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Comparison of Osteopontin, Megakaryocyte Potentiating Factor, and Mesothelin Proteins as Markers in the Serum of Patients with Malignant Mesothelioma
ORIGINAL ARTICLE
Comparison of Osteopontin, Megakaryocyte Potentiating Factor, and Mesothelin Proteins as Markers in the Serum of Patients with Malignant Mesothelioma Jenette Creaney, PhD,*† Deborah Yeoman, BSc,* Yvonne Demelker,* Amanda Segal, MBBS, FRCPA,‡ A. W. Musk, MBBS, FRACP,*§ Steven J. Skates, PhD,储 and Bruce W. S. Robinson MBBS, MD, FRACP*†
Introduction: There is increasing interest in identifying a bloodbased marker for the asbestos-related tumor, malignant mesothelioma. Three potential markers for mesothelioma are mesothelin, megakaryocyte potentiating factor (MPF) and osteopontin. The purpose of the current study was to directly compare these biomarkers in the same sample population, determining their sensitivity and specificity in establishing a diagnosis, and to determine if diagnostic accuracy for mesothelioma is improved by combining the data from all three markers. Methods: Serum levels of mesothelin, MPF and osteopontin were determined by commercially available assays in 66 samples from patients with pleural malignant mesothelioma, 20 healthy individuals, 21 patients with asbestos-related lung or pleural disease, 30 patients presenting with benign pleural effusions and 30 patients with other malignancies. Results: Serum levels of the three markers were elevated in mesothelioma patients. At a level of specificity of 95% relative to healthy controls and patients with benign asbestos related disease, the sensitivity for mesothelioma was 34% for MPF, 47% for osteopontin and 73% for mesothelin. Osteopontin and MPF were unable to differentiate patients with mesothelioma from patients with other malignancies or those presenting with transudative pleural effusions. Combining the data from the three biomarkers using a logistic regression model did not improve sensitivity for detecting mesothelioma above that of the mesothelin marker alone. *National Research Centre for Asbestos Related Diseases, Western Australian Institute of Medical Research, School of Medicine and Pharmacology, University of Western Australia, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; †The Australian Mesothelioma Tissue Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; ‡PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Hospital Ave., Nedlands, Western Australia, Australia; §Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; and 储Biostatistics Center, Massachusetts General Hospital, Boston, Massachusetts. Disclosure: Jenette Creaney and Bruce W. S. Robinson have received consultancy fees from Fujirebio Diagnositic Incorporated (FDI), Malvern, PA. The remaining authors have no conflicts to disclose. Address for correspondence: Jenette Creaney, PhD, Sir Charles Gairdner Hospital, University of Western Australia, Verdun St., Nedlands, WA 6009, Australia. E-mail: [email protected] Copyright © 2008 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/08/0308-0851
Conclusion: Serum mesothelin remains the most specific marker for the diagnosis of mesothelioma. Key Words: Mesothelioma, Tumor markers, Mesothelin, SMRP, MPF, Osteopontin, Diagnosis. (J Thorac Oncol. 2008;3: 851–857)
M
alignant mesothelioma is caused by exposure to asbestos and is a fatal disease with limited treatment options.1,2 In other forms of cancers, serum-based tumor markers have provided clinicians with a valuable aid in the management of patients. Markers may have different roles in diagnosis, monitoring, assessing prognosis, and in detection of early disease. There are no markers in routine clinical use for malignant mesothelioma, although the use of soluble mesothelin has received recent attention. Mesothelin is a differentiation marker of mesothelial cells. Differential post-transcriptional and post-translational processing of the mesothelin gene produces four protein products: megakaryocyte potentiating factor (MPF),3,4 mesothelin variant 1, mesothelin variant 2,5 and serum mesothelin-related protein (SMRP).6 – 8 MPF is a soluble ⬃32 kd protein with cytokine activity.3,4 Mesothelin is ⬃40 kd glycosylated protein predominately anchored to the cell surface of normal mesothelial cells, but also present in solution (the physiological mechanism for mesothelin release from the cell surface is not clear).9,10 Mesothelin variant 1 is the predominant form of the protein and differs from variant 2 by only eight amino acids. SMRP is a soluble protein with an identical N-terminal sequence to mesothelin but a unique C-terminal.6 Using an enzyme-linked immunosorbent assay (ELISA) which detects both mesothelin variant 1 and SMRP, we recently demonstrated increased levels of this biomarker in the serum of patients with mesothelioma,11 findings recently confirmed using the same assay12 and an independent13 assay. Serum mesothelin is highly specific for mesothelioma (specificity 98%) with a sensitivity at diagnosis of 49% (57/117)14 and 84% in advanced disease.11 Despite very encouraging findings showing serum mesothelin as a useful marker in mesothelioma, we found that at diagnosis only half of mesothelioma patients were mesothelin positive; therefore, there is a need for additional markers to improve diagnostic sen-
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sitivity. Possible candidate markers include proteins that are related to mesothelin, such as MPF, or those that are independent of mesothelin but associated with the disease such as osteopontin. Given the successful detection of mesothelin and SMRP in the serum of cancer patients, two groups recently, independently developed assays to measure levels of MPF.15,16 Onda and colleagues showing 51/56 patients with advanced mesothelioma had elevated serum MPF.15 As mesothelin must be cleaved or shed from the cell surface serum MPF may be at least as sensitive, if not more so, than serum mesothelin as a marker of mesothelioma. To clarify this, we measured and compared serum mesothelin and MPF levels in the same patient cohort using commercially available assays. Another strategy to improve diagnostic sensitivity is to incorporate the information from multiple markers. Pathologists use suites of immunohistochemical markers in tissue to improve diagnostic accuracy of the histopathologic findings17,18 and multiple serum markers have achieved greater sensitivity and specificity in the diagnosis of some cancers, i.e., ovarian19,20 and prostate cancer.21 Recently, we tested the hypothesis that combining data from two markers (mesothelin and CA125) would improve the sensitivity of the mesothelin marker used alone to distinguish mesothelioma patients from other asbestos-exposed individuals; however, we found that this combination was not significantly different to the results for mesothelin alone.14 Osteopontin is a secreted 44 kd glycoprotein with roles in cell-matrix interactions, cell migration, and other diverse functions. Serum osteopontin levels are elevated in breast, ovarian, lung, and prostrate cancer.22 Osteopontin has been shown to be a useful adjunct to CA125 in the detection of recurrent ovarian cancer.19 In mesothelioma serum, osteopontin has a sensitivity of 78% at a specificity of 85% relative to subjects exposed to asbestos.23 The purpose of the current study was to directly compare each of these biomarkers in the same sample population and to determine if diagnostic accuracy for mesothelioma is improved by combining these three markers.
PATIENTS AND METHODS
Serum Mesothelin Soluble mesothelin concentrations were determined in duplicate following the manufacturer’s instructions using a double-determinant ELISA assay, the MESOMARK kit, supplied by Fujirebio Diagnostics (Malvern, PA). Mesothelin concentrations were determined from a standard curve performed on each plate and expressed as nanomolar. Dilution of samples was carried out, if necessary, using the diluent supplied by the manufacturer. All assays were performed on coded samples by technical staff unaware of the patient’s diagnosis.
MPF Assay The Human N-ERC/Mesothelin Assay kit was purchased from Immuno-Biologic Laboratories (Gunma, Japan) and the manufacturer’s recommended protocol was followed. Serum samples were assayed undiluted and concentrations of MPF were determined from a standard curve performed in parallel. The manufacturer’s reported assay sensitivity was 0.024 ng/ml; the concentrations for samples from 0.024 ng/ml to 0.011 ng/ml were extrapolated from the standard curve, for the purposes of data analysis concentrations of samples below 0.011 ng/ml were assigned a value of 0.005 ng/ml.
Osteopontin Assay The human osteopontin assay, a double-determinant ELISA, was purchased from Immuno-Biologic Laboratories. The manufacturer’s recommended protocol was followed except serum was used as the assay substrate instead of plasma. Serum samples were diluted 1:3 in buffer supplied with the kit. The manufacturer’s reported assay sensitivity was 3.33 ng/ml, the concentrations for samples from 3.3 ng/ml to 0.5 ng/ml were extrapolated from the standard curve, and for the purposes of data analysis concentrations of samples below 0.5 ng/ml were assigned a value of 0.1 ng/ml.
Statistical Analysis
Patients and Controls Serum samples were collected from consecutive patients presenting at the respiratory clinics of either Sir Charles Gairdner Hospital or the Hollywood Specialist Centre in Perth, Western Australia and form part of the Australian Mesothelioma Tissue Bank. We obtained written and oral informed consent from participants. This study was approved by the human ethics committees of Sir Charles Gairdner and Hollywood Hospitals. The final diagnosis in all patients was confirmed by pathologists experienced in the diagnosis of mesothelioma and effusions and included clinical follow-up of all cases until death or for an average of 6.8 months (range, 1– 42 months) to confirm that the clinical pattern matched the diagnosis. Effusions were classified as being malignant or nonmalignant on the basis of cytologic and immunohistochemical features. Nonmalignant effusions were classified as exudates or transudates by Light’s criteria.24
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Blood samples were collected by routine venepuncture into clotted blood tubes. Samples were allowed to clot for at least 2 hours at room temperature, or alternatively were stored at 4°C before processing. Samples were centrifuged at 1200 rpm for 10 minutes, then the supernatant was removed, aliquoted, and stored at ⫺80°C until use.
Differences between groups of patients were assessed by Student t test after transforming the biomarker values to the log scale for which the distributions were closer to normality. For the same reason, median biomarker values were estimated from the mean on the log scale and exponentiated to provide the estimate of the median on the original scale. All reported p values are two sided. A level of p ⬍ 0.05 was accepted as significant. Receiver operating characteristic (ROC) curves display the trade-off between sensitivity and specificity for biomarkers differentiating between groups of patients. Area under the curve (AUC) for two biomarkers, including “markers” formed by combining multiple biomarkers, was compared using the method of DeLong et al.,25 which accounts for the correlation because of the markers being measured on the same set of serum samples. To combine biomarkers, we first transformed data with the natural logarithm, and then standardized the markers relative
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to asbestos-exposed controls. Logistic regression to predict case/control status was used to determine the weight given each marker. Markers were multiplied by their logistic regression coefficient and added to give a combined marker value. Pearson’s correlation coefficient was used to assess correlations between different biomarkers (log scale).
RESULTS Patient Characteristics Serum samples were collected from 66 patients with pleural malignant mesothelioma; 9 with predominantly sarcomatoid histology and 57 with predominantly epithelioid histology or from whom a diagnosis was made on immunocytological grounds.26 Sera were collected from 20 apparently healthy individuals, 10 of whom had no reported previous asbestos exposure, and 10 with exposure. Samples were also collected from 21 patients with benign asbestos-related pulmonary disease (asbestosis and/or pleural plaques); from 30 patients with nonmalignant effusions (10 had exudative effusions, 10 had effusions relating to an infection, and 10 had transudate effusions); from 20 people who presented with effusions because of other malignancies (10 with primary lung cancer, 3 with breast cancer, 2 each with colon and pancreatic cancer, and one each with melanoma, sarcoma, and non-Hodgkin lymphoma) and from 10 additional cases of lung cancer who did not present with an effusion (Table 1).
MPF Levels in Serum Serum MPF levels in patients with mesothelioma ranged from undetectable to 5.9 ng/ml (Figure 1). Levels ranged from undetectable to 0.7 ng/ml in patients with mesothelioma tumors of sarcomatoid histology, levels which
Comparison of Osteopontin, MPF, and Mesothelin Proteins
were lower than those observed in patients with epithelialtype mesotheliomas although not significantly so (p ⫽ 0.09). Serum MPF levels were significantly higher in the serum of patients with mesothelioma than the healthy control population (p ⫽ 0.007), the patients with benign asbestos-related disease (p ⫽ 0.01), and patients with nonmalignant exudative effusions (p ⫽ 0.01). There was no difference in MPF levels between those patients with mesothelioma, lung cancer, or with effusions of a transudate or malignant nature.
Osteopontin Levels in Serum Serum osteopontin levels in patients with mesothelioma ranged from undetectable to 438 ng/ml (Figure 1). There was no relationship between the levels of osteopontin and the histologic diagnosis of the patients’ mesothelioma (Table 1). Serum osteopontin levels were significantly higher in the serum of patients with mesothelioma than the healthy control population (p ⫽ 0.0002), the patients with benign asbestosrelated disease (p ⬍ 0.0001), and the patients with nonmalignant exudative effusions (p ⫽ 0.0002). There was no difference in osteopontin levels between patients with mesothelioma, lung cancer, or effusions of a transudate or malignant nature. There was no significant difference in serum osteopontin levels between healthy control individuals and individuals with benign asbestos-related disease.
Mesothelin Levels in Serum Serum mesothelin levels were significantly higher in patients with mesothelioma compared with healthy controls (p ⫽ 0.0001), individuals with benign asbestos-related disease (p ⫽ 0.0001), patients with benign effusions (p ⬍ 0.005), patients with malignant effusions (p ⫽ 0.0006), and patients with lung cancer (p ⫽ 0.03). Serum mesothelin levels
TABLE 1. Patient Characteristics and Biomarker Levels
MM—all Epithelial and cytology Sarcomatoid Healthy controls—all Nonasbestos exposed Asbestos exposed Benign controls—all Asbestosis Asbestosis and plaques Pleural effusions Transudate Exudate Exudate—infection Malignancy Lung cancer
No. of Cases
Median Age, yr (range)
66 (5F) 57 (5F)
69 (50–84) 70 (50–84)
9 (0F) 20 (7F) 10 (2F) 10 (5F) 21 (4F) 11 (3F) 10 (1F)
68 (53–77) 55 (33–64) 48 (33–58) 58 (40–64) 71 (49–86) 70 (49–86) 73 (60–86)
9 (2F) 10 (1F) 9 (5F) 20 (10F) 10 (2F)
76 (48–82) 76 (32–80) 64 (50–86) 74 (50–83) 74 (58–82)
Osteopontina,b 14.8 ⫾ 8.2 15.9 ⫾ 8 9.2 ⫾ 10.4 2 ⫾ 7.3** 1.2 ⫾ 10.5 3.2 ⫾ 4.6 1.8 ⫾ 8** 1.8 ⫾ 8.6 1.9 ⫾ 8.5 15.6 ⫾ 11 (NS) 1.8 ⫾ 12.1* 1.7 ⫾ 9** 20.7 ⫾ 8.1 (NS) 19 ⫾ 7.5 (NS)
No. of Positive Samplesc 30/66 26/57
MPFa,b,d 0.19 ⫾ 13.5 0.23 ⫾ 13.4
No. of Positive No. of Positive Samplesc Mesothelina,c Samplesc 21/66 21/57
4.7 ⫾ 3.4 5.7 ⫾ 3.3
48/66 47/57
4/9 0/20 0/10 0/10 3/20 1/10 2/10
0.05 ⫾ 10.5 0.05 ⫾ 5.8* 0.04 ⫾ 8.4 0.05 ⫾ 4.1 0.09 ⫾ 6.2 (NS) 0.06 ⫾ 6.6 0.14 ⫾ 5.5
0/9 1/20 1/10 0/10 1/20 0/10 1/10
1.3 ⫾ 1.3 0.4 ⫾ 3.4*** 0.24 ⫾ 5.1 0.65 ⫾ 1.32 0.87 ⫾ 2.4*** 0.76 ⫾ 3.1 1.0 ⫾ 1.6
1/9 0/20 0/10 0/10 1/20 1/10 0/10
5/10 0/10 1/10 10/20 7/10
0.2 ⫾ 8.9 (NS) 0.05 ⫾ 8.3 (NS) 0.08 ⫾ 6.1 (NS) 0.33 ⫾ 4.6 (NS) 0.36 ⫾ 3.6 (NS)
1/10 0/10 0/10 0/20 2/10
1.7 ⫾ 1.9* 0.8 ⫾ 1.8*** 0.7 ⫾ 3*** 1.6 ⫾ 2.5* 1.79 ⫾ 2.95*
5/10 0/10 2/10 9/20 5/10
Expontiated mean of log transformed data ⫾ standard error. Significant difference between indicated cohorts and the mesothelioma cohort as a whole (n ⫽ 66) as determined by Student t test (NS is not significant; *p ⬍ 0.05; **p ⬍ 0.001; ***p ⬍ 0.0001). c The number of samples above the 95% specificity level as determined for each marker and described in Table 2 below. d MPF concentrations determined from Human N-ERC/Mesothelin Assay (IBL). a b
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FIGURE 2. ROC curve showing accuracy of serum biomarker concentration in differentiating all patients with mesothelioma (n ⫽ 66) from healthy and benign controls (n ⫽ 41).
Comparison of Multiple Biomarker Levels When ROC curves were generated to assess the ability of the markers to distinguish patients with mesothelioma from the healthy controls and the patients with benign asbestos-related disease, the AUC for MPF was 0.614 (95% confidence interval [CI], 0.508 – 0.721), for osteopontin it was 0.757 (95% CI, 0.666 – 0.848) and for mesothelin it was 0.915 (95% CI, 0.869 – 0.968) (Figure 2). The AUC for mesothelin was significantly higher than for MPF or for osteopontin. From the curves, the sensitivities were determined for specificities of 85%, 90%, 95%, and 98% (Table 2). In the present study, at a specificity of 95%, the MPF assay had a sensitivity of 34%; the osteopontin assay had a sensitivity of 47%, and the mesothelin assay had a sensitivity of 73%. A bivariate scattergram of mesothelin versus MPF (Figure 3A) and osteopontin (Figure 3B) levels in individuals with mesothelioma and controls with the threshold values for 95% specificity indicated only one individual who had elevated MPF and osteopontin levels who was negative for mesothelin, and another individual with sarcomatoid variant with elevated osteopontin but negative mesothelin, demonstrating that neither MPF nor osteopontin add significant information about the presence of mesothelioma to mesotheTABLE 2. Sensitivity of Individual Biomarkers Estimated at Different Levels of Specificity for Differentiating all Patients with Mesothelioma (n ⫽ 66) from Healthy and Benign Controls (n ⫽ 41) Estimated Sensitivities
FIGURE 1. Biomarker concentrations in serum. Biomarker concentrations were determined at least in duplicate by ELISA and individual patient values are plotted on the graph. A, MPF; B, osteopontin; and C, mesothelin.
were significantly higher in patients with mesothelioma tumors of epithelial-like histology than those with predominately sarcomatoid histology (p ⫽ 0.0004) (Table 1).
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MPF Osteopontin Mesothelin
85% Specificity (threshold value)
90% Specificity (threshold value)
95% Specificity (threshold value)
98% Specificity (threshold value)
39 (0.6) 55 (10.5) 81 (1.25)
36 (0.7) 53 (12) 78 (1.4)
34 (1) 47 (18) 73 (1.6)
34 (1.3) 22 (77) 68 (1.8)
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Comparison of Osteopontin, MPF, and Mesothelin Proteins
FIGURE 4. ROC curve showing accuracy of serum biomarker concentrations individually and combined using a logistic regression model in differentiating all patients with mesothelioma (n ⫽ 66) from healthy and benign controls (n ⫽ 41).
teopontin significantly increased the AUC when added to a panel. All combined markers with mesothelin had AUCs in the range 90.7 to 91.5% which was not significantly different from the AUC for mesothelin alone, whereas the AUC for the combined marker formed from MPF and osteopontin had an AUC of 74.5% which was significantly lower than any AUC from a combined marker with mesothelin at p ⬍ 0.001.
DISCUSSION
FIGURE 3. Bivariate scattergram of biomarker concentrations determined in serum from individual patients with mesothelioma (䉬) and from healthy and benign controls (‚). Dashed lines represent the level at which the individual biomarker has a specificity of 95%. A, MPF concentrations plotted against mesothelin; B, osteopontin against mesothelin; and C, osteopontin against MPF.
lin. There was a group of approximately 40% and 34% mesothelioma patients positive for mesothelin and negative for MPF and osteopontin, respectively. Examining only the patients with mesothelioma, there was a significant correlation between mesothelin levels and MPF (r ⫽ 0.477; p ⬍ 0.01) and osteopontin (r ⫽ 0.373; p ⬍ 0.01) levels. A bivariate scattergram of MPF and osteopontin revealed that the majority of patients with elevated osteopontin had elevated MPF which was reflected in a correlation of r ⫽ 0.632 (p ⬍ 0.01) between these markers (Figure 3C). The results of using a logistic regression model to combine the data from the three markers are presented in Figure 4 and Table 3. Those combinations that include mesothelin all increase the AUC value compared with the panel without mesothelin; however, neither MPF nor os-
The development of multiple markers in disease diagnostics is the goal of a number of biomarker research centers. This study shows no improvement in combining serum osteopontin and MPF with mesothelin in determining the accuracy of diagnosis of malignant mesothelioma over the mesothelin marker used alone. MPF levels are reported to be elevated in mesothelioma patients.15,16 Using a commercial version of the assay developed by Shiomi et al., we found that, at a specificity of 95%, 34% of mesothelioma patients had elevated levels of MPF, less than the 7/716 and 51/56 positive mesothelioma patients previously reported.15 Such a difference may reflect differences in the stage distribution of the mesothelioma patients as Onda et al.15 reported that all their subjects had advanced disease. A diagnostic test with high sensitivity only in patients who have advanced cancer is clearly of little clinical value. One might expect that the MPF and mesothelin assays would have similar sensitivity and specificity. Our finding that this was not the case was contrary to the notion that MPF would be a more sensitive marker for mesothelioma than mesothelin because of the latter’s need to be cleaved or shed from the cell surface. For the same specificity, fewer mesothelioma patients had elevated MPF compared with the numbers with elevated mesothelin. The reason for the difference in sensitivity of the two markers of the same precursor protein may simply be a reflection of the relative stabilities of the molecules, though the physiology of their relative stabilities in vivo and clearance kinetics is yet to be defined. Osteopontin is a secreted glycol-phosphoprotein which is produced by many cell types including osteoclasts, osteoblasts, epithelial cells of the breast, kidney and skin, nerve
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TABLE 3. Area Under the ROC Curve for Individual and Combined Biomarkers, Discriminating All Patients with Mesothelioma (n ⫽ 66) from Healthy and Benign Controls (n ⫽ 41) Asymptotic 95% CI a
Biomarker Combination
AUC
SE
Combined mesothelin, OSN and MPF Combined mesothelin and OSN Combined mesothelin and MPF Combined OSN and MPF Mesothelin OSN MPF
0.920 0.926 0.916 0.745 0.905 0.768 0.605
0.026 0.024 0.026 0.048 0.028 0.045 0.054
Lower Bound
Upper Bound
pb
pc
pd
0.869 0.878 0.866 0.651 0.849 0.679 0.499
0.971 0.974 0.966 0.838 0.960 0.857 0.711
Reference 0.89 0.40 ⬍0.0001 NA NA NA
NA Reference 0.87 ⬍0.0001 NA NA NA
NA NA Reference ⬍0.0001 NA NA NA
a
Standard error under the nonparametric assumption. p values for comparing the AUC for combinations of biomarkers in a logistic regression model using the method of DeLong et al. AUC, area under the curve; CI, confidence interval; OSN, osteopontin.
b,c,d
cells, endothelial cells, and immune cells such as T cells, natural killer cells, and macrophages. In malignancy, osteopontin is believed to play a role in tumor progression, growth, invasiveness, and metastasis. It has been shown to be elevated in the serum of patients with melanoma, breast, lung, colorectal, stomach, and ovarian cancer22,27 and recently also in mesothelioma.23,28 The results of our current study confirm that serum levels of osteopontin are elevated in some mesothelioma patients. One concern that remains is the nature of the test sample. In the three mesothelioma-osteopontin studies to date where osteopontin was measured in the serum, the protein may potentially be cleaved by thrombin also present in the sera and results may not reflect the true levels of the protein in the blood. Grigoriu et al. reported that although osteopontin levels were higher in plasma, the ability of osteopontin to discriminate between patients with mesothelioma and healthy asbestos-exposed subjects was similar when using plasma or serum levels.28 We restricted the current study to serum as we needed to use the same sample for all patients. The need to use plasma, serum, or other collection methods needs further study. Although serum levels of osteopontin discriminate between mesothelioma patients and healthy controls,23,28 levels of osteopontin alone are unlikely to be of clinical value in mesothelioma diagnosis. Furthermore, serum osteopontin levels are elevated in other malignancies, as described above. In addition, some common nonmalignant, nonpleural effusionassociated conditions including coronary artery disease, interstitial pneumonia, and other benign pulmonary disease28 –30 can result in increased levels of osteopontin in the serum. We have previously found that individual patients with mesothelioma exhibit different biomarker profiles: either both CA125 and mesothelin, only one or neither marker. This was not found to be the case with the pattern of expression of osteopontin and mesothelin as virtually all osteopontin positive mesothelioma patients were positive for mesothelin. This high degree of concordance means that combining such markers would be unlikely to add to sensitivity. As the various combinations of markers discriminated between mesothelioma patients and controls, no better than when mesothelin was used alone, our current practice is to measure serum mesothelin as the
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preferred biomarker for suspected mesothelioma and not include assays for the other biomarkers. The mesothelin biomarker represents the best available serum biomarker for mesothelioma. An elevated mesothelin level is highly suggestive of malignancy, and particularly of mesothelioma. Other markers are needed to improve the sensitivity for mesothelioma in symptomatic patients, and possibly for use in any screening strategy for asbestosexposed individuals. Studies are therefore ongoing to identify new mesothelioma-specific markers.
ACKNOWLEDGMENTS Supported in part by research grants from the National Health and Medical Council of Australia and the Insurance Commission of Western Australia, and in part by FDI. The study sponsors had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication. We thank the staff of PathWest Laboratory Medicine, Sir Charles Gairdner Hospital, Hollywood Hospital and St. John of God Pathology for their assistance with this study. REFERENCES 1. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2005;353:1591–1603. 2. Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet 2005;366:397– 408. 3. Kojima T, Oh-eda M, Hattori K, et al. Molecular cloning and expression of megakaryocyte potentiating factor cDNA. J Biol Chem 1995;270: 21984 –21990. 4. Yamaguchi N, Hattori K, Oh-eda M, et al. A novel cytokine exhibiting megakaryocyte potentiating activity from a human pancreatic tumor cell line HPC-Y5. J Biol Chem 1994;269:805– 808. 5. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci USA 1996;93:136 –140. 6. Scholler N, Fu N, Yang Y, et al. Soluble member(s) of the mesothelin/ megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci USA 1999;96: 11531–11536. 7. Muminova ZE, Strong TV, Shaw DR. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer. BMC Cancer 2004;4:19.
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8. Hassan R, Bera T, Pastan I. Mesothelin: a new target for immunotherapy. Clin Cancer Res 2004;10:3937–3942. 9. Hellstrom I, Raycraft J, Kanan S, et al. Mesothelin variant 1 is released from tumor cells as a diagnostic marker. Cancer Epidemiol Biomarkers Prev 2006;15:1014 –1020. 10. Ho M, Onda M, Wang QC, et al. Mesothelin is shed from tumor cells. Cancer Epidemiol Biomarkers Prev 2006;15:1751. 11. Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins and diagnosis of mesothelioma. Lancet 2003;362:1612–1616. 12. Scherpereel A, Grigoriu B, Conti M, et al. Soluble mesothelin-related protein in the diagnosis of malignant pleural mesothelioma. Am J Respir Crit Care Med 2006;173:1155–1160. 13. Hassan R, Remaley AT, Sampson ML, et al. Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer. Clin Cancer Res 2006;12:447– 453. 14. Creaney J, van Bruggen I, Hof M, et al. Combined CA125 and mesothelin levels for the diagnosis of malignant mesothelioma. Chest 2007;132: 1239 –1246. 15. Onda M, Nagata S, Ho M, et al. Megakaryocyte potentiation factor cleaved from mesothelin precursor is a useful tumor marker in the serum of patients with mesothelioma. Clin Cancer Res 2006;12:4225– 4231. 16. Shiomi K, Miyamoto H, Segawa T, et al. Novel ELISA system for detection of N-ERC/mesothelin in the sera of mesothelioma patients. Cancer Sci 2006;97:928 –932. 17. Ordonez NG. The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol 2003;27:1031–1051. 18. Whitaker D. The cytology of malignant mesothelioma. Cytopathology 2000;11:139 –151. 19. Schorge JO, Drake RD, Lee H, et al. Osteopontin as an adjunct to CA125 in detecting recurrent ovarian cancer. Clin Cancer Res 2004;10: 3474 –3478. 20. Skates SJ, Horick N, Yu Y, et al. Preoperative sensitivity and specificity
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30.
Comparison of Osteopontin, MPF, and Mesothelin Proteins
for early-stage ovarian cancer when combining cancer antigen CA125II, CA 15-3, CA 72-4, and macrophage colony-stimulating factor using mixtures of multivariate normal distributions. J Clin Oncol 2004; 22:4059 – 4066. Etzioni R, Falcon S, Gann PH, et al. Prostate-specific antigen and free prostate-specific antigen in the early detection of prostate cancer: do combination tests improve detection? Cancer Epidemiol Biomarkers Prev 2004;13:1640 –1645. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer 2004;90:1877–1881. Pass HI, Lott D, Lonardo F, et al. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 2005;353:1564 –1573. Light RW, Macgregor MI, Luchsinger PC, et al. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77:507–513. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 1988;44:837– 845. Henderson DW, Shilkin KB, Whitaker D. Reactive mesothelial hyperplasia vs mesothelioma, including mesothelioma in situ: a brief review. Am J Clin Pathol 1998;110:397– 404. Wai PY, Kuo PC. The role of osteopontin in tumor metastasis. J Surg Res 2004;121:228 –241. Grigoriu BD, Scherpereel A, Devos P, et al. Utility of osteopontin and serum mesothelin in malignant pleural mesothelioma diagnosis and prognosis assessment. Clin Cancer Res 2007;13:2928 –2935. Kadota J, Mizunoe S, Mito K, et al. High plasma concentrations of osteopontin in patients with interstitial pneumonia. Respir Med 2005; 99:111–117. Ohmori R, Momiyama Y, Taniguchi H, et al. Plasma osteopontin levels are associated with the presence and extent of coronary artery disease. Atherosclerosis 2003;170:333–337.
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Comparison of Osteopontin, Megakaryocyte Potentiating Factor, and Mesothelin Proteins as Markers in the Serum of Patients with Malignant Mesothelioma Jenette Creaney, PhD,*† Deborah Yeoman, BSc,* Yvonne Demelker,* Amanda Segal, MBBS, FRCPA,‡ A. W. Musk, MBBS, FRACP,*§ Steven J. Skates, PhD,储 and Bruce W. S. Robinson MBBS, MD, FRACP*†
Introduction: There is increasing interest in identifying a bloodbased marker for the asbestos-related tumor, malignant mesothelioma. Three potential markers for mesothelioma are mesothelin, megakaryocyte potentiating factor (MPF) and osteopontin. The purpose of the current study was to directly compare these biomarkers in the same sample population, determining their sensitivity and specificity in establishing a diagnosis, and to determine if diagnostic accuracy for mesothelioma is improved by combining the data from all three markers. Methods: Serum levels of mesothelin, MPF and osteopontin were determined by commercially available assays in 66 samples from patients with pleural malignant mesothelioma, 20 healthy individuals, 21 patients with asbestos-related lung or pleural disease, 30 patients presenting with benign pleural effusions and 30 patients with other malignancies. Results: Serum levels of the three markers were elevated in mesothelioma patients. At a level of specificity of 95% relative to healthy controls and patients with benign asbestos related disease, the sensitivity for mesothelioma was 34% for MPF, 47% for osteopontin and 73% for mesothelin. Osteopontin and MPF were unable to differentiate patients with mesothelioma from patients with other malignancies or those presenting with transudative pleural effusions. Combining the data from the three biomarkers using a logistic regression model did not improve sensitivity for detecting mesothelioma above that of the mesothelin marker alone. *National Research Centre for Asbestos Related Diseases, Western Australian Institute of Medical Research, School of Medicine and Pharmacology, University of Western Australia, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; †The Australian Mesothelioma Tissue Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; ‡PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Hospital Ave., Nedlands, Western Australia, Australia; §Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; and 储Biostatistics Center, Massachusetts General Hospital, Boston, Massachusetts. Disclosure: Jenette Creaney and Bruce W. S. Robinson have received consultancy fees from Fujirebio Diagnositic Incorporated (FDI), Malvern, PA. The remaining authors have no conflicts to disclose. Address for correspondence: Jenette Creaney, PhD, Sir Charles Gairdner Hospital, University of Western Australia, Verdun St., Nedlands, WA 6009, Australia. E-mail: [email protected] Copyright © 2008 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/08/0308-0851
Conclusion: Serum mesothelin remains the most specific marker for the diagnosis of mesothelioma. Key Words: Mesothelioma, Tumor markers, Mesothelin, SMRP, MPF, Osteopontin, Diagnosis. (J Thorac Oncol. 2008;3: 851–857)
M
alignant mesothelioma is caused by exposure to asbestos and is a fatal disease with limited treatment options.1,2 In other forms of cancers, serum-based tumor markers have provided clinicians with a valuable aid in the management of patients. Markers may have different roles in diagnosis, monitoring, assessing prognosis, and in detection of early disease. There are no markers in routine clinical use for malignant mesothelioma, although the use of soluble mesothelin has received recent attention. Mesothelin is a differentiation marker of mesothelial cells. Differential post-transcriptional and post-translational processing of the mesothelin gene produces four protein products: megakaryocyte potentiating factor (MPF),3,4 mesothelin variant 1, mesothelin variant 2,5 and serum mesothelin-related protein (SMRP).6 – 8 MPF is a soluble ⬃32 kd protein with cytokine activity.3,4 Mesothelin is ⬃40 kd glycosylated protein predominately anchored to the cell surface of normal mesothelial cells, but also present in solution (the physiological mechanism for mesothelin release from the cell surface is not clear).9,10 Mesothelin variant 1 is the predominant form of the protein and differs from variant 2 by only eight amino acids. SMRP is a soluble protein with an identical N-terminal sequence to mesothelin but a unique C-terminal.6 Using an enzyme-linked immunosorbent assay (ELISA) which detects both mesothelin variant 1 and SMRP, we recently demonstrated increased levels of this biomarker in the serum of patients with mesothelioma,11 findings recently confirmed using the same assay12 and an independent13 assay. Serum mesothelin is highly specific for mesothelioma (specificity 98%) with a sensitivity at diagnosis of 49% (57/117)14 and 84% in advanced disease.11 Despite very encouraging findings showing serum mesothelin as a useful marker in mesothelioma, we found that at diagnosis only half of mesothelioma patients were mesothelin positive; therefore, there is a need for additional markers to improve diagnostic sen-
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sitivity. Possible candidate markers include proteins that are related to mesothelin, such as MPF, or those that are independent of mesothelin but associated with the disease such as osteopontin. Given the successful detection of mesothelin and SMRP in the serum of cancer patients, two groups recently, independently developed assays to measure levels of MPF.15,16 Onda and colleagues showing 51/56 patients with advanced mesothelioma had elevated serum MPF.15 As mesothelin must be cleaved or shed from the cell surface serum MPF may be at least as sensitive, if not more so, than serum mesothelin as a marker of mesothelioma. To clarify this, we measured and compared serum mesothelin and MPF levels in the same patient cohort using commercially available assays. Another strategy to improve diagnostic sensitivity is to incorporate the information from multiple markers. Pathologists use suites of immunohistochemical markers in tissue to improve diagnostic accuracy of the histopathologic findings17,18 and multiple serum markers have achieved greater sensitivity and specificity in the diagnosis of some cancers, i.e., ovarian19,20 and prostate cancer.21 Recently, we tested the hypothesis that combining data from two markers (mesothelin and CA125) would improve the sensitivity of the mesothelin marker used alone to distinguish mesothelioma patients from other asbestos-exposed individuals; however, we found that this combination was not significantly different to the results for mesothelin alone.14 Osteopontin is a secreted 44 kd glycoprotein with roles in cell-matrix interactions, cell migration, and other diverse functions. Serum osteopontin levels are elevated in breast, ovarian, lung, and prostrate cancer.22 Osteopontin has been shown to be a useful adjunct to CA125 in the detection of recurrent ovarian cancer.19 In mesothelioma serum, osteopontin has a sensitivity of 78% at a specificity of 85% relative to subjects exposed to asbestos.23 The purpose of the current study was to directly compare each of these biomarkers in the same sample population and to determine if diagnostic accuracy for mesothelioma is improved by combining these three markers.
PATIENTS AND METHODS
Serum Mesothelin Soluble mesothelin concentrations were determined in duplicate following the manufacturer’s instructions using a double-determinant ELISA assay, the MESOMARK kit, supplied by Fujirebio Diagnostics (Malvern, PA). Mesothelin concentrations were determined from a standard curve performed on each plate and expressed as nanomolar. Dilution of samples was carried out, if necessary, using the diluent supplied by the manufacturer. All assays were performed on coded samples by technical staff unaware of the patient’s diagnosis.
MPF Assay The Human N-ERC/Mesothelin Assay kit was purchased from Immuno-Biologic Laboratories (Gunma, Japan) and the manufacturer’s recommended protocol was followed. Serum samples were assayed undiluted and concentrations of MPF were determined from a standard curve performed in parallel. The manufacturer’s reported assay sensitivity was 0.024 ng/ml; the concentrations for samples from 0.024 ng/ml to 0.011 ng/ml were extrapolated from the standard curve, for the purposes of data analysis concentrations of samples below 0.011 ng/ml were assigned a value of 0.005 ng/ml.
Osteopontin Assay The human osteopontin assay, a double-determinant ELISA, was purchased from Immuno-Biologic Laboratories. The manufacturer’s recommended protocol was followed except serum was used as the assay substrate instead of plasma. Serum samples were diluted 1:3 in buffer supplied with the kit. The manufacturer’s reported assay sensitivity was 3.33 ng/ml, the concentrations for samples from 3.3 ng/ml to 0.5 ng/ml were extrapolated from the standard curve, and for the purposes of data analysis concentrations of samples below 0.5 ng/ml were assigned a value of 0.1 ng/ml.
Statistical Analysis
Patients and Controls Serum samples were collected from consecutive patients presenting at the respiratory clinics of either Sir Charles Gairdner Hospital or the Hollywood Specialist Centre in Perth, Western Australia and form part of the Australian Mesothelioma Tissue Bank. We obtained written and oral informed consent from participants. This study was approved by the human ethics committees of Sir Charles Gairdner and Hollywood Hospitals. The final diagnosis in all patients was confirmed by pathologists experienced in the diagnosis of mesothelioma and effusions and included clinical follow-up of all cases until death or for an average of 6.8 months (range, 1– 42 months) to confirm that the clinical pattern matched the diagnosis. Effusions were classified as being malignant or nonmalignant on the basis of cytologic and immunohistochemical features. Nonmalignant effusions were classified as exudates or transudates by Light’s criteria.24
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Blood samples were collected by routine venepuncture into clotted blood tubes. Samples were allowed to clot for at least 2 hours at room temperature, or alternatively were stored at 4°C before processing. Samples were centrifuged at 1200 rpm for 10 minutes, then the supernatant was removed, aliquoted, and stored at ⫺80°C until use.
Differences between groups of patients were assessed by Student t test after transforming the biomarker values to the log scale for which the distributions were closer to normality. For the same reason, median biomarker values were estimated from the mean on the log scale and exponentiated to provide the estimate of the median on the original scale. All reported p values are two sided. A level of p ⬍ 0.05 was accepted as significant. Receiver operating characteristic (ROC) curves display the trade-off between sensitivity and specificity for biomarkers differentiating between groups of patients. Area under the curve (AUC) for two biomarkers, including “markers” formed by combining multiple biomarkers, was compared using the method of DeLong et al.,25 which accounts for the correlation because of the markers being measured on the same set of serum samples. To combine biomarkers, we first transformed data with the natural logarithm, and then standardized the markers relative
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to asbestos-exposed controls. Logistic regression to predict case/control status was used to determine the weight given each marker. Markers were multiplied by their logistic regression coefficient and added to give a combined marker value. Pearson’s correlation coefficient was used to assess correlations between different biomarkers (log scale).
RESULTS Patient Characteristics Serum samples were collected from 66 patients with pleural malignant mesothelioma; 9 with predominantly sarcomatoid histology and 57 with predominantly epithelioid histology or from whom a diagnosis was made on immunocytological grounds.26 Sera were collected from 20 apparently healthy individuals, 10 of whom had no reported previous asbestos exposure, and 10 with exposure. Samples were also collected from 21 patients with benign asbestos-related pulmonary disease (asbestosis and/or pleural plaques); from 30 patients with nonmalignant effusions (10 had exudative effusions, 10 had effusions relating to an infection, and 10 had transudate effusions); from 20 people who presented with effusions because of other malignancies (10 with primary lung cancer, 3 with breast cancer, 2 each with colon and pancreatic cancer, and one each with melanoma, sarcoma, and non-Hodgkin lymphoma) and from 10 additional cases of lung cancer who did not present with an effusion (Table 1).
MPF Levels in Serum Serum MPF levels in patients with mesothelioma ranged from undetectable to 5.9 ng/ml (Figure 1). Levels ranged from undetectable to 0.7 ng/ml in patients with mesothelioma tumors of sarcomatoid histology, levels which
Comparison of Osteopontin, MPF, and Mesothelin Proteins
were lower than those observed in patients with epithelialtype mesotheliomas although not significantly so (p ⫽ 0.09). Serum MPF levels were significantly higher in the serum of patients with mesothelioma than the healthy control population (p ⫽ 0.007), the patients with benign asbestos-related disease (p ⫽ 0.01), and patients with nonmalignant exudative effusions (p ⫽ 0.01). There was no difference in MPF levels between those patients with mesothelioma, lung cancer, or with effusions of a transudate or malignant nature.
Osteopontin Levels in Serum Serum osteopontin levels in patients with mesothelioma ranged from undetectable to 438 ng/ml (Figure 1). There was no relationship between the levels of osteopontin and the histologic diagnosis of the patients’ mesothelioma (Table 1). Serum osteopontin levels were significantly higher in the serum of patients with mesothelioma than the healthy control population (p ⫽ 0.0002), the patients with benign asbestosrelated disease (p ⬍ 0.0001), and the patients with nonmalignant exudative effusions (p ⫽ 0.0002). There was no difference in osteopontin levels between patients with mesothelioma, lung cancer, or effusions of a transudate or malignant nature. There was no significant difference in serum osteopontin levels between healthy control individuals and individuals with benign asbestos-related disease.
Mesothelin Levels in Serum Serum mesothelin levels were significantly higher in patients with mesothelioma compared with healthy controls (p ⫽ 0.0001), individuals with benign asbestos-related disease (p ⫽ 0.0001), patients with benign effusions (p ⬍ 0.005), patients with malignant effusions (p ⫽ 0.0006), and patients with lung cancer (p ⫽ 0.03). Serum mesothelin levels
TABLE 1. Patient Characteristics and Biomarker Levels
MM—all Epithelial and cytology Sarcomatoid Healthy controls—all Nonasbestos exposed Asbestos exposed Benign controls—all Asbestosis Asbestosis and plaques Pleural effusions Transudate Exudate Exudate—infection Malignancy Lung cancer
No. of Cases
Median Age, yr (range)
66 (5F) 57 (5F)
69 (50–84) 70 (50–84)
9 (0F) 20 (7F) 10 (2F) 10 (5F) 21 (4F) 11 (3F) 10 (1F)
68 (53–77) 55 (33–64) 48 (33–58) 58 (40–64) 71 (49–86) 70 (49–86) 73 (60–86)
9 (2F) 10 (1F) 9 (5F) 20 (10F) 10 (2F)
76 (48–82) 76 (32–80) 64 (50–86) 74 (50–83) 74 (58–82)
Osteopontina,b 14.8 ⫾ 8.2 15.9 ⫾ 8 9.2 ⫾ 10.4 2 ⫾ 7.3** 1.2 ⫾ 10.5 3.2 ⫾ 4.6 1.8 ⫾ 8** 1.8 ⫾ 8.6 1.9 ⫾ 8.5 15.6 ⫾ 11 (NS) 1.8 ⫾ 12.1* 1.7 ⫾ 9** 20.7 ⫾ 8.1 (NS) 19 ⫾ 7.5 (NS)
No. of Positive Samplesc 30/66 26/57
MPFa,b,d 0.19 ⫾ 13.5 0.23 ⫾ 13.4
No. of Positive No. of Positive Samplesc Mesothelina,c Samplesc 21/66 21/57
4.7 ⫾ 3.4 5.7 ⫾ 3.3
48/66 47/57
4/9 0/20 0/10 0/10 3/20 1/10 2/10
0.05 ⫾ 10.5 0.05 ⫾ 5.8* 0.04 ⫾ 8.4 0.05 ⫾ 4.1 0.09 ⫾ 6.2 (NS) 0.06 ⫾ 6.6 0.14 ⫾ 5.5
0/9 1/20 1/10 0/10 1/20 0/10 1/10
1.3 ⫾ 1.3 0.4 ⫾ 3.4*** 0.24 ⫾ 5.1 0.65 ⫾ 1.32 0.87 ⫾ 2.4*** 0.76 ⫾ 3.1 1.0 ⫾ 1.6
1/9 0/20 0/10 0/10 1/20 1/10 0/10
5/10 0/10 1/10 10/20 7/10
0.2 ⫾ 8.9 (NS) 0.05 ⫾ 8.3 (NS) 0.08 ⫾ 6.1 (NS) 0.33 ⫾ 4.6 (NS) 0.36 ⫾ 3.6 (NS)
1/10 0/10 0/10 0/20 2/10
1.7 ⫾ 1.9* 0.8 ⫾ 1.8*** 0.7 ⫾ 3*** 1.6 ⫾ 2.5* 1.79 ⫾ 2.95*
5/10 0/10 2/10 9/20 5/10
Expontiated mean of log transformed data ⫾ standard error. Significant difference between indicated cohorts and the mesothelioma cohort as a whole (n ⫽ 66) as determined by Student t test (NS is not significant; *p ⬍ 0.05; **p ⬍ 0.001; ***p ⬍ 0.0001). c The number of samples above the 95% specificity level as determined for each marker and described in Table 2 below. d MPF concentrations determined from Human N-ERC/Mesothelin Assay (IBL). a b
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FIGURE 2. ROC curve showing accuracy of serum biomarker concentration in differentiating all patients with mesothelioma (n ⫽ 66) from healthy and benign controls (n ⫽ 41).
Comparison of Multiple Biomarker Levels When ROC curves were generated to assess the ability of the markers to distinguish patients with mesothelioma from the healthy controls and the patients with benign asbestos-related disease, the AUC for MPF was 0.614 (95% confidence interval [CI], 0.508 – 0.721), for osteopontin it was 0.757 (95% CI, 0.666 – 0.848) and for mesothelin it was 0.915 (95% CI, 0.869 – 0.968) (Figure 2). The AUC for mesothelin was significantly higher than for MPF or for osteopontin. From the curves, the sensitivities were determined for specificities of 85%, 90%, 95%, and 98% (Table 2). In the present study, at a specificity of 95%, the MPF assay had a sensitivity of 34%; the osteopontin assay had a sensitivity of 47%, and the mesothelin assay had a sensitivity of 73%. A bivariate scattergram of mesothelin versus MPF (Figure 3A) and osteopontin (Figure 3B) levels in individuals with mesothelioma and controls with the threshold values for 95% specificity indicated only one individual who had elevated MPF and osteopontin levels who was negative for mesothelin, and another individual with sarcomatoid variant with elevated osteopontin but negative mesothelin, demonstrating that neither MPF nor osteopontin add significant information about the presence of mesothelioma to mesotheTABLE 2. Sensitivity of Individual Biomarkers Estimated at Different Levels of Specificity for Differentiating all Patients with Mesothelioma (n ⫽ 66) from Healthy and Benign Controls (n ⫽ 41) Estimated Sensitivities
FIGURE 1. Biomarker concentrations in serum. Biomarker concentrations were determined at least in duplicate by ELISA and individual patient values are plotted on the graph. A, MPF; B, osteopontin; and C, mesothelin.
were significantly higher in patients with mesothelioma tumors of epithelial-like histology than those with predominately sarcomatoid histology (p ⫽ 0.0004) (Table 1).
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MPF Osteopontin Mesothelin
85% Specificity (threshold value)
90% Specificity (threshold value)
95% Specificity (threshold value)
98% Specificity (threshold value)
39 (0.6) 55 (10.5) 81 (1.25)
36 (0.7) 53 (12) 78 (1.4)
34 (1) 47 (18) 73 (1.6)
34 (1.3) 22 (77) 68 (1.8)
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Comparison of Osteopontin, MPF, and Mesothelin Proteins
FIGURE 4. ROC curve showing accuracy of serum biomarker concentrations individually and combined using a logistic regression model in differentiating all patients with mesothelioma (n ⫽ 66) from healthy and benign controls (n ⫽ 41).
teopontin significantly increased the AUC when added to a panel. All combined markers with mesothelin had AUCs in the range 90.7 to 91.5% which was not significantly different from the AUC for mesothelin alone, whereas the AUC for the combined marker formed from MPF and osteopontin had an AUC of 74.5% which was significantly lower than any AUC from a combined marker with mesothelin at p ⬍ 0.001.
DISCUSSION
FIGURE 3. Bivariate scattergram of biomarker concentrations determined in serum from individual patients with mesothelioma (䉬) and from healthy and benign controls (‚). Dashed lines represent the level at which the individual biomarker has a specificity of 95%. A, MPF concentrations plotted against mesothelin; B, osteopontin against mesothelin; and C, osteopontin against MPF.
lin. There was a group of approximately 40% and 34% mesothelioma patients positive for mesothelin and negative for MPF and osteopontin, respectively. Examining only the patients with mesothelioma, there was a significant correlation between mesothelin levels and MPF (r ⫽ 0.477; p ⬍ 0.01) and osteopontin (r ⫽ 0.373; p ⬍ 0.01) levels. A bivariate scattergram of MPF and osteopontin revealed that the majority of patients with elevated osteopontin had elevated MPF which was reflected in a correlation of r ⫽ 0.632 (p ⬍ 0.01) between these markers (Figure 3C). The results of using a logistic regression model to combine the data from the three markers are presented in Figure 4 and Table 3. Those combinations that include mesothelin all increase the AUC value compared with the panel without mesothelin; however, neither MPF nor os-
The development of multiple markers in disease diagnostics is the goal of a number of biomarker research centers. This study shows no improvement in combining serum osteopontin and MPF with mesothelin in determining the accuracy of diagnosis of malignant mesothelioma over the mesothelin marker used alone. MPF levels are reported to be elevated in mesothelioma patients.15,16 Using a commercial version of the assay developed by Shiomi et al., we found that, at a specificity of 95%, 34% of mesothelioma patients had elevated levels of MPF, less than the 7/716 and 51/56 positive mesothelioma patients previously reported.15 Such a difference may reflect differences in the stage distribution of the mesothelioma patients as Onda et al.15 reported that all their subjects had advanced disease. A diagnostic test with high sensitivity only in patients who have advanced cancer is clearly of little clinical value. One might expect that the MPF and mesothelin assays would have similar sensitivity and specificity. Our finding that this was not the case was contrary to the notion that MPF would be a more sensitive marker for mesothelioma than mesothelin because of the latter’s need to be cleaved or shed from the cell surface. For the same specificity, fewer mesothelioma patients had elevated MPF compared with the numbers with elevated mesothelin. The reason for the difference in sensitivity of the two markers of the same precursor protein may simply be a reflection of the relative stabilities of the molecules, though the physiology of their relative stabilities in vivo and clearance kinetics is yet to be defined. Osteopontin is a secreted glycol-phosphoprotein which is produced by many cell types including osteoclasts, osteoblasts, epithelial cells of the breast, kidney and skin, nerve
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TABLE 3. Area Under the ROC Curve for Individual and Combined Biomarkers, Discriminating All Patients with Mesothelioma (n ⫽ 66) from Healthy and Benign Controls (n ⫽ 41) Asymptotic 95% CI a
Biomarker Combination
AUC
SE
Combined mesothelin, OSN and MPF Combined mesothelin and OSN Combined mesothelin and MPF Combined OSN and MPF Mesothelin OSN MPF
0.920 0.926 0.916 0.745 0.905 0.768 0.605
0.026 0.024 0.026 0.048 0.028 0.045 0.054
Lower Bound
Upper Bound
pb
pc
pd
0.869 0.878 0.866 0.651 0.849 0.679 0.499
0.971 0.974 0.966 0.838 0.960 0.857 0.711
Reference 0.89 0.40 ⬍0.0001 NA NA NA
NA Reference 0.87 ⬍0.0001 NA NA NA
NA NA Reference ⬍0.0001 NA NA NA
a
Standard error under the nonparametric assumption. p values for comparing the AUC for combinations of biomarkers in a logistic regression model using the method of DeLong et al. AUC, area under the curve; CI, confidence interval; OSN, osteopontin.
b,c,d
cells, endothelial cells, and immune cells such as T cells, natural killer cells, and macrophages. In malignancy, osteopontin is believed to play a role in tumor progression, growth, invasiveness, and metastasis. It has been shown to be elevated in the serum of patients with melanoma, breast, lung, colorectal, stomach, and ovarian cancer22,27 and recently also in mesothelioma.23,28 The results of our current study confirm that serum levels of osteopontin are elevated in some mesothelioma patients. One concern that remains is the nature of the test sample. In the three mesothelioma-osteopontin studies to date where osteopontin was measured in the serum, the protein may potentially be cleaved by thrombin also present in the sera and results may not reflect the true levels of the protein in the blood. Grigoriu et al. reported that although osteopontin levels were higher in plasma, the ability of osteopontin to discriminate between patients with mesothelioma and healthy asbestos-exposed subjects was similar when using plasma or serum levels.28 We restricted the current study to serum as we needed to use the same sample for all patients. The need to use plasma, serum, or other collection methods needs further study. Although serum levels of osteopontin discriminate between mesothelioma patients and healthy controls,23,28 levels of osteopontin alone are unlikely to be of clinical value in mesothelioma diagnosis. Furthermore, serum osteopontin levels are elevated in other malignancies, as described above. In addition, some common nonmalignant, nonpleural effusionassociated conditions including coronary artery disease, interstitial pneumonia, and other benign pulmonary disease28 –30 can result in increased levels of osteopontin in the serum. We have previously found that individual patients with mesothelioma exhibit different biomarker profiles: either both CA125 and mesothelin, only one or neither marker. This was not found to be the case with the pattern of expression of osteopontin and mesothelin as virtually all osteopontin positive mesothelioma patients were positive for mesothelin. This high degree of concordance means that combining such markers would be unlikely to add to sensitivity. As the various combinations of markers discriminated between mesothelioma patients and controls, no better than when mesothelin was used alone, our current practice is to measure serum mesothelin as the
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preferred biomarker for suspected mesothelioma and not include assays for the other biomarkers. The mesothelin biomarker represents the best available serum biomarker for mesothelioma. An elevated mesothelin level is highly suggestive of malignancy, and particularly of mesothelioma. Other markers are needed to improve the sensitivity for mesothelioma in symptomatic patients, and possibly for use in any screening strategy for asbestosexposed individuals. Studies are therefore ongoing to identify new mesothelioma-specific markers.
ACKNOWLEDGMENTS Supported in part by research grants from the National Health and Medical Council of Australia and the Insurance Commission of Western Australia, and in part by FDI. The study sponsors had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication. We thank the staff of PathWest Laboratory Medicine, Sir Charles Gairdner Hospital, Hollywood Hospital and St. John of God Pathology for their assistance with this study. REFERENCES 1. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2005;353:1591–1603. 2. Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet 2005;366:397– 408. 3. Kojima T, Oh-eda M, Hattori K, et al. Molecular cloning and expression of megakaryocyte potentiating factor cDNA. J Biol Chem 1995;270: 21984 –21990. 4. Yamaguchi N, Hattori K, Oh-eda M, et al. A novel cytokine exhibiting megakaryocyte potentiating activity from a human pancreatic tumor cell line HPC-Y5. J Biol Chem 1994;269:805– 808. 5. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci USA 1996;93:136 –140. 6. Scholler N, Fu N, Yang Y, et al. Soluble member(s) of the mesothelin/ megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci USA 1999;96: 11531–11536. 7. Muminova ZE, Strong TV, Shaw DR. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer. BMC Cancer 2004;4:19.
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Journal of Thoracic Oncology • Volume 3, Number 8, August 2008
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