|Year : 2018 | Volume
| Issue : 3 | Page : 608-613
The clinical importance of serum urokinase plasminogen activator receptor and carbonic anhydrase IX levels and the effect of anthracycline-based adjuvant chemotherapy on these biomarkers in breast cancer
Turkan Ozturk Topcu1, Feyyaz Ozdemir1, Halil Kavgaci1, Meral Gunaldi2, Hakan Kocoglu2, Goksen Inanc Imamoglu3, Ahmet Mentese4, Serap Ozer Yaman4, Asim Orem4, Fazil Aydin1
1 Department of Medical Oncology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey
2 Department of Medical Oncology, Bakirkoy Dr. Sadi Konuk Education and Research Hospital, Istanbul, Turkey
3 Department of Medical Oncology, Diskapi Yildirim Beyazit Education and Research Hospital, Ankara, Turkey
4 Department of Biochemistry, School of Medicine, Karadeniz Technical University, Trabzon, Turkey
|Date of Web Publication||12-Jun-2018|
Dr. Turkan Ozturk Topcu
Department of Medical Oncology, School of Medicine, Karadeniz Technical University, Trabzon - 61080
Source of Support: None, Conflict of Interest: None
Introduction: Breast cancer mortality rates after metastasis is high. Urokinase plasminogen activator receptor (uPAR) and carbonic anhydrase IX (CAIX) play very important roles during tumor cell invasion and metastasis. The purpose of this study was to evaluate plasma levels of uPAR and CAIX and the effect of anthracycline-based chemotherapy on these biomarkers in patients with operable breast cancer.
Materials and Methods: Sixty-five patients and 25 age-matched healthy controls were enrolled. Levels of uPAR and CAIX were investigated before and after adjuvant chemotherapy. Basal (prechemotherapy) uPAR and CAIX levels in patients were compared with those in healthy controls and in patients after 3 cycles of chemotherapy. Levels of uPAR and CAIX were determined using the ELISA method.
Results: uPAR and CAIX levels were significantly higher in patients (P: 0.02 and P: 0.03, respectively). Postchemotherapy uPAR and CAIX levels were higher than basal levels (P: 0.645 and P < 0.001, respectively). A cut-off value of 27.99 pg/mL for uPAR was associated with 45.31% sensitivity and 84.62% specificity, and with a positive predictive value (PPV) of 87.9% and a negative predictive value (NPV) of 38.6%. A cut-off value of 777.84 pg/mL for CAIX was associated with 90.62% sensitivity and 30.77% specificity, and with a PPV of 76.3% and an NPV of 57.1%.
Conclusion: We determined that uPAR and CAIX levels were higher in the fluorouracil, epirubicin, and cyclophosphamide (FEC) chemotherapy group than in the control group, but there was no difference between the FEC and epirubicin/adriamycin chemotherapy groups in terms of basal and postchemotherapy uPAR, CAIX levels. Furthermore, uPAR is more specific, and CAIX is more sensitive in the diagnosis of breast cancer.
Keywords: Breast cancer, carbonic anhydrase IX, thrombosis, urokinase plasminogen activator
|How to cite this article:|
Topcu TO, Ozdemir F, Kavgaci H, Gunaldi M, Kocoglu H, Imamoglu GI, Mentese A, Yaman SO, Orem A, Aydin F. The clinical importance of serum urokinase plasminogen activator receptor and carbonic anhydrase IX levels and the effect of anthracycline-based adjuvant chemotherapy on these biomarkers in breast cancer. J Can Res Ther 2018;14:608-13
|How to cite this URL:|
Topcu TO, Ozdemir F, Kavgaci H, Gunaldi M, Kocoglu H, Imamoglu GI, Mentese A, Yaman SO, Orem A, Aydin F. The clinical importance of serum urokinase plasminogen activator receptor and carbonic anhydrase IX levels and the effect of anthracycline-based adjuvant chemotherapy on these biomarkers in breast cancer. J Can Res Ther [serial online] 2018 [cited 2020 Jul 9];14:608-13. Available from: http://www.cancerjournal.net/text.asp?2018/14/3/608/174547
| > Introduction|| |
Breast cancer is the most common form of cancer in women. It has high mortality and morbidity and is a major global health problem. More innovative methods for screening, diagnosis, and treatment need to be identified to prevent these high mortality levels associated with breast cancer. Breast cancer mortality levels after metastasis are high. The basic mechanism involved in cancer cell invasion and metastasis is proteolysis of the extracellular matrix (ECM) and basement membrane. The urokinase plasminogen activator (uPA) system composed of uPA and its receptor (uPAR) is one of the serine proteinase systems involved in ECM degradation. This combination performs a very important role in adhesion, migration, invasion, and metastasis. It is, therefore, a critical target for cancer treatment. Binding of uPA to its receptor uPAR on the cellular surface increased uPA activity. uPAR is motile in the cellular membrane. uPA/uPAR system activation is necessary for the effective invasion and metastasis of cancer cells. uPAR has been determined in many types of cancer, including breast, prostate, gastrointestinal, and lung cancers.
Plasmin is the main enzyme in fibrinolysis and forms via activation of plasminogen by the uPA in the ECM. The primary role of uPAR is degradation of the ECM and cell migration during wound healing, tissue repair, and physiological angiogenesis. uPAR is a glycoprotein found in most tissues and plasma. Measurement of plasma uPAR levels increases accurate prediction of disease risk or outcome in suspected cases of cancer.
Hypoxia and acidosis cause cancer cells to develop a more resistant and invasive character. Oxygen and nutrients are limited in the tumor microenvironment. Transcription factor produces new mechanisms in order to adapt the cancer cell to this microenvironment. Hypoxia-inducible factor-1 (HIF-1) is activated in hypoxia and acidosis settings., Activation of HIF-1 elevates transmembrane carbonic anhydrase IX (CAIX) levels and is associated with poor prognosis in breast cancer. CAIX increases the adaptation of tumor cells to a hypoxic environment, resulting in increased tumor cell survival. Elevated CAIX increases the potential for metastasis in various cancers. D-dimer is a well-known parameter indicating the relationship between coagulation activation and fibrinolysis. Thrombosis and D-dimer levels are closely linked. Activation of the coagulation system increases cancer progression.
The purpose of this study was to evaluate plasma levels of the biomarkers uPAR and CAIX, and the effect of chemotherapy regimens on these, in operated breast cancer patients. No previous studies have investigated these parameters together in terms of pre- and post-chemotherapy levels in operated breast cancer patients.
| > Materials and Methods|| |
Sixty-five consecutive patients with operable breast cancer admitted to the outpatient medical oncology clinic at our hospital were enrolled. Twenty-five age-matched controls were also enrolled in this case-control study for the comparison of serum levels of uPAR and CAIX. Pregnant patients, patients with renal and hepatic function impairment, coronary insufficiency or active infections, subjects using anticoagulants or antiaggregant drugs, and hospitalized patients were excluded. The Ethical Committee at our hospital approved the study, and informed consent was received from all patients and controls. Patients were staged according to the Sixth Edition of the American Joint Committee on Cancer. Patients received epirubicin/adriamycin (EC/AC) (EC 90 mg/m 2 or AC 60 mg/m 2/d1, intravenous [IV] and cyclophosphamide 600 mg/m 2 d1, IV) and fluorouracil, epirubicin, and cyclophosphamide (FEC) (5-fluorouracil 500 mg/m 2 d1, IV, EC 100 mg/m 2 d1, IV and cyclophosphamide 500 mg/m 2 d1, IV) as adjuvant chemotherapy regimens. FEC or EC/AC was administered in six or four cycles, respectively, 3 or 4 weeks after surgery, every 21 days. Patients receiving growth factor and dose reduction were excluded. Blood samples were collected at baseline and before the fourth cycles of chemotherapy regimens. Blood samples were collected in tubes containing 3.8% sodium citrate or tri-potassium ethylenediaminetetraacetic acid as an anticoagulant and serum separator. Samples were centrifuged at 3,000 rpm for 10 min in order to obtain plasma and serum supernatants. The plasma and serum samples were then stored at −80°C until biochemical analyses. We used a Roche Hitachi Cobas 8000 autoanalyzer (Roche, Germany) for routine biochemical parameters and a Beckman Coulter autoanalyzer for cell blood count analysis. Prothrombin time (PT), partial thromboplastin time (PTT), and D-dimer were assayed using an automatic coagulation analyzer (STA compact, Diagnostica Stago, Asnieres, France) in a routine setting.
Measurement of human urokinase plasminogen activator receptor
Levels of uPAR were determined using an enzyme-linked immunosorbent assay kit (Elabscience Biotechnology Co., Ltd. Wuhan, China), following the manufacturer's instructions. The absorbance of samples was measured at 450 nm using a VERSA max tunable microplate reader (designed by Molecular Devices in California, USA). The results were expressed as pg/mL.
Measurement of human carbonic anhydrase IX
Levels of human serum human CAIX were determined using an enzyme-linked immunosorbent assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, P.R.C.), according to the manufacturer's instructions. The absorbance of samples was measured at 450 nm using a VERSA max tunable microplate reader (designed by Molecular Devices in California, USA). The results were expressed as pg/ml.
The Kolmogorov–Smirnov test was used to determine data distribution. During analysis of differences between patient and control samples, the independent samples t-test was used for data with normal distribution and the Mann–Whitney U-test for nonnormally distributed data. Differences between groups before and after chemotherapy were determined using Wilcoxon's two related samples test. Pearson's correlation coefficient analysis was used to examine relationships between parameters. The area beneath the receiver operating characteristic (ROC) curves was used to demonstrate the power of a biomarker in the diagnosis or exclusion of breast cancer. Specificity, sensitivity, negative predictive value (NPV), and positive predictive value (PPV) were calculated.
| > Results|| |
Sixty-five patients with operable breast cancer and 25 age-matched healthy controls were enrolled. Median age at diagnosis was 48 years (range: 28–72 years). uPAR and CAIX levels were investigated in the two chemotherapy groups before and after chemotherapy. All patients underwent curative mastectomy surgery and were chemotherapy naive. Demographic and clinical characteristic of patients and controls are shown in [Table 1]. uPAR and CAIX levels were significantly higher in patients than in the controls (P: 0.02 and P: 0.039, respectively) [Table 2]. Postchemotherapy CAIX levels were higher than basal levels (P < 0.001). Postchemotherapy uPAR levels were higher than basal levels, although the difference was not statistically significant (P: 0.645) [Table 3] and [Figure 1].
|Table 2: Urokinase plasminogen activator receptor, carbonic anhydrase IX levels in prechemotherapy and control groups|
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|Table 3: Urokinase plasminogen activator receptor and carbonic anhydrase IX levels in the pre- - and post-chemotherapy groups|
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|Figure 1: Carbonic anhydrase IX levels in the pre- and post-chemotherapy and control groups|
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uPAR and CAIX levels were higher in the FEC chemotherapy groups than in the control group, but no statistical difference was determined between the FEC and EC/AC chemotherapy groups in terms of basal and postchemotherapy uPAR, CAIX or D-dimer levels [Table 4]. Patients' platelet counts, PT, PTT, and mean platelet volume (MPV) were within normal ranges. No negative or positive correlation was observed between patients' hemostatic markers (PT, PTT, and international normalization ration), platelet counts, MPV, and uPAR or CAIX levels. We determined moderate positive correlations between basal and postchemotherapy uPAR and CAIX levels (P < 0.001, r: 0.481 and P < 0.001, r: 0.631, respectively).
|Table 4: Urokinase plasminogen activator receptor, carbonic anhydrase IX and D-dimer levels in the groups|
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Strong positive correlations were observed between basal and postchemotherapy carcinoembryonic antigen (CEA) and cancer antigen (CA) 15–3 levels (P < 0.001, r: 0.737 and P < 0.001, r: 0.702, respectively). No significant differences were determined between triple negative and nontriple negative groups in terms of uPAR, CAIX or D-dimer. Results (median and range) of comparisons between assays and various clinical parameters in patients with breast cancers are shown in [Table 5]. uPAR levels in E-cadherin (+) groups were higher significantly than those in E-cadherin (−) groups (P: 0.004). Postchemotherapy CA 15–3 and CEA levels were statistically significantly lower than basal levels (P < 0.001 and P < 0.001, respectively) [Table 3].
|Table 5: Results (median and range) of comparisons between urokinase plasminogen activator receptor, carbonic anhydrase IX and D-dimer assays and various clinical parameters in patients with breast cancer|
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The area under the ROC curve, sensitivity, specificity, PPV, and NPV were calculated using ROC curve method analysis. A cut-off value of 27.99 pg/mL for uPAR was associated with 45.31% sensitivity and 84.62% specificity, and with PPV of 87.9% and NPV of 38.6%. A cut-off value of 777.84 pg/mL for CAIX was associated with 90.62% sensitivity and 30.77% specificity, and with PPV of 76.3% and NPV of 57.1%. The analysis results are shown in [Figure 2] and [Figure 3].
|Figure 3: Receiver operating characteristic of urokinase plasminogen activator receptor|
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| > Discussion|| |
Several studies have shown correlating levels of proteolytic factors and clinical prognosis in many types of cancer. The uPA/uPAR complex is capable of initiating proteolysis of the ECM and signal transduction. Plasminogen produces plasmin through these molecules. Plasmin is able to degrade the basement membrane and ECM and activate other zymogen proteases, thus increasing invasion and metastasis of cancer cells. uPAR plays a very important role in this process. The activated uPAR molecule can independently initiate signal transduction for cancer progression and metastasis. These markers can be identified not only in cancer tissue but also in plasma, making them easily available for assessment. They have also been shown to be significant predictors of overall survival (OS) and metastasis in colon cancer and lung cancer. The fibrinolytic markers uPA and uPAR indicate the invasive and metastatic potential of cancer. These markers should, therefore, be used in routine clinical practice to develop targeted treatment for the fibrinolytic pathway, including the uPA and uPAR system.
These molecules are glycoproteins and are found in most tissues and plasma. Measurement of the plasma level of uPAR can improve the prediction of disease risk or outcome in suspected cases of cancer. uPAR can interact with other partner proteins by signal transductions since it is a trans-membrane domain connected to the cell surface with a glycosylphophatidylinositol anchor. High uPAR levels have been reported to be correlated with clinical prognostic factors in ovarian, colonic, and hepatocellular cancers.,
Several studies have determined a correlation between the expression pattern and prognostic significance of uPAR in breast cancer and an association with OS and disease-free survival.,, In our study, uPAR levels in E-cadherin-positive groups were higher significantly than those in E-cadherin-negative groups.
The cancer cell phenotype becomes a resistant cell by acquiring more aggressive characteristics in a hypoxic and acidic microenvironment. These resistant cells exhibit greater invasive metastatic potential and resistance to treatment., Multidrug-resistant genes increase in low-oxygen settings that raise the frequency of point mutations, deletions, and inversions. A hypoxic environment activates HIF-1, which increases expression of the transmembrane CAIX that is associated with poor prognosis in breast cancer. Most patients with breast cancer die from metastatic diseases, and the metastatic phenotype is strongly linked to hypoxic adaptations. The target treatment for resistant tumor cells and the adaptive mechanism are therefore very important if breast cancer outcomes are to be improved.
Recent studies have shown CAIX is an appropriate target for treatment and for achieving better survival rates in breast cancer patients., CAIX adapts the tumor cell to a hypoxic setting and increases its survival, thus permitting ECM degradation and cell invasion. Several studies have reported that elevated CAIX expression is associated with poor prognosis and resistance to treatment in breast cancers. This is compatible with the results of our study. Two patients in our study with higher CAIX levels after adjuvant treatment had lung metastasis and short survival, and two other patients with higher CAIX levels after adjuvant treatment had a pulmonary embolism. CAIX plays an important role in increasing the metastatic potential of several cancers.
No correlations were determined between hemostatic markers and basal or postchemotherapy uPAR and CAIX levels. This may be attributed to our study including only patients with operable breast cancer and not metastatic patients, and to hemostatic markers levels being within normal ranges. There was no correlation between platelet count and uPAR and CAIX levels in our study. This may account for platelet counts being within normal ranges. We determined no correlations between basal D-dimer levels and basal uPAR and CAIX levels. The main limitations of this study are the low patient numbers and the fact that it excluded patients with metastatic diseases. In addition, we did not examine levels of uPAR and CAIX after completion of adjuvant treatment or simultaneous expression of uPAR and CAIX in tumor tissues.
Few data from an adjuvant setting have previously been available. It is unclear whether uPAR and CAIX levels fluctuate during adjuvant chemotherapy and show disease activity, or how these fluctuations can be manipulated in the management of cancer treatment. We investigated pre-and postchemotherapy uPAR and CAIX levels and determined that uPAR and CAIX levels were higher in the FEC chemotherapy groups than in the control group, but there was no difference between the FEC and EC/AC chemotherapy groups in terms of basal and postchemotherapy uPAR, CAIX levels.
| > Conclusion|| |
Measurement of plasma levels of uPAR and CAIX may be useful in assessing the risk of cancer metastasis, monitoring tumor recurrence, predicting response to chemotherapy, and diagnosing cancers. In the light of these benefits, further researches are now needed to standardize uPAR and CAIX levels so they can be used in the follow-up and diagnosis of cancers in routine clinical practice.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]