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© 2002 American Society for Clinical Oncology Minimal Residual Disease in Gastric Cancer: Evidence of an Independent Prognostic Relevance of Urokinase Receptor Expression by Disseminated Tumor Cells in the Bone MarrowByFrom the Department of Surgery, Klinikum Grosshadern, Ludwig Maximilians University of Munich, Munich, and Institute of Pathology and Cytology, Deggendorf, Germany. Address reprint requests to Heike Allgayer, MD, PhD, Department of Surgery, Klinikum Grosshadern, Ludwig Maximilians University of Munich, 81377 Munich, Germany; email: DAllgayer{at}aol.com
PURPOSE: To study the invasion-related molecule urokinase-type plasminogen activator receptor (u-PAR) expressed by disseminated tumor cells as a biologic predictor of poor survival in a large prospective series of patients with gastric cancer. PATIENTS AND METHODS: In 156 gastric cancer patients (prospective series), disseminated tumor cells in the bone marrow and the u-PAR expressed by these tumor cells were determined by cytokeratin (CK) 18 immunocytochemistry and u-PAR/CK18 double immunocytochemistry.
RESULTS: In contrast to the mere detection of disseminated tumor cells at primary surgery, the additional evidence of u-PAR on these cells correlated significantly with pathologic T stage (P = .0474) and the expression of u-PAR (P = .0093) and plasminogen-activator inhibitor 1 (P = .0145) in the primary tumor (immunohistochemistry, CONCLUSION: This is the first large study to show that u-PAR, detected on disseminated tumor cells in the bone marrow, is an independent prognostic parameter in gastric cancer, in contrast to the mere detection of minimal residual disease (MRD). u-PAR may be a promising marker to define a critical subpopulation of disseminated tumor cells and a target to eliminate MRD. Molecular phenotyping of MRD is critical for defining its individual clinical relevance.
MINIMAL RESIDUAL disease (MRD) has been called one of the major causes for tumor relapse after curative resection of the primary tumor in solid carcinomas. Even for early stages of carcinomas such as breast, gastric, colon, and lung cancers, investigations over the past decade have shown that single disseminated tumor cells can be detected easily with immunocytochemistry or polymerase chain reaction for epithelial cytokeratin markers.1-17 These methods were able to detect disseminated tumor cells within the mesenchymal background of the bone marrow at sufficient sensitivity and specificity. However, MRD is one of the issues in solid carcinomas that is still controversial as to its prognostic value and therapeutic implications. Especially for the detection of disseminated tumor cells at the time of primary cancer surgery, numerous studies have reported controversial results as to the prognostic relevance of these cells.1-3,8,12,16-20 One of the reasons for this certainly may be the fact that the pure detection of these cells is unable to reflect the biologic heterogeneity of these cells and, consequently, their individual potential to, for example, cause metastasis or overcome the immune system. Indeed, some studies, including ours, investigating the postoperative development of disseminated tumor cells in bone marrow after curative tumor resection observed substantial changes of the presence or absence of disseminated tumor cells over time.21,22 These studies suggest that microenvironment interactions and the phenotypic characteristics of these cells, rather than the mere evidence of MRD, will be able to indicate the stage of systemic disease that is of metastatic and prognostic relevance. First efforts using double immunocytochemistry techniques and, more recently, new molecular biology methods have been initiated to achieve MRD phenotyping. The objectives are to provide new models for molecular tumor staging and to develop molecular targeting strategies for biologically relevant disseminated tumor cells.4,23-30 Among these, our group focused on the urokinase-type plasminogen activator receptor (u-PAR) as one potential marker of metastatic phenotypes and as a potential new target on disseminated tumor cells.21,22,30 u-PAR is a 55- to 60-kd, heavily glycosylated, disulfide-linked cell surface receptor which, by its ligand—urokinase plasminogen activator (u-PA)—which accelerates plasmin-dependent proteolysis, leads to a very efficient degradation of extracellular matrix components such as fibrin and collagen IV.31-39 The glycosyl-phosphatidylinositol anchor of the u-PAR is thereby hypothesized to guarantee a high intramembrane flexibility of the receptor; this potentially could lead to a focus of proteolytic activity.37-41 Thus, u-PAR and its associated parameters—u-PA and the specific inhibitor plasminogen activator inhibitor (PAI)-1, which promotes internalization and re-expression of the receptor (the u-PA system36)—have been suggested to be key factors in invasion and metastasis of diverse cancers, such as gastric, colorectal, breast, and esophageal carcinomas.42-50 In our previous work, we suggested that u-PAR expression is one of the phenotypic markers that are critical for the establishment of MRD because it correlated with an increase of the number of disseminated tumor cells in gastric cancer patients over time after curative surgery of the primary tumor. In addition, preliminary results had suggested an association of this parameter with poor survival.21,22 The objective of the present study was to analyze u-PAR as a marker for the differentiation of a clinically and metastatically relevant MRD component at the time of tumor surgery, as judged by its prognostic relevance in a large patient series. In the present work, evidence is provided for the first time that u-PAR, investigated in a large prospective series of gastric cancer patients, is an independent predictor of a poor clinical prognosis when detected on disseminated tumor cells in the bone marrow at the time of primary surgery. This is in contrast to the mere detection of disseminated tumor cells at surgery, which had no prognostic impact in the same series. Moreover, u-PAR expression on disseminated tumor cells, in contrast to the mere presence of disseminated tumor cells, correlates with the expression of u-PA system parameters of the corresponding primary tumor. This supports the hypothesis that u-PAR is one of the first promising molecular targets for the elimination of a clinically relevant MRD component in solid cancers. In addition, it emphasizes that the ongoing detailed molecular characterization of MRD will potentially be the key to the biologic understanding of MRD and its therapeutic elimination.
Patients and Tumors A prospective series of 156 patients underwent tumor resection for primary gastric cancer at the Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians University of Munich, Munich, Germany, between April 1989 and October 1996. Their mean age was 63.1 years (SD, 12.0 years; range, 29 to 87 years); there were 84 men and 72 women. Of 156 patients, 144 underwent curative resection (R0), including radical lymph node dissection (compartments I and II). The remaining 12 patients were given palliative resection, five with microscopic (R1) and seven with macroscopic tumor residues (R2). According to the tumor-node-metastasis classification, 33 of the resected tumors were International Union Against Cancer (UICC) stage Ia, 31 were stage Ib, 29 were stage II, 32 were stage IIIa, 17 were stage IIIb, and 14 were stage IV. According to Laurén’s classification, 74 were intestinal, 67 were diffuse, and 15 were mixed-type carcinomas. Eight tumors were well differentiated (G1), 32 were G2-type tumors, and 116 were poorly differentiated (G3). According to Borrmann’s classification, 19 tumors were class I, 49 were class II, 37 were class III, 41 were class IV, and 10 were unclassified. In 32 cases, tumors were located at the cardia and/or the fundus, and in 123 cases, it was located at the corpus and/or the antrum. Four patients were given intraoperative radiation therapy (all received curative resection). Two patients received radiation therapy after noncurative resection. Five patients received chemotherapy after palliative resection. Prospective follow-up was scheduled 6, 12, 18, and 24 months after surgery and in 1-year intervals thereafter. Follow-up included physical examination, abdominal ultrasound, gastroscopy, chest x-ray, hematology, blood chemistry, and screening for the tumor markers carcinoembryonic antigen, CA 19-9, and CA 72-4. If tumor recurrence was suspected, a confirmation of the diagnosis by biopsy or explorative surgery was attempted if possible (two cases). Otherwise, imaging procedures had to be accepted for the diagnosis of a recurrence. Causes of death were evaluated clinically. Patients were included in the study after giving informed consent and after the study was approved by the national ethics board.
Bone Marrow Aspiration Biopsies
Algorithm of Immunocytochemical Staining
Immunocytochemical APAAP Staining for the Detection of Disseminated Tumor Cells in the Bone Marrow
Immunocytochemical Double Staining for the Detection of u-PAR on Disseminated Tumor Cells in the Bone Marrow Cytospins were fixed in acetone (7 minutes) and incubated with 20% AB serum/PBS (25 minutes). Antibodies were diluted in 10% AB/PBS, and preparations were washed carefully three times in PBS after each incubation step. Mouse monoclonal antibody against u-PAR, recognizing intracellular and membrane-bound receptor (no. 3937, 10 µg/mL, IgG2-alpha; American Diagnostica, Greenwich, CT), was applied for 60 minutes, followed by goat antimouse antibody conjugated with 1-nm gold particles (0.08 mg/mL, 1:50, Auroprobe One Reagent; Amersham, Braunschweig, Germany) for 30 minutes and 10% mouse serum/PBS (Dako) to avoid cross-reactions (25 minutes). For the second part of the staining procedure, biotinylated monoclonal antibody CK2 was incubated for 45 minutes (10 µg/mL), followed by alkaline phosphatase–conjugated streptavidin (1.1 mg/mL, 1:100, 30 minutes; Jackson Immuno Research, West Grove, PA). Specifically bound CK2 was visualized by new fuchsin dye (0.4 mg/mL; Serva, Heidelberg, Germany), sodium nitrite (0.04 mg/mL; Merck, Darmstadt, Germany), levamisole (0.36 mg/mL; Sigma), 0.2 m Tris buffer (pH 8.7), and naphthol-AS-BI-phosphate (0.08 mg/mL; Sigma) dissolved in dimethyl-formamide (Sigma). For completion of the immunogold step, slides were washed thoroughly in bidistilled water. Equal volumes of inducer and enhancer of a silver enhancement kit (Amersham) were mixed and incubated at room temperature to visualize specifically bound gold particles under microscopic control (maximum time, 40 minutes). After 20 minutes, the silver kit was completely exchanged to avoid unspecific precipitation of silver granules. After development of the silver staining, the slides were washed in bidistilled water and mounted with Kaiser’s glycerol gelatin (Merck).
Slides of the colon cancer cell lines SW 403 and HT 29 (ATCC) were stained under same conditions as the positive controls were. An equimolar replacement of either CK2 by nonspecific IgG1 (MOPC21; Sigma) or monoclonal antibody against u-PAR by IgG2
Immunohistochemical Staining for u-PA System Parameters in Primary Tumors Staining was performed at room temperature. Each incubation step was followed by washing in 0.001% Brij/PBS (Sigma). All slides were preincubated with 5% horse serum/PBS for 20 minutes. The mouse-derived monoclonal antibodies 394 (against u-PA), 3937 (against u-PAR), 3783 (against PAI-1), and 3750 (against PAI-2) were incubated for 60 minutes at room temperature. This was followed by incubation with horse-derived bridging antibody (7.5 µg/mL, 30 minutes) and the Vectastain ABC elite complex for 30 minutes (Vectastain, Burlingame, CA). After washing in PBS, the enzyme substrate aminoethylcarbazole (Sigma) was added for 15 minutes. Finally, the slides were counterstained with hematoxylin. One section of each tumor treated with nonspecific IgG and one section treated with antibody MLG/7S (Nordic, Tilburg, the Netherlands) directed against murine IgG instead of the primary antibody in equimolar protein concentration served as negative controls. A section of a routinely processed tumor with known strong expression of either u-PA, u-PAR, PAI-1, or PAI-2 served as a positive control. The slides were coded and evaluated without knowledge of patients’ histories and clinical status by an independent observer (B.C.M.B.) and were controlled by an experienced pathologist (R.B.). Analysis of staining was restricted to the reactions observed in tumor cells. Staining of stromal cells was not considered. On the basis of our previous experience with the scoring of this method,21,42,46,55-57 which had revealed a reproducible quantifiability of stained tumor cells and a significant correlation with staining intensity, the staining results were coded semiquantitatively into four groups according to number of positively stained tumor cells: 0 = negative; 1 = 30% or fewer positive tumor cells; 2 = 30% to 70% positive cells; and 3 = 70% or more positive tumor cells.
Statistical Analysis
Patients One hundred fifty-six patients (144 of whom underwent curative resection) with primary gastric cancer were followed prospectively after surgery for a median period of 34 months (range, 1 to 98 months). Of all 156 patients, 72 died, and 58 deaths were due to malignant disease. In the 144 curatively resected (R0) patients, 57 recurrences occurred. Twelve of the recurrences were peritoneal carcinoses, 18 were locoregional recurrences, eight were liver metastases, 11 were distant metastases (bone, lung, brain, and generalized metastases), and eight were secondary carcinomas.
Staining Results Regarding the Detection of Disseminated Tumor Cells in Bone Marrow With Single (CK18) and Double (CK18/u-PAR) Immunocytochemistry In these 81 patients (72 of whom underwent curative resection), additional evidence of u-PAR on at least one disseminated CK18-positive cell (example shown in Fig 1) was found in 47 patients (40 R0 resected, 58%). The other 34 patients (32 R0 resected) did have CK18-positive cells only, with no additional evidence of u-PAR. An example of a u-PAR–expressing tumor cell in the bone marrow is shown in Fig 1.
The median number of disseminated tumor cells detected with the single APAAP method was four per one million bone marrow cells examined (range, one to 280). The median number of cells detected with the double staining method was four (range, one to 253) per one million bone marrow cells. This corresponded to the slight difference in sensitivity between these two previously published methods.30 The median number of disseminated tumor cells with evidence of u-PAR was three (range, one to 222). CK18-negative bone marrow cells did show a weak specific staining for u-PAR in only a few cases, which corresponded to our analysis published earlier in which a weak expression of u-PAR had been detected in 6.5% of mature bone marrow precursor cells.30 Therefore, for all further analyses, only CK18-positive cells with additional evidence of u-PAR were included.
Immunohistochemical Results Regarding the u-PA System in Primary Tumors
Correlation of the Presence of Disseminated Tumor Cells in the Bone Marrow With Established Tumor Characteristics and u-PA System Parameters in the Primary Tumor As a first step, we analyzed potential correlations between the detection of disseminated tumor cells in the bone marrow (additional evidence of u-PAR not considered) and established tumor parameters. Because of the differences in sensitivity, the analysis was performed using the results of both the single APAAP staining technique and the double staining technique. As a result, no significant association was found between the mere evidence of CK18-positive cells in the bone marrow and pathologic T, pathologic N, and M stage, G stage, UICC stage, Borrmann’s stage, Laurén’s classification, or lymphangiosis carcinomatosa. In a subset of 105 patients, an additional analysis was conducted with regard to potential correlations between the evidence of CK18-positive cells and the expression of u-PA system parameters in the primary tumors. No significant association was seen between the qualitative evidence of disseminated tumor cells in the bone marrow and the high expression of u-PA, u-PAR, PAI-1, or PAI-2 in the primary tumor.
Correlation of the Detection of u-PAR on Disseminated Tumor Cells in the Bone Marrow With Established Tumor Characteristics and u-PA System Parameters in the Primary Tumor
Analysis of Univariate Prognostic Impact of Detection of Disseminated Tumor Cells in the Bone Marrow For a first univariate prognostic analysis, the qualitative detection of CK18-positive cells in the bone marrow was considered. Again, two separate analyses were performed using the results of the single staining method and the double staining method because of the difference in sensitivity of the methods regarding the detection of CK18-positive cells. However, in both analyses, there was no significant association between the mere presence of CK18-positive disseminated tumor cells and shorter disease-free or overall survival in Kaplan-Meier analysis (Mantel-Cox log-rank test). As an example, Figs 2 and 3 show the survival curves for disease-free and overall survival using the results of the APAAP method. These results indicate that the mere detection of disseminated tumor cells at the time of primary surgery is not necessarily indicative of a worse clinical prognosis in gastric cancer.
Analysis of Univariate Prognostic Impact of the Detection of u-PAR on Disseminated Tumor Cells in the Bone Marrow As a next step, the additional evidence of u-PAR on disseminated (CK18-positive) tumor cells in the bone marrow found with the double staining technique was included in the Kaplan-Meier survival analysis. In contrast to the above results, where no prognostic impact of the presence of disseminated tumor cells in the bone marrow was seen, the additional detection of u-PAR on at least one disseminated tumor cell was indicative of a significantly poorer disease-free survival in the 144 curatively resected patients (P < .0001, Mantel-Cox log-rank, Fig 4). Overall survival of curatively resected patients was also significantly shorter in patients with u-PAR on disseminated tumor cells (P = .0005). Finally, overall survival analysis of all 156 patients (P = .0002, Fig 5) demonstrated a significant disadvantage in survival for patients with evidence of u-PAR on disseminated tumor cells in the bone marrow at the time of primary surgery.
Analysis of Multivariate Prognostic Impact of the Detection of u-PAR on Disseminated Tumor Cells To correct the above-mentioned univariate results for the impact of established risk factors in gastric cancer, a multivariate analysis was performed for the detection of u-PAR on disseminated tumor cells in the bone marrow (Table 3). In the multivariate analysis model applied (Cox proportional hazards model), the additional parameters pathologic T stage, pathologic N stage, M stage, G stage, surgical curability, necessity of extended resections, Laurén’s classification, Borrmann’s stage, lymphangiosis carcinomatosa, vessel infiltration, tumor localization, and tumor diameter were considered. For disease-free survival, pathologic T status and pathologic N status were the dominant established independent parameters, and u-PAR on disseminated tumor cells turned out to be a new independent parameter with the relative risk of tumor recurrence enlarged by 2.35 (95% confidence interval, 1.72 to 3.21, P = .024, Table 3). For overall survival of all 156 patients, evidence of u-PAR on disseminated tumor cells was a new independent prognostic factor as well (P = .0049; relative risk, 2.89; 95% confidence interval, 1.92 to 4.30), in addition to surgical curability, pathologic T stage, and Laurén’s classification (Table 3). These results indicate that, in contrast to a mere detection of disseminated tumor cells at the time of primary surgery, the additional evidence of the invasion-related molecule u-PAR on these cells is a strong and independent biologic parameter for clinical prognosis in gastric cancer.
This is the first large study to demonstrate that immunocytochemical evidence of the expression of u-PAR on disseminated tumor cells in the bone marrow taken at the time of primary surgery is an independent predictor of a poor clinical prognosis in gastric cancer. Second, our study shows a positive and significant association of the expression of u-PAR on disseminated tumor cells with the expression of u-PAR and PAI-1 in the primary tumor, both which have been shown to predict patient subgroups at high risk for tumor recurrence and metastasis.45,47-49,56,62-68 In contrast, no prognostic relevance or correlation with invasion-related parameters of the primary tumor was seen when only the presence of disseminated tumor cells in the bone marrow was analyzed. This strongly implies that the mere detection of disseminated tumor cells at the time of surgery is not sufficient to represent a biologically relevant MRD component. Rather, the results propose that additional molecular phenotyping (in this case, by a metastasis-related gene) is able to define patient subgroups at high risk for relapse, indicating a clinically relevant MRD component that needs therapeutic intervention. Our results are supported by two of our previous studies in which we had suggested that disseminated tumor cells, when monitored over time after curative surgery for primary gastric cancer, undergo diverse quantitative developments individually, and that the over-time development of the number of tumor cells in the bone marrow correlated with clinical outcome rather than the mere presence of these cells at surgery.21 These data suggested that the establishment of a biologically relevant MRD occurs over time as a result of the interactions between individually heterogeneic tumor cells and their microenvironment. In these previous studies, u-PAR expression of these cells was associated significantly with a later increase of the number of tumor cells in the bone marrow.21,22 Also, the number of disseminated tumor cells expressing u-PAR increased over time, suggesting a positive selection of cells with this marker.22 These data, together with the clinical data presented here, propose u-PAR as one of the phenotypic characteristics critical for the development of a clinically relevant MRD. Certainly, one has to consider that there is a heterogeneity of the disseminated tumor cells regarding u-PAR detection. There are cases in which there are disseminated tumor cells with and without u-PAR in the same patient. This can be seen from the fact that the median number of disseminated tumor cells detected with the double staining method (four; range, one to 253) is higher than the median number of cells carrying u-PAR (three; range, one to 222). From our survival data, however, it becomes clear that only the subclones of disseminated tumor cells with evidence of u-PAR correlate with a significantly worse clinical prognosis. We are aware of the fact that both the immunocytochemical detection of disseminated tumor cells and the additional detection of u-PAR on them have a certain limit of sensitivity as published previously,30 and it is possible that some patients with u-PAR–expressing disseminated tumor cells are potentially not identified because they are below the level of sensitivity. However, in our large patient series, the definition of the status "u-PAR positive on disseminated tumor cells" (ie, as soon as at least one tumor cell with u-PAR had been detected among the pool of disseminated tumor cells) clearly turned out to be a convincing and independent parameter for predicting a poor clinical prognosis; this in contrast to the mere evidence of cells. Therefore, the method has been demonstrated to be valid and sensitive enough to give clinically relevant results.21,22,30 The biologic reasons for why u-PAR may be one of the molecular characteristics to promote the establishment of MRD, thus elevating the risk for clinical tumor recurrence, can only be speculated on. It is well known that u-PAR, via its ligand u-PA, promotes plasminogen-dependent proteolysis of extracellular matrix components, the binding of specific inhibitors like PAI-1 promoting internalization, and re-expression of the receptor.31-41 Thus, u-PAR is involved in diverse cellular processes, such as invasion, intravasation, migration, and metastasis.31-41 Hence, it is not surprising that the detection of u-PAR on disseminated tumor cells in the bone marrow correlated significantly with the immunohistochemical evidence of u-PAR and PAI-1 on the primary tumors, suggesting an advantage of the cells with invasive properties in the dissemination process. Moreover, more recent studies have implicated u-PAR as a molecule that induces diverse signal transduction pathways and leads to different cellular phenomena, such as proliferation.69-73 One of the most striking discoveries in this context might be that, on physical interaction of the u-PAR with integrins, the mitogen-activated protein kinase signaling cascade was favored in a study by Aguirre-Ghiso et al69 as leading to tumor cell growth and proliferation. In contrast, the interruption of the u-PAR/integrin interaction led to an activation of the p38 pathway, which resulted in tumor cell dormancy. Indeed, the phenomenon of dormancy has been discussed for disseminated tumor cells and micrometastases.25,26 It has been hypothesized that dormant tumor cells potentially can be reintroduced into the cell cycle and cause tumor recurrence years after the primary tumor has been resected. Future investigations should determine whether u-PAR and integrins colocalize in disseminated tumor cells, which would support the notion that u-PAR could act as a switch between proliferation and dormancy of minimal residual tumor cells. Other molecules that have been characterized differentially on minimal residual tumor cells in the bone marrow as shown by others are, for example, proliferation-associated molecules (such as p120 and Ki-67),5 the tyrosine kinase receptor ErbB-2, which has been shown to promote tumor cell growth, invasion, and metastasis,5,74 and MHC class I molecules, the loss of which is a potential mechanism to escape cytotoxic T cells and prevent an immune response.24,75,76 These data, together with our data, strongly suggest that minimal residual tumor cells are biologically diverse and that the clinical fact of MRD in solid cancer is not represented sufficiently by the mere detection of disseminated tumor cells in the bone marrow at the time of surgery. This is reflected by the numerous studies conducted in the past that, for some carcinoma types, gave conflicting or even disappointing results regarding the prognostic value of the perioperative detection of these cells in the bone marrow, including our data presented here. For example, the promising first studies on disseminated tumor cells in the bone marrow in mamma cancer and colon cancer1-3,8 have been questioned by a detailed meta-analysis and later studies.17,18 In our own earlier study on gastric cancer,12 the mere detection of perioperatively disseminated tumor cells was predictive in certain subgroups only, and in esophageal cancer, different studies on the prognostic value of MRD detected at the time of primary surgery were conflicting.16,19,20 This is plausible, especially since the biologic characteristics of the disseminated tumor cells were not considered. Rather, we hypothesize that a more detailed differentiation as to the molecular potential of these cells most likely will identify cell subpopulations that are biologically critical for establishing MRD and, consequently, that lead to a clinical risk of relapse. Our own method applied for the present study gave convincing results regarding the clinical applicability of using u-PAR as one critical antigen predicting poor prognosis. However, as the basis of a routine clinical procedure, this method might be too time-consuming. Therefore, it probably should be replaced by recent methods that have the potential to screen single cells on a routine basis for a pattern of biologically and clinically relevant phenotypic properties, such as, for example, microarrays. Recent work conducted by Klein et al27 and Gath et al77 has introduced new and promising molecular biology strategies for performing molecular phenotyping at the single-cell level, such as single-cell polymerase chain reaction, comparative genome analysis, and microarray technology. Ongoing studies need to demonstrate the potential clinical impact of this approach, which will enable broad molecular characterization of disseminated tumor cells in solid cancers and may lead, consequently, to the identification of individual molecular targeting strategies based on MRD components identified as clinically critical. It needs to be emphasized that the characterization of MRD will be of particular interest in patients whose tumors could be curatively resected rather than in patients with macroscopic tumor residues, for whom the prognosis is still limited, particularly by the surgical noncurability, as shown in our multivariate analysis (Table 3, all patients). Toward this end, our present study has suggested u-PAR as one of the first molecular targets on minimal residual tumor cells that might be used to improve the prognosis of gastric cancer patients. From own data, potential strategies to achieve a downregulation of u-PAR may be the inhibition of Src and K-Ras oncogene proteins (eg, by small molecular compounds) that regulate u-PAR in gastrointestinal cancer or even a direct targeting of the u-PAR promoter.78-80 Other groups have implicated exciting strategies at the protein level, such as interruption of the u-PAR/integrin interaction to prevent tumor cell growth,74 potent synthetic low-molecular-weight inhibitors directed at the proteolytic activity of u-PA,81,82 and a soluble recombinant form of u-PAR lacking the glycosyl-phosphatidylinositol anchor of the molecule, which acts as an efficient scavenger for the ligand u-PA and leads to inhibition of tumor cell proliferation and invasion.83 Investigations in the next few years will determine the clinical potential of these anti–u-PAR approaches in the treatment of cancer invasion and metastasis and, potentially, in targeting minimal residual tumor cells characterized by an expression of this invasion-related gene.
H.A. and M.M.H. are supported by the Dr Mildred Scheel Stiftung (Deutsche Krebshilfe), Bonn, and the Wilhelm Sander Stiftung, Munich, Germany. H.A. is also supported by the Faculty of Medicine of the Ludwig Maximilians University of Munich, Friedrich Bauer Stiftung, and Muenchener Medizinische Wochenschrift Board of Editors, Munich, Germany. R.B. was supported by the Wilhelm Sander Stiftung, Neustadt/Donau, Germany. We thank Jan Lich for his excellent help with the manuscript.
M.M.H. and E.H.S. share first authorship. This article contains parts of the dissertations of E.H.S. and B.C.M.B. performed in partial fulfillment of the requirements for the Dr Med at the Faculty of Medicine, Ludwig Maximilians University, Munich, Germany.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2). Kaplan-Meier analysis revealed no association with prognosis for the mere detection of disseminated tumor cells. In contrast, a significant association was seen between detection of u-PAR on these cells and shorter disease-free (P < .0001) and overall survival (P < .0001). Multivariate analysis revealed that u-PAR on disseminated tumor cells at the time of primary surgery is an independent prognostic factor for disease-free (95% confidence interval [CI], 1.72 to 3.21; P = .024) and overall survival (P = .0049; relative risk, 2.89; 95% CI, 1.92 to 4.30). 
-aminocaproyl-N-hydroxy-succinimide (Boehringer) and 50 µL of dimethyl-formamide, overnight incubation, and membrane ultrafiltration (Centricon microconcentrator; Amicon, Beverly, MA). 
(UPC 10; Sigma) served as the isotype control and the cross-control, respectively. In two other slides, the first bridge and the second primary antibody were left out. In two other slides, the first of the second bridge were left out, respectively. The bone marrow of a healthy donor was stained as another control after informed consent was obtained. All slides were coded and evaluated by two investigators. 
