Combined CA125 and Mesothelin Levels for the Diagnosis of Malignant Mesothelioma*

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1 CHEST Combined CA125 and Mesothelin Levels for the Diagnosis of Malignant Mesothelioma* Jenette Creaney, PhD; Ivonne van Bruggen, BSc; Michelle Hof, BSc; Amanda Segal, MBBS; Arthur W. Musk, MBBS, MD, FCCP; Nick de Klerk, PhD; Nora Horick, MSc; Steven J. Skates, PhD; and Bruce W. S. Robinson, MBBS, MD Original Research NEOPLASTIC DISEASES Background: Malignant mesothelioma is an aggressive, uniformly fatal tumor. Serum markers would be useful for the diagnosis of this disease. One potential marker is mesothelin. The purpose of this study was to study the mesothelin biomarker in a large patient cohort and to determine if another biomarker, CA125, improves on the sensitivity of mesothelin in the diagnosis of mesothelioma. Methods: Serum levels of mesothelin and CA125 were determined by commercially available assays in 117 samples obtained at diagnosis from patients with pleural malignant mesothelioma, 33 healthy, asbestos-exposed individuals, 53 patients with asbestos-related lung or pleural disease, and 30 patients presenting with benign pleural effusions. Cross-validated sensitivities were determined, and receiver operator characteristic curves were generated to compare the diagnostic accuracy of the biomarkers. Results: CA125 had a cross-validated sensitivity of 27% for mesothelioma patients at a specificity of 95% relative to asbestos-exposed individuals, or 50% relative to individuals with benign pleural effusions. Mesothelin had a cross-validated sensitivity of 52% for mesothelioma patients, at a sensitivity of 95% relative to individuals with benign lung or pleural disease. CA125 and mesothelin levels were discordant in > 50% of mesothelioma patients. Combining the data from the two biomarkers using a logistic regression model did not improve sensitivity for detecting mesothelioma above that of the mesothelin marker alone. Conclusion: Combining mesothelin and CA125 data does not improve the sensitivity of mesothelioma diagnosis over mesothelin alone. The use of both markers potentially increases the number of patients who can be monitored. (CHEST 2007; 132: ) Key words: CA125; malignant mesothelioma; mesothelin; SMRP; tumor markers Abbreviations: AUC area under the receiver operating characteristic curve; CI confidence interval; ROC receiver operator characteristic Malignant mesothelioma is an aggressive, uniformly fatal disease related to asbestos exposure. 1 Serum tumor markers are usually proteins associated with malignancy and have been useful in the clinical management of various forms of cancer. 2 4 CA125 is a well-characterized tumor marker. 5 It is a large transmembrane mucin 6 that localizes to the cell surface of epithelial cells of the fallopian tube, endometrium, other epithelia, and to mesothelial cells lining the pleura, pericardium, and peritoneum. 7 Serum levels of CA125 are elevated in a number of clinical and nonclinical settings, most notably ovarian cancer. 5 There have been few small-scale studies investigating serum CA125 levels in pleural mesothelioma; elevated levels have been reported in 3 of 10 cases, 8 and no conclusions could be drawn from such a small number of patients. Elevations have also been described in small numbers of patients with peritoneal mesothelioma. 9,10 A definitive study of CA125 levels in patients with pleural malignant mesothelioma has never been undertaken. CHEST / 132 / 4/ OCTOBER,

2 Table 1 Summary of Patient Characteristics and Serum Biomarker Levels Variables Patients, No. Female Gender, % Mean Age, yr CA125 Mesothelin Median SE 21 U/mL, No. Median SE 2.5 nmol/l, No. Malignant (42%) (48%) mesothelioma (all) Unspecified Epithelial Mixed Sarcomatoid Premorbid Asbestos exposed (all) (10%) (1%) Healthy Asbestosis Asbestosis and plaques Benign pleural effusion (53%) (3%) We therefore set out to determine, in a study of a large number of patients with different histologic types of pleural mesothelioma, the sensitivity of CA125 measurements. Also, because we have demonstrated that serum mesothelin is a sensitive marker for malignant mesothelioma, 11 we determined whether combining CA125 measurements with mesothelin improved the accuracy of either test alone. *From the National Research Centre for Asbestos Related Diseases (Dr. Creaney, Ms. van Bruggen, Ms. Hof, Dr. Musk, and Dr. Robinson), Western Australian Institute of Medical Research, University of Western Australia, Nedlands, Australia; PathWest Laboratory Medicine (Dr. Segal), Queen Elizabeth II Medical Centre, Perth, Western Australia; Biostatistics and Epidemiology Unit (Dr. de Klerk), Telethon Institute for Child Health Research, Subiaco, WA, Australia; and Biostatistics Center (Ms. Horick and Dr. Skates), Massachusetts General Hospital, Boston, MA. This work was performed at the National Research Centre for Asbestos Related Diseases, Western Australian Institute of Medical Research, University of Western Australia. This work was 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 Fujirebio Diagnostic Incorporated, Malvern, PA. Dr. Creaney and Dr. Robinson have received consultancy fees from Fujirebio Diagnostic Incorporated. The remaining authors have no conflicts of interest to disclose. Manuscript received January 9, 2007; revision accepted June 2, Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( org/misc/reprints.shtml). Correspondence to: Jenette Creaney, PhD, National Research Centre for Asbestos Related Diseases, 4th Floor, G Block, Sir Charles Gairdner Hospital, Verdun St, Nedlands, WA 6009, Australia; creaneyj@cyllene.uwa.edu.au DOI: /chest Sample Population Materials and Methods We studied sera from four groups of patients: (1) 117 consecutive patients with pleural malignant mesothelioma; the diagnosis of malignant mesothelioma was confirmed with published criteria using cytologic or histopathologic samples 12 ; samples were obtained at diagnosis (within 6 weeks before or after pathologically confirmed diagnosis); (2) 53 individuals with benign asbestosrelated disease (asbestosis, or asbestosis and pleural plaques): (3) 30 patients with benign pleural effusions classified as exudates or transudates 13 ; and (4) 33 healthy asbestos-exposed individuals. 14 The study was approved by the Human Research Ethics Committees of Sir Charles Gardiner and Hollywood Hospitals, and written consent was obtained from all participants. Biomarker Assay: CA125 concentrations were determined (Immunlite 2000 OM-MA assay; Diagnostic Products Corporation; Los Angeles, CA) following the instructions of the manufacturer. A result 21 U/mL was considered elevated. Mesothelin concentrations were determined (Mesomark assay; Fujirebio Diagnostics; Malvern, PA) following the instructions of the manufacturer. A mesothelin value 2.5 nmol/l was considered positive. All assays were performed on coded samples by investigators who were unaware of the patient s diagnosis. Immunohistochemistry: Immunohistochemistry was performed using standard techniques on a tissue microarray of 33 malignant mesothelioma tumor tissue samples, 26 mesothelioma pleural effusions, and a range of malignant and normal tissues including tonsil, kidney, liver, appendix, and colon. Primary antibodies, antimesothelin (clone 5B2; NovaCastra; Newcastle-on-Tyne, UK), and anti- CA125 (clone OC125; DakoCytomation; Glostrup, Denmark) were used at a dilution of 1:50. Staining was assessed by three independent observers (J.C., I.v.B., and A.S.). A positive result was defined as the presence of membranous stain in 33% of tumor cells. Staining intensity was graded semiquantitatively as negative, weak (1 ), moderate (2 ), or strong (3 ). Statistical Analysis: To test for statistical significant differences, biomarkers were transformed to the logarithmic scale on which normal theory statistical estimates (mean, SD) and tests (t tests) were applied. Receiver operator characteristic (ROC) curves were used to quantify marker performance and provide 1240 Original Research

3 Figure 1. CA125 levels in patients with malignant mesothelioma and control subjects. Top: CA125 levels were determined at least in duplicate, and individual patient values are plotted on the graph. CA125 levels in the serum were plotted for all patients with malignant mesothelioma (MM) [regardless of the histology], for individuals with a history of asbestos exposure with no evidence of disease (asbestos exposed), for patients with asbestosis, for patients with asbestosis and pleural plaques (Asb & Plaques), and also for patients with benign pleural effusion (PE). Bottom: CA125 levels were determined for patients with different mesothelioma histologies. Mesothelioma patients were characterized by the histology of the tumor (sarcomatoid, mixed histology, or epithelial) or in those cases when the diagnosis was made without histology being reported (cytology only). CA125 levels were determined in serum samples obtained from people with mesothelioma developing 1 to 7 years after sampling (premorbid). The manufacturer-defined cut-off value of 21U/mL is depicted by the hashed horizontal line. Figure 2. Mesothelin levels in patients with malignant mesothelioma and control subjects. Top: Mesothelin levels were determined at least in duplicate by enzyme-linked immunosorbent assay, and individual patient values are plotted. Serum mesothelin levels were plotted for all patients with mesothelioma (regardless of the histology), for individuals with a history of asbestos exposure, for individuals with no evidence of disease (asbestos exposed), for patients with asbestosis, for patients with asbestosis and pleural plaques, and for patients with benign pleural effusions. Bottom: Mesothelin levels were determined for patients with different mesothelioma histologies. Mesothelioma patients were characterized by the histology of the tumor (sarcomatoid, mixed histology, or epithelial) or in those cases where diagnosis was made without histology being reported (cytology only). Mesothelin levels were determined in serum samples obtained from people with mesothelioma developing 1 to 7 years after sampling (premorbid). The manufacturer-defined cut-off value of 2.5 nmol/l is depicted by the hashed horizontal line. See Figure 1 legend for expansion of abbreviations. estimates of sensitivity for a given specificity. The area under the ROC curve (AUC) was calculated as an overall performance indicator of the biomarkers. To combine CA125 and mesothelin levels, we first transformed data with the natural logarithm, and then standardized the markers relative to asbestos-exposed control subjects. Logistic regression to predict case/control status was used to determine the weight to be given each marker, which were then linearly added to give a combined marker value. For validation studies, univariate logistic regression sensitivities for CA125 alone and for mesothelin alone were calculated. Crossvalidation to reduce overfitting bias left out one observation at a time, and predicted the status of the left out observation. By repeating this process for each observation in the data set, a cross-validated estimate of sensitivity was obtained for given specificities (80%, 90%, 95%, and 98%). Results Patient Characteristics Serum samples were collected from 117 patients with pleural malignant mesothelioma: 35 patients with CHEST / 132 / 4/ OCTOBER,

4 predominantly epithelioid histology, 15 patients with sarcomatoid, 18 patients with mixed histology, and 49 patients with a diagnosis made on immunocytology grounds. 12 Premorbid serum samples were analyzed from four individuals with mesothelioma developing approximately 1, 2, 5, and 7 years after serum sampling, respectively. Sera were collected from 30 patients with nonmalignant effusions (10 had exudative effusions, 10 had effusions relating to an infection, and 10 had transudate effusions) [Table 1]. CA125 in Patients With Malignant Mesothelioma and Control Subjects Serum CA125 levels were elevated in 49 of 117 mesothelioma patients (42%); levels ranged from nondetectable to 1,072 U/mL (Table 1; Fig 1). Seven of 53 individuals with nonmalignant asbestos-related lung disease had elevated CA125 levels (range, 2.9 to 197 U/mL). In asbestos-exposed individuals without disease, 2 of 33 patients had elevated CA125 serum levels (range, 2 to 111 U/mL) [Fig 1, top] and remain alive and disease free 10 years and 11 years, respectively, after the positive sample was obtained. CA125 levels were elevated in 16 of 30 patients with nonmalignant pleural effusions. The median ( SE) serum CA125 concentration was U/mL at diagnosis for the mesothelioma patient cohort, significantly different from the asbestos-exposed cohort ( U/mL; p ), and from the patients with benign pleural effusions ( U/mL; p 0.05). The level of CA125 in patients with epithelial tumors was not statistically significantly different from those with tumors of other histologies (Fig 1, bottom). None of four premorbid serum samples from individuals with mesothelioma developing subsequently had elevated CA125 levels. Mesothelin in Patients With Malignant Mesothelioma and Control Subjects At diagnosis, 48% of mesothelioma patients had elevated levels of mesothelin (56 of 117 patients). Only 1 of the 53 individuals in this study with benign asbestos-related lung or pleural disease had a mesothelin level 2.5 nmol/l. None of the asbestos- Figure 3. Combined CA125 and mesothelin biomarker concentrations. Top: CA125 and mesothelin levels in 117 individual patients with malignant mesothelioma (closed diamonds) and 86 asbestos-exposed individuals (open diamonds) were correlated. Cut-off values for each assay are designated by the hashed lines. Center: ROC curves showing accuracy of CA125, mesothelin, and combined CA125 and mesothelin biomarker levels in differentiating patients with mesothelioma from individuals with a history of asbestos-exposure including those with benign lung and/or pleural disease and healthy individuals. Bottom: ROC curve of CA125, mesothelin, and combined biomakers for differentiating patients with mesothelioma from individuals with a history of asbestos-exposure and patients with benign pleural effusions. Numbers in parenthesis indicate AUC Original Research

5 Table 2 Cross-Validated Sensitivity at Different Levels of Specificity Markers 80% Specificity, % (Threshold) 90% Specificity (Threshold) 95% Specificity (Threshold) 98% Specificity (Threshold) Mesothelin 67 (1.4 nmol/l) 60 (1.7 nmol/l) 52 (2.2 nmol/l) 49 (2.5 nmol/l) CA (6 U/mL) 42 (8 U/mL) 27 (10 U/mL) 9 (25 U/mL) Mesothelin and CA exposed healthy individuals had elevated mesothelin levels. One of the 30 patients with benign pleural effusions had elevated serum levels of mesothelin (Table 1; Fig 2, top). Mesothelin levels were significantly higher in patients with mesothelioma (median, ), compared with individuals with a history of asbestos exposure (median, ; p ) and patients with benign pleural effusions (median, ; p ). No significant difference in mesothelin levels at the time of diagnosis was observed in mesothelioma patients with tumors of different histologies. Three of four premorbid samples from individuals with mesothelioma subsequently developing were mesothelin positive (Fig 2, bottom). Combining Mesothelin and CA125 A bivariate scatter plot of CA125 vs mesothelin levels in mesothelioma cases and control subjects revealed that 18% of patients were positive for both mesothelin and CA125, 30% were positive for only mesothelin, 24% were positive for only CA125, and 28% were negative for both markers (Fig 3, top). ROC curves were generated to distinguish patients with mesothelioma from the asbestos-exposed individuals with and without benign disease. The AUC for mesothelin was (95% confidence interval [CI], to 0.851) and for CA125 was (95% CI, to 0.759). Using a logistic regression model, the diagnostic capability of CA125 and mesothelin were combined, generating a ROC curve with an AUC of (95% CI, to 0.861) (Fig 3, center). The AUC of the combined markers was not significantly different from the AUC of the mesothelin marker alone. Using univariate logistic regression to fit sensitivities for CA125 alone and mesothelin alone, crossvalidated sensitivities were compared to those obtained using the combined CA125 and mesothelin marker (Table 2). The addition of information from the CA125 biomarker did not add value to the mesothelin marker at the moderate specificities of 80% and 90%. At the higher specificities of 95% and 98%, the mesothelin marker was more sensitive than the combined CA125 and mesothelin marker, with the mesothelin marker alone having a sensitivity of 49% compared to 42% for the combined marker at a specificity of 98%. Using data generated from the entire control study population, including those individuals with benign pleural effusions and the mesothelioma patient data generated ROC curves with an AUC of (95% CI, to 0.832) for mesothelin, (95% CI, to 0.683) for CA125, and (95% CI, to 0.807) for the combined markers (Fig 3, bottom). Tissue Localization of Mesothelin and CA125 Immunohistochemical staining for mesothelin and CA125 in tumor and effusion specimens showed strong membrane accentuation (Fig 4). Approximately 73% of malignant mesothelioma tissue samples stained with mesothelin and 62% stained for CA125; almost half of the samples stained positive with both antibodies. Similarly, 16 of 20 effusions (80%) from patients with mesothelioma stained with mesothelin, and 15 of 19 effusions (79%) stained with CA125. In 10 of 16 cases, nonmalignant mesothelial cells present in benign effusions were mesothelin positive, and 13 of 14 cases were CA125 positive (Table 3). Mesothelin staining of normal mesothelial cells in effusions was weak but clearly localized to the membrane. Approximately one half of the positively stained samples had elevated levels of the respective protein in the serum obtained at the time of tissue collection (Table 4). Of note, in those patients with samples available, elevated levels of serum markers were generally observed at a later time in those individuals with marker-positive tumors. Only one individual was observed with elevated serum levels of a marker in the setting of a marker-negative tumor. In approximately one third of cases, lack of detectable biomarker in the serum correlated with lack of tumor staining. Discussion Combining markers often improves the diagnostic performance of diagnosis and screening strategies, and the use of independent (or nonredundant) bio- CHEST / 132 / 4/ OCTOBER,

6 Figure 4. Immunohistochemical staining in tumor tissue from three patients with mesothelioma stained with antibodies to mesothelin (left panels) and antibodies to CA125 (right panels). The tumor from patient 10 (top panels) stained with both antibodies to mesothelin and CA125 and had elevated levels of both serum biomarkers. The tumor from patient 8 (center panels) stained with antibody to mesothelin and had a positive serum mesothelin level. The tumor from patient 53 (bottom panels) was positive for CA125 staining, and the patient had an elevated serum level of CA125. Control, nonmalignant tonsil, kidney, liver, appendix, and colon did not stain for either CA125 or mesothelin. markers can significantly enhance sensitivity. 15 This study shows that combining CA125 and mesothelin serum levels does not improve the accuracy of diagnosis of malignant mesothelioma over the mesothelin marker used alone. Although CA125 has previously been reported to be elevated in a small number of patients with mesothelioma, particularly those with substantial tumor burden, 8 10 a definitive study in a large number of cases was necessary to help define a 1244 Original Research

7 Table 3 Results of Immunohistochemical Staining* Tissue Type Mesothelin CA125 Both Malignant mesothelioma 24/33 20/32 16/32 epitheliod tissue Malignant mesothelioma 16/20 15/19 9/19 effusion BRM effusion 10/16 13/14 6/14 *Data are presented as No./total cases. BRM benign reactive mesothelial cell. Table 4 Correlation of Immunohistochemical Staining and Serum Biomarker Levels* Variables Mesothelin CA125 Tissue positive, serum negative 10 8 Tissue positive, serum negative 3 4 Tissue negative, serum positive 0 1 Tissue negative, serum positive 6 6 *Data are presented as No. of findings. potential clinical use for this assay. In this study, 42% of 117 mesothelioma patients had elevated levels of CA125 in their serum at the time of diagnosis. The number of patients with elevated CA125 increased in patients with advanced disease (data not shown). CA125 levels are elevated in a number of diverse cancerous and noncancerous conditions, 5,16 particularly those with serosal involvement. 17 The present study reports that approximately 50% of patients with benign effusions and 10% of patients with asbestos exposure had elevated CA125, confirming the lack of diagnostic specificity of the marker in the setting of mesothelioma diagnosis. The current study of mesothelin levels in mesothelioma advances our original study, 11 in that it uses a commercially prepared enzyme-linked immunosorbent assay and studies a much larger cohort of patients. We show that 48% of 117 mesothelioma patients were mesothelin positive at the time of diagnosis. This result is similar to that of Scherpereel et al, 18 who, using the same assay, found 50% of 60 mesothelioma patients had elevated mesothelin levels in serum (ie, 2 nmol/l). The high specificity of the mesothelin assay for mesothelioma in patients with clinical histories and/or symptoms of nonmesothelioma pleural effusion, benign pleural plaques, or asbestosis was confirmed. Mesothelin is elevated in few asbestosexposed individuals; Scherpereel et al 18 reported that none of 23 asbestos-exposed patients with benign disease had elevated mesothelin, and in the current study only 1 individual with asbestosis had an elevated level. Thus, an elevated level of mesothelin should be considered sufficient to warrant further more invasive diagnostic procedures, such as biopsy, and a negative mesothelin level does not exclude a diagnosis of mesothelioma. CA125 is not elevated in exactly the same patients as those with mesothelin elevations. Mesothelioma patients express either both, only one, or neither marker. This suggests that the processes regulating the production or release of each of these molecules from the malignant mesothelioma cells or effusions differ. Both CA125 and mesothelin are surface molecules, and both can be cleaved from the cell surface 5,16,19 ; therefore, the production of specific proteases by the tumor or tumor microenvironment may differ. There are other putative mechanisms governing their detectability in serum, including for mesothelin the generation of splice variants that induce a frame-shift mutation that results in the loss of the transmembrane portion of the molecule. 20 The observation that CA125 is commonly elevated in patients with benign effusions, and as observed in this study in patients with sarcomatous mesothelioma, whereas mesothelin is not, suggests that CA125 forms part of a normal exudative effusion and is absorbed into the circulation from the fluid, whereas elevated serum mesothelin levels are probably more related to the malignant process itself and/or reach the bloodstream from the tumor rather than the pleural fluid. We are currently analyzing these processes. Combining CA125 and mesothelin does not improve the accuracy of diagnosis of mesothelioma over the mesothelin marker used alone. This is in contrast to the results demonstrating improved sensitivity in predicting ovarian cancer cases from healthy control subjects when combining CA125 and mesothelin. 21 We have used a standard statistical approach to combining two markers, logistic regression, which is numerically stable and standardized, and has been used by other authors 15 to combine markers in ovarian cancer detection. Alternative approaches, such as support vector machines, may improve on the numerical strength of the data, but it appears that the clinical significance of such a small increase would probably not be worth the time and expense of the added test. The estimates of sensitivity and specificity obtained by cross-validation, as we provide in this report, are reduced compared to the full data approach, and ameliorate although do not completely eliminate overfitting. While mesothelioma patients can be distinguished by their profile of biomarker expression, no obvious survival advantage was associated with each of the different patterns (ie, elevated CA125, mesothelin, or both; data not shown). Taken together, 72% of mesothelioma patients have detectable CA125 or mesothelin biomarkers in CHEST / 132 / 4/ OCTOBER,

8 their serum. Therefore, in most cases the use of both of these biomarkers may represent a useful diagnostic tool in patients suspected of having mesothelioma. The potential role for each of these markers, or both, as an alternative to radiologic or nuclear medicine methods of assessing tumor regression or progression is evidenced by these preliminary studies and warrants further investigation. There is considerable interest in screening asbestosexposed individuals for the early detection of mesothelioma. Use of the CA125 biomarker to screen women for the early detection of ovarian cancer is being intensively evaluated. Although we have only been able to study four mesothelioma premorbid samples, the finding that CA125 levels are not elevated prior to diagnosis plus our finding that CA125 levels are elevated in approximately 10% of asbestos-exposed individuals without mesothelioma suggest that CA125 alone is unlikely to be useful as a screening tool for mesothelioma. Currently, methods of combining the biomarkers and a program that determines significant changes in biomarker values, rather than absolute values, are being evaluated. Tissue distribution of mesothelin and CA125 has previously been shown in immunohistochemical studies 22 to be similar but not identical. Indeed, when the antibody to mesothelin was first isolated, there was a concern that it was directed against another determinant on the CA125 molecule. 22 Both antigens are expressed on normal mesothelial cells, and in this study 73% of mesotheliomas were mesothelin positive and 62% were CA125 positive, levels less than previously reported Further work is needed to determine whether the combined use of the biomarkers mesothelin and CA125 will be useful as a component of the clinical care of mesothelioma patients and offers any clinical advantage to the patients. 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