Primary Tumor and Metastasis in Ovarian Cancer Differ in Their Content of Urokinase-Type Plasminogen Activator, Its Receptor, and Inhibitors Types 1 and 21

[C'ANC'ER K|-:S|-:AKC|| SS. .Â«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. KÃ¼hn,U. Reuning, L. Pache, P. Dettmar, M. Se-limiti, F. JÃ¤nicke, H. HÃ¶fler,and H. Graeff

Frauenklinik der Technischen UniversitÃ¤t MÃ¼nchen. Klinikum rechts tier Isar. Ismaninger Strasse 22. D-KI675. Munich Â¡B.S.. W. K., U. R., L P.. M. S., F. J., H. G.I. and Institut fÃ¼rAllgemeine Pathologie und Pathologische Anatomie, Technische UniversitÃ¤t MÃ¼nchenÂ¡P.D., H. H. I, Munich, Germany

Abstract metastasis is controlled by a series of successive events. After local proteolysis of the surrounding extracellular matrix, detachment and The relevance of urokinase-type plasminoceli activator (uPA) and plas- spread of tumor cells is facilitated followed by the invasion of the minogen activator inhibitor (PAI) type I in predicting the survival prob adjacent tissue, crossing of tissue boundaries and the basement mem ability of patients with advanced ovarian cancer after radical surgery and adjuvant chemotherapy by assessing the patients' primary tumors has brane. Intravasation and extravasation are followed by reimplantation recently been shown by us (W. KÃ¼hnelal., Gynecol. Oncol., 55: 401-409, of tumor cells into the respective target organ, remodeling of a new 1994). In the present study, we determined uPA, uPA receptor, PAI-1, and tumor stroma, and angiogenesis in order to consolidate a secondary PAI-2 concentrations in primary tumors and tumor-infiltrated omentum 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 FÃ©dÃ©ra tion Internationale de GynÃ©cologieet d'ObstÃ©triqueil-Ã•GOiHic or IV; for tors, oncoproteins, and growth factors has to be adjusted (2, 3). Basic and clinical research in tumor invasion and metastasis have comparison 7 patients with early carcinoma stage FIGO I were also focused on the role of tumor-associated proteases such as metallopro- included. In mÃ©tastasesof the omentum from ovarian cancer stage FIGO lile or IV patients, we noted a 4-fold elevated uPA content, a 2-fold teinases and the serine proteases of the plasmin/plasminogen activator increase in PAI-I, and also a significant increase in uPA receptor and system (2). Metalloproteinases seem to be correlated with tumor cell PAI-2 over primary tumors. In mÃ©tastasesof the lymph nodes the levels of invasion and metastasis in animal models; however, conclusive data the respective antigens were also increased when compared to primary on their clinical significance in humans are still lacking. In contrast, tumors. These data may indicate that elevated levels of components of the for several different solid cancers a strong prognostic impact of the fihrinolytic system at sites of mÃ©tastasesmay contribute to the aggressive scrine protease uPA1 and/or its inhibitor PAI-1 as predictors for the potential of cancer cells by favoring their rcimplantation and/or the course and the outcome of the cancer has been reported. Besides their consolidation of a new tumor stroma. statistically independent prognostic impact in cancer of the breast (4, Introduction 5), lung (6), colon (7), and the gastrointestinal tract (8), significantly elevated levels of both uPA and PAI-1 have been demonstrated in Ovarian carcinoma is one of the most severe gynecological malig ovarian cancer tissues compared to those in normal ovarian tissue nancies and because of a long asymptomatic course of the disease specimens. Patients with advanced ovarian cancer can be subgrouped displays already advanced stages at diagnosis with extensive cancer on the basis of computer-optimized "cutoff values for uPA and spread. This is reflected by the high incidence (70% of the cases) of PAI-1: patients with low uPA and PAI-1 (uPA < 0.9 ng/mg protein; mortality within 5 years, a fact that stresses the necessity of an early PAI-1 < 13.5 ng/mg protein) had a statistically better prognosis than diagnosis for risk selection and the prediction of the course of the patients with high uPA and PAI-1 antigen levels (9). First indications disease. Several investigators have demonstrated that the prognosis of for a role of the uPA-mediated proteolytic system in tumor invasion patients with advanced ovarian cancer could be improved by radical and metastasis came in 1976 from Astedt and Holmberg (10), who surgery followed by adjuvant chemotherapy. Although the absence of demonstrated for the first time high uPA concentrations in cultured residual tumor after cytoreductive surgery proved to be a strong ovarian carcinoma tissue. In the following years increased uPA and prognostic factor for survival, the complete resection of the tumor PAI-1 levels were also found in tumor tissue extracts of patients with mass was not sufficient to protect all patients from the recurrence of ovarian carcinoma (11, 12) and in malignant ascites (13). the disease and subsequent death. Several attempts have been under uPA is synthesized and secreted by normal and tumor cells. Both taken to improve the prognosis of cancer patients and to establish the inactive proenzyme form of uPA, pro-uPA, and protcolytically predictive factors that are related to tumor biology. Numerous factors active uPA bind to a specific glycanlipid-anchored receptor (uPA-R, have been screened, among them the receptors for estrogen, proges CD 87) on the tumor cell surface. Upon binding of pro-uPA, its terone, and epidermal growth factors, DNA ploidy, S-phase, and activation by other proteases (e.g., plasmin) is facilitated. uPA acti oncogenes ( 1). In recent years special attention has been paid to tumor cell surface- vates the proenzyme plasminogcn into the broad spectrum serine protease plasmin, which may in turn either directly degrade extracel associated proteolytic enzyme systems regarding their impact on lular matrix proteins such as fibrin, fibronectin, laminin, and proteo- cancer progression and metastasis. The process of tumor invasion and glycans or activates certain matrix-degrading enzymes such as pro- collagenase. The activation of uPA is controlled by its specific- Received ft/15/95; accepted 8/4/95. inhibitors PAI-1 and PAI-2. Binding of PAI-1 or PAI-2 to uPA-R- 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 bound uPA results in the subsequent internalization of the ternary 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). 1The abbreviations used are: uPA. urokinase-type plasminogcn activator; PAI, plas- : To whom requests for reprints should be addressed. minogen activator inhibitor; uPA-R. uPA receptor. 3958

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1995 American Association for Cancer Research. iil'A. iil'A-R. AND PAI-1/2 IN PRIMARY TUMOR AND MUTASI ASÃ•S complex, thereby regulating cell surface plasmin generation. uPA-R minorversusTable 2 Owi/ximwi ofiiPA. uPA-R. PAI-1.anil PAI-2 utilicen /nr/.v//( primary IVIAntigen(ng/mgtuimir-injiliratal iimenlum nuijus in iul\tinced tmirian cunÃ¯mmii Ãr'lÃiOlile Â¡ir in combination with specific plasmin(ogen) receptors focalizes the FIGOIlle/IVtumor metastasis(Â« plasmin/plasminogen activator system to the tumor cell surface, thus (11=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=33)"3.65*(0.66-16.58)4.3o(1.15-8.71)34.14(4.60-125.3)1.72(0.01-12.39)Statisticalsignificance protein)uPA(median (P)II.IKIOI0.050.00090.1104 constituting an effective proteolytic enzyme system (2). Tumor-associated proteolytic enzyme systems are not only impor range)uPA-R(median tant for tumor cell spread from the primary tumor but seem to be crucial parameters for the events at the site of mÃ©tastases.In this report we investigated the levels of uPA, uPA-R, PAI-1, and PAI-2 in range)PAI-1(median tissue extracts of primary tumors, tumor-infiltrated omentum, and tumor-infiltrated lymph nodes of patients suffering from advanced range)PAI-2(median ovarian carcinoma. range)Primary Patients and Methods " il. number of patients. Forty-six patients with early (FIGO I) or advanced ovarian cancer stages '' n = 34. ' n = 38. (FIGO lile or IV) were enrolled as par! of a prospective study on ovarian cancer patients undertaken at the Frauenklinik der Technischen UniversitÃ¤t MÃ¼nchen(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 assisted analysis (EIA program; ICN-Flow Laboratories, Meckenheim. Ger advanced ovarian carcinoma received the following radical surgical treatment: many). Protein concentrations were determined using the BCA Protein Assay longitudinal laparotomy with hysterectomy, bilateral adenectomy. appendec Reagent kit (Pierce, Rockford, IL). Values for uPA, uPA-R, PAI-1, and PAI-2 tomy, infragastric omentectomy, and bilateral pelvic and paraaortic lymphad- concentrations are expressed as ng/mg protein of the tissue extracts. In our enectomy. Among the group of patients, seven patients received limited laboratory the interassay variation coefficient for uPA was 5.8%, for uPA-R surgical treatment because of their had health condition. In younger patients 5.6%, for PAI-1 4.0%, and for PAI-2 1.4%, thus fulfilling the criteria for a (<35 years) with tumor stage FIGO I. less radical surgery was performed in reliable test. Immunohistochemical staining for uPA, its receptor (uPA-R), and order to preserve the fertility of the patients. HistolÃ³gica! examinations of all the inhibitor PAI-1 was performed as described in detail by Janicke el al. (4). of the tumor tissue sections were performed at the Institut fÃ¼rAllgemeine The following antibodies supplied by American Diagnostica were used at 10 Pathologie und Pathologische Anatomie, Technische UniversitÃ¤t MÃ¼nchen /ng/ml: uPA (mAb 3689), uPA-R (mAb 3639), and PAI-1 (mAb 3785). (Munich, Germany). Benign ovarian tumors (n = 21), e.g., benign ovarian cysts, serous cystadenoma, or cystadenof'ibroma served as controls. Statistical Analyses. All results are given as median values. Antigen determinations were performed in duplicate. The significance level of differ Tissue Collection and Extraction. Ovarian cancer tissue specimens from ences between the antigen levels in cancer tissue extracts versus controls was primary tumors, omentum majus, and lymph nodes were collected during evaluated using the Mann-Whitney (/ test. Differences in antigen concentra surgery, classified by the pathologist, and stored in liquid nitrogen until used. tions between specimens from the same patient were tested for significance Dcep-fro/.en specimens of 200-500 mg wet weight were pulverized (30 s) using the Wilcoxon test for combined samples (5). Differences were consid using the Micro-Dismembrator (Braun, Melsungen, Germany) set to maximum ered to be significant when P < 0.05. 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, Results Germany), and extracted at 4Â°Cfor 12 h followed by an ultracentrifugation step (1()0,(XK) x K, 45 min, 4Â°C)to separate cell debris. Due to our recent findings that both uPA and PAI-1 may serve as Laboratory Assay Systems. uPA, uPA-R, PAI-1, and PAI-2 concentra predictors for survival in patients with advanced ovarian carcinoma tions were determined by using the commercially available ELISA kits Imu- (9), we determined in the present study the content of uPA, uPA-R, bind 821, 822, 823, and 893, respectively (American Diagnostica, Greenwich. PAI-1, and PAI-2 in primary tumors and typical sites of metastasis in CT; Refs. 1, 10, 12, and 14). The data obtained were subjected to computcr- ovarian 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;?, Ãiisl(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

VariableMedian ELISA. The important new findings in this study can be summarized (range)StageFIGOage yr (20-85)72415284595II23713267322597as follows. 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 IVHistologySerousMucinousrindomctroidUnditÃ¯erenliatcdGradingGlG2G3G4Nodal In extracts of tumor-infiltrated omentum majus, the uPA content was approximately 4-fold higher than in primary tumors exhibiting statis tical significance (P = 0.0001, Table 2). The corresponding values for 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 statusNilNlNXLvmphadenectomyPelvicParaaorticPelvic 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 andparaaorticSamplingNone60 significant (P = 0.05, Table 2). Values for corresponding uPA-R concentrations in primary tumor and omentum metastasis from each 3959

100 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 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 0.1 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 0,01 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 Primary Omentum tica; data not shown). As for uPA, stromal cells of different origin Tumor MetÃ¡stasis were found to react with mAbs to uPA-R or PAI-1. 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 Discussion IV. Note that there is an increase for the uPA antigen concentration in omentum metastasis over primary tumor in 30 of 34 patients (P â€”0.0001). 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 mÃ©tastases(tumor-infiltrated lymph nodes, 35.42 ng/mg pro tein; mÃ©tastasesof 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 iQ. primary tumors and omentum majus was statistically significant (P = 0.0009, Table 2), in contrast to only an insignificant increase in CO extracts of tumor-infiltrated lymph nodes (P = 0.11). The PAI-1 concentration in primary tumor tissue and the corresponding one in I 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 Primary Omentum statistical correlation was noticed among uPA, uPA-R, PAI-1, and Tumor Metastasis PAI-2 content, neither in the primary tumor nor in the mÃ©tastases. 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 The concentrations of uPA, uPA-R, and PAI-1 extracts from pri lile or IV. Note that there is an increase for the uPA-R antigen concentration in omentum mary tumor tissue of patients with advanced ovarian cancer FIGO lile metastasis over primary tumor in 20 of 33 patients (P = 0.05). 3960

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). 100- 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 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

0,1 Primary Omentum Tumor 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 i 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). Ã›L primary versus secondary ovarian carcinomas. Comparing the plas- 0,1 minogen 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 mÃ©tas tases has not been reported (14). Information on PAI-I and PAI-2 antigen levels in primary tumor versus metastasis is lacking in ovarian 0,01 cancer. In breast carcinoma, significantly elevated PAI-1 levels in mÃ©tastaseshave been reported (4). These findings are consistent with Primary Omentum our data on ovarian carcinoma. However, the uPA concentration in Tumor Metastasis lymph node mÃ©tastasesof breast tumors was not significantly altered Fig. 4. PAI-2 antigen in tissue extracts of primary tumors and omentum metastasis. compared to its content in primary tumors. Similar data have been Paired samples are displayed from each of the 33 patients with ovarian cancer stage FIGO reported for colon cancer showing no increase in uPA activity in Mie or IV. Note that there is an increase for the PAI-2 antigen concentration in omentum contrast to increased PAI-1 levels in liver metastasis (7). Increased metastasis over primary tumor in 24 of 33 patients (P = 0.004). levels of PAI-1 and also PAI-2 at the site of mÃ©tastasesmight locally down-regulate uPA activity, thus favoring the process of tumor cell Table 3 Comparison ofam.of tif'A. uPA-R. PAI-1. levelsovarianPAI-2 antigen tissueextractsStatisticalsignificance(P)0.00010.0001O.IXHMn.s.' reimplantation and the formation of a new tumor stroma. In the malignantAntigen(ng/mg benign and tumorsOvarian present study on ovarian carcinoma, we found both uPA and PAI-1/2 cancer(Primary levels elevated at the sites of metastasis. A possible explanation for ovariantumors tumor;,1 protein)uPA(median (n =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)/=.39)"0.91(0.05-10.16)2.87(0.55-8.66)16.99(0.14-1(15.1)0.25*(0.01-11.34)in the simultaneous increase of the protease uPA as well as its inhibitors

in mÃ©tastasesof ovarian versus breast and colon carcinomas is that in range)uPA-R(median ovarian cancer a different type of tumor growth and metastasis is displayed compared to other cancers. In breast and colon carcinomas, range)PAI-1(median 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 range)PAI-2(median arising from the tumor cells within the thrombus may play a role. According to this so-called "microthrombus theory," an increase in range)"

fibrinolytic activity via the action of uPA in the tumor cell vicinity patients.*;i, number of might prevent metastasis (16). In contrast, in ovarian cancer, tumor n =38.' cells spread on and invade the peritoneum, a process in which a n.s., not significant.Benign 3961

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 mÃ©tastasesin 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 mÃ©tastasesa 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 mÃ©tastasesduring 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 mÃ©tastasescompared 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.

Acknowledgments Fig. 5. A. immunohistochemical staining of uPA in formalin-fixed, paraffin-embedded human kidney. The region showed displays the glomerulus and Ihe tubules oÃthe cortical We thank Dr. Richard Hart (American Diagnostica, Greenwich, CT) for his 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 generous support. The expert technical assistance of Erika Scdlac/ck. Brigitte in red (APAAP); nuclei (light blue) are stained with hemaloxylin. lÃ¬,immunohistochem Jaud-MÃ¼nch.and Hildegard Seihold is acknowledged. ical staining of uPA in formalin-fixed, paraffin-embedded benign cystadenoma of the mucinous type. The epithelial layer (ÃÃrrmv/jtW.v)doesnot react with mAb 36S9, whereas References 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, 1. Friedlander, M. L., and Dcmbo, A. J. Prognostic factors in ovarian cancer. Semin. paraffin-embedded ovarian carcinoma (primary tumor, FIGO Mie) of the mucinous type. Oncol., IK: 205-212, 19" I. Both tumor cells (intense red cytoplasmic slain) and occasionally stroma cells (light red) 2. Schmid. M., Wilhelm, O.. JÃ¤nicke.F.. Magdolen. V.. Reuning. U., Ohi. II.. Moniwa, react with mAb 36X9. uPA staining in red (APAAP); nuclei (light blue) are stained with N.. Kobayashi, II.. Weidle, U., and Graeff, H. Urokinase-type plasminogen activator hematoxylin. A-C, X 2(K). (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 gynÃ¤kologischen 3962

Onkologie, Gcburlsh. Fraucnhcilkd., .5.5;MI7-M19. 1995. terology. 101: 1522-1528. 1991. JÃ¡nicke. F., Schmilt. M.. Hafter. R.. Hollrieder. A., Bahic. R., Ulm. K.. Gossner. W.. 16. Markus. G.. Camiolo. M.. Kohga. S.. Madeja. J. M., and Mittclman. A. Plasminogen and Ciracff. M. Urokinase-typc plasminogcn activator (uPA) antigen is a predictor of activator secretion of human tumors in short-term organ culture, including a com early relapse in breast cancer. Fibrinolvsis. 4: 69-78, WO. parison of primary and metastatic colon tumors. Cancer Res., 43: 5517-5525. 1983. JÃ¤nicke.F., Schmill. M.. Pache, L., Ulm. K., llarhcck. N.. HÃ¶fler,H., and Gracff. H. 17. Cassieri. B.. Bossmar. T.. Lecander. !.. and Asledt. B. Plasminogen activators and Urokinase (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors plasminogen activator inhibitors in Nun,I and tumor fluids of patients with ovarian in node-negative breast cancer. Breasl Cancer Res. Treat., 24: 195-208, 1993. cancer. Eur. J. Cancer. 3ÃœA:1302-1311, 1994. Chambers, S. K., Gertz, R. E., Ivins. C'. M.. and Kacinski. B. M. The significance of Pederscn. H.. (irondahl-Hansen. J.. Francis. I).. Osterlind. K- Hansen, H. H., DaÃ±o, 18. K., and BrÃ¼nner.N. Urokinase and plasminogen activator inhibitor type I in pulmo urokinase-type plasminogen activator, its inhibitors and its receptor in ascites of nary adenocarcinoma. Cancer Res., 54: 120-123, 1994. patients with epithelial ovarian cancer. Cancer (Phila.). 75: 1627-1633, 1995. Sier, C. F. M., Vloedgraven. J. M., Gancsh, S., Griffioen, G.. Ouax. P. H. A., 19. Foekens, J. A.. Buessecker, F., Peters, H. A.. Krainick. U.. van Putten. W. L. J., Look, Verheijen. J. J., Dooijewaard, (1.. Welvaart, K.. Van De Velde, C. J. H.. Lamcrs. M. P.. Klijn, J. (i. M., and Kramer, M. I). Plasminogen activator inhibilor-2: C".B. II.. and Verspaget. H. W. Inactive Urokinase and increased levels of its inhibitor prognostic relevance in 1012 patients with primary breast cancer. Cancer Res., 55: type 1 in colorÃ©ela)cancer liver metastasis. Gastroenterology, 107: 1449-1456. 1994. 1423-1427, 1995. Nekarda. H.. Siewert. J. R.. Schmilt. M., and Ulm, K. Tumor-associated proteolytic 20. Bouchet. C.. Spyratos, F., Martin. P. M.. Ilacene. K., Gentile. A., and Oglobine. J. factors uPA and PAI-1 and survival in totally resected gastric cancer. Lancet. 343: Prognostic value of urokinase-type plasminogen activator (uPA) and plasminogen 117, 1994. activator inhibitors PAI-1 and PAI-2 in breast carcinomas. Br. J. Cancer. 69: 398- KÃ¼hn.W.. Pache. L... Schmalleldl. B.. Dcttmar. P.. Schmilt. M.. JÃ¤nicke,F., and 405. 1994. GrÃ¡cil. H. Urokinasc (uPA) und PAI-1 predict survival in advanced ovarian cancer 21. Sier, C. F. M.. Ouax. P. H. A.. Vloedgraven. H. J. M.. Verheijen, J. J., Griffioen, G., palients (FIGO III) after radical surgery anil platinum-based chemotherapy. Gynccol. Ganesh, S.. Lamers C'.. B. H. W.. and Verspaget. H. W. Increased urokinase receptor Oncol., 55.- 401-409, 1994. levels in human gastrointestinal neoplasia related liver mÃ©tastases.Invasion Metas 10. Astedt. B.. anil Holmberg. L. Ininnmological identity ol urokinase and ovarian tasis, 13: 277-288. 1993. Gancsh. S., Sier, C. F.. Heerding. M. M.. Griffioen. G., (.amers. C".B.. and Verspaget, carcinoma plasminogcn activator released in tissue culture. Nature (I.ond.). 261: 595-547 m7(, H. W. Urnkinase receptor and colorectal cancer survival. Lancet, 344: 401-402, Pujade-Lauraine. E., Lu, IL. Mirshahi. S., Soria, J., Soria, C., Bernadou, A.. 1994. Kruilholf. F. K. O.. I.ijnen. 11. R.. and Burtin. P. The plasminogen-activation system 23. Pedersen. N.. Schmilt. M.. Ronne. E.. Nicolelli. M. L, Hoyer-Hansen. G.. Conese. M., in ovarian tumors. Int. J. Cancer, 55: 27-31. 1993. Giavazzi. R.. Dano. K.. KÃ¼hn.W.. Jiinickc. F., and Blasi. F. A ligand-ircc. soluble Berns. F.. M. J. J.. Klijn, J. Ci. M., Schmitt. M.. Hen/en-Utgmans, S. C., van der Burg. urokinase receptor is present in the ascitic tluid from patients with ovarian cancer. J. M. E. L., JÃ¤nicke. F., and Fockens J. A. Occurrence and interrelationships of Clin. Invest., 92: 2160-2167, 1993. urokinase-type plasminogcn activator, plasminogen activator inhibitor l and cathepsin 24. Young, T. N.. Rodriguez, G. C., Moscr, T. L., Basi, R. C.. Jr., Pizzo. S. V., and Stack, D in human ovarian tissues. Proc. Am. Assoc. Cancer Res.. 25: (XXI,1994. M. S. Coordinate expression of urinary-type plasminogen activator and its receptor Oh, Dâ€žHwang. S. G.. Hong. S. Y.. Suh, C. L. Lee, H. B., Kye, K. C., Park. S., and accompanies malignant transformation of the ovarian surface epithelium. Am. J. Kim. N. K. Fibrinolytic changes in malignant ascites. Fibrinolysis,

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B. Schmalfeldt, W. Kuhn, U. Reuning, et al.

Cancer Res 1995;55:3958-3963.

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