Abstract
Rationale: Soluble mesothelin-related protein (SMRP) is raised in epithelial-type malignant mesothelioma (MM), but the utility of SMRP in screening for MM is unknown.
Objectives: We aimed to evaluate SMRP in an asbestos-exposed cohort.
Methods: A total of 538 subjects were studied. Those with elevated SMRP (≥2.5 nM) underwent further investigation including positron emission tomography/computed tomography.
Measurements and Main Results: Mean (±SD) SMRP in healthy subjects exposed to asbestos (n = 223) was 0.79 (±0.45) nM. Fifteen subjects had elevated SMRP, of whom one had lung cancer, which was successfully resected. Another with lung cancer was undetected by SMRP. No subjects were diagnosed with MM. Mean SMRP in healthy subjects was significantly lower than in subjects with pleural plaques alone (P < 0.01).
Conclusions: This is the first large-scale prospective study of SMRP for screening for malignancy in asbestos-exposed individuals. A high false-positive rate was observed. SMRP seems unlikely to prove useful in screening for MM.
Soluble mesothelin-related protein (SMRP) is a biomarker reported to be useful in the diagnosis of malignant mesothelioma (MM). Its use a screening tool in a population at risk of MM has not been evaluated.
This is the first large-scale prospective study of SMRP for screening for malignancy in asbestos-exposed individuals. A high false-positive rate was observed. SMRP seems unlikely to prove useful in screening for MM.
Malignant mesothelioma (MM) is an aggressive and fatal cancer of the pleura causally related to asbestos exposure (1–3), with a latency period of between 15 and 40 years (4, 5). Patients with MM have a median survival of fewer than 18 months (6–8). MM is difficult to diagnose in its early stages and current treatment is largely ineffective in controlling disease. However, recently, several new drugs have become available and trials are now underway to evaluate whether earlier treatment (including combined surgery and radio-/chemotherapy) will be useful in improving survival. It is hoped that early detection will allow combination therapy to control or eradicate this neoplasm, but this is far from proven. The total number of MM cases in NSW between 1972 and 2004 was 3,099 (8), and we predict that the MM annual incidence in NSW will peak at 196 in 2014 (9).
Currently, there is considerable interest in the use of biomarkers to allow early detection of several malignancies, including MM. Promising biomarkers include osteopontin and soluble mesothelin-related protein (SMRP) (10–14). Mesothelin is a 40-kD glycoprotein that has a putative role in cell-to-cell adhesion, recognition, and signaling (15–17) and is highly expressed in MM (10, 12–14, 18) as well as in some other malignancies (16, 18, 19), including some gastrointestinal and pulmonary carcinomas (16). Elevated levels of SMRP correlate with MM of an epithelioid subtype rather than the sarcomatoid form (10, 12, 13). Epithelioid-type MM has a better prognosis, however, and is more likely to be potentially resectable or respond to chemotherapy.
There have been several studies evaluating SMRP in the diagnosis of MM (12, 20, 21), but to date there have been no large studies evaluating this test for diagnosing MM in a healthy asbestos-exposed population. SMRP is increasingly being used as a screening tool (12–14, 20), and has recently been approved in the United States for diagnosis and monitoring of MM. To study this issue, we set up a cohort study in asbestos-exposed subjects screening for MM and correlated SMRP levels with diagnosis. Those with raised SMRP levels were further evaluated with positron emission tomography (PET) and computed tomography (CT) scans. Some of these results have already been presented in the form of abstracts (22, 23).
This study was conducted at the Workers' Compensation (Dust Diseases) Board (DDB) of New South Wales, Sydney, Australia. This is a statutory authority that provides compensation to workers with dust diseases employed in NSW. An award is made after the diagnosis has been established by the DDB Medical Authority, a panel of three respiratory physicians specializing in occupational lung disease. Detailed clinical and pathologic information is available to the Medical Authority. Lifetime occupational histories are collected. The DDB regularly screens a large number of workers with previous asbestos exposure for potential respiratory disease.
Briefly, asbestos-exposed workers and workers with other dust exposures attending the DDB between January and November 2006 for a routine examination for screening and compensation purposes were invited to participate in the study. The routine examination included a standardized questionnaire, radiology, lung function, and a clinical examination by a thoracic physician. Subjects were consecutively recruited and, if they agreed to participate, provided signed informed consent followed by blood collection. Subjects were monitored for 12 months. The study was approved by the Human Research Ethics Committee of St. Vincent's Hospital, Sydney, Australia. Participants were not compensated for their participation.
Respiratory symptoms and physical examination were recorded from a physician-administered clinical card. Data collection included the following: smoking history; Medical Research Council (MRC) scales of exercise capacity, chest pain, cough, and dyspnea; and details of the physical examination. A chest radiograph was mandatory and chest CT scan and other investigations were performed if clinically indicated. For study purposes, the presence or absence of asbestos-related or other diseases was classified according to the determination of the Medical Authority comprising three respiratory physicians. Classification of such disorders was set up before the study was initiated and categorization was performed according to American Thoracic Society diagnostic criteria (24).
Ten milliliters of venous blood were collected by venipuncture. Serum was separated immediately and stored at −80°C until further analysis. Serum SMRP concentrations were measured by a specific ELISA assay (Mesomark; Fujirebio Diagnostics, Malvern, PA) according to the manufacturer's guidelines, and results were expressed in nanomoles. The limit of detection of the assay was 0.3 nM. All samples were coded and were analyzed “blinded” to the clinical information. The threshold for an abnormal result was calculated from the mean plus 3 standard deviations of the mean from control non–asbestos-exposed healthy individuals and was set at 2.5 nM (20).
Duplicate specimens (n = 59) were prepared for a random selection of at least 10% of the total collected serum samples. These samples were assigned a unique code, and thus treated as independent samples and analyzed “blind”. A small number of randomly selected serum samples (n = 21) were re-assayed on two different days.
Subjects who had an elevated SMRP level (≥2.5 nM) were invited to undergo further tests, including 18F-fluorodeoxyglucose (FDG) PET/CT scans of the thorax to assess any evidence of MM or other malignant diseases (see the online supplement for details). PET/CT scans were evaluated “blind” to the results of the SMRP analysis.
Values are reported as means ± SD. SMRP levels were compared between groups using one-way analysis of variance and Student's t test. The Bonferroni correction was applied for multiple comparisons in post hoc tests. All statistical analyses were performed in GraphPad Prism (version 4; Graphpad Software, San Diego, CA). A P value less than 0.05 was considered significant.
A total of 621 subjects (47.2%) from a potential total of 1,315 subjects agreed to participate in the study, with successful blood collection in 538 subjects. In 47 subjects it was not possible to collect blood for technical reasons, and 35 subjects had insufficient time to participate after initially agreeing to be in the study (Figure 1). One subject withdrew from the project. The majority (98.3%) of participants were male (n = 529), with only 1.7% female (n = 9). Demographic details are shown in Table 1. Mean age (±SD) of participants was 66.9 (±10) years and few participants reported current cigarette smoking (7.8%, n = 42). Although details on symptoms were collected, these will be reported separately.
Figure 1. Study flow chart. CT = computed tomography; FDG-PET/CT = 18F-fluorodeoxyglucose positron emission tomography/computed tomography. SMRP = soluble mesothelin-related protein.
[More] [Minimize]Overall Distribution (n = 538) | Healthy Asbestos-exposed Population (n = 223) | Silicosis (n = 20) | Asbestosis (n = 24) | DPT (n = 113) | Asbestosis/DPT (n = 13) | Pleural Plaques (n = 142) | |
|---|---|---|---|---|---|---|---|
| Age, mean yr (±SD) | 66.9 (±10.1) | 61.2 (±10.2) | 70.7 (±6.4) | 73.0 (±6.9) | 71.7 (±6.9) | 73.8 (±6.0) | 69.7 (±8.6) |
| Body mass index, % (n) | |||||||
| <18.5 | 0.6 (3) | 0.5 (1) | 0 (0) | 0 (0) | 0.9 (1) | 0 (0) | 0.7 (1) |
| 18.5–24.9 | 18.8 (101) | 20.6 (46) | 15.0 (3) | 12.5 (3) | 11.5 (13) | 15.4 (2) | 23.9 (34) |
| 25–29.9 | 52.2 (281) | 48.9 (109) | 60.0 (12) | 66.7 (16) | 53.1 (60) | 69.2 (9) | 51.4 (73) |
| ≥30 | 28.4 (153) | 30.1 (67) | 25.0 (5) | 20.8 (5) | 34.5 (39) | 15.4 (2) | 23.9 (34) |
| Smoking status, % (n) | |||||||
| Never-smoker | 36.4 (196) | 46.6 (104) | 40.0 (8) | 20.8 (5) | 22.1 (25) | 15.4 (2) | 35.9 (51) |
| Ex-smoker | 55.8 (300) | 42.6 (95) | 60.0 (12) | 70.8 (17) | 73.5 (83) | 84.6 (11) | 56.3 (80) |
| Current smoker | 7.8 (42) | 10.8 (24) | 0 (0) | 8.3 (2) | 4.4 (5) | 0 (0) | 7.8 (11) |
Eight groups were categorized in this study: (1) asbestosis (n = 24), (2) diffuse pleural thickening (DPT; n = 113), (3) asbestosis and DPT (n = 13), (4) silicosis (n = 20), (5) silicosis and asbestosis (n = 1), (6) silicosis and DPT (n = 2), (7) pleural plaques alone (PPs; n = 142), and (8) the healthy but asbestos-exposed population with no apparent asbestos-related disease (n = 223). SMRP levels are presented in Figures 2 and 3. Groups with small sample sizes (silicosis with asbestosis [n = 1], silicosis with DPT [n = 2]) were omitted. The SMRP level in the case with both silicosis and asbestosis (n = 1) was 1.15 nM, and in those with both silicosis and DPT (n = 2) the SMRP levels were 0.66 and 1.15 nM, respectively. SMRP levels differed between the six groups (P = 0.002; Figure 2). Mean (±SD) SMRP levels in healthy subjects exposed to asbestos (n = 223) and silica (n = 20) were 0.79 (±0.45) and 0.90 (±0.35) nM, respectively. Mean (±SD) SMRP levels in those with asbestosis (n = 24), DPT (n = 113), asbestosis and DPT (n = 13), and PPs (n = 142) were 1.14 (±0.99), 0.89 (±0.55), 1.08 (±0.68), and 1.06 (±0.92) nM. Mean SMRP level in the healthy subjects was significantly lower than in subjects with PPs (P < 0.01) and other asbestos-related disorders (P = 0.0003; Figure 3).
Figure 2. Soluble mesothelin-related protein (SMRP) levels (nM) in (1) healthy individual exposed to asbestos, or with (2) silicosis, (3) asbestosis, (4) diffuse pleural thickening (DPT), (5) combined asbestosis and DPT, or (6) pleural plaques alone (PPs). Horizontal scale bars denote mean levels. There were significant differences between the groups (analysis of variance, P = 0.002).
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Figure 3. Soluble mesothelin-related protein (SMRP) levels (nM) in healthy individuals exposed to asbestos or subjects with asbestos-related disorders. Horizontal scale bars denote mean level. There were significant differences between the groups (Student's t test, P = 0.0003).
[More] [Minimize]There were 15 subjects (2.8%) with SMRP levels of 2.5 nM or greater, and who were therefore classified as having abnormally elevated results. One subject had a raised SMRP level of 9.34 nM and chronic renal failure on dialysis, but was investigated together with the others. Diagnoses in the other subjects were as follows: asbestosis (n = 3), DPT (n = 3), asbestosis and DPT (n = 1), PPs (n = 6), and healthy but asbestos-exposed subjects (n = 2). One individual had a CT scan of the thorax before a PET/CT scan was available. This was suggestive of malignancy and he subsequently underwent a resection of the right lower lobe, which revealed an adenocarcinoma of the lung. Currently, he is still alive and well with no evidence of recurrence. All other individuals (n = 14) had a PET/CT scan of the thorax. None had evidence of MM, but four had abnormal PET/CT scans. One scan showed increased uptake in the heart, whereas three showed hilar lymphadenopathy, with one also showing increased mediastinal uptake. The patient with the cardiac abnormality underwent a transthoracic echocardiogram, which was suggestive of a left atrial tumor, but refused further investigation in view of his age (86 yr) and other comorbidities. The two patients with increased hilar uptake had follow-up contrast CTs, which did not reveal any malignancy. The third declined further investigation. No demonstrable abnormality was evident in the other 10 patients, who are still being monitored.
Of the 538 subjects studied, six died during the study period: one died of lung cancer and one of metastatic carcinoma of the pancreas; another subject died of asbestosis; however, the remaining deaths were not attributable to dust disease. The subject with lung cancer had an SMRP level of 1.14 nM, whereas the subject with carcinoma of the pancreas had a level of 0.79 nM.
Asbestos was used extensively in a variety of applications in Australia until the 1980s. Asbestos inhalation may cause diffuse parenchymal pulmonary fibrosis (asbestosis), DPT, and PPs, as well as two malignancies: carcinoma of the bronchus and MM (25–27). The latter two neoplasms are usually fatal, with a median survival of MM in NSW of 9 months (8). Despite changes in work practices, the incidence of MM continues to rise in NSW (9).
It is theoretically possible that early treatment of MM could improve survival, although this is by no means proven. Curative treatment would probably involve combined surgical resection at an early stage of the cancer with other treatments, because MM is relatively chemo- and radioinsensitive. However, early detection of the tumor is not easy and radiologic surveillance is imperfect. Therefore, biomarkers could be useful to detect the development of a MM at a resectable stage, probably in conjunction with conventional methods such as chest radiology, CT, and PET scanning. Examples of promising biomarkers are SMRP (10, 12–14, 20, 21), osteopontin (11), and megakaryocyte potentiation factor (28). Biomarkers such as these have been suggested for use in screening for MM in asbestos-exposed populations, but no prospective cohort studies exist.
Robinson and colleagues first reported the association of raised SMRP levels with MM (10). In a retrospective study, they found that 84% of patients with MM (n = 44) in Western Australia had significantly elevated serum SMRP on testing stored samples compared with asbestos-exposed and non–asbestos-exposed control subjects. As an indicator of MM, SMRP had a sensitivity of 84% and a specificity of 100%. Scherpereel and coworkers (12) later reported that mean serum SMRP levels in patients with MM (n = 66) were higher than in patients with pulmonary metastases from carcinomas (n = 35) or benign pleural diseases associated with asbestos exposure (n = 28). These findings have subsequently been confirmed by several other investigators (13, 14, 21).
In our study, mean SMRP levels were very similar to other reports (14, 29), but were slightly higher than in the few other studies that have evaluated SMRP in asbestos-related disorders (14, 20). Different investigators have used different cutoff levels for abnormality, producing different sensitivities and specificities for the diagnosis of MM, ranging from sensitivities of 67% (13) to 80% (12), and specificities of 83% (12) to 99% (13). Limitations of these studies include small sample sizes, particularly of non-MM cases exposed to asbestos, retrospective analysis of stored samples, and the selection bias inherent in hospital-based populations (10, 12–14).
Ours is the first prospective cohort study to evaluate SMRP levels in a large number of individuals with asbestos exposure. The SMRP assay provided satisfactory results with good reproducibility in duplicate serum samples (n = 59, R2 = 0.90), and on repeat assay (n = 21, R2 =0.85). Between-day assay variation was also satisfactory with less than 8% coefficient of variation during the study period (see the online supplement). This confirms a previous report that SMRP is stable under freeze/thaw cycles, and at a temperature of 2–8°C, and that the interassay coefficient of variation does not exceed 5.3% (13).
A small proportion (2.8%) of our study participants had elevated SMRP levels. Our threshold for abnormality was higher than that used in several other studies (0.9 and 1.5 nM) (12–14). However, if we had used these thresholds, 210 and 61 subjects, respectively, would have been considered abnormal. Considering that in our study only one subject with an elevated SMRP had a neoplasm on CT scanning, it would be difficult to justify CT scanning in this number of patients. Other studies have examined subjects who had been referred to hospitals and therefore were mainly symptomatic, so sensitivities are likely to be higher than in our study in which subjects were coming for routine surveillance and assessment for compensation.
We detected one lung cancer on SMRP screening and found one probable cardiac malignancy. One lung cancer was not detected by SMRP screening (level, 1.14 nM), and one other participant was later found to have pancreatic carcinoma. None of our cohort have yet been diagnosed with MM. Of the six subjects who died during the study period, one died of lung cancer and one of pancreatic carcinoma (SMRP, 0.79 nM). Thus, this subject with lung cancer would have been detected had a lower cutoff point been used, but not the subject with pancreatic carcinoma. It is premature to attempt to calculate sensitivities and specificities for our study, because follow-up has been limited to 1 year. Thus, the true incidence of MM and other malignancies in our cohort is not yet known. However, it is probable that the false-positive rate for SMRP screening will be high. Our subjects represent a biased group because they were not randomly selected from the general population and were being assessed for compensation. However, the rate of disease in a truly asymptomatic population is likely to be even lower.
One unexpected finding was the significant difference in SMRP levels between healthy asbestos-exposed individuals and those with asbestos-related disorders. This difference was due to higher mean SMRP in those with PPs, because SMRP levels were not raised in other asbestos-related disorders. However, the difference between the healthy asbestos-exposed individuals and those with asbestos-related disorders was small (0.79 ± 0.45 vs. 1.00 ± 0.79 nM, respectively), and fell well below the cutoff value (2.5 nM) for our study. The reasons for these slightly increased levels remain uncertain. It seems unlikely that the mild ongoing pleural inflammation that occurs in PPs is responsible, because SMRP levels were not raised in subjects with DPT or asbestosis, in which pleural and parenchymal inflammation is acknowledged to be more intense (30). Also, these slightly increased levels have not been seen in other studies in subjects with PPs. It is possible that this finding is a type II error, but also possible that some subjects have occult disease, which will manifest itself on future follow-up.
In conclusion, our study examined the clinical utility of measuring SMRP in an asbestos-exposed cohort. It is the first to prospectively evaluate SMRP as a potential screening tool for workers in a high-risk population with occupational exposure to asbestos. Of our 538 subjects, 15 (2.8%) had a level that was higher than the estimated cutoff point (≥2.5 nM), and one malignancy was identified and successfully treated. Two subjects died of malignancies that were undetected by our study. Several probable false positives were observed, but this categorization could change in the future, and these subjects will be monitored carefully for development of disease. If, in the future, a raised SMRP level is found to be a good indicator of early MM development, then there is a manageable proportion of patients in whom PET/CT-guided investigation can be used using 2.5 nM as a cutoff point for abnormality. Thus, a combination of screening with SMRP and direct CT scanning and/or other imaging such as PET would be feasible. However, our data to date indicate that SMRP is unlikely to prove useful for screening, and is less useful than in diagnosing MM in symptomatic patients. The subjects in this study will provide a cohort suitable for longitudinal follow-up.
The authors thank all subjects whose participation enabled them to complete the study.
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