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[C'ANC'ER K|-:S|-:AKC|| SS. .«SU-MM. September 15, l")95| Advances in Brief Primary Tumor and Metastasis in Ovarian Cancer Differ in Their Content of Urokinase-type Plasminogen Activator, Its Receptor, and Inhibitors Types 1 and 21 B. Schmalfeldt,- W. Kühn,U. Reuning, L. Pache, P. Dettmar, M. Se-limiti, F. Jänicke, H. Höfler,and H. Graeff Frauenklinik der Technischen Universität München. Klinikum rechts tier Isar. Ismaninger Strasse 22. D-KI675. Munich ¡B.S.. W. K., U. R., L P.. M. S., F. J., H. G.I. and Institut fürAllgemeine Pathologie und Pathologische Anatomie, Technische Universität München¡P.D., H. H. I, Munich, Germany Abstract The relevance of urokinase-type plasminoceli activator (uPA) and plas- minogen activator inhibitor (PAI) type I in predicting the survival prob ability of patients with advanced ovarian cancer after radical surgery and adjuvant chemotherapy by assessing the patients' primary tumors has recently been shown by us (W. Kühnel al., Gynecol. Oncol., 55: 401-409, 1994). In the present study, we determined uPA, uPA receptor, PAI-1, and PAI-2 concentrations in primary tumors and tumor-infiltrated omentum metastasis is controlled by a series of successive events. After local proteolysis of the surrounding extracellular matrix, detachment and spread of tumor cells is facilitated followed by the invasion of the adjacent tissue, crossing of tissue boundaries and the basement mem brane. Intravasation and extravasation are followed by reimplantation of tumor cells into the respective target organ, remodeling of a new tumor stroma, and angiogenesis in order to consolidate a secondary tumor at a distant locus. For these events a fine-tuned balance between and rctroperitoneal lymph nodes of ovarian cancer patients. The group proteolytic factors, their inhibitors, adhesive proteins, angiogenic fac consisted of 39 patients with advanced ovarian carcinoma stages Fédéra tors, oncoproteins, and growth factors has to be adjusted (2, 3). tion Internationale de Gynécologieet d'Obstétriquei l-ÕGOiHic or IV; for comparison 7 patients with early carcinoma stage FIGO I were also included. In métastasesof the omentum from ovarian cancer stage FIGO lile or IV patients, we noted a 4-fold elevated uPA content, a 2-fold increase in PAI-I, and also a significant increase in uPA receptor and PAI-2 over primary tumors. In métastasesof the lymph nodes the levels of the respective antigens were also increased when compared to primary tumors. These data may indicate that elevated levels of components of the fihrinolytic system at sites of métastasesmay contribute to the aggressive potential of cancer cells by favoring their rcimplantation and/or the consolidation of a new tumor stroma. Introduction Ovarian carcinoma is one of the most severe gynecological malig nancies and because of a long asymptomatic course of the disease displays already advanced stages at diagnosis with extensive cancer spread. This is reflected by the high incidence (70% of the cases) of mortality within 5 years, a fact that stresses the necessity of an early diagnosis for risk selection and the prediction of the course of the disease. Several investigators have demonstrated that the prognosis of patients with advanced ovarian cancer could be improved by radical surgery followed by adjuvant chemotherapy. Although the absence of residual tumor after cytoreductive surgery proved to be a strong prognostic factor for survival, the complete resection of the tumor mass was not sufficient to protect all patients from the recurrence of the disease and subsequent death. Several attempts have been under taken to improve the prognosis of cancer patients and to establish predictive factors that are related to tumor biology. Numerous factors have been screened, among them the receptors for estrogen, proges terone, and epidermal growth factors, DNA ploidy, S-phase, and oncogenes ( 1). In recent years special attention has been paid to tumor cell surfaceassociated proteolytic enzyme systems regarding their impact on cancer progression and metastasis. The process of tumor invasion and Received ft/15/95; accepted 8/4/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C'. Section 1734 solely to indicate this fact. 1This work was supported by the Deutsche Forschungsgemeinschaft (Klinische For schergruppe GR 280/4-1) and the BIOMF.D I program (BMH1CTO1346). : To whom requests for reprints should be addressed. Basic and clinical research in tumor invasion and metastasis have focused on the role of tumor-associated proteases such as metalloproteinases and the serine proteases of the plasmin/plasminogen activator system (2). Metalloproteinases seem to be correlated with tumor cell invasion and metastasis in animal models; however, conclusive data on their clinical significance in humans are still lacking. In contrast, for several different solid cancers a strong prognostic impact of the scrine protease uPA1 and/or its inhibitor PAI-1 as predictors for the course and the outcome of the cancer has been reported. Besides their statistically independent prognostic impact in cancer of the breast (4, 5), lung (6), colon (7), and the gastrointestinal tract (8), significantly elevated levels of both uPA and PAI-1 have been demonstrated in ovarian cancer tissues compared to those in normal ovarian tissue specimens. Patients with advanced ovarian cancer can be subgrouped on the basis of computer-optimized "cutoff values for uPA and PAI-1: patients with low uPA and PAI-1 (uPA < 0.9 ng/mg protein; PAI-1 < 13.5 ng/mg protein) had a statistically better prognosis than patients with high uPA and PAI-1 antigen levels (9). First indications for a role of the uPA-mediated proteolytic system in tumor invasion and metastasis came in 1976 from Astedt and Holmberg (10), who demonstrated for the first time high uPA concentrations in cultured ovarian carcinoma tissue. In the following years increased uPA and PAI-1 levels were also found in tumor tissue extracts of patients with ovarian carcinoma (11, 12) and in malignant ascites (13). uPA is synthesized and secreted by normal and tumor cells. Both the inactive proenzyme form of uPA, pro-uPA, and protcolytically active uPA bind to a specific glycanlipid-anchored receptor (uPA-R, CD 87) on the tumor cell surface. Upon binding of pro-uPA, its activation by other proteases (e.g., plasmin) is facilitated. uPA acti vates the proenzyme plasminogcn into the broad spectrum serine protease plasmin, which may in turn either directly degrade extracel lular matrix proteins such as fibrin, fibronectin, laminin, and proteoglycans or activates certain matrix-degrading enzymes such as procollagenase. The activation of uPA is controlled by its specificinhibitors PAI-1 and PAI-2. Binding of PAI-1 or PAI-2 to uPA-Rbound uPA results in the subsequent internalization of the ternary 1The abbreviations used are: uPA. urokinase-type minogen activator inhibitor; uPA-R. uPA receptor. plasminogcn activator; PAI, plas- 3958 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research. iil'A. iil'A-R. AND PAI-1/2 IN PRIMARY TUMOR AND MUTASI ASÕS complex, thereby regulating cell surface plasmin generation. uPA-R Table 2 Owi/ximwi ofiiPA. uPA-R. PAI-1.anil PAI-2 utilicen /nr/.v//( primary minorversus IVIAntigen(ng/mg tuimir-injiliratal iimenlum nuijus in iul\tinced tmirian cunïmmii Ã-r'lÃ-iOlile ¡ir in combination with specific plasmin(ogen) receptors focalizes the plasmin/plasminogen activator system to the tumor cell surface, thus constituting an effective proteolytic enzyme system (2). Tumor-associated proteolytic enzyme systems are not only impor tant for tumor cell spread from the primary tumor but seem to be crucial parameters for the events at the site of métastases.In this report we investigated the levels of uPA, uPA-R, PAI-1, and PAI-2 in tissue extracts of primary tumors, tumor-infiltrated omentum, and tumor-infiltrated lymph nodes of patients suffering from advanced ovarian carcinoma. protein)uPA(median FIGOIlle/IV tumor metastasis(« 39)"(UM(0.05-10.16)2.87(0.55-8.66)16.99(0.14-105.1)0.25'(0.01-11.34)Omentum (11= 33)"3.65*(0.66-16.58)4.3o(1.15-8.71)34.14(4.60-125.3)1.72 = (P)II.IKIOI0.050.00090.1104 range)uPA-R(median range)PAI-1(median range)PAI-2(median range)Primary Patients and Methods " il. number of patients. '' n = 34. ' n = 38. Forty-six patients with early (FIGO I) or advanced ovarian cancer stages (FIGO lile or IV) were enrolled as par! of a prospective study on ovarian cancer patients undertaken at the Frauenklinik der Technischen Universität München(Table 1). Patients with stages FIGO II, Ilia, and Illh were excluded from the present analysis because of a too small number of eases. Patients with advanced ovarian carcinoma received the following radical surgical treatment: longitudinal laparotomy with hysterectomy, bilateral adenectomy. appendec tomy, infragastric omentectomy, and bilateral pelvic and paraaortic lymphadenectomy. Among the group of patients, seven patients received limited surgical treatment because of their had health condition. In younger patients (<35 years) with tumor stage FIGO I. less radical surgery was performed in order to preserve the fertility of the patients. Histológica! examinations of all of the tumor tissue sections were performed at the Institut fürAllgemeine Pathologie und Pathologische Anatomie, Technische Universität München (Munich, Germany). Benign ovarian tumors (n = 21), e.g., benign ovarian cysts, serous cystadenoma, or cystadenof'ibroma served as controls. Tissue Collection and Extraction. Ovarian cancer tissue specimens from primary tumors, omentum majus, and lymph nodes were collected during surgery, classified by the pathologist, and stored in liquid nitrogen until used. Dcep-fro/.en specimens of 200-500 mg wet weight were pulverized (30 s) using the Micro-Dismembrator (Braun, Melsungen, Germany) set to maximum power. The resulting powder was immediately suspended in 2 ml TBS (0.02 M Tris-HCI, 0.125 M NaCI, pH 8.5), 1% (v/v) Triton X-IOO (Sigma. Munich, Germany), and extracted at 4°Cfor 12 h followed by an ultracentrifugation step (1()0,(XK) x K, 45 min, 4°C)to separate cell debris. assisted analysis (EIA program; ICN-Flow Laboratories, Meckenheim. Ger many). Protein concentrations were determined using the BCA Protein Assay Reagent kit (Pierce, Rockford, IL). Values for uPA, uPA-R, PAI-1, and PAI-2 concentrations are expressed as ng/mg protein of the tissue extracts. In our laboratory the interassay variation coefficient for u PA was 5.8%, for uPA-R 5.6%, for PAI-1 4.0%, and for PAI-2 1.4%, thus fulfilling the criteria for a reliable test. Immunohistochemical staining for uPA, its receptor (uPA-R), and the inhibitor PAI-1 was performed as described in detail by Janicke el al. (4). The following antibodies supplied by American Diagnostica were used at 10 /ng/ml: uPA (mAb 3689), uPA-R (mAb 3639), and PAI-1 (mAb 3785). Statistical Analyses. All results are given as median values. Antigen determinations were performed in duplicate. The significance level of differ ences between the antigen levels in cancer tissue extracts versus controls was evaluated using the Mann-Whitney (/ test. Differences in antigen concentra tions between specimens from the same patient were tested for significance using the Wilcoxon test for combined samples (5). Differences were consid ered to be significant when P < 0.05. Results Due to our recent findings that both uPA and PAI-1 may serve as predictors for survival in patients with advanced ovarian carcinoma (9), we determined in the present study the content of uPA, uPA-R, PAI-1, and PAI-2 in primary tumors and typical sites of metastasis in bind 821, 822, 823, and 893, respectively (American Diagnostica, Greenwich. CT; Refs. 1, 10, 12, and 14). The data obtained were subjected to computcrovarian cancer patients, e.g., omentum majus and, in addition, in retropcritoneal lymph nodes. The respective tissues were extracted in the presence of the nonionic detergent Triton X-100, and the antigen Tahlc 1 Patient dilla tm a^e. Iunior stai;?, Ã-iisl(ilt>u\.grading, nodal status, unti l\ini>liadi'ni'ctomy content of uPA, uPA-R, PAI-1, and PAI-2 was determined using the ELISA. The important new findings in this study can be summarized VariableMedian as follows. yr (20-85)72415284595II23713267322597 (range)StageFIGO age uPA. The median concentration of uPA in primary tumor tissue of IFIGO patients with tumor stage FIGO lile and IV was 0.91 ng/mg protein. lileFIGO In extracts of tumor-infiltrated omentum majus, the uPA content was IVHistologySerousMucinousrindomctroidUnditïerenliatcdGradingGlG2G3G4Nodal approximately 4-fold higher than in primary tumors exhibiting statis tical significance (P = 0.0001, Table 2). The corresponding values for Laboratory Assay Systems. uPA, uPA-R, PAI-1, and PAI-2 concentra tions were determined by using the commercially available ELISA kits Imu- statusNilNlNXLvmphadenectomyPelvicParaaorticPelvic paraaorticSamplingNone60 and the uPA content in primary tumor tissue and omentum metastasis of patients with ovarian carcinoma FIGO lile or IV are depicted as paired samples in Fig. 1. In tumor-infiltrated retroperitoneal lymph nodes, an approximately 2-fold increase in the median value of uPA was noted compared to the uPA content in tissue extracts from primary tumors; however, statistical significance was lacking (P = 0.68). uPA-R. The median value for the receptor of uPA, uPA-R. in extracts of primary tumor tissues of the same patients was 2.87 ng/mg protein compared to a median value of 4.36 ng uPA-R/mg protein in tumor-infiltrated omentum majus. This difference was statistically significant (P = 0.05, Table 2). Values for corresponding uPA-R concentrations in primary tumor and omentum metastasis from each 3959 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research. uPA. uPA-R, AND PA1-1/2 IN PRIMARY TUMOR AND METÕSTASIS or IV were significantly higher than in extracts prepared from benign ovarian tumors. PAI-2 levels were almost comparable in extracts of benign and malignant tumors (Table 3). uPA, uPA-R. and PAI-1 levels in extracts of tumor-infiltrated lymph nodes and omentum 100 majus of patients with advanced ovarian carcinoma stages FIGO lile or IV were significantly higher than in extracts of tumor-free lymph nodes and omentum majus of patients with early ovarian cancer stage FIGO I. However, PAI-2 levels were not significantly increased in extracts of tumor-infiltrated lymph nodes and omentum majus com pared to their tumor-free counterparts of FIGO I patients. To demonstrate the cellular origin of uPA in advanced ovarian cancer, immunohistochemical stainings were performed in compari son to benign cystadenoma of the ovary (Fig. 5, B and C). All primary tumor specimens (FIGO IIIc/IV) examined in the present study by an ELISA displayed a positive reaction of tumor and stromal cells with mAb 3689 (American Diagnostica) directed to uPA when assessed by immunohistochemistry. A typical example is shown in Fig. 5C. In contrast, although stromal cells of benign ovarian tumors were reac tive with the uPA antibody, epithelial cells failed to react (Fig. 5B). To demonstrate the specificity of mAb 3689, uPA-rich kidney tissue specimens were also stained with mAb 3689 (Fig. 5A). As expected, mAb 3689 intensively stained the renal tubule cells, whereas the glomeruli were negative. Tumor cells of primary advanced ovarian carcinomas were also found to be positive for uPA-R (mAb 3639; American Diagnostica) and PAI-1 (mAb 3785; American Diagnos tica; data not shown). As for uPA, stromal cells of different origin were found to react with mAbs to uPA-R or PAI-1. 0.1 0,01 Primary Tumor Omentum Metástasis Fig. 1. uPA antigen in tissue extracts of primary tumor and omentum metastasis. Paired samples are displayed for each of the 34 patients with ovarian cancer stage FIGO lile or IV. Note that there is an increase for the uPA antigen concentration in omentum metastasis over primary tumor in 30 of 34 patients (P —0.0001). Discussion Thus far only little information is available concerning differences in the content of uPA, uPA-R, and the inhibitors PAI-1 and PAI-2 in of the ovarian cancer patients are depicted as paired samples in Fig. 2. As for uPA. the median uPA-R content in extracts of tumor-infiltrated lymph nodes was increased compared to primary tumors, but lacked statistical significance (P = 0.53). PAI-1. The median value of PAI-1 in extracts obtained from pri mary tumor tissues was 16.99 ng/mg protein; in the corresponding sites of métastases(tumor-infiltrated lymph nodes, 35.42 ng/mg pro tein; métastasesof the omentum majus, 34.14 ng/mg protein), an approximately 2-fold increase over extracts of primary tumors was noted. The difference in the PAI-1 concentrations in extracts of primary tumors and omentum majus was statistically significant (P = 0.0009, Table 2), in contrast to only an insignificant increase in extracts of tumor-infiltrated lymph nodes (P = 0.11). The PAI-1 concentration in primary tumor tissue and the corresponding one in omentum metastasis tissue are depicted as paired samples for each of the patients with ovarian cancer FIGO lile or IV in Fig. 3. PAI-2. In extracts obtained from primary tumor tissues of patients with advanced ovarian carcinoma, a low PAI-2 antigen content was determined and reflected by a median value of 0.91 ng/mg protein. In extracts of tumor-infiltrated omentum majus, the median PAI-2 con centration was approximately 7-fold higher than in extracts of primary tumors. This difference was statistically significant (P = 0.004, Table 2). Corresponding values for PAI-2 in primary tumor and omentum metastasis from each of the 33 ovarian cancer patients (FIGO lile or IV) are depicted as paired samples in Fig. 4. PAI-2 antigen measure ments in extracts of tumor-infiltrated retroperitoneal lymph nodes and primary tumors did not reveal significant differences (P = 0.82). No statistical correlation was noticed among uPA, uPA-R, PAI-1, and PAI-2 content, neither in the primary tumor nor in the métastases. The concentrations of uPA, uPA-R, and PAI-1 extracts from pri mary tumor tissue of patients with advanced ovarian cancer FIGO lile i Q. CO I Primary Tumor Omentum Metastasis Fig. 2. uPA-R antigen in tissue extracts of primary tumor and omentum metastasis. Paired samples are diplayed from each of the 33 patients with ovarian cancer stage FIGO lile or IV. Note that there is an increase for the uPA-R antigen concentration in omentum metastasis over primary tumor in 20 of 33 patients (P = 0.05). 3960 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research. uPA. uPA-R, AND PAI-1/2 IN PRIMARY TUMOR AND METASTASIS 1000-1 fine-tuned balance among uPA, uPA-R, and PAI-1/2 might be impli cated. With respect to PAI-2, a significant increase was observed in cystic fluids of patients with malignant ovarian tumors compared to benign tumors (17). In ascites of patients with ovarian cancer, PAI-2 was found to be an independent factor indicating poor prognosis; however, measurements in tumor tissues were not performed (18). PAI-2 levels are also elevated in tissue extracts from breast carcino mas compared to benign breast tumors, although this increase is of no clinical relevance (19). We observed only low expression of PAI-2 in 100- extracts of primary tumors of ovarian carcinoma and of benign tissue with almost comparable concentrations. These data are in good ac cordance with data from colon and breast carcinoma (18-20). 1CH 100 10 0,1 Primary Tumor Omentum Metastasis Fig. 3. PAI-I antigen in tissue extracts of primary tumor and omentum metastasis. Paired samples are displayed from each of the 33 patients with ovarian cancer stage FIGO IIIc or IV. Note that there is an increase for the PAI-1 antigen concentration in omentum metastasis over primary tumor in 26 of 33 patients (P = 0.0009). i ^ ÛL primary versus secondary ovarian carcinomas. Comparing the plasminogen activator activity in tissue extracts from primary tumors and metastatic ovarian carcinomas as early as 1987, it has been shown that this activity was mainly due to the action of uPA; however, a repre sentative comparison of uPA activity in primary tumor versus métas tases has not been reported (14). Information on PAI-I and PAI-2 antigen levels in primary tumor versus metastasis is lacking in ovarian cancer. In breast carcinoma, significantly elevated PAI-1 levels in métastaseshave been reported (4). These findings are consistent with our data on ovarian carcinoma. However, the uPA concentration in lymph node métastasesof breast tumors was not significantly altered compared to its content in primary tumors. Similar data have been reported for colon cancer showing no increase in uPA activity in contrast to increased PAI-1 levels in liver metastasis (7). Increased levels of PAI-1 and also PAI-2 at the site of métastasesmight locally down-regulate uPA activity, thus favoring the process of tumor cell reimplantation and the formation of a new tumor stroma. In the present study on ovarian carcinoma, we found both uPA and PAI-1/2 levels elevated at the sites of metastasis. A possible explanation for the simultaneous increase of the protease uPA as well as its inhibitors in métastasesof ovarian versus breast and colon carcinomas is that in ovarian cancer a different type of tumor growth and metastasis is displayed compared to other cancers. In breast and colon carcinomas, tumor cells mainly spread hematogenously and/or lymphogenously, a process in which the formation of tumor cell-loaded microthrombi in small vessels and the consecutive generation of a new tumor stroma arising from the tumor cells within the thrombus may play a role. According to this so-called "microthrombus theory," an increase in fibrinolytic activity via the action of uPA in the tumor cell vicinity might prevent metastasis (16). In contrast, in ovarian cancer, tumor cells spread on and invade the peritoneum, a process in which a 0,1 0,01 Primary Tumor Omentum Metastasis Fig. 4. PAI-2 antigen in tissue extracts of primary tumors and omentum metastasis. Paired samples are displayed from each of the 33 patients with ovarian cancer stage FIGO Mie or IV. Note that there is an increase for the PAI-2 antigen concentration in omentum metastasis over primary tumor in 24 of 33 patients (P = 0.004). Table 3 Comparison of am.of tif'A. uPA-R. PAI-1. malignantAntigen(ng/mg benign and protein)uPA(median levelsovarian PAI-2 antigen tumorsOvarian extractsStatisticalsignificance(P)0.0 tissue cancer(Primary ovariantumors tumor;,1 21)"0.31(0.01-2.54)1.63(0.41-2.83)4.50(1.69-15.30)0.18(0.01-2.47)/ (n = .39)"0.91(0.05-10.16)2.87(0.55-8.66)16.99(0.14-1(15.1)0.2 = range)uPA-R(median range)PAI-1(median range)PAI-2(median range)" patients.* ;i, number of 38.' n = n.s., not significant.Benign 3961 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research. ul'A. iil'A-R. ANI) PAl-l/a IN PRIMARY TUMOR AND Ml-TASTASIS In the present study an increase in the uPA-R concentration in tumor-infiltrated omcntum and lymph nodes of advanced ovarian tumor stages has been observed. In primary colonie carcinomas and their métastasesin the liver, an increased presence of uPA-R has been found, too (21). For colon carcinomas it has been demonstrated that a high content of uPA-R in tissue extracts correlated with a poor prognosis for the patients (22). For ovarian carcinomas it remains to be established whether the amount of uPA-R correlates with Ihe progression of tumor growth and malignancy. Interestingly, in ascitic fluid, but also in tumor tissue and blood, a soluble, ligand-free form of uPA-R has been detected in invasive carcinomas. uPA-R may derive from the tumor via a phospholipase and/or proteascs-mediated shedding of cell surface-attached uPA-R (23). High concentrations of uPA, uPA-R, and PAI-1 in malignant ovar ian tumor tissue reflect an increased expression of these factors in tumor cells or nonmalignant cells of the tumor stroma. In the present investigation on protcolytic factors in advanced ovarian cancer, by using the same mAbs (3689 and 3639) to uPA and uPA-R, respec tively, we could confirm the results by Young et al. (24) that both tumor cells and stromal cells in fact react with the respective anti bodies. Moreover, our immunohistochemical results support the as sumption that ovarian cancer cells not only contain uPA and uPA-R but also the inhibitor PAI-1. However, it is still an open question whether the process of metas tasis might depend on the selection of tumor cell clones with increased expression of plasminogen activators, or whether the synthesis of uPA and PAI-1/2 is due to regulatory mechanisms or malignant transfor mation. In several metastasis models, it has been shown that primary tumors display a heterogeneity with respect to their metastatic poten tial (25). The occurrence of several types of ovarian carcinomas eliciting different aggressive potential has been reported: in patients with tumor stage FIGO IV accompanied by extensive métastasesa smaller intraabdominal tumor mass had frequently been observed compared to patients with tumor stage FIGO III, although they had a comparable rctropcritoneal lymph node involvement. These observa tions might be related to differences in the expression of plasminogen activators and their inhibitors. Thus far only little information is available about the different patterns of protein expression in primary tumors versus métastasesduring the progression of cancer. In a recent publication on breast cancer, it has been reported that the CD44 variant expression was increased by approximately 15-35% in lymph node métastasescompared to primary tumors of breast cancer patients (26). Likewise, elevated levels of uPA, its inhibitors, and/or uPA-R at sites of metastasis may contribute to the spread and invasion of ovarian cancer cells and their capability to reimplant in the perito neum. The elucidation of the concerted action of the different com ponents of the plasmin/plasminogen activator system and their inter dependence involved in tumor metastasis will help to further the understanding of tumor progression and the promotion of malignancy in ovarian carcinoma. Fig. 5. A. immunohistochemical staining of uPA in formalin-fixed, paraffin-embedded human kidney. The region showed displays the glomerulus and Ihe tubules oÃ-the cortical area. The glomerulus is devoid of uPA staining, in contrast, the cytoplasm of the epithelial cells of the renal tubules shows a strong positive reaction with mAh 3689. uPA staining in red (APAAP); nuclei (light blue) are stained with hemaloxylin. lì,immunohistochem ical staining of uPA in formalin-fixed, paraffin-embedded benign cystadenoma of the mucinous type. The epithelial layer (Ã-Ã-rrmv/jtW.v) does not react with mAb 36S9, whereas stronial cells are found to be positive. uPA staining in red (APAAP); nuclei (light blue) arc stained with hematoxylin. C. immunohistochemical staining of uPA in formalin-fixed, paraffin-embedded ovarian carcinoma (primary tumor, FIGO Mie) of the mucinous type. Both tumor cells (intense red cytoplasmic slain) and occasionally stroma cells (light red) react with mAb 36X9. uPA staining in red (APAAP); nuclei (light blue) are stained with hematoxylin. A-C, X 2(K). Acknowledgments We thank Dr. Richard Hart (American Diagnostica, Greenwich, CT) for his generous support. The expert technical assistance of Erika Scdlac/ck. Brigitte Jaud-Münch. and Hildegard Seihold is acknowledged. References 1. Friedlander, M. L., and Dcmbo, A. J. Prognostic factors in ovarian cancer. Semin. Oncol., IK: 205-212, 19" I. 2. Schmid. M., Wilhelm, O.. Jänicke.F.. Magdolen. V.. Reuning. U., Ohi. II.. Moniwa, N.. Kobayashi, II.. Weidle, U., and Graeff, H. Urokinase-type plasminogen activator (uPA) and its receptor (C'DK7). A new target in tumor invasion and metastasis. J. Obstet. Gynaecol. (Basingslokc), 21: 151-165, 1995. 3. Graeff, H. Tumorbiologie-orientierte Bchandlungskon/epte in der gynäkologischen 3962 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research. ul'A. iiPA-R. ANI) PAI-]/: IN PRIMARY TUMOR AND MlilASIASIS Onkologie, Gcburlsh. Fraucnhcilkd., .5.5;MI7-M19. 1995. Jánicke. F., Schmilt. M.. Hafter. R.. 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