Background: Diagnosis of malignant pleural mesothelioma is a challenging issue. Potential markers in mesothelioma diagnosis include soluble mesothelin–related peptides (SMRPs) and osteopontin, but no subsequent validation has been published yet.
Methods: We prospectively evaluated SMRPs in serum and pleural effusion from patients with mesothelioma (n = 74), pleural metastasis of carcinomas (n = 35), or benign pleural lesions associated with asbestos exposure (n = 28), recruited when first suspected for mesothelioma.
Findings: Mean serum SMRP level was higher in patients with mesothelioma (2.05 ± 2.57 nM/L [median ± interquartile range]) than in patients with metastasis (1.02 ± 1.79 nM/L) or benign lesions (0.55 ± 0.59 nM/L). The area under the receiver operating characteristic curve (AUC) for serum SMRP was 0.872 for differentiating mesothelioma and benign lesions, cut-off = 0.93 nM/L (sensitivity = 80%, specificity = 82.6%). The AUC for serum SMRP differentiating metastasis and mesothelioma was 0.693, cut-off = 1.85 nM/L (sensitivity = 58.3%, specificity = 73.3%). SMRP values in pleural fluid were higher than in serum in all groups (mesothelioma: 46.1 ± 83.2 nM/L; benign lesions: 6.4 ± 11.1 nM/L; metastasis: 6.36 ± 21.73 nM/L). The AUC for pleural SMRP-differentiating benign lesions and mesothelioma was 0.831, cut-off = 10.4 nM/L (sensitivity = 76.7%, specificity = 76.2%). The AUC for pleural SMRP-differentiating metastasis and mesothelioma was 0.793.
Interpretation: We show that SMRPs may be a promising marker for mesothelioma diagnosis when measured either in serum or pleural fluid. The diagnostic value of SMRPs was similar in both types of samples, but pleural fluid SMRPs may better discriminate mesothelioma from pleural metastasis.
Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a poor survival rate that arises from the surface cells of the pleura. Previously considered as a rare tumor, MPM has become a very important public health issue, and its incidence is expected to continue to increase for at least the next 10 yr (1). Asbestos exposure is the main factor involved in MPM pathogenesis (2). The screening and the diagnosis of MPM in subjects exposed to asbestos are difficult because of the following factors: (1) the disease may occur up to 30 to 40 yr after exposure and (2) the differential diagnosis on pleural biopsy between MPM and pleural benign disease, also frequently induced by asbestos exposure, or pleural metastasis of adenocarcinoma may be difficult in some cases, even with the use of immunohistochemistry (3). Management of patients with MPM remains difficult because they are often referred for evaluation late in the evolution of the disease. Moreover, MPM exhibits a high resistance to radiotherapy and chemotherapy; only a few patients are candidates for radical surgery in a multimodal treatment scheme. New therapeutic strategies, such as gene or cell therapy (4–6), and new drugs, such as histone deacetylase inhibitors (7), are still on clinical trial. Finally, there is no single current marker to help MPM diagnosis or to predict response to treatment or prognosis.
Mesothelin is a 40-kD cell surface glycosylated phosphatidylinositol (GPI)-anchored glycoprotein, which has putative functions in cell-to-cell adhesion (8). Mesothelin is expressed by normal mesothelial cells (9); however, it is highly overexpressed in cancers such as malignant mesothelioma (10, 11), pancreatic (12–14) or ovarian carcinoma (10, 15–18), sarcomas (13), and in some gastrointestinal (16, 19) or pulmonary carcinomas (3, 16, 18, 20). The mechanisms of release of mesothelin from the mesothelial cell surface are not yet established. The release of soluble mesothelin–related peptides (SMRPs) 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 is that SMRP is a proteolytically cleaved fragment of membrane-bound mesothelin (21). SMRPs can be detected in blood, and have been found highly increased in the blood of patients with mesothelioma (22) or ovarian (23) tumors. A first report of SMRPs as a marker of mesothelioma suggested excellent values for sensitivity (84%) and specificity (virtually 100%) (22). This retrospective study evaluated 44 mesotheliomas (of which 15 had a diagnosis based on cytology only) and a large panel of 160 patients with various diseases from only two referral hospitals. The pleural fluid SMRP level, which could eventually be more discriminant than blood SMRP level, was not assessed in this study (21).
Therefore, in a search of new tools for MPM screening and management, we studied SMRPs in serum and pleural fluid from a large panel of patients with benign pleural disease related to asbestos exposure, MPM, or pleural metastasis of carcinomas, in a prospective, multicentric study. Differences in SMRP values were also evaluated in the various pathologic subtypes of MPM.
Starting May 2003, all consecutive patients suspected of, or recently diagnosed with, MPM were recruited in a prospective study in participating pulmonary or thoracic surgery departments in northern and western regions of France (from 20 different hospitals). Exclusion criteria were the presence or suspicion of any concomitant infectious disease, previous radical surgery, radiotherapy, or chemotherapy for the MPM, or patient refusal to participate in the study. Most of the patients had documented asbestos exposure history. Pathologic diagnosis by trained pathologists was done on pleural biopsies obtained by thoracoscopy or thoracotomy, or more rarely by tomodensitometry or ultrasound-guided biopsy. Final diagnosis divided the patients into three groups: confirmed MPM, benign pleural lesions associated with asbestos exposure (BPLAE group, diagnosis compliant with the 2004 American Thoracic Society recommendations ), and pleural metastasis of carcinomas (Mets group; Table 1). This last group of patients had a lower incidence of documented asbestos exposure, but was used as a comparison group for MPM subjects. Patients were included only if they had not received any prior antitumor treatment.
Mets (n = 35)
BPLAE (n = 28)
MPM (n = 74)
|Age, yr (mean ± SD)||65.3 ± 10.8||61.4 ± 8.6||63.8 ± 10.6|
|Male sex, n (%)||16 (45.7)||24 (85.7)||53 (71.6)|
|Pleural fluid, median (QR25–QR75)|
|Total protein, g/L||44.5 (32.5–48)||40 (34.5–45)||46 (40–48.5)|
|Glucose, g/L||0.93 (0.82–1.02)||1.39 (1.13–1.52)||0.84 (0.57–1.16)|
|LDH (IU/L)||676 (404–806)||654 (275–835)||542 (353–801)|
|Blind pleural biopsy||—||—||1 (1.35%)|
|Thoracoscopy||24 (68.6%)||20 (71.4%)||49 (66.2%)|
|Surgery||7 (20%)||8 (28.6%)||23 (31.1%)|
|Guided biopsy (TDM or US)||4 (10.4%)||—||1 (1.35%)|
|Asbestos exposure, n (%)|
|Yes||9 (25.8)||22 (78.6)||56 (75.7)|
|No||22 (74.2)||4 (14.3)||8 (10.8)|
|Likely (but not confirmed)||0 (0)||2 (7.1)||10 (13.5)|
|Mixed type||13 (17.6%)|
|Survival: median (95% CI), mo*||7 (4–10)||—||16 (12–20)|
|Primary tumor, n (%)|
|Lung adenocarcinoma||14 (40)||—||—|
|Breast adenocarcinoma||9 (25.7)|
|Ovarian adenocarcinoma||2 (5.7)|
|Digestive adenocarcinoma||3 (8.6)|
|Unknown origin||3 (8.6)|
| Other||4 (11.4)|
A standard operating procedure concerning patient sampling and data retrieval was set in place: at inclusion, blood and pleural fluid samples, if available, were collected from each patient and stored at −80°C in aliquots until analyzed. Clinical data and outcome of the patients were also collected.
Serum and pleural levels of SMRP levels were determined in a single laboratory (INSERM unit 774) using a newly available sandwich-type ELISA (Mesomark; CIS-Bio International, Gif/Yvette, France; Fujirebio Diagnostics, Inc., Malvern, PA) according to manufacturers' instructions, and results were expressed in nmol/L.
All data are reported as median and interquartile range. Comparisons between groups were performed using both Kruskal-Wallis test and a nonparametric analysis of variance (ANOVA) after rank transformation using the methodology suggested by Conover and Iman (25). The Bonferroni correction was applied for multiple comparisons in post hoc tests. Area and standard errors of receiver operating curves (ROC) were calculated using standard techniques. The best statistical “cut-off” was calculated by minimizing the distance between the point with specificity = 1 and sensitivity = 1 and the points on the ROC curve. Areas under ROC curves (AUC) are reported with their 95% confidence intervals. Comparisons of AUC were done with the methodology suggested by Hanley and McNeil (26) using values available for both parameters. Unavailable data were coded as missing. Sensitivity and specificity for each cut-off were calculated using standard definitions. Correlations between serum and pleural SMRP values were calculated using Spearman's rank test. In an attempt to combine serum and pleural values to obtain a better classification of the patients, we built a score using discriminant analysis. Statistical calculations were performed with SPSS statistical package version 12.0F (SPSS, Inc., Chicago, IL) and the SAS system (version 8.2; Cary, NC).
We recruited a total of 137 patients, of whom 74 had MPM, 35 had Mets, and 28 had BPLAE. Serum was available from 30 patients with Mets, 23 patients with BPLAE, and 60 patients with MPM, and pleural fluid from 28 Mets, 21 BPLAE, and 43 MPM cases. The characteristics of patients are reported in Table 1.
Serum SMRP levels were different between the three groups (p < 0.0001, Kruskal-Wallis test; Table 2, Figure 1); the median values measured in patients with MPM (2.05 ± 2.57 nM/L) were significantly higher than in patients with either Mets (1.02 ± 1.79 nM/L) or BPLAE (0.55 ± 0.59 nM/L; p <0.002, ANOVA after rank transformation). We also found a marginal difference between Mets and BPLAE groups (p = 0.05). ROC analysis showed an AUC of 0.872 (0.798–0.946) for differentiating between BPLAE and MPM with a best statistical cut-off of 0.93 nM/L (sensitivity = 80%, specificity = 82.6%; Figure 2A). The AUC for differentiating Mets and MPM was 0.693 (0.576–0.810) with a best cut-off of 1.85 nM/L (sensitivity = 58.3% and specificity =73.3%; Figure 2B). The AUC for MPM versus all patients was 0.771 (0.683–0.858) with a cut-off greater than 1.1 nM/L (sensitivity = 71.7%, specificity = 69.8%).
Mets (n = 30)
BPLAE (n = 23)
MPM (n = 60)
|Serum SMRP (median), nM/L||1.02||0.55*||2.05†|
|(n = 28)||(n = 21)||(n = 43)|
|Pleural SMRP (median), nM/L||6.36||6.4||46.1†|
As expected, in the three groups of subjects, SMRP values in pleural fluid were much higher than respective serum values (Table 2). Median pleural level was significantly higher in patients with MPM (46.1 ± 83.2 nM/L) compared with either patients with Mets (6.36 ± 21.73 nM/L) or patients with BPLAE (6.4 ± 11.1 nM/L; p < 0.001). Pleural values were low and not significantly different between patients with BPLAE and patients with Mets. When differentiating between BPLAE and MPM, ROC curve analysis showed an AUC of 0.831 (0.734–0.927) with a best statistical cut-off of 10.4 nM/L (sensitivity = 76.7%, specificity = 76.2%; Figure 2C). The AUC for pleural SMRP for differentiating Mets and MPM was 0.793 (0.691–0.894; Figure 2D). No single “best statistical cut-off” has been found in this case because SMRP values between 11.4 nM/L (sensitivity = 76%, specificity = 64%) and 36.8 nM/L (sensitivity = 58.1%, specificity = 93%) were all at about the same distance from the ideal classification point of sensitivity = 1 and specificity = 1. The AUC for MPM versus all patients was 0.809 (0.718–0.900) with two “best” cut-offs: 11.4 nm/L (sensitivity = 76.7%, specificity = 69.4%) or 20.8 nM/L (sensitivity = 65.1%, specificity = 83.7%).
Comparison between AUC of serum and pleural values showed similar diagnostic performance for differentiating BPLAE and MPM. Despite the fact that pleural SMRP had an AUC higher for differentiating MPM and Mets, this difference was not statistically significant when considering only the cases for which both values were available. We found a significant correlation between serum and pleural values of SMRP (Spearman's rho = 0.666; p < 0.001). When running a discriminant analysis combining serum and pleural SMRP values to obtain a better classification of the patients, we found no statistically significant advantage over each variable taken separately. However, this analysis was done on only 73 patients from whom both serum and pleural samples were available.
We also found that the MPM epithelioid subtype had significantly higher values of serum SMRP (2.47 ± 2.63 nM/L; n = 42) than mixed subtype (0.892 ± 4.22 nM/L; n = 9; p = 0.026) or sarcomatoid subtype (1.05 ± 1.29 nM/L, p=0.06 due to the low number of patients in this group, n=5; Table 3, Figure 3). Pleural SMRP was also significantly higher in epithelioid mesothelioma compared with sarcomatoid type (p < 0.001) and mixed MPM subtype (p = 0.019). The differences between sarcomatoid MPM and mixed subtype were not statistically significant in either serum or pleural fluid (Table 3, Figure 3).
Epithelioid (n = 42)
Mixed type (n = 9)
Sarcomatoid (n = 5)
|Serum SMRP, median||2.47*||0.892||1.05|
|(n = 31)||(n = 6)||(n = 4)|
|Pleural SMRP, median||72.7†||10.48||5.96|
MPM is still a very challenging problem because its incidence is increasing all over the world, linked to a widespread exposure to asbestos. To date, there is no efficient or established curative treatment strategy of the tumor. To increase treatment effectiveness, an early and reliable diagnosis of MPM is needed. This has been difficult to achieve using only clinical and radiologic data. The management of MPM would be improved if guided by reliable markers (27). A few markers have been proposed in MPM, but none are used routinely (28). By evaluating new biological markers, such as SMRPs in both blood and pleural fluid in a large panel of patients, our goal was to address a crucial clinically question: Can we find new tools to easily differentiate patients with MPM from those with benign asbestos exposure–related lesions or pleural metastasis of carcinoma?
In agreement with other reports, we confirmed here that serum SMRP level is significantly higher in patients with MPM than in subjects exposed to asbestos with pleural benign disease, or in those with Mets. The median value of serum SMRPs in our series is similar for the BPLAE group to the one reported by Pass and colleagues (29) (1.166 μM/L; range, 0.004–2.821 μM/L), but is lower in the group of MPM (2.05 compared with 17.27 nM/L). However, the ROC curve analysis for differentiating MPM and BPLAE shows an AUC of 0.872, which is higher than reported by Pass and colleagues (29) (i.e., 0.793), especially if we take into account that these authors compared their MPM group versus a “normal volunteers” group and an “asbestos-exposed non-MPM” group together. The characteristics of these two last groups are not clearly described because the “normal volunteers” group presented pleural effusion, and it was not specified if patients of the “asbestos-exposed non-MPM” group had benign pleural disease. Because SMRP serum measures reported by Robinson and coworkers (22) were only expressed as optic absorbance values, we were unable to directly compare their results with our data. However, in our study, sensitivity and specificity of the SMRP test were much lower than those reported by Robinson and coworkers' study (22). When we tried in our study to optimize both sensitivity and specificity of the test, the best cut-off for serum SMRPs was 0.93 μM/L, with sensitivity = 80% and specificity = 82.6%. To achieve similar specificity as reported by Robinson and colleagues (22) (i.e., higher than 95%), we had to set the cut-off at 1.3 μM/L, which resulted in only 63.3% sensitivity.
Pleural values of SMRPs from non-MPM patients in our study were lower than those previously reported (29) (18.997 nM/L; range, 0.291–140.121), but similar in both studies for patients with MPM (65.573 nM/L; range, 0.421–255.166, compared with values in Table 2). Areas under ROC curves were slightly higher than reported by Pass and colleagues (29). Pleural SMRP assessment exhibited diagnostic power similar to serum SMRP. The AUC for pleural SMRPs was slightly higher than for serum SMRPs but the difference was not statistically significant. However, it is important to stress that both pleural fluid and serum were not available in all patients; therefore, pleural SMRPs were compared with serum SMRPs in a limited number of patients and thus the difference did not reach significance.
The ability to discriminate the MPM group vs. the secondary pleural carcinomas (Mets) group by serum SMRP was low. This is due, in part, to high serum levels of SMRP in some patients with Mets. When we tried to maximize both sensitivity and specificity of this marker, we obtained values of sensitivity = 58.3% and specificity = 73.3%, for a cut-off of 1.85 nM/L. Because specificity is the most desired feature in this goal, we would need to choose a cut-off of 3.2 μM/L to obtain at least 90% specificity, which gives a sensitivity of only 33.3%. In addition to serum SMRP assessment, evaluation of pleural fluid SMRPs could be helpful to separate MPM and Mets groups, since a cut-off of 41 nM/L in pleural fluid resulted in a specificity of over 90% and a sensitivity of 53% (for 100% specificity, the cut-off is 55 nM/L, with 50% sensitivity). Thus, our data suggest that serum SMRPs could be a useful marker for diagnosis of pleural mesothelioma in patients with pleural abnormalities associated with asbestos exposure. However, negative results should be interpreted with caution.
As previously suggested from the observation of a few asbestos-exposed subjects, an initially high serum level of SMRPs in patients with BPLAE may predict development of mesothelioma in the subsequent years of follow-up (22). However, mesothelioma may develop slowly (30), and high levels of serum SMRPs in these three “healthy” patients could reflect the presence of small foci of mesothelioma that were not initially detected. In our opinion, association of pleural abnormalities and high SMRP levels in asbestos-exposed patients should be managed “aggressively” to exclude MPM or Mets diagnosis. Such strategy would need to be validated by a thorough prospective trial. On the basis of our study, the use of serum SMRP as an MPM screening marker may not reach sufficient sensitivity with adequate specificity; to reach a sensitivity of more than 90 to 95% (a cut-off between 0.5 and 0.6 μM/L.), the specificity falls down to very low levels (between 30 and 50%). However, established tumor markers, such as prostate-specific antigen (PSA), exhibit similar or even lower test performances: PSA has a sensitivity for detecting cancer of approximately 75% but a specificity of only 40%, even if the identification of other molecular forms of PSA within serum has recently led to a new era in PSA markers (31).
Robinson and colleagues have already suggested that the operative characteristics of the test would likely be less favorable when investigating a larger number of patients in conditions similar to day-to-day practice (22). This prediction has been confirmed in our multicenter study where all patients were consecutively recruited over a short period of time in 20 different hospitals. This could result in minimizing potential recruiting bias compared with a selected retrospective study (29) or evaluation of patients only recruited in MPM-dedicated centers (22). Second, our series included about 50% more patients than both previous reports (22, 29) with more patients with Mets.
Previous reports have shown that mesothelin is significantly expressed at the mRNA and protein levels in a variety of tumoral localizations. Thus, our results were consistent with previous studies from McIntosh and colleagues (23), Scholler and colleagues (16), and Pass and colleagues (29) who reported high levels of SMRP in ovarian, breast, colon, and non–small cell lung carcinomas. However, it should be taken into account that mesothelin is a membrane-bound protein and the mechanism of release of its soluble form is still unknown. Therefore, every cancer expressing mesothelin on the surface of tumor cells would not automatically be associated with elevated levels of SMRPs.
Another finding is that a significantly elevated level of SMRPs was found only in epithelioid mesothelioma but not in sarcomatoid type, as already suggested by Robinson and colleagues (22). Interestingly, Ordonez has reported that only epithelioid mesothelioma cells are positive for mesothelin staining (32). Some patients with mixed-type MPM also had elevated values of serum SMRPs as probed by the high interquartile range. It can be speculated that the level of serum SMRPs is correlated to the percentage of the epithelioid component in the tumor.
Recently, Pass and coworkers suggested that serum osteopontin may be used as a screening marker for MPM in an asbestos-exposed population, with a sensitivity of 77.6% and a specificity of 85.5%, at a cut-off value of 48.3 ng/ml of osteopontin (33). However, serum osteopontin level is not specific to MPM as it has been found elevated in other cancers or nonmalignant diseases (34). Thus, the diagnostic value of serum osteopontin in MPM remains to be investigated in larger series of patients, including subjects with BPLAE and in patients with Mets (27).
In conclusion, we evaluated the performance of serum SMRPs as a diagnostic marker of patients with MPM using a newly available commercial kit. The operational characteristics of the kit were lower than previously obtained with the laboratory test. We showed that pleural fluid SMRP is a biological marker at least as interesting as serum SMRP, and potentially better able to differentiate MPM from pleural metastatic carcinomas. However, the pathologic study of pleural biopsies remains the gold standard in the diagnosis of MPM. Additional investigations in a larger panel of subjects will be necessary to demonstrate the usefulness of blood and pleural fluid SMRPs in the management of MPM treatment.
The following medical centers and investigators were involved in the study: University Hospital of Lille (J-.J. Lafitte, C-.H. Marquette, A-.B. Tonnel, P. Ramon, A. Scherpereel, S. Leroy, A. Wurtz, H. Porte, M. Conti, R. Akkad), University Hospital of Nantes (M. Gregoire), Clinique du Bois-Lille (J-.M. Faillon, S. Jaillard, Y. Rogeaux, C. Croxo), Clinique de la Louvière-Lille (T. Gentina, J-.C. Bout, B. Douay, E. Mensier, M. Debaert), Denain (J-.P. Grignet, L. Kedziora, P. Desmarest), Roubaix (N. Just, F. Steenhouwer), Valenciennes (B. Stach, A. Courbot, G. Demarcq, J-.P. Roux, F. Radenne), Armentieres (L. Boudoux, N. Boumaad), Lens (J-.Y. Tavernier, B. Chevalon, F. Desliers, H. Guenanen, I. Barrage), Dunkerque (E. Lelieur-Lepoivre, C. Deroubaix, L. Brillet, C. Rousselot, F. Dohen-Becue), Calais (V. Cliquennois, V. Tack), Saint-Omer (P. Richard, A. Boileau), Henin-Beaumont (R. Roboubi, E. Fournier), Lievin (D. Musielak), Bethune (J. Soots, C. Mordacque), Douai (M-.C. Florin, E. Maetz, S. Desurmont), Cambrai (F. Le Baron), Centre Oscar Lambret-Lille (E. Dansin), Saint-Philibert Hospital-Lomme (P. Mulliez), Tourcoing (X. Ficheroule), Maubeuge (M. Derollez). The authors also thank Dr. Steven M. Albelda from University of Pennsylvania (Philadelphia) for his helpful suggestions.
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