Comparison of Osteopontin, Megakaryocyte Potentiating Factor, and Mesothelin Proteins As Markers in the Serum of Patients with Malignant Mesothelioma

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector 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,ʈ and Bruce W. S. Robinson MBBS, MD, FRACP*†

Conclusion: Serum mesothelin remains the most specific marker for Introduction: There is increasing interest in identifying a blood- the diagnosis of mesothelioma. based marker for the asbestos-related tumor, malignant mesothelioma. Three potential markers for mesothelioma are mesothelin, Key Words: Mesothelioma, Tumor markers, Mesothelin, SMRP, megakaryocyte potentiating factor (MPF) and osteopontin. The pur- MPF, Osteopontin, Diagnosis. pose of the current study was to directly compare these biomarkers (J Thorac Oncol. 2008;3: 851–857) 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 alignant mesothelioma is caused by exposure to asbes- all three markers. Mtos and is a fatal disease with limited treatment op- Methods: Serum levels of mesothelin, MPF and osteopontin were tions.1,2 In other forms of cancers, serum-based tumor mark- determined by commercially available assays in 66 samples from ers have provided clinicians with a valuable aid in the patients with pleural malignant mesothelioma, 20 healthy individu- management of patients. Markers may have different roles in als, 21 patients with asbestos-related lung or pleural disease, 30 diagnosis, monitoring, assessing prognosis, and in detection patients presenting with benign pleural effusions and 30 patients of early disease. There are no markers in routine clinical use with other malignancies. for malignant mesothelioma, although the use of soluble Results: Serum levels of the three markers were elevated in me- mesothelin has received recent attention. sothelioma patients. At a level of specificity of 95% relative to Mesothelin is a differentiation marker of mesothelial cells. healthy controls and patients with benign asbestos related disease, Differential post-transcriptional and post-translational process- the sensitivity for mesothelioma was 34% for MPF, 47% for os- ing of the mesothelin gene produces four protein products: teopontin and 73% for mesothelin. Osteopontin and MPF were megakaryocyte potentiating factor (MPF),3,4 mesothelin variant unable to differentiate patients with mesothelioma from patients 1, mesothelin variant 2,5 and serum mesothelin-related protein with other malignancies or those presenting with transudative pleu- (SMRP).6–8 MPF is a soluble ϳ32 kd protein with cytokine ral effusions. Combining the data from the three biomarkers using a activity.3,4 Mesothelin is ϳ40 kd glycosylated protein predom- logistic regression model did not improve sensitivity for detecting inately anchored to the cell surface of normal mesothelial cells, mesothelioma above that of the mesothelin marker alone. but also present in solution (the physiological mechanism for mesothelin release from the cell surface is not clear).9,10 Me- sothelin variant 1 is the predominant form of the protein and *National Research Centre for Asbestos Related Diseases, Western Austra- lian Institute of Medical Research, School of Medicine and Pharmacol- differs from variant 2 by only eight amino acids. SMRP is a ogy, University of Western Australia, Sir Charles Gairdner Hospital, soluble protein with an identical N-terminal sequence to me- Nedlands, Western Australia, Australia; †The Australian Mesothelioma sothelin but a unique C-terminal.6 Tissue Bank, Sir Charles Gairdner Hospital, Nedlands, Western Austra- Using an enzyme-linked immunosorbent assay (ELISA) lia, Australia; ‡PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Hospital Ave., Nedlands, Western Australia, Australia; which detects both mesothelin variant 1 and SMRP, we §Department of Respiratory Medicine, Sir Charles Gairdner Hospital, recently demonstrated increased levels of this biomarker in Nedlands, Western Australia, Australia; and ʈBiostatistics Center, Mas- the serum of patients with mesothelioma,11 findings recently sachusetts General Hospital, Boston, Massachusetts. confirmed using the same assay12 and an independent13 assay. Disclosure: Jenette Creaney and Bruce W. S. Robinson have received Serum mesothelin is highly specific for mesothelioma (spec- consultancy fees from Fujirebio Diagnositic Incorporated (FDI), 14 Malvern, PA. The remaining authors have no conflicts to disclose. ificity 98%) with a sensitivity at diagnosis of 49% (57/117) Address for correspondence: Jenette Creaney, PhD, Sir Charles Gairdner and 84% in advanced disease.11 Despite very encouraging Hospital, University of Western Australia, Verdun St., Nedlands, WA findings showing serum mesothelin as a useful marker in 6009, Australia. E-mail: [email protected] mesothelioma, we found that at diagnosis only half of me- Copyright © 2008 by the International Association for the Study of Lung Cancer sothelioma patients were mesothelin positive; therefore, there ISSN: 1556-0864/08/0308-0851 is a need for additional markers to improve diagnostic sen-

Journal of Thoracic Oncology • Volume 3, Number 8, August 2008 851 Creaney et al. Journal of Thoracic Oncology • Volume 3, Number 8, August 2008 sitivity. Possible candidate markers include proteins that are Blood samples were collected by routine venepuncture related to mesothelin, such as MPF, or those that are inde- into clotted blood tubes. Samples were allowed to clot for at pendent of mesothelin but associated with the disease such as least 2 hours at room temperature, or alternatively were osteopontin. stored at 4°C before processing. Samples were centrifuged at Given the successful detection of mesothelin and SMRP 1200 rpm for 10 minutes, then the supernatant was removed, in the serum of cancer patients, two groups recently, indepen- aliquoted, and stored at Ϫ80°C until use. dently developed assays to measure levels of MPF.15,16 Onda and colleagues showing 51/56 patients with advanced me- Serum Mesothelin sothelioma had elevated serum MPF.15 As mesothelin must Soluble mesothelin concentrations were determined in be cleaved or shed from the cell surface serum MPF may be duplicate following the manufacturer’s instructions using a dou- at least as sensitive, if not more so, than serum mesothelin as ble-determinant ELISA assay, the MESOMARK kit, supplied a marker of mesothelioma. To clarify this, we measured and by Fujirebio Diagnostics (Malvern, PA). Mesothelin concentra- compared serum mesothelin and MPF levels in the same tions were determined from a standard curve performed on each patient cohort using commercially available assays. plate and expressed as nanomolar. Dilution of samples was Another strategy to improve diagnostic sensitivity is to carried out, if necessary, using the diluent supplied by the incorporate the information from multiple markers. Patholo- manufacturer. All assays were performed on coded samples gists use suites of immunohistochemical markers in tissue to by technical staff unaware of the patient’s diagnosis. improve diagnostic accuracy of the histopathologic find- 17,18 MPF Assay ings and multiple serum markers have achieved greater The Human N-ERC/Mesothelin Assay kit was pur- sensitivity and specificity in the diagnosis of some cancers, chased from Immuno-Biologic Laboratories (Gunma, Japan) 19,20 and prostate cancer.21 i.e., ovarian and the manufacturer’s recommended protocol was followed. Recently, we tested the hypothesis that combining data Serum samples were assayed undiluted and concentrations of from two markers (mesothelin and CA125) would improve MPF were determined from a standard curve performed in the sensitivity of the mesothelin marker used alone to distin- parallel. The manufacturer’s reported assay sensitivity was guish mesothelioma patients from other asbestos-exposed 0.024 ng/ml; the concentrations for samples from 0.024 ng/ml individuals; however, we found that this combination was not to 0.011 ng/ml were extrapolated from the standard curve, for significantly different to the results for mesothelin alone.14 the purposes of data analysis concentrations of samples below Osteopontin is a secreted 44 kd glycoprotein with roles 0.011 ng/ml were assigned a value of 0.005 ng/ml. in cell-matrix interactions, cell migration, and other diverse functions. Serum osteopontin levels are elevated in breast, Osteopontin Assay ovarian, lung, and prostrate cancer.22 Osteopontin has been The human osteopontin assay, a double-determinant shown to be a useful adjunct to CA125 in the detection of ELISA, was purchased from Immuno-Biologic Laboratories. recurrent ovarian cancer.19 In mesothelioma serum, osteopon- The manufacturer’s recommended protocol was followed tin has a sensitivity of 78% at a specificity of 85% relative to except serum was used as the assay substrate instead of subjects exposed to asbestos.23 plasma. Serum samples were diluted 1:3 in buffer supplied The purpose of the current study was to directly com- with the kit. The manufacturer’s reported assay sensitivity pare each of these biomarkers in the same sample population was 3.33 ng/ml, the concentrations for samples from 3.3 and to determine if diagnostic accuracy for mesothelioma is ng/ml to 0.5 ng/ml were extrapolated from the standard curve, improved by combining these three markers. and for the purposes of data analysis concentrations of samples below 0.5 ng/ml were assigned a value of 0.1 ng/ml. PATIENTS AND METHODS Statistical Analysis Patients and Controls Differences between groups of patients were assessed Serum samples were collected from consecutive pa- by Student t test after transforming the biomarker values to tients presenting at the respiratory clinics of either Sir Charles the log scale for which the distributions were closer to Gairdner Hospital or the Hollywood Specialist Centre in normality. For the same reason, median biomarker values Perth, Western Australia and form part of the Australian were estimated from the mean on the log scale and exponen- Mesothelioma Tissue Bank. We obtained written and oral tiated to provide the estimate of the median on the original informed consent from participants. This study was approved scale. All reported p values are two sided. A level of p Ͻ 0.05 by the human ethics committees of Sir Charles Gairdner and was accepted as significant. Receiver operating characteristic Hollywood Hospitals. The final diagnosis in all patients was (ROC) curves display the trade-off between sensitivity and confirmed by pathologists experienced in the diagnosis of specificity for biomarkers differentiating between groups of mesothelioma and effusions and included clinical follow-up patients. Area under the curve (AUC) for two biomarkers, of all cases until death or for an average of 6.8 months (range, including “markers” formed by combining multiple biomar- 1–42 months) to confirm that the clinical pattern matched the kers, was compared using the method of DeLong et al.,25 diagnosis. Effusions were classified as being malignant or which accounts for the correlation because of the markers nonmalignant on the basis of cytologic and immunohisto- being measured on the same set of serum samples. To chemical features. Nonmalignant effusions were classified as combine biomarkers, we first transformed data with the exudates or transudates by Light’s criteria.24 natural logarithm, and then standardized the markers relative

852 Copyright © 2008 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology • Volume 3, Number 8, August 2008 Comparison of Osteopontin, MPF, and Mesothelin Proteins to asbestos-exposed controls. Logistic regression to predict were lower than those observed in patients with epithelial- case/control status was used to determine the weight given type mesotheliomas although not significantly so (p ϭ 0.09). each marker. Markers were multiplied by their logistic re- Serum MPF levels were significantly higher in the serum of gression coefficient and added to give a combined marker patients with mesothelioma than the healthy control popula- value. Pearson’s correlation coefficient was used to assess tion (p ϭ 0.007), the patients with benign asbestos-related correlations between different biomarkers (log scale). disease (p ϭ 0.01), and patients with nonmalignant exudative effusions (p ϭ 0.01). There was no difference in MPF levels RESULTS between those patients with mesothelioma, lung cancer, or with effusions of a transudate or malignant nature. Patient Characteristics Serum samples were collected from 66 patients with Osteopontin Levels in Serum pleural malignant mesothelioma; 9 with predominantly sar- Serum osteopontin levels in patients with mesotheli- comatoid histology and 57 with predominantly epithelioid oma ranged from undetectable to 438 ng/ml (Figure 1). There histology or from whom a diagnosis was made on immuno- was no relationship between the levels of osteopontin and the cytological grounds.26 Sera were collected from 20 appar- histologic diagnosis of the patients’ mesothelioma (Table 1). ently healthy individuals, 10 of whom had no reported pre- Serum osteopontin levels were significantly higher in the vious asbestos exposure, and 10 with exposure. Samples were serum of patients with mesothelioma than the healthy control also collected from 21 patients with benign asbestos-related population (p ϭ 0.0002), the patients with benign asbestos- pulmonary disease (asbestosis and/or pleural plaques); from related disease (p Ͻ 0.0001), and the patients with nonma- 30 patients with nonmalignant effusions (10 had exudative lignant exudative effusions (p ϭ 0.0002). There was no effusions, 10 had effusions relating to an infection, and 10 difference in osteopontin levels between patients with me- had transudate effusions); from 20 people who presented with sothelioma, lung cancer, or effusions of a transudate or effusions because of other malignancies (10 with primary malignant nature. There was no significant difference in lung cancer, 3 with breast cancer, 2 each with colon and serum osteopontin levels between healthy control individuals pancreatic cancer, and one each with melanoma, sarcoma, and individuals with benign asbestos-related disease. and non-Hodgkin lymphoma) and from 10 additional cases of lung cancer who did not present with an effusion (Table 1). Mesothelin Levels in Serum Serum mesothelin levels were significantly higher in MPF Levels in Serum patients with mesothelioma compared with healthy controls Serum MPF levels in patients with mesothelioma (p ϭ 0.0001), individuals with benign asbestos-related dis- ranged from undetectable to 5.9 ng/ml (Figure 1). Levels ease (p ϭ 0.0001), patients with benign effusions (p Ͻ ranged from undetectable to 0.7 ng/ml in patients with me- 0.005), patients with malignant effusions (p ϭ 0.0006), and sothelioma tumors of sarcomatoid histology, levels which patients with lung cancer (p ϭ 0.03). Serum mesothelin levels

TABLE 1. Patient Characteristics and Biomarker Levels No. of Median Age, No. of Positive No. of Positive No. of Positive Cases yr (range) Osteopontina,b Samplesc MPFa,b,d Samplesc Mesothelina,c Samplesc MM—all 66 (5F) 69 (50–84) 14.8 Ϯ 8.2 30/66 0.19 Ϯ 13.5 21/66 4.7 Ϯ 3.4 48/66 Epithelial and 57 (5F) 70 (50–84) 15.9 Ϯ 8 26/57 0.23 Ϯ 13.4 21/57 5.7 Ϯ 3.3 47/57 cytology Sarcomatoid 9 (0F) 68 (53–77) 9.2 Ϯ 10.4 4/9 0.05 Ϯ 10.5 0/9 1.3 Ϯ 1.3 1/9 Healthy controls—all 20 (7F) 55 (33–64) 2 Ϯ 7.3** 0/20 0.05 Ϯ 5.8* 1/20 0.4 Ϯ 3.4*** 0/20 Nonasbestos exposed 10 (2F) 48 (33–58) 1.2 Ϯ 10.5 0/10 0.04 Ϯ 8.4 1/10 0.24 Ϯ 5.1 0/10 Asbestos exposed 10 (5F) 58 (40–64) 3.2 Ϯ 4.6 0/10 0.05 Ϯ 4.1 0/10 0.65 Ϯ 1.32 0/10 Benign controls—all 21 (4F) 71 (49–86) 1.8 Ϯ 8** 3/20 0.09 Ϯ 6.2 (NS) 1/20 0.87 Ϯ 2.4*** 1/20 Asbestosis 11 (3F) 70 (49–86) 1.8 Ϯ 8.6 1/10 0.06 Ϯ 6.6 0/10 0.76 Ϯ 3.1 1/10 Asbestosis and plaques 10 (1F) 73 (60–86) 1.9 Ϯ 8.5 2/10 0.14 Ϯ 5.5 1/10 1.0 Ϯ 1.6 0/10 Pleural effusions Transudate 9 (2F) 76 (48–82) 15.6 Ϯ 11 (NS) 5/10 0.2 Ϯ 8.9 (NS) 1/10 1.7 Ϯ 1.9* 5/10 Exudate 10 (1F) 76 (32–80) 1.8 Ϯ 12.1* 0/10 0.05 Ϯ 8.3 (NS) 0/10 0.8 Ϯ 1.8*** 0/10 Exudate—infection 9 (5F) 64 (50–86) 1.7 Ϯ 9** 1/10 0.08 Ϯ 6.1 (NS) 0/10 0.7 Ϯ 3*** 2/10 Malignancy 20 (10F) 74 (50–83) 20.7 Ϯ 8.1 (NS) 10/20 0.33 Ϯ 4.6 (NS) 0/20 1.6 Ϯ 2.5* 9/20 Lung cancer 10 (2F) 74 (58–82) 19 Ϯ 7.5 (NS) 7/10 0.36 Ϯ 3.6 (NS) 2/10 1.79 Ϯ 2.95* 5/10

a Expontiated mean of log transformed data Ϯ standard error. b Significant difference between indicated cohorts and the mesothelioma cohort as a whole (n ϭ 66) as determined by Student t test (NS is not significant; *p Ͻ 0.05; **p Ͻ 0.001; ***p Ͻ 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).

FIGURE 2. ROC curve showing accuracy of serum biomarker concentration in differentiating all patients with mesothelioma (n ϭ 66) from healthy and benign controls (n ϭ 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, demon- strating that neither MPF nor osteopontin add significant information about the presence of mesothelioma to mesothe-

TABLE 2. Sensitivity of Individual Biomarkers Estimated at Different Levels of Specificity for Differentiating all Patients with Mesothelioma (n ϭ 66) from Healthy and Benign Controls (n ϭ 41) Estimated Sensitivities FIGURE 1. Biomarker concentrations in serum. Biomarker concentrations were determined at least in duplicate by 85% 90% 95% 98% ELISA and individual patient values are plotted on the graph. Specificity Specificity Specificity Specificity A, MPF; B, osteopontin; and C, mesothelin. (threshold (threshold (threshold (threshold value) value) value) value) MPF 39 (0.6) 36 (0.7) 34 (1) 34 (1.3) were significantly higher in patients with mesothelioma tu- Osteopontin 55 (10.5) 53 (12) 47 (18) 22 (77) mors of epithelial-like histology than those with predomi- Mesothelin 81 (1.25) 78 (1.4) 73 (1.6) 68 (1.8) nately sarcomatoid histology (p ϭ 0.0004) (Table 1).

854 Copyright © 2008 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology • Volume 3, Number 8, August 2008 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 ϭ 66) from healthy and benign controls (n ϭ 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 Ͻ 0.001.

DISCUSSION 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 devel- FIGURE 3. Bivariate scattergram of biomarker concentra- oped by Shiomi et al., we found that, at a specificity of 95%, tions determined in serum from individual patients with me- 34% of mesothelioma patients had elevated levels of MPF, sothelioma (᭜) and from healthy and benign controls (‚). less than the 7/716 and 51/56 positive mesothelioma patients Dashed lines represent the level at which the individual bi- previously reported.15 Such a difference may reflect differ- omarker has a specificity of 95%. A, MPF concentrations ences in the stage distribution of the mesothelioma patients as plotted against mesothelin; B, osteopontin against mesothe- Onda et al.15 reported that all their subjects had advanced lin; and C, osteopontin against MPF. disease. A diagnostic test with high sensitivity only in patients who have advanced cancer is clearly of little clinical value. lin. There was a group of approximately 40% and 34% One might expect that the MPF and mesothelin assays mesothelioma patients positive for mesothelin and negative would have similar sensitivity and specificity. Our finding for MPF and osteopontin, respectively. Examining only the that this was not the case was contrary to the notion that MPF patients with mesothelioma, there was a significant correla- would be a more sensitive marker for mesothelioma than tion between mesothelin levels and MPF (r ϭ 0.477; p Ͻ mesothelin because of the latter’s need to be cleaved or shed 0.01) and osteopontin (r ϭ 0.373; p Ͻ 0.01) levels. from the cell surface. For the same specificity, fewer me- A bivariate scattergram of MPF and osteopontin re- sothelioma patients had elevated MPF compared with the vealed that the majority of patients with elevated osteopontin numbers with elevated mesothelin. The reason for the differ- had elevated MPF which was reflected in a correlation of r ϭ ence in sensitivity of the two markers of the same precursor 0.632 (p Ͻ 0.01) between these markers (Figure 3C). protein may simply be a reflection of the relative stabilities of The results of using a logistic regression model to the molecules, though the physiology of their relative stabil- combine the data from the three markers are presented in ities in vivo and clearance kinetics is yet to be defined. Figure 4 and Table 3. Those combinations that include Osteopontin is a secreted glycol-phosphoprotein which mesothelin all increase the AUC value compared with the is produced by many cell types including osteoclasts, osteo- panel without mesothelin; however, neither MPF nor os- blasts, epithelial cells of the breast, kidney and skin, nerve

TABLE 3. Area Under the ROC Curve for Individual and Combined Biomarkers, Discriminating All Patients with Mesothelioma (n ϭ 66) from Healthy and Benign Controls (n ϭ 41) Asymptotic 95% CI Biomarker Combination AUC SEa Lower Bound Upper Bound pb pc pd Combined mesothelin, OSN and MPF 0.920 0.026 0.869 0.971 Reference NA NA Combined mesothelin and OSN 0.926 0.024 0.878 0.974 0.89 Reference NA Combined mesothelin and MPF 0.916 0.026 0.866 0.966 0.40 0.87 Reference Combined OSN and MPF 0.745 0.048 0.651 0.838 Ͻ0.0001 Ͻ0.0001 Ͻ0.0001 Mesothelin 0.905 0.028 0.849 0.960 NA NA NA OSN 0.768 0.045 0.679 0.857 NA NA NA MPF 0.605 0.054 0.499 0.711 NA NA NA

a Standard error under the nonparametric assumption. b,c,d 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.

cells, endothelial cells, and immune cells such as T cells, preferred biomarker for suspected mesothelioma and not include natural killer cells, and macrophages. In malignancy, os- assays for the other biomarkers. teopontin is believed to play a role in tumor progression, The mesothelin biomarker represents the best available growth, invasiveness, and metastasis. It has been shown to be serum biomarker for mesothelioma. An elevated mesothelin elevated in the serum of patients with melanoma, breast, lung, level is highly suggestive of malignancy, and particularly of colorectal, stomach, and ovarian cancer22,27 and recently also mesothelioma. Other markers are needed to improve the in mesothelioma.23,28 The results of our current study confirm sensitivity for mesothelioma in symptomatic patients, and that serum levels of osteopontin are elevated in some me- possibly for use in any screening strategy for asbestos- sothelioma patients. One concern that remains is the nature of exposed individuals. Studies are therefore ongoing to identify the test sample. In the three mesothelioma-osteopontin stud- new mesothelioma-specific markers. ies to date where osteopontin was measured in the serum, the protein may potentially be cleaved by thrombin also present ACKNOWLEDGMENTS in the sera and results may not reflect the true levels of the Supported in part by research grants from the National protein in the blood. Grigoriu et al. reported that although Health and Medical Council of Australia and the Insurance osteopontin levels were higher in plasma, the ability of Commission of Western Australia, and in part by FDI. The osteopontin to discriminate between patients with mesotheli- study sponsors had no role in study design, in the collection, oma and healthy asbestos-exposed subjects was similar when analysis, and interpretation of data, in the writing of the using plasma or serum levels.28 We restricted the current manuscript, and in the decision to submit the manuscript for study to serum as we needed to use the same sample for all publication. patients. The need to use plasma, serum, or other collection We thank the staff of PathWest Laboratory Medicine, methods needs further study. Sir Charles Gairdner Hospital, Hollywood Hospital and St. Although serum levels of osteopontin discriminate be- John of God Pathology for their assistance with this study. tween 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 REFERENCES 1. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl are elevated in other malignancies, as described above. In J Med 2005;353:1591–1603. addition, some common nonmalignant, nonpleural effusion- 2. Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet associated conditions including coronary artery disease, in- 2005;366:397–408. terstitial pneumonia, and other benign pulmonary disease28–30 3. Kojima T, Oh-eda M, Hattori K, et al. Molecular cloning and expression of megakaryocyte potentiating factor cDNA. J Biol Chem 1995;270: can result in increased levels of osteopontin in the serum. 21984–21990. We have previously found that individual patients with 4. Yamaguchi N, Hattori K, Oh-eda M, et al. A novel cytokine exhibiting mesothelioma exhibit different biomarker profiles: either both megakaryocyte potentiating activity from a human pancreatic tumor cell CA125 and mesothelin, only one or neither marker. This was line HPC-Y5. J Biol Chem 1994;269:805–808. not found to be the case with the pattern of expression of 5. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. osteopontin and mesothelin as virtually all osteopontin posi- Proc Natl Acad Sci USA 1996;93:136–140. tive mesothelioma patients were positive for mesothelin. This 6. Scholler N, Fu N, Yang Y, et al. Soluble member(s) of the mesothelin/ high degree of concordance means that combining such markers megakaryocyte potentiating factor family are detectable in sera from would be unlikely to add to sensitivity. As the various combi- patients with ovarian carcinoma. Proc Natl Acad Sci USA 1999;96: 11531–11536. nations of markers discriminated between mesothelioma patients 7. Muminova ZE, Strong TV, Shaw DR. Characterization of human me- and controls, no better than when mesothelin was used alone, sothelin transcripts in ovarian and pancreatic cancer. BMC Cancer our current practice is to measure serum mesothelin as the 2004;4:19.

856 Copyright © 2008 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology • Volume 3, Number 8, August 2008 Comparison of Osteopontin, MPF, and Mesothelin Proteins

8. Hassan R, Bera T, Pastan I. Mesothelin: a new target for immunother- for early-stage ovarian cancer when combining cancer antigen CA- apy. Clin Cancer Res 2004;10:3937–3942. 125II, CA 15-3, CA 72-4, and macrophage colony-stimulating factor 9. Hellstrom I, Raycraft J, Kanan S, et al. Mesothelin variant 1 is released using mixtures of multivariate normal distributions. J Clin Oncol 2004; from tumor cells as a diagnostic marker. Cancer Epidemiol Biomarkers 22:4059–4066. Prev 2006;15:1014–1020. 21. Etzioni R, Falcon S, Gann PH, et al. Prostate-specific antigen and free 10. Ho M, Onda M, Wang QC, et al. Mesothelin is shed from tumor cells. prostate-specific antigen in the early detection of prostate cancer: do Cancer Epidemiol Biomarkers Prev 2006;15:1751. combination tests improve detection? Cancer Epidemiol Biomarkers 11. Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins and Prev 2004;13:1640–1645. diagnosis of mesothelioma. Lancet 2003;362:1612–1616. 22. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. 12. Scherpereel A, Grigoriu B, Conti M, et al. Soluble mesothelin-related Br J Cancer 2004;90:1877–1881. protein in the diagnosis of malignant pleural mesothelioma. Am J Respir 23. Pass HI, Lott D, Lonardo F, et al. Asbestos exposure, pleural mesothelioma, Crit Care Med 2006;173:1155–1160. and serum osteopontin levels. N Engl J Med 2005;353:1564–1573. 13. Hassan R, Remaley AT, Sampson ML, et al. Detection and quantitation 24. Light RW, Macgregor MI, Luchsinger PC, et al. Pleural effusions: the of serum mesothelin, a tumor marker for patients with mesothelioma and diagnostic separation of transudates and exudates. Ann Intern Med ovarian cancer. Clin Cancer Res 2006;12:447–453. 1972;77:507–513. 14. Creaney J, van Bruggen I, Hof M, et al. Combined CA125 and mesothe- 25. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas lin levels for the diagnosis of malignant mesothelioma. Chest 2007;132: under two or more correlated receiver operating characteristic curves: a 1239–1246. 15. Onda M, Nagata S, Ho M, et al. Megakaryocyte potentiation factor nonparametric approach. Biometrics 1988;44:837–845. cleaved from mesothelin precursor is a useful tumor marker in the serum 26. Henderson DW, Shilkin KB, Whitaker D. Reactive mesothelial hyper- of patients with mesothelioma. Clin Cancer Res 2006;12:4225–4231. plasia vs mesothelioma, including mesothelioma in situ: a brief review. 16. Shiomi K, Miyamoto H, Segawa T, et al. Novel ELISA system for Am J Clin Pathol 1998;110:397–404. detection of N-ERC/mesothelin in the sera of mesothelioma patients. 27. Wai PY, Kuo PC. The role of osteopontin in tumor metastasis. J Surg Cancer Sci 2006;97:928–932. Res 2004;121:228–241. 17. Ordonez NG. The immunohistochemical diagnosis of mesothelioma: a 28. Grigoriu BD, Scherpereel A, Devos P, et al. Utility of osteopontin and comparative study of epithelioid mesothelioma and lung adenocarci- serum mesothelin in malignant pleural mesothelioma diagnosis and noma. Am J Surg Pathol 2003;27:1031–1051. prognosis assessment. Clin Cancer Res 2007;13:2928–2935. 18. Whitaker D. The cytology of malignant mesothelioma. Cytopathology 29. Kadota J, Mizunoe S, Mito K, et al. High plasma concentrations of 2000;11:139–151. osteopontin in patients with interstitial pneumonia. Respir Med 2005; 19. Schorge JO, Drake RD, Lee H, et al. Osteopontin as an adjunct to 99:111–117. CA125 in detecting recurrent ovarian cancer. Clin Cancer Res 2004;10: 30. Ohmori R, Momiyama Y, Taniguchi H, et al. Plasma osteopontin levels 3474–3478. are associated with the presence and extent of coronary artery disease. 20. Skates SJ, Horick N, Yu Y, et al. Preoperative sensitivity and specificity Atherosclerosis 2003;170:333–337.