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a Department of Cardiothoracic Surgery, Division of Thoracic Surgery, New York University School of Medicine, New York, New York
b Karmanos Cancer Institute, Detroit, Michigan
c Cancer Research Center of Hawaii, Honolulu, Hawaii
d Fujirebio Diagnostics Inc, Malvern, Pennsylvania
Accepted for publication July 16, 2007.
* Address correspondence to Dr Pass, Department of Cardiothoracic Surgery, Division of Thoracic Surgery, NYU School of Medicine, 530 First Avenue, 9V, New York, NY 10016 (Email: harvey.pass{at}med.nyu.edu).
Presented at the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007.
| Dr Allard discloses a financial relationship with Fujirebio Diagnostics, Inc.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Methods: We evaluated SMRP in serum from MPM patients (n = 90), lung cancer patients (n = 170), age and tobacco-matched asbestos-exposed individuals (n = 66), and in MPM pleural effusions (n = 45), benign effusions (n = 30), and non-MPM effusions (n = 20) using the MesoMark enzyme-linked immunosorbent assay kit (Fujirebio Diagnostics, Malvern, PA). Receiver operating characteristic (ROC) curves were used to define true and false positive rates at various cutoffs.
Results: Mean serum SMRP levels were higher in MPM compared with lung cancer (5.67 ± 0.82 nM [mean ± standard error of the mean vs 1.99 ± 0.43 nM, p < 0.001), and stage I MPM SMRP levels (n = 12; 2.09 ± 0.41 nM) were significantly higher than those in asbestos-exposed individuals (0.99 ± 0.09 nM, p = 0.02, respectively). Stage 2 to 4 SMRP serum levels were significantly higher than those for stage 1 MPM. The area under the ROC curve for serum SMRP was 0.81 for differentiating MPM and asbestos-exposed individuals; cutoff = 1.9 nM (sensitivity = 60%, specificity = 89%). The MPM pleural effusion SMRP was significantly higher than benign or other non-MPM pleural effusions (65.57 ± 11.33 nM vs 27.46 ± 11.25 nM [p = 0.003] and 18.99 ± 7.48 nM [p = 0.044], respectively).
Conclusions: These data support SMRP as a promising marker for MPM in both serum and pleural effusion fluid, and justify prospective screening studies of SMRP in combination with other markers for screening of asbestos-exposed cohorts.
Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related tumor which is increasing in incidence and causes an estimated 15,000 to 20,000 deaths per annum worldwide. Malignant pleural mesothelioma has a median survival of seven to ten months and a clinical pattern that usually involves substantial pain and dyspnea. It presents at a clinically advanced stage in most patients so there is a need for new methods of early detection. The fact that asbestos is the main etiologic agent for MPM means that at-risk populations can be readily identified and studied, and these populations represent ideal cohorts in which to undertake early cancer detection studies.
There have been a number of studies attempting to define biomarkers that could predate symptoms in a "high risk for MPM" population and also distinguish MPM from other malignancies. Unfortunately, the majority of these studies have had very few patients of various stages of MPM and the markers have not been prospectively evaluated. Some of these biomarkers include tissue polypeptide antigen, carcinoembryonic antigen, hyaluronic acid, and ferritin [1] as well as hyaluronic acid levels [2]. Other markers such as cytokeratins [3] and cancer antigen 125 (CA-125) [4] have been evaluated in MPM but have been inconclusive. One must conclude, therefore, that until recent reports of soluble mesothelin-related protein (SMRP) [5] and osteopontin [6] in MPM, there have been no reliable, validated serum or pleural effusion markers that can distinguish a high-risk, asbestos-exposed population without MPM from patients with established MPM, or to distinguish other malignancies from MPM.
The SMRP is related to the mesothelin family of molecules. Mesothelin is a 40-kD cell surface glycosylated phosphatidylinositol-anchored glycoprotein, which functions in cell-to-cell adhesion [7]. Data from our laboratory using serial analysis of gene expression revealed that the mesothelin levels were 49-fold increased over normal peritoneum [8]. Mesothelin is expressed by normal mesothelial cells [9]; however, it is highly overexpressed in cancers such as MPM [10, 11], pancreatic [12], or ovarian carcinoma [10, 13]. In 1999, Scholler and colleagues [14] described a monoclonal antibody 569, which binds to a 40-kDa molecule with an NH2-terminal sequence similar to mesothelin and is overexpressed in ovarian cancers and certain other tumors. Scholler and colleagues speculated that a mesothelin-like molecule must be released from tumor cells and demonstrated that this molecule was released into culture supernatants, sera, and malignant effusions from patients with certain tumors. The release of these soluble mesothelin-related proteins could be due to an abnormal splicing event resulting in a frameshift mutation of the protein, making it unable to stay attached at the cell surface. Another hypothesis for the origin of SMRP is that it represents a proteolytically cleaved fragment of membrane-bound mesothelin.
Evidence has been published indicating that an enzyme-linked immunosorbent assay using two antibodies to SMRP, OV569 and 4H3, holds promise for detection of MPM and that it favorably complements CA-125 for detection of ovarian carcinoma. Robinson and colleagues [5] were the first to investigate SMRP using a nonquantitative assay in the serum of MPM patients and a limited numbers of controls. He published that 37 (84%) of 44 patients with MPM had raised concentrations of SMRP compared with a limited number of non-MPM cancers (n = 30), asbestos-exposed individuals (n = 40), and nonasbestos-exposed individuals (n = 28). The SMRP was elevated in 1 of 30 cancers, 0 of 28 nonasbestos-exposed individuals, and 7 of 40 asbestos patients. In a recent report from Scherpereel and colleagues [7], serum SMRP level was significantly higher in patients with MPM than in subjects exposed to asbestos with pleural benign disease, or in those with metastatic disease to the pleura.
The Karmanos and New York University Thoracic Oncology Laboratories have had an interest in mesothelioma and has developed a large archive of serum, plasma, and pleural effusions from patients having surgery for the disease. As such, we felt it was important to corroborate the data regarding MESOMARK (Fujirebio Diagnostics, Malvern, PA) by defining the sensitivity and specificity of the marker in distinguishing patients with mesothelioma from patients exposed to asbestos but not having the cancer. By having a surgical archive with complete staging, we wanted to establish whether SMRP serum levels varied between early and late stage mesothelioma. Moreover, we wanted to define whether SMRP is elevated in other thoracic malignancies such as lung cancer. Finally, we felt that an investigation of SMRP levels as a biomarker for differentiating pleural malignancies was warranted.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Patients with mesothelioma were staged according to the International Mesothelioma Interest Group Staging System (IMIG). Sera from lung cancer patients having surgery at the Karmanos Cancer Institute (n = 120) and from the archives at Fujirebio Diagnostics (Malvern, PA [n = 50]), as well as serum from consenting normal individuals (n = 409) were analyzed for SMRP.
Clinical demographics including age, sex, histology, stage, time to recurrence, and survival time were available on patients, except for the lung cancer sera provided by Fujirebio for whom only sex and age were available. All specimens were obtained using the same model serum tubes and all sera were stored at –80°C.
Serum also was obtained from 66 consenting individuals with a history of asbestos exposure and (or) radiographic changes consistent with asbestosis at the Center for Occupational and Environmental Medicine, Royal Oak, Michigan from July 2004 to September 2004. Entry criteria for this cohort were similar to that described by Cullen and colleagues [15]. Briefly, exposure was documented by (1) employment in a trade with established regular asbestos exposure and published risk of asbestos-related diseases, or (2) occupational asbestos exposure in any job or occupation and evidence of radiographic changes consistent with a diagnosis of nonmalignant asbestos-related disease. These radiographic findings included (1) benign pleural disease, defined as thickening or fibrotic plaques on pleural surfaces of the lung bilaterally, and (or) (2) asbestosis, defined as diffuse lung scarring based on small irregular shadows bilaterally. Each consenting asbestos-exposed individual had a plain chest radiograph that was interpreted by a single trained B-reader (National Institute for Occupational Safety and Health [NIOSH] B Reader Program). The B-reader specifically commented on the presence of pleural changes including plaques as well as the presence in lung fibrosis. Lung fibrosis was interpreted according to the to International Labor Organization scoring process [16].
Pleural Effusions
Nonmesothelioma pleural effusions were obtained from 30 individuals with a benign etiology (postoperative day 3 effluent after benign thoracotomy or bypass [n = 20], pneumothorax treatment [n = 2], Wegeners granulomatosis, granulomatous disease, asbestosis, congestive heart failure, postinterleukin-2 effusion, sympathetic effusion, postchemoradiotherapy effusion, renal failure, [n = 1 each]), and from 20 individuals with nonmesothelioma malignant effusions (adenocarcinoma [n = 16]; lymphoma, breast, renal cell carcinoma, thymoma [1 each]). Effusions were processed by centrifugation with storage of the liquid component at –80°C.
MESOMARK Assay
The MESOMARK assay was performed according to the manufacturer’s instructions. Briefly, patient serum samples were diluted 1:101 using the assay diluent provided and 100 µL of the diluted samples were added in duplicates to a 96-well plate precoated with the 4H3 antibody. The samples were incubated on a plate-shaker for 60 minutes followed by a 5x rinse with wash buffer. The OV569-HRP conjugate was next added to the sample wells and the microwell plate incubated for a further 60 minutes on a plate-shaker. After a wash step, 100 µL of substrate was added to the reaction wells for 15 minutes before adding 100 µL of stop solution. The absorbance at 450 nm was used to quantify the SMRP levels by comparison to a six-point calibration curve. The MESOMARK values are expressed as nM (nanomolar).
Statistical Analysis
Kaplan-Meier survival plots and log-rank tests were used to assess differences in survival for the MPM patients. All survival data were calculated from the date of the operation. The performance of SMRP in detecting MPM from other groups was evaluated by descriptive statistics and by receiver operating characteristic (ROC) curves. The ROC curves defined the sensitivity and specificity for distinguishing between subgroups of patients with MPM and from the asbestos-exposed individuals and those with lung cancer [17, 18] by illustrating the true positive rate as a function of false positive rate at different cutoff points. The area under the ROC curve (AUC) was calculated, and 95% confidence intervals for the AUC were used to test the hypothesis that the theoretical area is 0.5. Areas under the curve whose confidence interval did not include the 0.5 value were considered evidence that the laboratory test had some ability to distinguish between the groups studied [18, 19]. As with any analyses using ROC curves, the AUC must for relevance had to be not only different than 0.5 but ideally significantly greater than 0.5. Differences between groups were calculated using the Kruskal-Wallis test and analysis of variance (ANOVA). All statistical analyses were performed using MedCalc Software (Mariakerke, Belgium).
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Results |
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Comment |
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TopAbstractIntroductionPatients and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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The SMRP investigation conducted by our group used archives that were accumulated by one of the authors (HIP) over a 16-year period, with supplementation of 50 lung cancers from a commercial source. A portion of these archives were previously used to investigate osteopontin as a marker for mesothelioma in the hope of combining multiple markers for the early detection and classification of mesothelioma. All of the serum cohorts used for these investigations had well-documented demographics. For the mesotheliomas all of the patients had attempted cytoreduction of the disease with careful intraoperative staging, and 120 of 170 lung cancer patients had resection of their tumor with mediastinal lymph node dissection by one of the authors (HIP). The asbestos- exposed population, although small, was specifically recruited for this study in 2004 not only to compare the values with cancer bearing populations, but also to see if any of the mesothelioma markers being investigated were influenced by parameters such as age, sex, or duration of asbestos exposure (by history). One could argue that this is not a representative group of study subjects because all of the groups had sera or pleural effusion collected in the past, and do not represent a new, prospective collection. Such a prospective collection is being planned by the Early Detection Research Network as a validation for markers such as SMRP and will serve as a reference set for determining how robust combinations of markers can be in detecting or classifying mesothelioma.
Our study reveals, like those of Robinson and colleagues [5] and Scherpereel and colleagues [7], that serum SMRP levels are significantly higher in patients with MPM than in an asbestos-exposed cohort which either does or does not have radiographic evidence of the exposure. In this surgically staged group of mesotheliomas, SMRP levels rise dramatically comparing early stage I mesotheliomas with those serum levels seen with more advanced disease, which confirms data from the Robinson group in noncytoreduced patients [5]. Normal individuals (n = 409) with a median age of 46 years have significantly lower levels for serum SMRP than asbestos-exposed or MPM bearing individuals. The marker is different from osteopontin, in that SMRP serum levels are not significantly different among asbestos-exposed individuals based on their duration of exposure, and although the marker is elevated in individuals with pleural plaques, there is no further elevation as the degree of radiographic changes increases (ie, with addition of interstitial fibrosis). These findings would imply that SMRP should be a more robust marker for mesothelioma because it seems not to be influenced by inflammatory cascades, as osteopontin may be.
The discriminatory power of SMRP between MPM and asbestos-exposed cohorts in our study compared with those of Scherpereel and colleagues [7] and Robinson and colleagues [5] is not as clearly defined, and our AUC, although very respectable at 0.81, is less than that seen in the study by Scherpereel and colleagues (0.89) and the most recent publication by Robinson and colleagues (0.89). One possible explanation for this is that the patients in our series, all of which had blood taken prior to cytoreductive surgery, had a different stage distribution than those seen in the other studies in the literature. The possibility exists then that our series of patients had less bulk of disease, and hence had lower levels of SMRP (some closer to levels seen in asbestos-exposed individuals). This is difficult to sort out considering that the published data of Robinson and colleagues is not described as nM levels and the study by Scherpereel and colleagues uses median values for serum SMRP as opposed to mean. Moreover, the number of asbestos-exposed individuals and the characterization of the asbestos-exposed population are more detailed in the present study than in the others. The ideal cutoff for differentiating the asbestos group from the mesothelioma group in our study was 1.9 nM, with a sensitivity of 60% and a specificity of 89%. Using Scherpereel and colleagues’ cutoff of 0.93, we would have had a sensitivity of 83% and a specificity of 60% with our data. In order for this marker to be used for early detection, the sensitivity must obviously be close to 100% at an acceptable specificity. Our serum data suffer from the same limitations as Scherpereel and colleagues’ in that adjusting our sensitivity to 95% at a cutoff of 0.36 nM would lead to a specificity of 17%. The only way in which such a low specificity marker could be adapted presently into the clinical arena would be to demonstrate conclusively that the marker rises with time as the degree of disease increases, or that the marker functions effectively as a monitor of treatment of the disease. For SMRP, there are only anecdotal, but extremely interesting data from Robinson and colleagues at this time that SMRP will rise months in advance of the development of clinical symptoms of mesothelioma (Robinson, personal communication). Validation studies are presently being designed using retrospective collections of sera from well-described asbestos-associated archives, which were generated during large prospective screening or chemoprevention studies in the United States. By measuring the longitudinal changes in a given patient known to have developed mesothelioma who was entered on these trials, we will better be able to define whether SMRP or any other marker for mesothelioma alone or in combination can signal the development of the disease.
This study tries to distinguish mesothelioma from lung cancer specifically. Mesothelin has been described to be associated with a number of cancers including pancreatic and ovarian cancer, and mesothelin staining has been identified in non-small cell lung cancers in up to 27%. The data are encouraging that discrimination exists between lung cancer and mesothelioma serum SMRP in that the AUC was 0.820 with a respectable specificity of 76.4% and sensitivity of 78.9% at 1.1 nM. This is especially important for asbestos-exposed cohorts who use tobacco products and will have an elevated risk not only for mesothelioma but for lung cancer. A clinical scenario characterized by a developing pleural effusion in a smoker with or without obvious pleural metastases in which the SMRP is high would probably point to the development of mesothelioma, while a low SMRP could not rule out stage IIIB lung cancer. Future prospective studies which combine SMRP with novel markers for both mesothelioma and lung cancer could further enhance this discrimination.
Short of video-assisted thoracoscopy with tissue biopsy, the characterization of an isolated new effusion in a patient without a previous history of malignancy depends on cytologic description with or without specialized immunopanels. The pleural effusion data which attempt to characterize such pleural effusion presented in the manuscript should be considered very preliminary but concordant with the results described by Scherpereel and colleagues [7]. One of the problems with performing such an analysis is the characterization of the comparator groups, and the definition of a benign pleural effusion. The description of these effusions is only tangentially alluded to in the article by Scherpereel and colleagues, and the benign effusions used in our data represent a wide variety of pathologic conditions that may very well be influenced by pleural homeostasis. The obvious benign conditions that must be represented in future studies include effusions related to the following: (1) primary organ failure including congestive heart failure, renal failure, and cirrhosis; (2) inflammatory-infectious conditions including postpneumonic exudative effusions; and (3) autoimmune-related effusions. These conditions are not well-represented in this manuscript and a different approach was taken (ie, collection of postthoracotomy or postcardiopulmonary bypass effluents from uncomplicated futile or benign thoracotomies). Despite some variation, over 75% of the 30 benign pleural effusions had an SMRP level less than 20 nM. The majority of the cancer effusions were weighted toward adenocarcinoma of the lung, and further discrimination between mesothelioma effusions and effusions from mesothelin expressing tumors, including ovarian cancer, should be performed. Nevertheless, the 82% specificity and 76% sensitivity discriminating mesothelioma from all the "other" effusions is encouraging enough to warrant a multiinstitutional United States prospective study assessing the role of SMRP in classifying new onset pleural effusions.
These preliminary North American data investigating SMRP demonstrate the need for prospective reference samples that can be used for the validation of markers associated with mesothelioma. The design of prospective trials in which the level of SMRP may decide intervention must, by definition, incorporate reference ranges for "nonpathologic" levels of SMRP in the serum or in the pleural effusion. Whether these nonpathologic ranges are influenced by such factors as geographic location of the subjects, type of fiber the individual is exposed to, and duration of exposure to the fiber, is not clearly defined such that SMRP levels can be used to guide the definitive management of the patient. In the interim such prospective reference samples are being formulated and will be used to insure that SMRP has the desired characteristics as a putative marker for mesothelioma either alone or in combination with other novel proteins.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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DR PASS: Sure, but in the context of a screening trial, those are the numbers you want to use. You don’t want to have a lot of sensitivity that’s going to be too low because you’re going to have a lot of patients that you’re going to work up, so you want good specificity. So what I was shooting for was to have a high specificity with whatever the sensitivity is, and those are the ideal numbers. You can lower the two and then even it out, but then you’re losing the value of the marker as an early detection marker.
DR LAWRENCE R. GLASSMAN (Manhasset, NY): I have a question regarding the availability of the assay. Is it generally available, and if it’s not, when will it be available?
DR PASS: Well, a week ago, based on these data from our laboratory in which it showed that you could actually use this marker for monitoring of mesothelioma. Here you see mesothelioma levels before and then after surgery and then rising after recurrence in patients who have had operations. This marker is now going to be available through a reference laboratory. The physician will be able to see a patient with mesothelioma, patients who are under treatment. So the indication for this marker now is not for screening. It will be to follow patients who have had treatment for mesothelioma to see if the marker decreases or if the marker then starts to rise while the patient is on therapy.
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