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Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.
Testicular germ cell tumors (TGCTs) represent the most common type of cancer in young men (
It is well established that hypoxia and an hypoxic microenvironment are key factors in cancer pathogenesis (
Peripheral blood is easily accessible and enables repeated examination in the course of the disease. In several studies evaluating the clinical utility of soluble CA IX levels in cancer patients, high serum or plasma CA IX levels were associated with an inferior outcome in cervical and vulvar cancer, oral squamous cell carcinoma, non-small cell lung cancer and metastatic breast cancer (
The current prospective translational study (protocol IZLO1, chair Dr M. Mego) was approved by the Institutional Review Board (IRB) of the National Cancer Institute (Bratislava, Slovakia), and was conducted between March 2011 and April 2013. All consecutive patients with TGCTs treated with ≥1 cycle of cisplatin-based chemotherapy in the National Cancer Institute or St. Elizabeth Cancer Institute (Bratislava, Slovakia) were enrolled in the study. Serum samples from 35 healthy individuals were used as a control group. Data regarding age, tumor histological subtype, clinical stage, type and number of sites of metastasis and type of chemotherapy regimen were recorded for all patients and compared with CA IX expression. TGCTs patients and controls were recruited and provided informed consent according to the IRB approved protocol.
Peripheral blood samples were collected from all participants in the present translational study. Samples were collected into Vacutainer® Rapid Serum Tubes (BD Biosciences, Franklin Lakes, NJ, USA) containing silica clot activator polymer gel at baseline in the morning on days 1 or 0 of the first cycle of chemotherapy (n=73) and prior to the second cycle of chemotherapy (n=37), or before starting a new line of salvage chemotherapy in patients with relapsed disease (n=10). Patients' blood samples (1 ml) were centrifuged at 2,800 × g for 10 min to separate the serum from the blood cells. Serum aliquots were stored at −80°C until further analysis.
ELISA for the quantitative determination of serum CA IX concentration was performed at room temperature. The microplate wells were coated overnight with 100 µl/well of the anti-CA IX ectodomain (ECD) monoclonal antibody (MAb) that had been purified in a previous study (
The study included available tumor specimens resected from 17 patients prior to the administration of cisplatin-based chemotherapy, namely from 13 (76.5%) patients with primary testicular tumors and 4 (23.5%) patients with retroperitoneal germ cell tumors. All specimens were classified according to the most recent World Health Organization classification from 2004 (
A pathology review was performed at the Department of Pathology, Faculty of Medicine, Comenius University (Bratislava, Slovakia), by two pathologists (Z.C. and P.B.) associated with the study. All specimens were assessed by light microscopy following hematoxylin and eosin (HE) staining, and with the use of immunohistochemical markers typical for germ cell tumors.
From each histological subtype, 1–2 representative tumor areas were identified on HE-stained sections, according to tumor histology. If normal testicular tissue samples were present, they were also marked. Sections were matched to the donor blocks (corresponding wax blocks). Tumor cores (3-mm diameter) were removed from the donor blocks using the multipurpose sampling tool Harris Uni-Core (Sigma-Aldrich) and inserted into the recipient master block. The recipient block was cut into 5-µm sections, and sections were transferred to FLEX IHC Microscope slides (cat. no. K8020; Dako, Glostrup, Denmark).
Deparaffinized slides were rehydrated and immersed in PBS (10 mM, pH 7.2). Tissue epitopes were demasked using the automated water bath heating process in PT Link (Dako). Briefly, the slides were incubated in Tris-ethylenediaminetetraacetic acid (EDTA) retrieval solution (10 mM Tris, 1 mM, pH 9.0) at 98°C for 20 min. CA IX expression was detected by IHC using the in-house generated monoclonal antibody M75 against the N-terminal domain of human CA IX, as described previously (
Two observers (Z.C. and P.B.), who were blinded to the patients clinicopathological data, independently assessed the tumor cores. In cases of disagreement, the result was reached by consensus. CA IX expression was stratified as negative or positive (any staining).
Patients' data were tabulated. The patients' characteristics were summarized using the mean ± standard error of the mean or median (range) for continuous variables, and frequency (percentage) for categorical variables. Statistical analysis was performed using non-parametric tests, as the distribution of CA IX expression was significantly different from a normal distribution (Shapiro-Wilk test). The Mann-Whitney U test was used for analysis of the association between serum CA IX expression and clinicopathological variables in 2 groups of patients, while the Kruskal-Wallis test was used to compare >2 groups. The Wilcoxon test was used to compare the serum CA IX level prior to the first and second cycles of chemotherapy.
The median follow-up period was calculated as a median observation time among all patients and among those who were still alive at the time of their last follow-up. Progression-free survival (PFS) was calculated from the date of treatment initiation with cisplatin-based chemotherapy to the date of progression, mortality or last follow-up. Overall survival (OS) was calculated from the date of treatment initiation with cisplatin-based chemotherapy to the date of mortality or last follow-up. Survival rates were estimated using the Kaplan-Meier product limit method, and were compared with the log-rank test to determine their significance. CA IX expression data were dichotomized into high and low groups based on the CA IX expression median value of all samples. The Spearman correlation coefficient was used to examine a potential correlation between CA IX serum concentration and lactate dehydrogenase (LDH), human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP) levels as well as CA IX expression in tumor specimens. All statistical tests were two-sided, and P<0.05 was considered to indicate a statistical significant difference. Statistical analyses were performed using NCSS 2007 software (NCSS, LLC, Kaysville, UT, USA).
From March 2011 to April 2013, 83 patients, including 16 non-metastatic patients subjected to orchiectomy with no evidence of disease (group 1), 57 metastatic chemotherapy-naïve patients (group 2) and 10 metastatic relapsed chemotherapy-pretreated patients (group 3), who were starting adjuvant and/or a new line of chemotherapy were registered to participate in the present study at the National Cancer Institute and St. Elizabeth Cancer Institute of Slovakia. The characteristics of the patients are shown in
The majority of patients had non-seminoma histology and primary testicular cancer. The majority of chemotherapy-naïve patients (61; 83.6%) were treated with the BEP (bleomycin, etoposide, cisplatin) regimen; 8 patients (11.0%) received EP (etoposide, cisplatin) chemotherapy, while 2 patients (2.7%) received VIP (ifosfamide, etoposide, cisplatin) chemotherapy and 2 patients (2.7%) were treated with dose-dense chemotherapy, as described previously (
The mean serum level of CA IX in TGCT patients was significantly higher compared with healthy controls (405.2±90.1 vs. 249.6±100.0 pg/ml; P=0.007). Metastatic chemotherapy-naïve patients had significantly higher mean serum levels of CA IX compared with serum samples of an independent group of healthy individuals (490.6±111.8 vs. 249.6±100.0 pg/ml; P=0.005), whereas there was no significant difference in the mean serum CA IX levels between metastatic relapsed chemotherapy-pretreated patients and healthy controls (216.0±170.2 vs. 249.6±100.0 pg/ml; P=0.370). Similarly, the mean serum levels of CA IX in non-metastatic upon orchiectomy with no evidence of disease patients were not significantly different compared with those in healthy donors (218.9±128.8 vs. 249.6±100.0 pg/ml; P=0.080).
There was no significant difference in the mean CA IX serum levels between non-metastatic, metastatic chemotherapy-naïve and chemotherapy-pretreated TGCT patients (
The associations between the histological subtypes of the primary germ cell tumors and the mean CA IX levels detected by ELISA in the patients' serum samples are summarized in
Intratumoral CA IX expression was evaluated by IHC in 17 tumor tissue specimens collected on tissue microarray. CA IX protein was detected in 8 specimens (47.1%) and exhibited mostly a focal expression pattern. CA IX staining was present in cancer cells, but in certain specimens, it was visible in the stroma (
In the median follow-up time of 31.5 months (range, 0.3–46.8 months), 11 patients (13.3%) experienced disease progression (4 in the chemotherapy-naïve group and 7 in the chemotherapy-pretreated group) and 10 patients (12.0%) succumbed to the disease (4 in the chemotherapy-naïve group and 6 in the chemotherapy-pretreated group).
No significant differences in PFS were observed based on the CA IX levels in serum of the TGCT patients enrolled in the present study [hazard ratio (HR)=0.81, 95% confidence interval (CI)=0.25–2.65, P=0.730). Similarly, there was no difference in OS according to the serum CA IX level (HR=0.64, 95% CI=0.18–2.20, P=0.48) (
TGCTs belong to the most chemosensitive group of tumors, and represent a model for the cure of cancer (
The absence of a significant correlation between CA IX levels in the serum of the analyzed TGCT patients and their treatment outcome is not an unusual finding. Recently published data suggest that the potential clinical value of the CA IX ECD shed from the surface of tumor cells to the serum of patients is inconsistent. While the studies by Műller
For survival analysis, the CA IX level was dichotomized based on the median values of all analyzed samples. Although it cannot be excluded that using different cut-off values may be able to discriminate good vs. poor prognosis patients, there was no correlation between serum CA IX level and disease stage, International Germ Cell Consensus Classification Group prognostic group, disease burden or any other known prognostic factors, thus supporting the limited prognostic value of serum CA IX in TGCTs. To increase the statistical power, chemotherapy-naïve and chemotherapy-pretreated patients were combined. However, when separate analyses of these subgroups were performed, the study results remained the same. It is also possible that, due to the good prognosis of TGCTs, all pathophysiological aspects associated with the activation of CA IX and HIF pathways do not affect the chemosensitivity of TGCTs. This would be consistent with the findings of Vranic
A significant association between CA IX expression in tumor samples and CA IX values in serum was observed. However, there were several cases of CA IX positivity in serum samples and CA IX negativity in the corresponding tumors. Since the hypoxia-related expression of CA IX in tumors is generally very heterogeneous and often focal, it is probable that the available region encompassed in the tissue microarray subjected to IHC staining was actually devoid of CA IX, but this does not exclude the presence of CA IX in another tumor region. Thus, the correlation observed in the present study may actually be an underestimation of the real association. The limited sample size (n=17) and time period between orchiectomy and administration of chemotherapy could represent limitations that may affect the study results. The association between CA IX expression in tumor samples and the CA IX values in serum could be explained by intratumoral hypoxia, which leads to the release of the soluble form of CA IX into the bloodstream. Studies on the association of intratumoral CA IX expression and detection of its soluble isoform are limited. A study by Zhou
In a previous study, CA IX expression was identified in the flat surface epithelium (modified mesothelium) of all male and female genital organs (
In the present study, there were no differences in serum CA IX levels between non-metastatic, metastatic chemotherapy-naïve and chemotherapy-pretreated TGCT patients, but significant differences were observed between CA IX serum levels in TGCT patients and healthy controls. Unexpectedly, there was no difference when comparing only relapsed chemotherapy-pretreated patients, suggesting a different biology and role of hypoxia in relapsed TGCTs compared with chemotherapy-naïve patients. Analysis of serum CA IX levels revealed no significant changes during therapy (prior to chemotherapy and prior to the initiation of the second cycle of chemotherapy). However, in the cohort of 37 patients prior to receiving the second line of chemotherapy, 3 patients experienced disease progression and succumbed to the disease. All these patients had CA IX levels above the median values. However, the limited sample size precludes the drawing of any definitive conclusions from these data.
In summary, the present study is the first aimed to assess the prognostic role of serum CA IX level in TGCTs. The present data suggest that there is an increased level of serum CA IX in metastatic chemotherapy-naïve TGCTs compared with healthy controls, and that a weak correlation exists between serum CA IX levels and tissue CA IX expression. These data suggest neither a prognostic value for serum CA IX levels nor an association between serum CA IX levels and patients/tumor characteristics. Despite the limited clinical utility of serum CA IX, the biological and clinical value of CA IX expression in TCGT tissues cannot be excluded, and therefore, further research into this area is warranted.
The authors would like to acknowledge Mrs. Daniela Jantekova from the Population Registry of Slovak Republic (Bratislava, Slovakia) for helping to update the patient database; Dr Maria Reckova from the 2nd Department of Oncology, Faculty of Medicine, Comenius University for discussion and critical input; Mrs. Zlatica Pekova from the Department of Oncology, National Cancer Institute for administration support; and Mrs. Emilia Klincova from the Department of Pathology, Faculty of Medicine, Comenius University for the excellent technical assistance. The present study was supported by the Slovak Research and Development Agency (Bratislava, Slovakia; contract nos. APVV-0016-11 and APVV-15-0086), the European Regional Development Fund (Brussels, Belgium), the State Budget of the Slovak Republic (Bratislava, Slovakia; project no. ITMS 26240220071) and the Slovak Scientific Grant Agency (Bratislava, Slovakia; grant no. VEGA-2/0108/16).
Serum CA IX in different groups of TGCT patients (group 1, adjuvant chemotherapy-naïve patients, n=16; group 2, metastatic chemotherapy-naïve patients, n=57; group 3, relapsed chemotherapy-pretreated patients, n=10). The median level of CA IX in serum in groups 1, 2 and 3 was 93.7, 186.7 and 141.7 pg/ml, respectively (P=0.630). CA IX, carbonic anhydrase IX; TGCT, testicular germ cell tumor.
Serum CA IX level before (day 1) starting chemotherapy and before (day 22) the second cycle of chemotherapy. Serum CA IX levels displayed no significant changes during therapy (median serum level before and after chemotherapy, 157.8 and 52.1 pg/ml, respectively, P=0.360). CA IX, carbonic anhydrase IX.
Immunohistochemical staining of carbonic anhydrase IX (CA IX) in testicular germ cell tumor tissue specimens using the M75 monoclonal antibody targeting the N-terminal extracellular proteoglycan domain of both native and denatured CA IX. Focal staining pattern includes (A) seminoma cells and (B) stroma. Immunoperoxidase and 3,3-diaminobenzidine staining. Magnification, ×200.
Kaplan-Meier estimates of PFS according to CA IX expression in serum. Patients with ‘low’ CA IX had similar PFS to patients with ‘high’ CA IX (hazard ratio=0.81, P=0.730). CA IX, carbonic anhydrase IX; PFS, progression-free survival.
Kaplan-Meier estimates of OS according to CA IX expression in serum. Patients with ‘low’ CA IX had similar OS to patients with ‘high’ CA IX (hazard ratio=0.64, P=0.480). CA IX, carbonic anhydrase IX; OS, overall survival.
Patients' characteristics (n=83).
Chemotherapy-naïve TGCTs | Chemotherapy-pretreated relapsed TGCTs | |||
---|---|---|---|---|
Characteristics | N=73 | % | N=10 | % |
Age, years | ||||
Median (range) | 34 (19–67) | 34 (24–50) | ||
Primary tumor | ||||
Gonadal | 70 | 95.9 | 9 | 90.0 |
Retroperitoneal | 3 | 4.1 | 1 | 10.0 |
Histology |
||||
Seminoma | 12 | 16.4 | 3 | 30.0 |
Non-seminoma | 60 | 82.2 | 7 | 70.0 |
Stage of TGCTs | ||||
I.A-I.B | 16 | 21.9 | 0 |
0.0 |
I.S | 6 | 8.2 | 0 | 0.0 |
II.A-III.A | 27 | 37.0 | 2 | 20.0 |
III.B | 13 | 17.8 | 2 | 20.0 |
III.C | 11 | 15.1 | 6 | 60.0 |
Sites of metastases |
||||
Retroperitoneum | 48 | 84.2 | 10 | 100.0 |
Mediastinum | 9 | 15.8 | 0 | 0.0 |
Lung | 18 | 31.6 | 7 | 70.0 |
Liver | 4 | 7.0 | 1 | 10.0 |
Brain | 2 | 3.5 | 0 | 0.0 |
Other | 5 | 8.8 | 2 | 20.0 |
Visceral non-pulmonary | 7 | 12.3 | 1 | 10.0 |
IGCCCG risk group |
||||
Good prognosis | 36 | 63.2 | NA | NA |
Intermediate prognosis | 11 | 19.3 | NA | NA |
Poor prognosis | 10 | 17.5 | NA | NA |
Mean (range) | ||||
AFP, mIU/ml | 869.9 (0.9–13,936.0) |
10,376.2 (1.6–89,954.0) | ||
HCG, IU/ml | 81,602.5 (0.0–1,840,510.0) |
53,742.2 (0.1–480,259.0) | ||
LDH, mkat/l | 11.8 (1.8–76.0) |
11.3 (2.3–33.2) |
In 1 patient, chemotherapy started without histological confirmation due to very advanced disease.
Only patients with metastatic disease (n=57).
Initial stage of disease. HCG, human chorionic gonadotropin; AFP, alpha-fetoprotein; LDH, lactate dehydrogenase; IGCCCG, International Germ Cell Consensus Classification Group; NA, not applicable; TGCT, testicular germ cell tumor.
CA IX concentration in serum of metastatic chemotherapy-naïve patients with different histological subtypes of primary germ cell tumors (n=55).
Serum CA IX level (pg/ml) | |||||
---|---|---|---|---|---|
Histological subtype |
N | Mean | SEM | Median | P-value |
Seminoma | 25 | 417.5 | 196.3 | 207.8 | 0.830 |
Embryonal carcinoma | 26 | 644.8 | 189.7 | 198.7 | 0.570 |
Yolk sac tumor | 25 | 372.1 | 195.7 | 186.7 | 0.570 |
Choriocarcinoma | 12 | 261.7 | 281.8 | 152.3 | 0.410 |
Teratoma | 20 | 304.1 | 217.9 | 77.9 | 0.510 |
In 1 patient, chemotherapy started without histological confirmation due to very advanced disease. The percentages of certain histological types of non-seminoma germ cell tumors were as follows: Pure embryonal carcinoma occurred in 14.5% (8 patients), pure choriocarcinoma in 7.3% (4 patients), pure form of yolk sac tumor in 5.5% (3 patients) and immature teratoma in 1.8% (1 patient). Mixed germ cell tumors were the most commonly presented histological subtype of non-seminomas. CA IX, carbonic anhydrase IX; SEM, standard error of the mean.
Association between serum CA IX level and patients/tumor characteristics in metastatic chemotherapy-naïve testicular germ cell tumor patients (n=57).
Serum CA IX level (pg/ml) | |||||
---|---|---|---|---|---|
Variable | N | Mean | SEM | Median | P-value |
All patients | 57 | 490.6 | 127.9 | 186.7 | NA |
Primary tumor |
0.750 | ||||
Seminoma | 12 | 315.0 | 282.6 | 142.2 | |
Non-seminoma | 43 | 510.4 | 149.3 | 157.8 | |
IGCCCG risk group | 1.000 | ||||
Good prognosis | 36 | 590.4 | 162.2 | 169.4 | |
Intermediate prognosis | 11 | 382.6 | 231.7 | 231.7 | |
Poor prognosis | 10 | 250.1 | 78.9 | 78.9 | |
Number of metastatic sites | 0.200 | ||||
0–1 | 31 | 409.5 | 174.3 | 86.7 | |
>2 | 26 | 587.3 | 190.3 | 219.8 | |
Retroperitoneal lymph nodes metastases | 0.420 | ||||
Present | 9 | 266.1 | 323.1 | 221.3 | |
Absent | 48 | 532.7 | 139.9 | 209.3 | |
Mediastinal lymph nodes metastases | 0.770 | ||||
Present | 48 | 534.6 | 139.9 | 172.2 | |
Absent | 9 | 255.9 | 323.0 | 231.7 | |
Lung metastases | 0.670 | ||||
Present | 39 | 419.1 | 155.1 | 210.8 | |
Absent | 18 | 645.7 | 228.3 | 149.4 | |
Non-pulmonary visceral metastases | 0.310 | ||||
Present | 50 | 494.8 | 137.8 | 149.4 | |
Absent | 7 | 460.7 | 368.3 | 286.9 | |
S-stage | 0.460 | ||||
0 | 11 | 378.1 | 290.7 | 285.7 | |
1 | 22 | 773.6 | 205.6 | 197.3 | |
2 | 16 | 290.0 | 241.1 | 221.3 | |
3 | 8 | 268.3 | 341.0 | 0.0 |
In 1 patient, chemotherapy started without histological confirmation due to very advanced disease, and in 1 patient, histology data were not available. NA, not applicable; SEM, standard error of the mean; CA IX, carbonic anhydrase IX; IGCCCG, International Germ Cell Consensus Classification Group.