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Originally published online as doi:10.2353/ajpath.2009.080785 on May 12, 2009

Published online before print May 12, 2009
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(American Journal of Pathology. 2009;174:2246-2253.)
© 2009 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.080785

Amplification of the Urokinase-Type Plasminogen Activator Receptor (uPAR) Gene in Ductal Pancreatic Carcinomas Identifies a Clinically High-Risk Group

Ralf Hildenbrand*{dagger}, Marco Niedergethmann{ddagger}, Alexander Marx{dagger}, Djeda Belharazem{dagger}, Heike Allgayer§, Christiane Schleger{dagger} and Philipp Ströbel{dagger}

From the Institute of Pathology,* Bonn; the Department of Surgery,{dagger} Institute of Pathology,{ddagger} and the Department of Experimental Surgery and Molecular Oncology of Solid Tumors,§ Medical Faculty Mannheim, University Heidelberg and German Cancer Research Center, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are known to be involved in the invasion and metastasis of many solid tumors. In this study, we analyzed the role of the uPAR/uPA system in both the development and progression of pancreatic cancer in invasive ductal adenocarcinomas of the pancreas (PDA) and their premalignant precursors (PanIN lesions) in 50 patients with long-term clinical follow-up. We found overexpression of the uPAR in 48 of 50 invasive carcinomas as well as in a large proportion of high-grade PanIN lesions by immunohistochemistry and in situ hybridization. Fluorescence in situ hybridization analysis showed both high- and low-level amplification of the uPAR gene in ~50% of cases with strictly identical patterns between invasive cancers and their accompanying precursor lesions. These results suggest that PDA may develop from PanIN lesions along an alternative rather than a sequential molecular pathway. The detection of the gene amplification of uPAR was a highly significant, adverse prognostic parameter (P < 0.001) because it likely renders the tumors more sensitive to uPA and its proproliferative and anti-apoptotic signals. We conclude that the activation of the uPAR/uPA system is an early event in the development of PDA and that uPAR gene amplifications identify a subgroup of particularly aggressive tumors, making the uPAR/uPA system a critical and highly promising target for therapeutic interventions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Invasive ductal adenocarcinoma of the pancreas (PDA) represents the fourth most common cause of cancer-related death and its incidence is still increasing.1 With an estimated number of 232,000 new cases per year, pancreatic cancer is among the most common malignancies worldwide.2 Moreover, it is one of the most lethal cancers, as indicated by a mortality incidence ratio of 98%.2 Once pancreatic cancer is clinically evident, it progresses at a rapid rate, and metastasis is often present already at the time of diagnosis.3 The mechanisms that regulate this aggressive growth behavior in pancreatic cancer are still poorly understood.

Several studies indicate that during cancer cell invasion and metastasis proteolytic enzymes participate in the degradation of extracellular matrix components. The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR, CD87) are involved in the control of extracellular matrix turnover, cell migration, invasion, and cell signaling leading to a variety of different responses under both physiological and pathological conditions.4-6 uPAR is a highly glycosylated 45- to 60-kDa protein that is attached to the cell membrane via glycosylphosphatidylinositol anchor. It consists of three homologous domains of which the N-terminal one represents the uPA-binding domain.7 The urokinase receptor that binds to the growth factor-like domain of uPA directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating tissue regeneration, angiogenesis, cancer growth, and metastasis.6,8-10 Furthermore, uPA and amino-terminal fragment of uPA (ATF) induce proliferation in cancer cells by binding to uPAR. Integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the mitogen-activated protein kinase (MAPK) pathway.11 The ability of uPA to activate the MAPK pathway by binding to uPAR was reported previously.6,12-14 There appears to exist a positive feedback loop between uPAR/uPA expression and MAPK: the uPAR/uPA system plays a critical role in maintaining a high level of activated MAPK and at the same time, activated MAP kinases are necessary to maintain uPA and uPAR expression.15

The present study was conducted to analyze uPAR expression and uPAR gene amplification in pancreatic cancer and precursor lesions and to further evaluate the role of uPAR/uPA system in pancreatic carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor Samples and Patient Data

A prospective database of patients with pancreatic tumors has been developed in the Department of Surgery, University Hospital Mannheim, since 1987 with approval of the ethical committee of the medical faculty. One fraction of freshly removed pancreatic tissue samples was fixed in 4% buffered paraformaldehyde for 24 hours and paraffin-embedded for histological analysis, another fraction was frozen in liquid nitrogen immediately on surgical removal and maintained at –80°C until use. Some of the fresh-frozen specimens had been stored for more than 10 years. In this study, 50 R0 resected invasive ductal adenocarcinomas of the pancreas (20 female, 30 male patients) with available fresh-frozen tissue of sufficient quality operated at the Department of Surgery, Medical Faculty Mannheim, between 1994 and 2004 were enrolled. Ampullary, duodenal and biliary cancers were excluded from this study. The median age of the patients was 66.5 years (range, 41 to 79 years). Surgical procedure consisted of either a partial duodenopancreatectomy (48 patients) or a left resection of the pancreas (2 patients). Seven patients had tumor stage I, forty patients had tumor stage II, and three patients had a tumor stage III (UICC 2003). Four tumors were grade 1, thirty-three grade 2, and thirteen grade 3. Overall survival was defined as time between surgery and death. The mean overall survival (OS) was 24.3 ± 5.1 months (range, 2 to 156 months). All patients were examined every 3 to 6 months during the first 5 years of follow-up and once a year thereafter. Follow-up was performed either through direct contact with the patient or with the patients' primary physician and was terminated at patients' death. Recurrence was diagnosed either by ultrasound, computed tomography, magnetic resonance imaging, or through a palliative surgery with biopsy. Of the 50 patients examined 43 patients showed evidence of disease during follow-up (local relapse and/or distant metastasis) and subsequently died of their cancer. The seven remaining patients died without evidence of disease. In 28 cases the invasive adenocarcinoma was associated with low-grade pancreatic intraepithelial neoplasia (PanIN-1), in 16 cases with moderate-grade pancreatic intraepithelial neoplasia (PanIN-2), and in 26 cases with high-grade pancreatic intraepithelial neoplasia (PanIN-3). Furthermore, tissues from eight male patients (between 56 and 74 years of age, median 69 years) (n = 8) undergoing pancreatic resection for chronic pancreatitis were examined as controls.

Fluorescence in Situ Hybridization (FISH) of Interphase Nuclei

Three to five paraffin sections of 10 µm thickness were stained with hematoxylin and eosin. Under microscopic control tumor tissue was selectively collected with fine forceps. Extraction of interphase nuclei from microdissected tumor material was performed as described.16 Interphase FISH was performed as described previously.17 Hybridization was performed using a cosmid probe for uPAR (clone R28316 was kindly provided by the Lawrence Livermore National Laboratory Genome Center, Livermore, CA) and an {alpha}-satellite probe for the centromeric regions of chromosome 19 (Vysis Inc., Downers Grove, IL). The cosmid probe was labeled with digoxigenin by nick translation (Roche Diagnostics GmbH, Mannheim, Germany). Detection of cosmid probe was performed by tyramide signal amplification (TSA; Invitrogen GmbH, Karlsruhe, Germany). The {alpha}-satellite probe was purchased directly labeled (SpectrumGreen) from Vysis Inc.

Microscopy and Image Acquisition

At least 100 nonoverlapping interphase nuclei from each tumor sample were evaluated by using a fluorescence microscope (Axiophot, C; Zeiss, Göttingen, Germany) fitted with a triple band pass filter set (Vysis Inc.) allowing for the simultaneous visualization of green and red signals. Images were acquired with a cooled charge-coupled device camera (Nu200; Photometrics Inc., Tucson, AZ) and the Quips-XL FISH-imaging software (Vysis. Inc). Each tumor cell was scored for the number of centromeric and uPAR signals. uPAR copy gain was defined as more uPAR signals than centromere 19 signals in more than 10% of cells. This was based on two controls of nonneoplastic pancreatic tissue showing more uPAR signals than centromere 19 signals in not more than 5% of cells. Hybridization efficiency was tested in lymphocytes, which were always present in interphase nuclei preparations. Only FISH experiments with high hybridization efficiency (showing at least 95% of lymphocyte nuclei with two signals for uPAR and two signals for centromere 19) were considered adequate for evaluation. Low-level amplification was defined as up to three more uPAR signals than centromere 19 signals, high-level amplification was scored by a ratio of more than three uPAR signals than centromere 19 signals. FISH experiments were repeated at least twice. Nuclei without any uPAR or centromere 19 signals were excluded from analysis.

In Situ Hybridization

Nonradioactive in situ hybridization for human uPAR mRNA applying digoxigenin-labeled oligodeoxinucleotides was performed as described previously.18 Briefly, paraffin sections of 10 µm thickness were deparaffinized and incubated with Proteinase K (5 µg/ml in 50 mmol/L Tris/HCl, pH 7.6; Roche) for 45 minutes at 37°C. After prehybridization (1 hour at 37°C; in 50 µl 4x standard saline citrate, 50% formamide, 1x Denhardt’s solution, 10% dextran sulfate, 150 µg/ml salmon sperm DNA) 50 µl of an equimolar mixture of five different 5'- and 3'-digoxigenin-labeled antisense or sense oligodeoxynucleotides to uPAR mRNA (total concentration: 25 ng/ml in hybridization solution) was added to the slides and incubated at 37°C overnight. The antisense nucleotides (MWG Biotech, Ebersberg, Germany) were complementary to nucleotides 121 to 150, 321 to 350, 521 to 550, 717 to 746, and 918 to 947 of uPAR mRNA (according to the EMBL data base accession number X51675). Control slides received the corresponding sense oligodeoxynucleotides. After hybridization, slides were washed in standard saline citrate and blocked for 30 minutes at room temperature (3% bovine serum albumin in Tris buffer). Detection was performed with an alkaline phosphatase-conjugated anti-dig antibody (30 minutes room temperature, 1:600; Roche) and subsequent immersion in Fast Red solution containing 0.1 mmol/L levamisole (Sigma, München, Germany) as chromogenic solution.

Immunohistochemistry

Immunohistochemistry was performed on 2-µm-thick paraffin sections using a chicken polyclonal antibody (IgY) directed to affinity-purified recombinant human uPAR1-277 expressed in Chinese hamster ovary (CHO) cells reacting with native and denaturated cell-associated uPAR antigen18-20 (pAb HU277, 1:100 dilution; kindly provided by Viktor Magdolen and Manfred Schmitt; Klinische Forschergruppe der Frauenklinik, TU-München, Germany). This antibody was used because we found superior sensitivity compared with a commercially available mouse anti-uPAR monoclonal antibody (clone 3B10, IgG2a, no. 3936, 1:100 dilution; American Diagnostica, Stamford, CT) when staining the same series (data not shown). Immunostaining was based on an alkaline phosphatase-conjugated streptavidin-biotin detection system (Amersham Pharmacia Biotech Inc., Piscataway, NJ), using Fast Red (Roche) as a chromogen. Antigen retrieval was achieved by microwave treatment (3 x 5 minutes, 600 W). Incubation of the primary antibody was performed for 60 minutes at 37°C. Negative controls were performed by substituting nonimmune IgG and by preincubation of pAb HU277 with an excess of the CHO-uPAR1-277 before the staining reaction. For the detection of macrophages, endothelial cells, fibroblasts, myofibroblasts, smooth muscle cells, and lymphocytes, consecutive tissue sections were stained with mAb to CD34, CD68, CD45, smooth muscle antigen (SMA), and to fibroblast antigen (all from DAKO, Hamburg, Germany).

Proliferation and Apoptosis

For detection of proliferating cells mAb anti-Ki-67 (clone SP6; Lab Vision, Fremont, CA) was used. In each case 2000 tumor cell nuclei were evaluated. Apoptosis is achieved through activation of caspases. Recently, it has been shown that cytokeratins, in particular cytokeratin 18, are affected in early events of apoptosis. The mAb M30 (CytoDeath, Roche), which recognizes a specific caspase cleavage site within cytokeratin 18 that is not detectable in native cytokeratin 18 of normal cells, was used to detect early apoptosis in epithelial cells. In each case 2000 tumor cells were evaluated.

Tissue Extraction and Enzyme-Linked Immunosorbent Assay (ELISA)

Pancreas cancer tissue specimens were obtained at surgery and stored at –80°C until extraction. From each tissue specimen frozen sections were stained (H&E) to ensure that only cancer tissue was used. Tissue extraction and ELISA was performed as previously reported.21 Briefly, deep-frozen specimens of 300 to 400 mg wet weight were pulverized by a microdismembrator (Satorius, Göttingen, Germany). The resulting powder was suspended in 1.8 ml of TBS (0.002 mol/L Tris-HCl, 0.125 mol/L NaCl, pH 8.5) and 0.2 ml of the nonionic detergent Triton X-100 10% (Sigma) yielding a 1% Triton X-100 final preparation. After gentle stirring for 12 hours at 4°C, the suspension was subjected to centrifugation (21,000 x g for 60 minutes, 4°C) to separate cell debris. The total protein content of the extract was measured by using a conventional biuret protein reaction assay (BCA protein assay kit; Pierce, Rockford, IL). We performed an uPA and uPAR ELISA using a commercially available ELISA kit (American Diagnostica, Pfungstadt, Germany) and a conventional ELISA reader (Multiscan EX; Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. The measurements were performed in duplicates. uPA content was determined in the Triton X-100 extract and calculated per mg of tissue protein.

DNA Extraction from Paraffin Material and Quantitative Polymerase Chain Reaction (PCR)

DNA was isolated from selected tumor blocks using the Trizol method. Gene quantification was performed on the ABI Step One Plus PCR system (Applied Biosystems, Foster City, CA). Fifty ng of genomic paraffin-isolated DNA were quantified using Power Syber green master mix (Applied Biosystems) according to the manufacturer’s specifications. The primers used were uPAR forward 5'-TCAGAGAAGACCAACAGGAC-3' and uPAR reverse, 5'-GGATGATGATGAGGTTGAGC-3'. The thermal cycling conditions were 10 minutes at 95°C denaturation, followed by 40 repeats of 15 seconds at 95°C, and 1 minute at 60°C annealing. Melting curves were achieved after a denaturation time of 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C with a temperature increase of 0,3°C/second. Data collection was performed during each annealing phase. During each run, a standard dilution of the DNA with a known quantity was included to permit gene quantification using the supplied software according to the instructions of the manufacturer. In each run a negative control (distilled water) was included. The measurements of gene quantification were taken three times, and the mean of these values was used for further analysis.

Statistical Analysis

Correlations between noncategoric variables were calculated using the Spearman rank sum correlation (r = Spearman’s correlation coefficient). Comparisons between different groups were performed using the Mann-Whitney U-test. Correlations between amplification status of the uPAR gene, UICC tumor stage, grading, and survival were calculated using the Kaplan-Meier method and tested using Cox-Mantel test. For all tests, a P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
uPAR Is Expressed in the Majority of Pancreatic Ductal Adenocarcinomas and Their Precursor Lesions

Fifty invasive ductal adenocarcinomas of the pancreas and associated precursor lesions were studied for expression and synthesis of uPAR by in situ hybridization, immunohistochemistry, and ELISA (Tables 1 and 2) . Previous results have suggested that endothelial cells may serve as an internal staining control21 and were positive also in all cases analyzed here. By in situ hybridization, cancer cells, stromal cells (macrophages, lymphocytes, and fibroblasts), and endothelial cells showed a positive reaction with the antisense probe in all cases. In six cases also normal ductal/ductular and acinus cells showed positive uPAR mRNA signals.


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  		Table 1. Results of Anti-uPAR Immunohistochemistry and uPAR mRNA in Situ Hybridization in 50 Invasive Ductal Adenocarcinoma of the Pancreas

 
By immunohistochemistry, the antibody pAb HU277 stained cancer cells in 48 of the cases (96%) (Figure 1, A–D) . In two cases with positive internal controls, no uPAR-positive tumor cells were detected. Normal, nonneoplastic ductal/ductular and acinar cells exhibited anti-uPAR immunoreactions in eight cases including the same six cases that were positive also on in situ hybridization. Tumor cells of all 26 PanIN-3 lesions showed uPAR expression on immunohistochemistry and in situ hybridization. Of the 16 PanIN-2 lesions, 8 cases were reactive on immunohistochemistry and 9 cases showed positive signals on in situ hybridization. Of 28 PanIN-1 lesions, 4 cases were positive using both methods (Tables 1 and 2) .


Figure 1
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  		Figure 1. Immunohistochemical staining of uPAR in pancreatic carcinomas and pancreatic intraepithelial neoplasia. A: Anti-uPAR (pAb HU277) immunoreaction in an invasive ductal adenocarcinoma of the pancreas adjacent to nonneoplastic (normal) pancreas tissue. Cancer cells express the uPAR antigen, whereas nonneoplastic acinus cells show only a faint anti-uPAR immunoreaction. B: Higher magnification shows strong staining in tumor cells (arrowheads) and endothelial cells (arrows). Macrophages and fibroblasts also stain positive. C: Strong uPAR staining in this pancreatic intraepithelial neoplasia grade 1. D: Moderate uPAR staining in this example of pancreatic intraepithelial neoplasia grade III. Atypical epithelial cells as well as myofibroblasts (arrows) are stained. Alkaline phosphatase immunohistochemical stainings of paraffin sections.

 
In six of eight tissue samples with chronic pancreatitis, small ductules showed faint uPAR and uPA immunoreactivity. Only in two cases acinar cells revealed weak cytoplasmic uPA immunoreaction and no anti-uPAR immunoreaction at all. In areas of chronic inflammation and tissue remodeling, however, all cases exhibited a strong anti-uPA and anti-uPAR immunoreaction in fibroblasts, macrophages, as well as endothelial cells, in good accordance with previous reports.22

Amplification of the uPAR Gene Is a Frequent Event in Pancreatic Adenocarcinomas and Their Precursor Lesions

Interphase FISH was performed on cancer cell nuclei isolated from 50 invasive adenocarcinomas of the pancreas. Among these cases, 8 (16%) showed high-level amplification of uPAR (Figure 2A) , 18 cases (36%) an uPAR low-level amplification, and 21 cases (42%) no uPAR gene amplification (Table 3) . Polysomy of chromosome 19 was detected in three carcinomas. uPAR gene amplification as detected by FISH was verified by quantitative PCR (qPCR) amplification of genomic tumor DNA in 16 randomly selected cases (n = 5 no amplification, n = 7 low level, n = 4 cases with high-level amplification on FISH) and by uPAR ELISA (Figure 3, A and B) . There was a significant statistical correlation between the FISH amplification status and the qPCR results (mean copy number in tumors without uPAR amplification on FISH 52 ± 44.2 versus 188 ± 295.4 in tumors with low-level amplifications versus 620 ± 317.7 in tumors with high-level amplifications; Spearman test; r = 0.57, P < 0.05). Moreover, there was also a statistically significant correlation between the FISH amplification status and the uPAR protein level on ELISA (all 50 cases included, r = 0.042, P = 0.002). uPAR levels in eight chronic pancreatitis whole tissue extracts intermediate between PDA with and without uPAR gene amplifications [uPAR (ELISA) in chronic pancreatitis 3.01 ± 1.19 ng/mg versus 2.71 ± 0.5 ng/mg in PDA without gene amplification versus 4.36 ± 2.28 ng/mg in PDA with gene amplification].


Figure 2
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  		Figure 2. uPAR fluorescence in situ hybridization (uPAR-FISH) in pancreatic carcinomas and pancreatic intraepithelial neoplasia. A: High-level uPAR gene amplification (red signals) in this example of an invasive pancreatic carcinoma. {alpha}-Satellite DNA probes (green signals) were used to detect the centromeric regions of chromosome 19. Only a few nuclei exhibit a polysomy of chromosome 19. DAPI stain (blue) was used to highlight cell nuclei. B: Concomitant high-level uPAR gene amplification (red signals) also in the adjacent pancreatic intraepithelial neoplasia grade 3.

 

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  		Table 3. Results of uPAR Gene Fluorescence in Situ Hybridization in 50 Samples of Invasive Ductal Adenocarcinoma of the Pancreas and in Tumor Cells of Pancreatic Intraepithelial Neoplasia Grade 1, 2, and 3 (panIN-1, panIN-2, panIN-3)

 

Figure 3
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  		Figure 3. Verification of uPAR FISH results by quantitative PCR and ELISA. Statistically significant difference between tumors with and without uPAR gene amplification as determined by FISH with respect to uPAR gene content as assessed by quantitative PCR from genomic tumor DNA (A) and uPAR protein as assessed by ELISA (B) Box-whisker plots showing mean, SE, and SD; P < 0.05.

 
The 26 PanIN-3, 16 PanIN-2, and 28 PanIN-1 lesions were also evaluated for uPAR gene amplification (Figure 2B) . The morphological grading system of PanIN lesions was partially reflected by the uPAR gene amplification status: 8 of 26 (31%) PanIN-3 lesions showed high-level amplification of uPAR, 9 cases (35%) a low-level amplification. Of the 16 PanIN-2 lesions, none showed a high-level amplification and 7 cases (44%) showed a low-level amplification. No uPAR gene amplification was detected in any of the 28 PanIN-1 lesions. Of note, the alterations detected in the PanIN lesions in all cases with no exceptions were identical to the alterations detected in the respective invasive tumor of the same patient. The same eight cases with high-level amplifications in the PanIN-3 lesions showed also a high-level amplification in the invasive carcinoma, and, likewise, the nine cases of PanIN-3 lesions with low-level amplification of uPAR showed also an uPAR low-level amplification in the invasive carcinoma. All of the seven cases with uPAR low-level amplification in PanIN-2-lesions also showed uPAR low-level amplification in the carcinoma (Table 3) .

Amplification of the uPAR Gene in Pancreatic Adenocarcinomas Is a Prognostically Adverse Parameter

On Kaplan-Meier analysis (Cox-Mantel test), presence of either a low (P = 0.017) or a high level (P = 0.0009) uPAR gene amplification was an adverse prognostic parameter compared with cases without detectable amplifications (median survival in patients without detectable uPAR gene amplifications 24.5 months, patients with low-level amplifications 12.0 months, patients with high-level amplifications 7.5 months) (Figure 4) . The difference in mortality between cases with low- and with high-level amplifications was statistically not significant (P = 0.25). All other parameters tested (tumor stage, lymph node metastasis, tumor grading) were statistically not significant.


Figure 4
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  		Figure 4. Statistical correlation between uPAR gene amplification status and survival in patients with pancreatic carcinomas. Significantly shorter survival in patients with tumors with either low- or high-level amplifications of the uPAR gene compared with tumors without. Kaplan-Meier-analysis, Cox-Mantel-test, P = 0.0009. The difference between the two tumor groups with either low- or high-level gene amplifications was statistically not significant.

 
uPA Expression Correlates with Increased Proliferation and Reduced Apoptosis in Pancreatic Adenocarcinomas

Protein extracts from the 50 invasive adenocarcinomas were analyzed for uPA content by ELISA and correlated with proliferation and apoptosis rates of the same tumors as assessed by quantitative immunohistochemistry. The mean uPA expression level in these tumors was 10.46 ± 2.41 ng/mg (mean ± SEM; range, 0.7 to 25.6 ng/mg). The mean Ki-67-associated proliferation rate was 20.1 ± 4.3% (mean ± SEM; range, 3 to 57%). The mean M30-associated apoptosis rate was 14.1 ± 2.7% (mean ± SEM; range: 2 to 33%). There was a positive correlation between uPA expression and proliferation (r = 0.77, 95% confidence interval (95% CI), 0.61 to 0.87; P < 0.001) (Figure 5A) and a negative correlation between uPA expression and apoptosis (r = –0.41; 95% confidence interval (95% CI), –0.69 to –0.11 (P < 0.001) (Figure 5B) . The proliferation level was significantly higher in tumors with high-level amplifications of the uPAR gene than in tumors without amplification (Ki-67: 14 ± 9.6 versus 30 ± 14; P < 0.01), while the rate of apoptosis was not statistically different.


Figure 5
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  		Figure 5. Correlation between uPA content measured by ELISA and proliferation and apoptosis rates determined by quantitative immunohistochemistry. A: Strong positive correlation between uPA level in tumor tissue extracts and Ki-67-associated proliferation rate [r = 0.77; 95% confidence interval (95% CI): 0.61 to 0.87; P < 0.001]. Significantly higher proliferation rate in tumors with high-level amplifications of the uPAR gene compared with tumors without amplification. B: Moderate negative correlation between uPA level and M30-associated apoptosis rate (r = –0.41; 95% confidence interval (95% CI): –0.69 to –0.11; P < 0.001). The difference in apoptosis was not different between tumors with and without uPAR gene amplifications.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The reasons for the aggressiveness and dismal prognosis of pancreatic cancer are still poorly understood. Several studies indicated that the urokinase system plays a central role in the control of extracellular matrix turnover, cell migration, invasion, cell signaling, angiogenesis, and metastasis.23 A moderate to strong expression of uPA and uPAR in pancreatic cancer has been previously described24 Our studies using immunohistochemistry and in situ hybridization confirm a strong expression of uPAR in virtually all pancreatic carcinomas and their precursors. Previous work from our and other groups demonstrated a high frequency of high-level amplifications of the chromosomal region 19q12-13.2 in pancreatic cancer by using comparative genomic hybridization.25,26 Using several complementary approaches, we here show that the uPAR gene (located on chromosome 19q13.2) is a likely biological target of these amplification events. In this study, 58% of cases showed either a low or a high level uPAR gene amplification. Importantly, uPAR gene amplifications were already detectable in precursor lesions and the uPAR gene amplification status in invasive carcinomas and the precursor lesions in the same specimens was remarkably homogeneous. Within a given sample, the pattern of high- or low-level uPAR gene amplifications was identical in the invasive carcinoma and the precursor lesion in all cases. Moreover, the morphological grading system of PanIN lesions was reflected by the uPAR gene amplification status: uPAR gene amplifications were observed only in PanIN-2 and -3 lesions and high-level amplifications only in PanIN-3 lesions. This was somewhat paralleled by immunohistochemistry because all of the high-grade precursor lesions (PanIN-3) and a substantial proportion of PanIN-2 lesions showed overexpression of uPAR, whereas PanIN-1 lesions and nonneoplastic tissues were usually negative. Similar results have been described in precancerous lesions of the breast (DCIS).18 Our interpretation of these observations is that uPAR gene amplification is an early event that is obviously not indispensable for carcinogenesis (given the 40% of carcinomas without detectable gene amplifications). Moreover, our findings are difficult to reconcile with a linear or sequential model of carcinogenesis, in which low-grade PanIN lesions acquire several hits and thereby progress to high-grade lesions and finally invasive carcinoma.27 By contrast, our findings fit better to an alternative model in which pancreatic carcinomas develop along different molecular pathways: preneoplastic lesions without uPAR gene amplifications appear to progress into carcinomas without uPAR gene amplifications rather than acquiring this alteration later in the process of carcinogenesis. In this respect, PDA would resemble the situation in breast carcinomas, in which many clinicomorphological and molecular data suggest that low-grade ductal carcinoma in situ (DCIS) tends to progress to low-grade invasive breast cancer, and high-grade DCIS tends to progress to high-grade invasive cancers.28-31

The detection of uPAR gene amplification (low and high level) was the only statistically significant predictor in this analysis and had an adverse prognostic impact, suggesting that uPAR gene amplifications identify a particularly aggressive subgroup of PDA. As shown here, tumors with high-level amplifications of the uPAR gene showed also a concomitant increase of uPAR protein and increased proliferation. Remarkably, the mean uPAR levels in pancreatic cancers appear much higher than in many other tumors, such as breast carcinomas.32 Increased uPAR protein expression (ELISA) were also observed in cases of chronic pancreatitis comparable with PDA without uPAR gene amplification.

Expression of the uPAR ligand uPA was correlated with increased proliferation and reduced apoptosis. These findings suggest that uPAR gene amplifications render the tumor more sensitive to the prosurvival and anti-apoptotic signals of uPA. Recent reports showed that uPAR/uPA-induced proliferation involves activation of ERKs and p38 MAP kinases11,12 either directly or via release of different growth factors and through dynamic control of cell-matrix interactions. In agreement with previous reports,24 we found strong uPAR expression in tumor-associated macrophages and fibroblasts of the tumor stroma. This increased expression in the stroma as well as in tumor cells implies a specific stromal response to tumor infiltration that may contribute to tumor invasiveness. Another possible mechanism involves tumor neovascularization. We have previously described a good correlation between microvessel density and uPA expression in breast carcinomas.21

In summary, our findings show that the urokinase system plays an important role in the biology and clinical behavior of PDA. Amplifications of the uPAR gene appear to be an early event in carcinogenesis and may be helpful to identify a distinct molecular pathway of PDA development, associated with a particularly poor prognosis. Interference with the urokinase system may be a promising therapeutic strategy to prolong survival of patients with PDA.


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  		Table 2. Results of Anti-uPAR Immunohistochemistry and in Situ Hybridization in Pancreatic Intraepithelial Neoplasia Grade 1, 2, and 3 (panIN-1, panIN-2, panIN-3)

 

   Footnotes
 
Address reprint requests to Dr. Ralf Hildenbrand, Institut für Pathologie, Heilsbachstrasse 15, 53123 Bonn, Germany. E-mail: hildenbrand{at}patho-bonn.de

Supported by the Deutsche Krebshilfe (to R.H. and grant 106430 to P.S. and A.M); the Dr. Mildred-Scheel-Stiftung für Krebsforschung (10-1507H to R.H.); the Faculty of Clinical Medicine Mannheim, University of Heidelberg (research funds to R.H.); the Alfried Krupp von Bohlen und Halbach Foundation (to H.A.); the Wilhelm Sander Stiftung (to H.A.); the Auguste-Schaedel-Dantscher-Stiftung (to H.A.); the Ingrid zu Solms Foundation (to H.A.); the Dr. Hella-Buehler-Foundation (to H.A.); and the European Union [grants LSHB-CT-2003-503410 (Euro-Thymaide) and 2005105 (European Myasthenia Gravis Network) to P.S. and A.M.).

Accepted for publication February 10, 2009.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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