Abstract
Background: We evaluated the prognostic value of the new serum biomarkers mesothelin (cell surface glycoprotein and tumor differentiation antigen), L1 cell adhesion molecule (L1CAM) and afamin (vitamin D-binding protein) alone and in combination with cancer antigen 125 (CA125) in serum samples of 154 patients with first-diagnosis of primary ovarian cancer, before surgery and after platinum-based chemotherapy. We correlated these findings with clinical parameters and evaluated their prognostic value with regard to overall survival (OS). Materials and Methods: Blood (9 ml) was obtained before surgery (n=154) and after chemotherapy (n=82) for the measurement of serum markers using commercial Enzyme Linked Immunosorbent Assay (ELISA) kits for mesothelin, L1CAM, afamin and CA125. Mesothelin positivity was defined as >2.0 nM, L1CAM as >10 ng/ml, afamin as <45 mg/l and CA125 as >35 U/ml, respectively. Results: Before surgery, mesothelin positivity significantly correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.002), residual postoperative tumor (p<0.0001), serous histological subtype (p<0.0001) and higher age (p=0.013). Elevated CA125 levels significantly correlated with advanced FIGO stage (p<0.0001) and grading (p=0.012). After chemotherapy, mesothelin as well as CA125 levels, were significantly associated with FIGO stage (p=0.041 and p=0.017) and residual tumor (p=0.022 and p=0.002) while L1CAM correlated with platinum sensitivity (p=0.041). In contrast, afamin at all determined time points showed no correlation with any of these parameters. The combination of markers did not add any significant power to their use. Conclusion: Mesothelin and L1CAM appear to correlate with clinical prognostic parameters and might be useful biomarkers for therapy monitoring and, thus, could serve as attractive targets for therapy of ovarian cancer.
Ovarian cancer is the leading cause of death among women with gynaecological malignancies. At the time of primary diagnosis, 70% of patients with ovarian cancer have advanced International Federation of Gynecology and Obstetrics (FIGO) stages III/IV, with peritoneal carcinosis and ascites (1). Although there are high response rates to carboplatin-based chemotherapy, the majority of patients with ovarian cancer cannot be cured. About 75% of patients suffer from recurrent disease. Standard treatment of advanced ovarian cancer constitutes primary surgery, aiming at macroscopic complete tumor resection, and subsequent platinum and paclitaxel-based chemotherapy (2). Postoperative residual tumor burden is one of the most important prognostic factors in ovarian cancer (3). However, despite advances in treatment, more than half of all patients will experience recurrence (4), resulting in a poor overall survival (OS). Innovative therapy strategies are necessary to improve patient prognosis.
Cancer antigen 125 (CA125) is the most commonly used, currently available, biomarker and is reported to have one of the highest sensitivities and specificities among biomarkers for ovarian cancer (5). It is widely applied to assess response to ovarian cancer treatment and to monitor patients for recurrence. Nevertheless, false-positive results have often been reported among women with endometriosis, fibroids and some other benign conditions (6). Thus, the identification of new ovarian cancer biomarkers to replace, or complement CA125 is urgently needed. A broad variety of serum markers have been evaluated, alone and in combination with CA125, including Human Epidedymal Secretory Protein4 (HE4), osteopontin and kallikrein (7, 8). In this regard, mesothelin, a cell-surface glycoprotein and tumor differentiation antigen, has been reported to be a new promising tumor-associated marker in a variety of human cancer types including ovarian carcinoma (6). By binding to CA125, mesothelin mediates cell adhesion and peritoneal spread of ovarian cancer, which suggests a potential supplemental role in combination with CA125 for monitoring these patients. Furthermore, a small amount of cell-bound mesothelin is shed into the serum and has been shown to be elevated in patients with mesothelioma and ovarian cancer (9, 10).
Using a differential proteomics approach, afamin was identified as a potential novel biomarker for ovarian cancer (11). Afamin is a member of the albumin family comprising albumin, alpha-fetoprotein, and vitamin D-binding protein, and plays a role as a vitamin E carrier in body fluids such as human plasma and follicular fluid under physiological conditions. Lysates from ovarian cancer cell lines have been shown to contain afamin (12). Recently, low concentration of serum afamin has been identified as a novel marker for the specific diagnosis of ovarian cancer (13).
Another promising marker is L1 cell adhesion molecule (L1CAM), a type 1 member glycoprotein of 200-220 kDa, structurally belonging to the Ig superfamily that initially was found to play an important role in the development of the nervous system (14). It has also been found to be expressed in human carcinomas, including melanoma (15, 16), renal carcinoma (17) and gastrointestinal cancer (18-20) and appears to be involved in tumor progression, apoptosis and chemoresistance (21). In ovarian cancer, overexpression of L1CAM in tumor tissue has been reported to predict poor outcome (22). Furthermore, it has been demonstrated that L1CAM is released from the cell membrane by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) (23, 24). The soluble L1CAM ectodomain can be detected in the serum and ascites from patients with ovarian carcinoma (22).
It was the purpose of the study to a) determine the levels of mesothelin, L1CAM, afamin and CA125 before surgery and after platinum-based chemotherapy in serum samples of patients with primary ovarian cancer and b) correlate these novel biomarkers alone and in combination with clinical parameters and evaluate their prognostic value with regard to OS.
Materials and Methods
Patients and volunteers. The present retrospective study was conducted at the Department of Gynecology and Obstetrics at the University Hospital in Essen, Germany. In total, 154 patients with histologically-confirmed FIGO stages I-IV ovarian cancer, aged 18 years or older, were included. All patients had been treated between October 2003 and August 2010. After primary surgery, patients received adjuvant chemotherapy with carboplatinum and paclitaxel. Total abdominal hysterectomy, bilateral salpingo-oophorectomy, infragastric omentectomy, peritoneal stripping and pelvic and para-aortic lymphadenectomy were performed when feasible. The most important aim of surgery was to achieve macroscopic complete tumor resection. Patients underwent radical pelvic and para-aortic lymphadenectomy when complete tumor resection was achieved following actual guidelines. Patients who had a disease-free survival (DFS) between one and 10 months and a treatment-free interval of up to five months were defined as suffering from platinum-resistant disease. The patients' mean age was 60 years (range=24-92 years), and the median follow-up time was 25 months, ranging from 1 to 78 months. The clinical data of the patients are summarized in Table I. The controls consisted of 24 healthy age-matched female voluntary donors with no history of cancer. Approval for this study was obtained from the local Ethics Committee (05-2870).
Sampling of serum. Nine milliliters of blood were collected with an S-Monovette® (Sarstedt AG & Co., Nürnbrecht, Germany) from each patient and from 24 healthy volunteers, stored at 4°C and processed within 4 h to avoid blood cell lysis. Blood fractionation was carried out by centrifugation for 10 min at 2500 ×g. Subsequently, 3 to 4 ml of the supernatant, constituting the blood serum, were removed and subjected to the determination of mesothelin, L1CAM, afamin and CA125.
Determination of mesothelin. Mesothelin was determined by application of the two-step quantitative sandwich Enzyme Linked Immunosorbent Assay (ELISA) MESOMARK™ (IBA, Berlin, Germany) using two separate monoclonal antibodies (4H3 and OV569) for capturing and detection of soluble mesothelin-related peptides (SMRP) in human serum. All reactions were carried out at room temperature with shaking at 700 rpm and all standards and controls were prepared according to the manufacturer's instructions. One hundred microliters of each standard, control and undiluted serum sample were dispensed into the wells coated with 4H3 murine monoclonal antibody and incubated for 60 min. Subsequently, after washing five times, 100 μl of the OV569 murine monoclonal antibodies conjugated to horseradish peroxidase were added for a further 60 min protected from light. After five additional washing steps, 100 μl of substrate solution containing TMB were added for 15 min and, subsequently, the reaction was stopped by the addition of 1% hydrochloric acid. The absorbance was measured at 450 nm by an automatic ELISA reader (Bio-Rad, Hercules, CA, USA), blanked using air. A direct relationship exists between the amount of SMRP in the sample and the optical density detected by the plate reader. Results are expressed in nM according to the established standard curve. The limit of detection is 0.16 nM. Considering the manufacturer's instructions and previous data, concentrations at or below 1.5 nM were considered normal.
Determination of L1CAM. L1CAM was determined using the DRG (Marburg, Germany) solid-phase sandwich ELISA for the quantitative in vitro measurement of L1CAM in serum. The assay was performed in flat-bottomed microtiter ELISA plates coated with a monoclonal mouse antibody, directed towards a unique antigenic site on the L1CAM molecule. All reactions were carried out at room temperature with shaking at 300 rpm. Standards and controls were prepared according to the manufacturer's instructions for further use. Fifty microliters of each standard, control and undiluted serum sample, and 100 μl of biotinylated L1CAM antibody were dispensed into the wells and incubated for 60 min. After incubation, the unbound conjugate was washed off five times before adding 100 μl of streptavidin peroxidase for a further 30 min. After five additional washing steps, 100 μl of substrate solution containing TMB were added for 15 minutes and subsequently, the reaction was stopped by 0.5 M sulfuric acid and the absorbance at 450 nm was measured by an automatic ELISA reader (Bio-Rad). The amount of bound biotin-streptavidin complex was proportional to the concentration of L1CAM in the sample. Having added the substrate solution, the intensity of developed color was proportional to the concentration of L1CAM in the sample. Results are expressed in ng/ml according to the established standard curve. The limit of detection is 2.7 ng/ml. Following the manufactures' instructions, reference concentrations were 2.7-16.9 ng/ml.
Patients' characteristics.
Determination of afamin. Afamin was determined using the DRG solid-phase sandwich ELISA for the quantitative in vitro measurement of afamin in serum. The assay was performed in flat-bottomed microtiter ELISA plates at room temperature with shaking at 300-500 rpm. Standards and controls were prepared according to the manufacturer's instructions and serum samples were diluted at 1:2000 for further use. Fifty microliters of each standard, control and sample were dispensed into the wells and subsequently, 100 μl of assay buffer was added. All samples were incubated for 60 min in wells coated with polyclonal rabbit anti-human afamin antibody. After shaking out the contents and washing five times with the supplied washing buffer, the wells were incubated for another 60 min with an anti-afamin antibody conjugated with horseradish peroxidase. Subsequently, the unbound conjugate was washed off five times and the wells were incubated with 100μl tetramethylbenzidine (TMB) for 15 min. After the addition of 100 μl stop solution (0.5 M sulfiric acid), the absorbance was measured at 450 nm by an automatic ELISA reader (Bio-Rad). The amount of bound peroxidase was proportional to the concentration of afamin in the sample. Having added the substrate solution, the intensity of developed color was proportional to the concentration of afamin in the patient sample. Results were expressed in mg/l according to the established standard curve. The limit of detection is 0.01 μg/l for this method. Reference concentrations were 45-99 mg/l according to the manufacturer's guidelines.
Determination of CA125. CA125 was determined using the Elecsys CA125 immunoassay (Roche, Mannheim, Germany) for the quantitative determination of CA125 in human serum and plasma. The serial measurement of CA125 was intended to aid in the management of patients with cancer. This assay was performed in the central laboratory of our University Hospital on Cobas® immunoassay analyzers, according to the manufacturer's instructions. The central laboratory has a valid certification for the performance of these assays following international guidelines. The lower limit of detection is 0.09 U/ml. CA125 concentrations >35 U/ml were considered positive in accordance to established reference concentrations.
Statistical analysis. Comparison of the clinical and laboratory parameters (pre- and post-treatment mesothelin, L1CAM, afamin and CA125 concentrations) was performed using the Mann Whitney U-test for continuous variables and the χ2 test for categorical data. Correlations between serum levels and categorical clinical data were tested using analysis of variance. Differences were regarded as statistically significant at p<0.05. Statistical analyses were performed using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA) and SPSS17.0 for Windows® (SPSS, Chicago, IL, USA). Positivity for a marker was defined when marker concentrations were above the accordant reference range.
Results
Determination of serum levels of mesothelin, L1CAM, afamin and CA125. The reference serum concentrations of mesothelin, L1CAM and afamin were approved in our collective of healthy volunteers. The mean concentrations were 0.87 nM (range=0.45-2.05 nM), 10.56 ng/ml (range=1.46-28.76 ng/ml) and 47.4 mg/l (range=32.5-63.2 mg/l), respectively. The statistical distribution of these concentrations was normal according to the Kolmogorow-Smirnow test.
Before surgery, the median concentration of mesothelin was 1.55 nM (range=0.22-30 nM), 4.250 ng/ml (range=0.51-35.6 ng/ml) for L1CAM, 46 mg/l (range=7.42-999 mg/l) for afamin and 322 U/ml (5-32,403 U/ml) for CA125. After chemotherapy, the concentrations were 0.85 nM (range=0.30-16.38 nM), 8.190 ng/ml (range=1-3,940 ng/ml), 57.78 mg/l (range=25.36-153.9 mg/l) and 16 U/ml (range=4-11,056 U/ml), respectively. Comparing concentrations before surgery and after treatment, a significant rise of afamin and L1CAM levels and a significant decrease of mesothelin levels were noted (p<0.0001; Figure 1).
Correlations between analyzed markers and clinical characteristics of the patients. A summary of the correlations between the analyzed markers and clinical characteristics of the patients is shown in Tables II and III. Mesothelin positivity before surgery significantly correlated with advanced FIGO stage (p=0.002), residual tumor (p<0.0001), serous histological subtype (p<0.0001) and higher age (p=0.013); elevated CA125 levels significantly correlated with advanced FIGO stage (p<0.0001) and grading (p=0.012). After chemotherapy, elevated mesothelin as well as CA125 levels were significantly associated with advanced FIGO stage (p=0.041 and p=0.017) and residual tumor (p=0.022 and p=0.002); after chemotherapy, persistent CA125 positivity indicated platinum resistance (p=0.001). L1CAM positivity after chemotherapy showed an inverse association with platinum resistance (p=0.041): out of 28 patients with persistent L1CAM positivity, only one patient did not have an adequate response to platinum-based chemotherapy. Afamin at all time points determined showed no correlation with any of these parameters.
Serum levels of mesothelin, afamin and L1CAM before surgery and after chemotherapy. Concentrations are depictetd in means.
The use of a combination of markers did not add any further significance to the results (data not shown). A total of 21/154 (13.6%) patients were found to have platinum-resistant disease with a significantly lower mean OS of 27 months (95% confidence interval=19-36 months) versus 55 months (95% CI=49-61 months) for patients with platinum-sensitive disease.
Correlation of analyzed markers with clinical outcome. Figure 2 illustrates the relationship between marker expression and clinical outcome. OS was significantly lower in cases of mesothelin positivity (p=0.001) and CA125 (p=0.028) elevation before surgery (p=0.001). No significant associations were found for L1CAM and afamin before surgery, nor for any markers analyzed after chemotherapy.
Discussion
In the current study, we demonstrated that besides CA125, mesothelin and L1CAM appear to correlate with clinical prognostic parameters and might be useful biomarkers for therapy monitoring and could serve as attractive targets for the therapy of patients with ovarian cancer. Before surgery, high mesothelin concentrations significantly correlated with advanced FIGO stage, residual postoperative tumor, serous histological subtype and higher age. L1CAM was significantly associated with platinum sensitivity, and for CA125, significant correlations were found for advanced FIGO stage and histopathological grading.
After chemotherapy, mesothelin and CA125 levels were associated with FIGO stages and residual tumor load.
Mesothelin. Here we demonstrate that elevated mesothelin concentrations can be detected in the sera of nearly 40% of patients with ovarian carcinoma at primary diagnosis. This significantly correlated with advanced FIGO stage, residual tumor, serous histological subtype, higher age and reduced OS. Cheng et al. showed significant correlations of mesothelin expression with pathological stage, tumor grade, status of optimal surgery and chemoresistance as well as shorter DFS and OS, as they analyzed tumor tissue samples from 139 patients with epithelial ovarian cancer for mesothelin expression by reverse transcription-polymerase chain reaction (RT-PCR) (25). Interestingly, they found the expression of mesothelin in chemosensitive patients with residual tumor (up to one centimetre or more than one centimetre) to be significantly lower than those in the chemoresistant groups. The authors concluded that cancer cells containing greater levels of mesothelin can resist cytotoxic drug-induced apoptosis and will continue to progress unless other tumor cells that fail to express mesothelin are produced. Serum analysis in these patients was not performed. Hassan et al., found elevated serum mesothelin levels in 14/21 (67%) patients which were increased in 16/21 (75%) of cases in which the tumors expressed mesothelin by immunohistochemistry. However, the serum mesothelin levels were not statistically significantly different in patients with mesothelin-positive from these with mesothelin-negative tumors (26). In another study published in 1999, a murine monoclonal antibody binding to membrane-bound mesothelin was generated and tested in various carcinomas. It bound to 20/21 ovarian tumor tissue samples and serum positivity was shown in 23/30 (76%) samples (27). Summarizing these results, mesothelin seems to be a promising candidate marker for ovarian cancer. Furthermore, cell-bound mesothelin is a promising target for antibody-based treatment of tumors overexpressing mesothelin. The combination of an anti-mesothelin immunotoxin in combination with tumor-directed radiation resulted in enhanced antitumor activity against mesothelin-expressing tumor xenografts (26, 27).
Significant associations of the analyzed markers with clinical parameters of the patients before surgery.
Significant associations of the analyzed markers with clinical parameters of the patients after chemotherapy.
Relationship between marker expression and clinical outcome.
Functionally, mesothelin appears to interact with CA125. McIntosh et al. proposed that it might be beneficial to use mesothelin as a composite marker with CA125 (28). The interaction between mesothelin and CA125 may facilitate the implantation and metastasis of tumors in the peritoneal cavity (29). This interaction potentially impacts upon experimental therapeutic options. In this regard, a novel high-affinity human monoclonal antibody, HN1, has been shown to bind to cell surface-associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. HN1 functionally blocks the interaction of mesothelin and CA125 on cancer cells and it has been concluded that it has significant potential for mesothelin-expressing cancer treatment and diagnosis (30). Our findings are in line with results of previous studies and, in our eyes, confirm the prognostic relevance of mesothelin in ovarian cancer. It will be interesting to analyze whether elevated serum mesothelin concentrations are of prognostic relevance for the use of these targeted therapies.
L1CAM. The expression and function of L1CAM has mainly been studied in ovarian cancer cell lines and mouse models, showing that its expression augments resistance to apoptosis and chemotherapy and that depletion of L1CAM sensitized cells to apoptosis. In addition, long-term treatment with cisplatin up-regulated L1CAM expression, which might be of clinical interest since this process could enrich for tumor cells with enhanced motility, invasiveness and better growth characteristics (23). Important results were published regarding tissue samples of 400 patients with ovarian cancer showing that L1CAM in normal ovarian surface epithelium supported cell-cell adhesion, enhanced apoptosis and had no effect on cell proliferation and invasion, whereas in ovarian carcinoma cells, it promoted all these malignancy-related properties. Furthermore, its expression significantly correlated with poor outcome and unfavourable clinicopathological features (30). To our knowledge, only one study so far determined L1CAM expression in serum samples of 28 patients with ovarian cancer indicating that L1CAM serum concentrations were raised in 80% of patients at stage III-IV whereas the levels in normal controls were usually very low (16). In our study, L1CAM positivity after chemotherapy significantly correlated with chemosensitivity. Admittedly, the association is weak. Still, it can be assumed that L1CAM is released on induced cell death and might thus be an indicator for chemotherapy response. It will be interesting to see whether there will be comparable observations in future analyses.
Recent work has shown that antibodies against L1CAM were shown to have therapeutic potential and can reduce cell proliferation in vivo and in vitro growth of SKOV3ip human ovarian carcinoma cells and xenografts (31-35). Thus, L1CAM might be a novel target for antibody-based therapy as second-line therapy against aggressive ovarian tumors.
Afamin. Few studies have been published for the determination of afamin in body fluids of patients with gynecological disorders. Seeber et al. showed significantly altered afamin levels in serum and peritoneal fluid of 242 reproductive-age women with and without endometriosis (36) and Dieplinger et al. evaluated the diagnostic utility of afamin in a large case control study of 181 patients with ovarian cancer of various clinical stages, 399 patients with benign gynecological diseases, including endometriosis, and 177 controls and compared results with those for the conventional ovarian cancer tumor marker CA125 (13). Afamin concentrations decreased from a median of 70.7 mg/l in healthy controls to 65.2 mg/l in patients with benign gynecological diseases to 56.0 mg/l in patients with ovarian cancer. Afamin added independent diagnostic information to that of CA125 and age for differentiating ovarian cancer from benign and healthy samples. In our study, the median values for healthy donors as well as patients with ovarian cancer in general were lower and did not differ relevantly between these groups. In addition, although nearly 50% of the patient samples were defined as normal ranging from 45-98 μg/ml as defined by the manufacturer, in many patients, marked changes occurred within the reference range, indicating that individual profiles have to be established in patient follow-up. This has also been reported for CA125, where an increase within the reference range was highly predictive of relapse (37).
In conclusion, besides significant associations found for CA125 before surgery and after chemotherapy, the use of mesothelin could add further information to assess response to ovarian cancer treatment. The fact that a high mesothelin concentration before surgery, as well as after chemotherapy, correlates with residual postoperative tumor is of special interest as the probability of a complete tumor resection might be estimated by this parameter. The correlation of soluble L1CAM with platinum sensitivity, although statistically weak, is an interesting phenomenon which also demands for further evaluation. In contrast, afamin does not seem to be a useful biomarker for ovarian cancer although marked changes occurred within the reference range.
Acknowledgements
We thank Gisela Koestner for her excellent technical assistance.
Footnotes
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Conflicts of Interest
The Authors declare that there are no conflicts of interest with regard to this study.
- Received October 8, 2012.
- Revision received November 26, 2012.
- Accepted November 27, 2012.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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