p = 0.002). Tumour cell proliferation assessed by Ki67 expression correlated to a shorter recurrence free survival in the total patient cohort. In conclusion, liver metastases from patients treated with neo-adjuvant chemotherapy and bevacizumab had significantly lower microvessel densities and tumour regression grades when compared to liver metastases from untreated or chemotherapy treated patients. This may indicate that bevacizumab treatment results in altered vascular biology and tumour viability, with possible tumour reducing effect.

Tissue inhibitor of matrix metalloproteinase-1 expression in colorectal cancer liver metastases is associated with vascular structures.

Metastatic growth by colorectal cancer cells in the liver requires the ability of the cancer cells to interact with the new microenvironment. This interaction results in three histological growth patterns of liver metastases: desmoplastic, pushing, and replacement. In primary colorectal cancer several proteases, involved in the degradation of extracellular matrix components, are up-regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a strong prognostic marker in plasma from colorectal cancer patients, with significant higher levels in patients with metastatic disease. We therefore wanted to determine the expression pattern of TIMP-1 in primary colorectal cancers and their matching liver metastases. TIMP-1 mRNA was primarily seen in α-smooth-muscle actin (α-SMA)-positive cells. In all primary tumors and liver metastases with desmoplastic growth pattern, TIMP-1 mRNA was primarily found in α-SMA-positive myofibroblasts located at the invasive front. Some α-SMA-positive cells with TIMP-1 mRNA were located adjacent to CD34-positive endothelial cells, identifying them as pericytes. This indicates that TIMP-1 in primary tumors and liver metastases with desmoplastic growth pattern has dual functions; being an MMP-inhibitor at the cancer periphery and involved in tumor-induced angiogenesis in the pericytes. In the liver metastases with pushing or replacement growth patterns, TIMP-1 was primarily expressed by activated hepatic stellate cells at the metastasis/liver parenchyma interface. These cells were located adjacent to CD34-positive endothelial cells, suggesting a function in tumor-induced angiogenesis. We therefore conclude that TIMP-1 expression is growth pattern dependent in colorectal cancer liver metastases.

Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes.

BACKGROUND: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. METHODS: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). RESULTS: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. CONCLUSIONS: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.

Plasmin-driven fibrinolysis facilitates skin tumor growth in a gender-dependent manner.

Rearrangement of the skin during wound healing depends on plasmin and plasminogen, which serve to degrade fibrin depositions in the provisional matrix and thereby facilitate keratinocyte migration. In the current study, we investigated whether plasmin and plasminogen likewise played a role during the development of skin cancer. To test this, we set up a chemically induced skin tumor model in a cohort of mice and found that skin tumor growth in Plg(-/-) male mice was reduced by 52% compared with wild-type controls. Histological analyses suggested that the growth-restricting effect of plasminogen deficiency was due to thrombosis and lost patency of the tumor vasculature, resulting in tumor necrosis. The connection between plasmin-dependent fibrinolysis, vascular patency, and tumor growth was further substantiated as the effect of plasminogen deficiency on tumor growth could be reverted by superimposing heterozygous fibrinogen deficiency on Plg(-/-) mice. Tumors derived from these Fib(-/+);Plg(-/-) mice displayed a significantly decreased level of tumor thrombosis compared with Plg(-/-) mice. In summary, these data indicate that plasmin-driven fibrinolysis facilitates tumor growth by maintaining patency of the tumor vasculature.

Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma.

The protein C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor, is a potential new biomarker in non-small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.

Prognosis in adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia evaluated by uPAR-immunohistochemistry.

Adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia in humans are highly invasive tumours with poor prognosis. The localisation of urokinase-type plasminogen activator receptor (uPAR) was determined in 66 patients; 60 with adenocarcinomas and six cases with Barrett's oesophagus. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts at both the invasion front and the tumour core. In areas with high-grade dysplasia or with Barrett's metaplasia adjacent to the tumour tissue, no uPAR-immunoreactivity was found. High local expression of uPAR, therefore, appears to be a characteristic marker for invasive behaviour in this tumour, suggesting that uPAR's contribution to matrix degradation during invasive growth is a late event in carcinogenesis. Using a scoring system for semiquantitative estimation of uPAR-positivity on immmunohistochemically stained specimens, a significant association was found between poor overall survival and high uPAR-score for cancer cells in the tumour core and for macrophages peripherally at the tumour invasion zone. In multivariate analysis, these two uPAR-scores were confirmed as highly significant prognostic parameters independent of Tumour, Node, Metastasis (TNM)-stage and World Health Organization (WHO) classification. The proteolytic action of these malignant and nonmalignant accessory cells thus seemed to follow two main patterns: one dominated by uPAR positive cancer cells and one by uPAR-positive macrophages. Scoring of uPAR-positivity might be a useful parameter for onset of invasion and prognosis in these adenocarcinomas.

Urokinase plasminogen activator receptor on invasive cancer cells: a prognostic factor in distal gastric adenocarcinoma.

Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. uPAR was expressed by neoplastic cells, macrophages, myofibroblasts and neutrophils in both intestinal and diffuse subtypes. No association was demonstrated between the expression of uPAR on cancer cells and histological subtype (p = 0.64) or TNM stage (p = 0.75). Univariate analysis revealed a significant association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor for overall survival in gastric cancer (HR = 2.39; 95% CI: 1.22-4.69; p = 0.011). In consequence, scoring of uPAR-positive cancer cells may be a direct measure for the invasive potential of gastric adenocarcinomas.

Activation of Notch signaling in cardiomyocytes during post-infarction remodeling.

OBJECTIVE: Notch signaling is crucial for cell-to-cell interaction during cardiovascular development and may influence differentiation, proliferation, and apoptotic events. We investigated whether Notch signaling is activated during myocardial remodeling in heart failure (HF). DESIGN: Myocardial gene expression and localization of Notch receptors (Notch1-4) and ligands (Jagged1-2, and Delta-like (Dll)-1 and 4) were investigated in rats with HF after induction of myocardial infarction and in humans with HF. RESULTS: All Notch receptors and ligands investigated and Notch intracellular domain (NICD) were present in rat and human myocardial tissue and in cardiomyocytes with differences in their relative expression levels and altered expression levels in failing vs. non-failing myocardium. In isolated rat cardiomyocytes, Notch3 and Notch4 appeared to be the major Notch receptors, and Notch3 and Notch4 mRNA levels and NICD-3 and -4 in cardiomyocytes were upregulated in chronic HF (p < 0.05), indicating increased Notch3 and Notch4 signaling. CONCLUSION: The Notch signaling system is present in the cardiomyocytes and activated in HF, indicating a role of Notch signaling during myocardial remodeling in HF.

Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model.

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm(3) in Galardin-treated mice compared with 0.56 mm(3) in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application.

Circadian variations in clock gene expression of human bone marrow CD34+ cells.

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow.

OBJECTIVE: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver. METHODS: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction. RESULTS: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver. CONCLUSIONS: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.

HPV subtypes in cervical cancer biopsies between 1930 and 2004: detection using general primer pair PCR and sequencing.

Our objective was to investigate the practicability of sequencing DNA from formalin fixed, paraffin embedded tissue stored for up to 75 years and to study human papillomavirus subtype distribution in cervical neoplasias between 1931 and 2004. Three protocols for DNA retrieval were tested, and magnetic bead DNA extraction proved advantageous, as it gave superior specimen purity and effortless sequencing. Successful sequencing was achieved in more than 70% of the specimens from 1931 to 1960. This technique was utilized in the study of papillomavirus subtypes using general primer pair PCR with sequencing of the products in a series of 97 cases of neoplastic and non-neoplastic cervical specimens from 1931 to 1960 and 73 similar cases from 1992 to 2004. HPV was detected in 61% of neoplastic specimens from 1931 to 1960, and in 89% of those from 1992 to 2004. In specimens from 1931 to 1934, only HPV type 16 was detected, whereas in the specimens from 1940 and up, other HPV subtypes were identified in one-third of the cases. The difference was significant and suggests an increase in papillomavirus subtype heterogeneity in Western Norway during 1930-2000. The results strongly support the feasibility of using DNA from paraffin-embedded specimens for studying cancer etiology and genotypes over extended time periods.

Clock gene expression in purified mouse hematopoietic stem cells.

OBJECTIVE: Circadian genes have recently been characterized in many tissues, but not in hematopoietic stem cells. These cells are rare in the bone marrow (BM), which makes it difficult to collect enough cells for detailed molecular analysis in a short period of time without reduced RNA quality. The aim was to improve methodology and reliability of clock gene expression analysis in purified mouse hematopoietic stem cells. METHODS: Stem cells were highly enriched by high-speed flow cytometric cell sorting of the side population (SP) cells from Hoechst 33342 (Hoechst)-stained mouse BM. Total RNA was isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative mRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb alpha were measured with real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay. RESULTS: Clock gene activity in sorted SP cells showed pronounced relative differences compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not influence clock gene expression or cell clonogenity, even when this was performed with a delay period up to 24 hours. CONCLUSIONS: We demonstrated expression of six clock genes in mouse hematopoietic stem cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be useful and reliable for analysis of clock gene activity in small stem cell fractions.

Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma.

The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer.

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

Tumour spread in serosal cavities: what have we learned? Montebello Conference--PSC, Lillehammer, Norway, June 18-22, 2004.

During the Montebello Conference on malignant serosal tumours at Lillehammer, Norway, in June 2004, a group of 30 international experts addressed the biologic and genetic aspects of malignant tumours affecting serosal cavities in the human body. Three neoplasms were mainly dealt with: mesotheliomas arising locally, ovarian carcinomas developing in close proximity to the serosa, and breast tumours in which the spread came from some distance. New, important data on the tumour microenvironment and the process of carcinogenesis with progression and acquisition of invasive properties shed new lights on the mechanisms, including proliferative properties, alterations of signal transduction pathways, and tissue remodelling by proteolytic enzymes in the metastasizing cells. Several of these markers have considerable diagnostic and clinical interest. In addition, new aspects of morphologic and immunocytochemical characteristics of the cells as well as genetic markers may soon become powerful tools for practical use. The molecular fingerprint of the individual tumours may also give guidelines for chemotherapy as well as biologic therapies, including induction of apoptosis. The easy accessibility of tumours from serosal fluids and possibilities for specific discrimination of the neoplastic cells from admixed leukocytes and other cells are promising avenues for cytodiagnostics.

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder--Possible Clinical Implications.

The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling during cancer invasion and metastasis and is an established prognostic marker in various cancer diseases other than bladder cancer. Formalin-fixed and paraffin-embedded tumour-tissue blocks from 186 patients treated with radical cystectomy were analysed. uPAR expression was scored as either negative or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated by Immunohistochemistry as well as a significant association between uPAR positivity and increasing tumour stage and tumour grade. This demonstrates the robustness of our previous and current findings. In addition the association between uPAR positive myofibroblasts and poor survival was reproduced. The highest hazard ratios for survival were seen for uPAR positive myofibroblasts both at the invasive front and in tumour core. Evaluating uPAR expression by the actual score showed a significant association between uPAR positive myofibroblasts in tumour core and an increased risk of cancer specific mortality. Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system.