MONITORING OF CHEMOTHERAPY SUCCESSFUL-NESS OF PLATINA / TAXOL CHEMOTHERAPY PROTOCOL BY USING DETERMINATION OF SERUM UROKINASE PLASMINOGEN ACTIVATOR ( UPA ) AND SOLUBLE UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR ( SUPAR ) IN PATIENTS WITH OVARIAN CARCINOMA FIGO II AND III STAGE

In about  of cases, ovarian carcinoma has been diagnosed at an advanced stage. Invasion and metastasis of solid tumors request protease activity resulting in basal membrane destruction and surrounding matrix. In that process, urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (suPAR) play a key role, that via plasmin activation lead to basal membrane and matrix degradation in surrounding of the tumor, enable to its invasion and metastasis. Determination of serum concentration of those tumor markers can be useful in preoperative as well as in postoperative period. Th eir serum concentrations in ovarian cancer patients may help in good monitoring of remission or progression during chemotherapy treatment. In late s and eary s, when it was found out that malignant ovarian tumors were chemosensitive, their chemotherapy treatment has begun. In the beginning it was used only mono-therapy, and by discovering new cytostatics it was replaced by poly-chemotherapy. Now days, in the therapy of advanced stages of ovarian carcinoma combination of cisplatine or carboplatine with paclitaxel is considering as standard treatment. Aim of this study was to determine serum uPA, suPAR and CEA in FIGO II and III patients with diff erent histological type (serous, mucinous, clear cell tumor) before and after PT chemotherapy protocol during following three cycles. In this prospective study we have analyzed  patients with ovarian carcinoma, those have been after surgery treated by chemotherapy. Serum levels of uPA and suPAR have been determined by ELISA-test (Imubind uPA, Imubind uPAR, American Diagnostica), and CEA by OPUS Imunoassay method. Results of this study have shown that uPA, suPAR and CEA met criteria for prognostic markers for monitoring of successfulness of platina/taxol chemotherapy protocol for serous, mucinous and clear cell tumor FIGO II and III stage of ovarian carcinoma. In case of PT chemotherapy protocol suPAR was better prognostic marker for monitoring of chemotherapy successfulness (Pearson coeffi cient , do ,; p<,l) than uPA (Pearson coeffi cient between , and ,; p<,) and CEA (Pearson coeffi cient , do ,; p<,).


Introduction
Ovarian carcinomas make  out of all malignant tumors in women, and as a death cause in the same population are on th place ().Despite aggressive treatment applying late years, a fi ve-year survival rate is se  ().However, a fi ve-year survival rate has been signifi cantly increasing (about ), even disease is discovered and treated in early stage ().Malignant ovarian epithelial tumors consist of  out of all malignant ovarian tumors ().In most of the patients malignant ovarian tumors has been diagnosed at advanced stage (), because of anatomic position of ovarium and poor clinical symptoms.Just  out of  well-known enzymes are useful in diagnostics and monitoring of development of ovarian carcinomas.Plasmin is an enzyme which is formed by cleavage of plasminogen and plays important role in proteolytic degradation surrounding intercellular matrix, allowing invasion and metastasis.Over few late years, it has been found an association between urokinase plasminogen activator (uPA) and its soluble receptors (suPAR) and development of diff erent malignant tumors, among them ovarian carcinomas (, , , , ).Beside in preoperative diagnostics of malignant tumors, uPA and suPAR may be determined in postoperative diagnostics and be useful in monitoring of remission and progression of the disease, and successfulness of certain chemotherapy protocol, as well.In late s and s, when it has been found that malignant tumors of the ovarium are chemosensitive, the application of chemotherapy had been began.In the beginning, just mono-chemotherapy has been applied.After discovering new cytostatics, mono-therapy has been replaced by poly-chemotherapy.In late s it has been discovered very powerful drug platina, which is today the base of all successful protocols.Successfulness of the treatment by chemotherapy depends on administered dose ().Th ere are a numerous chemotherapy protocols that have diff erent response rate with adverse eff ects such as alopecia, vomiting, myelosuppression, nephrotoxicity and etc.Standard chemotherapy application is cyclic repeating every - days, in order to recovering immunobiological system of the body.U patients with early stage of ovarian carcinoma the chemotherapy protocol is based on platina.In the treatment of advanced stage of ovarian carcinoma combination of cisplatina or carboplatina with paclitaxel is considered as standard treatment (, ).Aim of this study was to determine serum uPA, suPAR and CEA in FIGO II and III patients with diff erent histological type (serous, mucinous, clear cell tumor) before and after PT chemotherapy protocol during following three cycles.

Patients and Methods
In this prospective study we have analyzed  patients with ovarian carcinoma, those have been after surgery treated by chemotherapy and intraoperatively staged according FIGO classifi cation.Serum levels of uPA and suPAR have been determined by ELISA-test (Imubind uPA, Imubind uPAR, American Diagnostica), and CEA by OPUS Imunoassay method.Eight out of  patients were FIGO II, and nine were FIGO III stage.According to histological type nine of them had mucinous, fi ve clear cell tumor and three serous type.Chemotherapy was administered every three weeks in one dose (Platina  mg/m) and (Taxol  mg/m  ).Biological material was serum taken from the patients immediately before the application of certain chemotherapy cycle and always under same conditions by experienced medical staff in the morning between  and  am.Blood sample  ml plus , ml Na-citrate was immediately centrifuged at  rpm in  min at room temperature.Th e samples were preserved at - o C until use.In this study were included just those patients who had toxicity rate less then .All patients were introduced about this study and they agreed with it.Control group make patients prior chemotherapy application, and experimental one the same patients after chemotherapy application.In serum of those patients we have determined level of uPA, suPAR and CEA during three following chemotherapy cycles.Process of remission or progression for measurable and immeasurable lesions has been monitored according definition of objective chemotherapy successfulness (Table ) by CT (Toshiba  HQ; SOMATOM AR STAR-Siemens), trans-vaginal ultrasound (Toshiba

Conclusion
Analyzing serum level of tested markers in patients after PT chemotherapy application and comparing them with histological type and FIGO stage it has been found that suPAR and uPA are good indicators of successfulness of this chemotherapy protocol for serous, mucinous and clear cell tumor FIGO II and FIGO III, and CEA for mucinous type.

TABLE 1 .
Defi nitions of objective successfulness of chemotherapy DŽENITA LJUCA ET AL.: MONITORING OF CHEMOTHERAPY SUCCESSFULNESS OF PLATINA/TAXOL CHEMOTHERAPY PROTOCOL BY USING DETERMINATION OF SERUM UROKINASE PLASMINOGEN ACTIVATOR UPA AND SOLUBLE UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR SUPAR IN PATIENTS WITH OVARIAN CARCINOMA FIGO II AND III STAGE Sonolyer-L SAL- B) and laboratory fi ndings (Whole blood, ALT, AST, creatinine, urea, urine and ERS).Patients according to age have been divided into fi ve groups: <, -, -, ¬- and > years.Urokinase plasminogen activator (uPA) has been determined in serum by ELISA kit Imubind uPA  (American Diagnostica, Greenwich, CT, USA).Th is test is very sensitive and allows possibility for detection of uPA even very low level such as  pg/ml.Serum level of uPA has been determined by BIORAD MICROPLATE ME-NAGER with program IZODATA.Serum level of uPA is quantifi ed by measuring of level of absorption at  nm and comparing with values from standard curve.urokinase plasminogen activator receptor-suPAR has been determined by ELISA kit Imubind suPAR  (American Diagnostica, Greenwich, CT, USA).Th is test is very sensitive and allows possibility for detection of suPAR even very low level , ng/ml.Data have been analyzed by Student's t-test and Pearson's correlation test.LJUCA ET AL.: MONITORING OF CHEMOTHERAPY SUCCESSFULNESS OF PLATINA/TAXOL CHEMOTHERAPY PROTOCOL BY USING DETERMINATION OF SERUM UROKINASE PLASMINOGEN ACTIVATOR UPA AND SOLUBLE UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR SUPAR IN PATIENTS WITH OVARIAN CARCINOMA FIGO II AND III STAGE LJUCA ET AL.: MONITORING OF CHEMOTHERAPY SUCCESSFULNESS OF PLATINA/TAXOL CHEMOTHERAPY PROTOCOL BY USING DETERMINATION OF SERUM UROKINASE PLASMINOGEN ACTIVATOR UPA AND SOLUBLE UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR SUPAR IN PATIENTS WITH OVARIAN CARCINOMA FIGO II AND III STAGE We have found suPAR to be better prognostic marker for successfulness than uPA in patients treated by PT chemotherapy protocol (Pearson's coeffi cient between , and ,; p<,) comparing to uPA (Pearson's coefficient between , and ,; p<,) and CEA (Pearson's coefficient between , and ,; p<,).
ResultsIn this study  patients ( FIGO II and  FIGO III stage) who had pathohistologicaly verified ovarian carcinoma and who have been treated by Platina/ Taxol chemotherapy protocol after surgery.In serum of the patients with these stages, mucinous type, it has been found statistically significant difference between level of uPA after (, ±, ng/ml) Platinal/Taxol chemotherapy application for certain cycles comparing with level of uPA prior to application (,±, ng/ml) (t-test-,; p<,) (Figure).Decrease uPA serum level of the patients with FIGO IIc mucinous type (Figure) after PT chemotherapy protocol application (,±, ng/ml) was statisti-cally significant comparing with that one prior to application (,±, ng/ml) (t-test: ,; p<,).In serum of the patients with FIGO IIc stage type clear cell tumor (Figure) it has been found statistically signifi cant decrease of uPA level after application of chemotherapy PT protocol (,±, ng/mL) comparing control group (,±, ng/ml) (t-test: ,; p<,).condition of successfulness of chemotherapy, it can be said that PT chemotherapy application for mucinous and clear cell tumor was completely successful.Analyzing serum uPA levels after PT chemotherapy protocol and comparing them in patients with different histological types, we have noticed that uPA DŽENITA is good indicator for monitoring of successfulness of chemotherapy protocol for mucinous and clear cell tumor (Pearson's coeffi cient ,-,) (Figure ).DŽENITA

TABLE 2 .
Correlation serum level of uPA, suPAR and CEA with FIGO stage and hystological type of ovarian carcinoma in patients treated by PT chemotherapy protocol