Abstract
Rationale: Soluble mesothelin (SM) is currently the reference serum biomarker of malignant pleural mesothelioma (MPM). Megakaryocyte potentiating factor (MPF), which originates from the same precursor protein, is potentially more sensitive, yet lacks validation.
Objectives: To analyze the diagnostic performance of MPF as an MPM biomarker and compare this performance with SM.
Methods: A total of 507 participants were enrolled in six cohorts: healthy control subjects (n = 101), healthy asbestos-exposed individuals (n = 89), and patients with benign asbestos-related disease (n = 123), benign respiratory disease (n = 46), lung cancer (n = 63), and MPM (n = 85). Sera were analyzed for SM and MPF levels using the Mesomark and Human MPF ELISA kit, respectively.
Measurements and Main Results: SM and MPF levels differed significantly between patients with MPM and participants from each other cohort (P < 0.001). Receiver operating characteristics curve analysis did not reveal a significant difference between both markers in area under curve (AUC) for distinguishing MPM from all cohorts jointly (SM = 0.871, MPF = 0.849; P = 0.28). At 95% specificity, SM and MPF had a sensitivity of 64% (cutoff = 2.00 nmol/L) and 68% (cutoff = 12.38 ng/ml), respectively. Combining both markers did not improve the diagnostic performance.
Conclusions: In this prospective multicenter study, MPF is validated as a highly performant MPM biomarker. The similar AUC values of SM and MPF, together with the limited difference in sensitivity, show that both serum biomarkers have an equivalent diagnostic performance.
Soluble mesothelin (SM) is currently the reference serum biomarker of malignant pleural mesothelioma (MPM). Megakaryocyte potentiating factor (MPF), another fraction of the mesothelin precursor protein, is potentially more sensitive, yet lacks validation.
We compared MPF with SM in a prospective multicenter study and found that both MPM markers have an equivalent diagnostic performance.
Tumor-related serum biomarkers, such as soluble mesothelin (SM) and megakaryocyte potentiating factor (MPF), can be useful aids in the management of MPM (6). These two markers originate from the mesothelin gene product, a precursor protein cleaved into a membrane-bound 40-kD C-terminal glycoprotein, mesothelin (7), and a soluble 31-kD N-terminal fraction, MPF (8, 9), also denominated “N-ERC/mesothelin” (10). Subsequent cleavage of membrane-bound mesothelin sheds this fraction into the bloodstream as SM, also denominated “soluble mesothelin-related protein” or “C-ERC/mesothelin” (10–12).
Mesothelin is believed to play a role in cell adhesion and signaling (7, 13) and facilitates the metastasis of mesothelin-expressing cancers by binding CA125 (14, 15). MPF was originally identified as a cytokine with megakaryocyte-stimulating activity (8). Further research, including mesothelin knockout mice (16), did not reveal additional functional information.
Since serum SM was identified as an MPM marker (17), the marker has been extensively validated (18–24) using the Food and Drug Administration–approved Mesomark ELISA kit (25). Because of its high specificity, SM is currently considered as the reference serum biomarker of MPM. However, solely epithelioid mesothelioma cells stain positive for mesothelin (26), and elevated serum SM levels are present in only 50 to 60% of the patients with epithelioid MPM (18–24). The search for more sensitive (combinations of) MPM markers, including CA125, CYFRA 21.1, hyaluronic acid, and osteopontin, remains unsuccessful so far (18, 20, 27, 28).
Unlike SM, MPF does not require cleavage and shedding from the cell surface, and therefore could challenge the sensitivity of SM (29). However, the recently developed MPF ELISA kits require further validation (10, 30–32), and reports comparing SM and MPF are contradictory (32, 33). To establish whether SM or MPF is a more sensitive marker, we compared the performance of a recently developed MPF ELISA kit (32) with the standard SM assay, Mesomark, in a prospective multicenter study. Some of the results of this study have been previously reported in the form of abstracts (34, 35).
Between June 2004 and October 2009, eligible individuals were prospectively and consecutively recruited in one of the following six cohorts: (1) healthy control subjects, aged 50 years or older, without reported asbestos exposure, from which 15 individuals provided a second blood sample 2 days after inclusion; (2) individuals with documented asbestos exposure, without asbestos-related lesions on a standard chest X-ray; (3) patients with benign asbestos-related disease: pleural plaques, diffuse pleural thickening, or asbestosis; (4) patients with benign non–asbestos-related respiratory disease, without pleural effusion; (5) patients with lung cancer; and (6) patients with MPM. Exclusion criterion was previous antitumor therapy. MPM diagnosis was based on pleural biopsies, validated by an expert panel of Belgian pathologists.
Cohorts 2 and 3 were enrolled at two Belgian industrial companies with a history of asbestos use (Kapelle-op-den-Bos, Ghent) and at the Occupational Diseases Fund (Brussels). The other cohorts were recruited at the participating departments of respiratory medicine.
In patients with MPM, staging was done according to the International Mesothelioma Interest Group criteria (36), resulting in either a clinical tumor-node-metastasis stage, based on a recent computed tomography scan, or a pathological tumor-node-metastasis stage when surgery was performed shortly after inclusion.
This study was approved by the ethics committee of all participating hospitals. Participants gave written informed consent before inclusion, after which serum samples were obtained, coded, and stored in aliquots at −80°C until analysis.
Serum SM and MPF levels were measured in all participants using the Mesomark (Cisbio International, Gif sur Yvette, France) and Human MPF ELISA kit (32) (Medical & Biological Laboratories, Nagano, Japan), respectively, according to the manufacturer's instructions. SM and MPF concentrations were assayed in a single run in duplicate and expressed in nmol/L and ng/ml, respectively. Assays were performed in a single laboratory, blinded to the coded sample data.
The MPF assay reproducibility was determined by assessing the within- and between-run component of the total imprecision. Assays were prepared in a nonautomated manner at three subsequent days, with two runs daily. In each run, six serum samples, with low, moderate, and high MPF levels, were analyzed in duplicate. The Mesomark reproducibility was previously assessed (25).
When stratifying biomarker levels according to cohort, histology, or tumor stage, the assumption of a normal distribution and homogeneity of variances was not fulfilled, despite logarithmic transformation. Consequently, biomarker levels were compared using the nonparametric Mann-Whitney U test. The 2-day interval levels were normally distributed, and biomarker stability was evaluated using the paired t test and Bland-Altman analysis. Spearman rank analysis determined the correlation between biomarker levels (rs). Diagnostic performance was assessed with receiver operating characteristics (ROC) curve analysis. Area under curve (AUC) values, reported with their 95% confidence interval, were compared as proposed by Hanley and McNeil (37). The Youden index was used to obtain optimal cutoff levels (38).
Markers were combined into a cumulative marker by standardizing the logarithmically transformed biomarker levels relative to the non-MPM cohorts. For each marker, the logistic regression coefficient was assigned as a weight factor, and the cumulative marker was obtained by adding these values.
Two-sided P values less than 0.05 were considered significant. Statistical analysis was done with statistical software SPSS (version 17; Chicago, IL) and GraphPad Prism (version 4.0; San Diego, CA).
Sera were obtained from a total of 507 participants: 101 healthy control subjects, 89 healthy asbestos-exposed individuals, 123 patients with benign asbestos-related disease (69 patients with pleural plaques, 39 patients with diffuse pleural thickening, and 15 patients with predominately asbestosis), 46 patients with benign respiratory disease (mainly asthma, chronic obstructive pulmonary disease, and pneumonia), 63 patients with lung cancer (mainly non–small cell lung cancer), and 85 patients with MPM (73 epithelioid, 4 sarcomatoid, and 8 biphasic histology) (Table 1). Sixteen patients with MPM had tumor stage I, 22 stage II, 28 stage III, and 19 stage IV.
Number (Female) | Median Age yr (Range) | SM (nmol/L) | MPF (ng/ml) | |||||
|---|---|---|---|---|---|---|---|---|
| Cohort | Mean ± SD | Median (IQR 25–75) | Mean ± SD | Median (IQR 25–75) | ||||
| Healthy control subjects | 101 (21) | 56 (50–73) | 0.95 ± 0.33 | 0.95 (0.72–1.12) | 6.71 ± 2.26 | 6.16 (5.24–7.78) | ||
| Healthy asbestos-exposed subjects | 89 (13) | 52 (41–58) | 0.90 ± 0.39 | 0.82 (0.65–1.05) | 6.91 ± 2.90 | 6.32 (5.14–7.98) | ||
| Benign asbestos-related disease | 123 (4) | 64 (42–85) | 1.27 ± 0.65 | 1.12 (0.84–1.56) | 8.09 ± 4.50 | 7.16 (5.44–9.36) | ||
| Benign respiratory disease | 46 (13) | 62 (18–83) | 1.08 ± 0.53 | 0.99 (0.72–1.45) | 7.45 ± 3.23 | 6.66 (5.12–9.52) | ||
| Lung cancer | 63 (16) | 65 (40–86) | 1.09 ± 0.83 | 0.99 (0.73–1.30) | 8.81 ± 7.04 | 7.04 (6.32–9.48) | ||
| Malignant pleural mesothelioma | 85 (6) | 65 (38–89) | 7.70 ± 15.44 | 2.52 (1.43–5.99) | 50.83 ± 98.71 | 18.10 (9.28–53.10) | ||
| Epithelioid | 73 (7) | 64 (38–89) | 8.49 ± 16.51 | 2.73 (1.43–6.47) | 62.85 ± 130.30 | 26.48 (9.38–56.54) | ||
| Biphasic | 8 (1) | 66 (57–76) | 3.74 ± 2.87 | 2.43 (1.84–5.27) | 23.74 ± 18.85 | 17.58 (11.90–28.96) | ||
| Sarcomatoid | 4 (0) | 66 (57–73) | 1.31 ± 0.374 | 1.29 (0.98–1.67) | 7.10 ± 2.87 | 6.28 (5.00–10.02) | ||
Serum samples of 15 healthy individuals, obtained at inclusion and after 2 days, did not differ significantly for MPF (P = 0.75) and SM levels (P = 0.84). The Bland-Altman plot revealed a mean difference between the 2-day interval samples of 0.06 for SM and 0.09 for MPF. In addition, clinically acceptable limits of agreement, with a coefficient of repeatability of 0.24 for SM and 1.49 for MPF, were obtained (Figure 1). The reproducibility of the MPF ELISA kit was assayed in six samples with levels between 5.93 and 75.33 ng/ml. Within this broad range of MPF levels, the total imprecision of the assay was less than or equal to 12.2% (see Table E1 in the online supplement).
Figure 1. Bland-Altman plot of biomarker stability. Difference of the biomarker levels between the 2-day interval samples in 15 healthy control subjects plotted versus the average biomarker level. The horizontal solid line is the mean difference; the dotted lines are the limits of agreement. (A) Soluble mesothelin (SM). (B) Megakaryocyte potentiating factor (MPF).
[More] [Minimize]SM levels ranged from undetectable to 96.32 nmol/L. Levels below the detection limit (0.30 nmol/L) were arbitrary set at 0.15 nmol/L. In patients with MPM, SM levels were significantly higher than healthy control subjects (P < 0.001), healthy asbestos-exposed individuals (P < 0.001), and patients with benign asbestos-related disease (P < 0.001), benign respiratory disease (P < 0.001), and lung cancer (P < 0.001) (Figure 2A). Patients with sarcomatoid MPM had significantly lower SM levels compared with the epithelioid (P < 0.05) and biphasic subtypes (P < 0.05), whereas no difference was observed between the biphasic and epithelioid subtypes (P = 0.99) (Table 1). SM levels were significantly lower in MPM tumor stage I, compared with stage II (P < 0.01), stage III (P < 0.001), and stage IV (P < 0.05) (Figure 3A).
Figure 2. Participants' biomarker levels. Dashed lines represent the 95% specificity cutoffs of both markers. (A) Soluble mesothelin (SM), cutoff = 2.00 nmol/L. (B) Megakaryocyte potentiating factor (MPF), cutoff = 12.38 ng/ml. ARD = benign asbestos-related diseases; BRD = benign respiratory diseases; HAE = healthy asbestos-exposed individuals; MPM = malignant pleural mesothelioma.
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Figure 3. Mesothelioma patient biomarker levels, stratified according to International Mesothelioma Interest Group tumor stage. Vertical bars represent median levels. (A) Soluble mesothelin (SM). (B) Megakaryocyte potentiating factor (MPF). * P < 0.05; ** P < 0.01, *** P < 0.001. IQR = interquartile range.
[More] [Minimize]ROC curve analysis for differentiating MPM from the different cohorts revealed AUC values between 0.859 and 0.864 (Table 2). Distinguishing patients with MPM from all cohorts jointly gave an AUC of 0.871 (0.825–0.916) (Figure 4). The optimal cutoff of 1.89 nmol/L revealed a sensitivity of 66%, a specificity of 94%, and a positive and negative likelihood ratio of 11.00 and 0.36, respectively.
Figure 4. Receiver operating characteristics curves for differentiating mesothelioma from all cohorts. Solid line: soluble mesothelin (SM); dashed line: megakaryocyte potentiating factor (MPF). AUC = area under the curve.
[More] [Minimize]AUC (95% CI) | Sensitivity % (Cutoff) | ||||||
|---|---|---|---|---|---|---|---|
| Cohort | SM | MPF | P Value† | SM | MPF | ||
| Asbestos-exposed* | 0.859 (0.811–0.908) | 0.847 (0.789–0.905) | 0.54 | 62 (2.05) | 68 (12.66) | ||
| Benign respiratory disease | 0.864 (0.805–0.924) | 0.849 (0.786–0.913) | 0.51 | 64 (2.03) | 68 (12.98) | ||
| Lung cancer | 0.863 (0.806–0.921) | 0.816 (0.747–0.886) | 0.06 | 64 (2.03) | 68 (13.46) | ||
| All | 0.871 (0.825–0.916) | 0.849 (0.792–0.906) | 0.28 | 64 (2.00) | 68 (12.38) | ||
MPF levels ranged between 1.96 and 837.76 ng/ml, differed significantly between patients with MPM and each other cohort (P < 0.001) (Figure 2B), and were significantly lower in patients with sarcomatoid MPM, compared with those with epithelioid (P < 0.01) and biphasic histology (P < 0.05). Levels did not differ between the biphasic and epithelioid subtypes (P = 0.51) (Table 1). Patients with stage I MPM had significantly lower MPF levels compared with patients with stage II (P < 0.05), stage III (P < 0.0001), and stage IV (P < 0.01) (Figure 3B).
AUCs for distinguishing patients with MPM from the different cohorts ranged between 0.816 and 0.849 (Table 2). When differentiating from all cohorts jointly, an AUC of 0.849 (0.792–0.906) was obtained (Figure 4). At the optimal cutoff of 13.46 ng/ml, a sensitivity of 68%, a specificity of 97%, and a positive and negative likelihood ratio of 22.67 and 0.33, respectively, were obtained.
SM and MPF levels highly correlated in patients with MPM (rs = 0.88; P < 0.001) and in the other cohorts (rs = 0.67; P < 0.001). When differentiating all cohorts, both individually and jointly, from the patients with MPM, SM and MPF ROC curves crossed and closely ran together (Figure 4). Consequently, the obtained AUCs for SM and MPF did not differ significantly (Table 2). The AUC of the cumulative marker for differentiating MPM from all cohorts jointly was 0.869 (0.820−0.919), which gave no improvement compared with either SM (P = 0.89) or MPF (P = 0.10).
When comparing both markers at 95% specificity, MPF had a higher sensitivity than SM in each cohort, although the improvement was limited. Overall, the sensitivity for differentiating MPM from all cohorts for SM was 64% (cutoff = 2.00 nmol/L) and 68% for MPF (cutoff = 12.38 ng/ml) (Table 2). A bivariate scatter plot of SM versus MPF showed that 5 out of the 31 patients with MPM without elevated SM levels were picked up by MPF. In contrast, only 1 of the 27 MPF-negative patients had an elevated SM level (Figure 5). From the 26 patients with MPM who were negative for both markers, 35% had an advanced stage III or IV and 85% an epithelioid or biphasic histology.
Figure 5. Bivariate scatter plot of soluble mesothelin (SM) versus megakaryocyte potentiating factor (MPF). Dashed lines represent the 95% specificity cutoffs of SM (2.00 nmol/L) and MPF (12.38 ng/ml). Shaded squares: patients with MPM with or without elevated MPF and SM levels; solid squares: SM-negative patients with MPM with elevated MPF levels, or vice versa.
[More] [Minimize]To establish whether serum SM or MPF is a more sensitive MPM biomarker, we have set up a comparative multicenter study. The obtained diagnostic performance of SM was in agreement with previous reports (18–25). The few MPF validation studies available used different MPF ELISA kits, resulting in a difficult comparison. Onda and colleagues found that their MPF assay had a sensitivity of 91% and a specificity of 100%. However, results were based on samples from patients with advanced-stage MPM and healthy control subjects (30). Shiomi and colleagues optimized and validated an MPF ELISA in 39 patients with MPM and 254 patients with clinically suspected MPM (10, 31). The AUC of 0.850, with a sensitivity and specificity of 72 and 92%, respectively, was in close agreement with our findings. Creaney and colleagues evaluated a commercial MPF assay, based on the initially developed assay of Shiomi and colleagues (10), in 167 individuals, including 66 patients with MPM (33). At a specificity of 95%, only 34% of the patients with MPM were detected, compared with the 68% we found in the present series. The MPF assay applied in our study was recently developed by Iwahori and colleagues and validated in 156 individuals: 38 healthy control subjects, 9 healthy asbestos-exposed individuals, 35 patients with non–pleural epithelioid cancer, 47 patients with lung cancer, and 27 patients with MPM (32). The AUC for distinguishing MPM from all cohorts was 0.879, and at a cutoff level of 19.10 ng/ml, a sensitivity of 74% and a specificity of 89% were obtained (32). Although we confirmed the diagnostic performance of this assay in 507 individuals, mean MPF values were lower in our series, and the cutoff of 19.10 ng/ml resulted in a sensitivity and specificity of 53 and 99%, respectively. This is likely attributable to differences in study design and cohort composition.
Besides validating MPF as an MPM biomarker, our findings refute previous concerns regarding the stability of MPF (33), because a high short-interval stability with a low coefficient of repeatability was obtained. In addition, the MPF assay imprecision was acceptable in an extensive range of MPF levels.
Stratification of the patients with MPM according to histology and tumor stage revealed similar results for SM and MPF. The absence of elevated biomarker levels in patients with sarcomatoid MPM concurred with previous reports (19–25, 31), and with the lack of mesothelin overexpression in this histological subtype (26). However, the number of patients with sarcomatoid MPM in our series was limited and results should be interpreted with caution. Tumor staging revealed significantly increased biomarker levels in more advanced stages compared with the early stage I. Although for both biomarkers similar trends were previously observed (19, 23, 31), significant differences were rare (22). This is possibly due to the limited number of patients with MPM per stage, and also illustrative for the current challenges in MPM staging.
Two reports previously compared the performance of SM and MPF. Iwahori and colleagues found that MPF (AUC = 0.865) had a significantly higher performance than SM (AUC = 0.712) (32). However, SM levels were obtained with a newly developed ELISA kit, and the superior performance of MPF can be attributable to an inferior performance of this SM ELISA kit compared with the Mesomark kit. Creaney and colleagues compared a commercial MPF assay with Mesomark and found that the performance of SM (AUC = 0.915) was significantly higher than MPF (AUC = 0.614) (33). However, their control population was limited, and the applied MPF assay was recently significantly optimized (31). The conflicting results in both reports are likely attributable to the use of different ELISA kits, illustrating the need for a further head-to-head comparison of the available SM and MPF assays.
In our series, comparing the MPF assay of Iwahori and colleagues (32) with Mesomark did not reveal a difference in AUC values. Although the sensitivity of MPF was slightly higher than SM at 95% specificity, MPF detected only 16% of the SM-negative patients, and thus did not substantially improve MPM detection. As such, a substantial number of patients with MPM, including advanced-stage epithelioid tumors, remained undetected by both markers.
The clinical application of serum biomarkers of MPM is still under debate. Besides a potential role in therapy monitoring (6), the high positive likelihood ratios of SM and MPF suggest that both markers can be of use in the early diagnosis of MPM.
In asbestos-exposed individuals, the positive predictive value of any diagnostic tool is limited due to the low prevalence of MPM (39). This makes a screen-based early detection strategy only worthwhile if the target population is better selected. Biomarker levels can triage asbestos-exposed individuals, resulting in an enriched population with an increased risk of developing MPM. However, the outcome of the ongoing longitudinal follow-up studies of asbestos-exposed individuals is still required to validate this hypothesis (40).
In patients with symptomatic MPM in the differential list, SM and MPF levels can assess the pretest probability of disease and efficiently steer the ensuing diagnostic approaches (41), reducing the diagnostic delay and hastening a symptom-based early diagnosis. As such, the application of serum MPM biomarkers in today's clinical practice is likely more useful in guiding diagnostic and therapeutic decisions than in risk assessment of asbestos-exposed populations.
In conclusion, we have validated MPF in a large, prospective multicenter study as an MPM biomarker and found that SM and MPF have an equivalent diagnostic performance. Because combining both markers did not improve the detection of MPM, further biomarker development should primarily focus on the diverse group of patients with MPM who are undetected by SM and MPF.
The authors thank all study participants and the investigators involved in the study at Ghent University Hospital (F. Vande Walle, T. Verstraete, V. Stove, S. De Craene), University Hospital Gasthuisberg (B. Nemery, L. Peeters), Occupational Diseases Fund (A. Marin, M. Vandeweerdt), Antwerp University Hospital (S. Kohl), Erasme Hospital ULB (G. Amand), CHU Sart Tilman (S. Maccan, K. Mobarak), AZ Nikolaas (K. Deschepper, M. Van Hooste), AZ Sint-Maarten (M. Lambrechts), Eternit N.V. (J. Van Cleemput, M. Geeroms, C. Verheyen, M. Flies), and Vyncolit N.V. (A. Schockaert, P. Tomme). They also thank Y. Fujii and R. Uehara (Medical & Biological Laboratories) for preparing and providing the MPF ELISA kits. MBL has an exclusive right to prepare and provide the MPF assay on a noncommercial basis. Cisbio International and MBL had no role in performing the assays, data analysis, writing, or approval of the manuscript.
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