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Soluble Mesothelin Related Protein in Mesothelioma
MINI-SYMPOSIUM
Soluble Mesothelin Related Protein in Mesothelioma Jenette Creaney, PhD,* Hanne Christansen, PhD,* Richard Lake, PhD,*† A. Bill Musk, MBBS,‡ Nick de Klerk, PhD,§ and Bruce W.S. Robinson, MD*†
(J Thorac Oncol. 2006;1: 172–174)
T
he worldwide incidence of malignant mesothelioma is increasing. This is a highly aggressive asbestos-related tumor with a median survival of less than a year.1 Treatment options have been limited and have done little to extend the median survival, although some strategies, such as complete tumor resection in patients with early disease, seem promising.2,3 Serum markers have been useful for the clinical management of different cancers.4 Currently no serum markers are in routine use for the clinical management of patients with mesothelioma. We have therefore evaluated a newly described tumor marker, soluble mesothelin related protein (SMRP),5 in patients with mesothelioma. SMRP is a member of the mesothelin family of proteins. Little is known of the function of this family of proteins. Mesothelin itself is a 40kDa cell surface protein that is present on normal mesothelial cells and on some cancers including mesothelioma, ovarian, and pancreatic cancer.6 It is believed that alternative splicing of the mesothelin gene results in the production of SMRP, a putatively soluble protein with an N-terminal region identical to that of mesothelin but with a different C-terminal. Using a doubledeterminant enzyme-linked immunosorbent assay, SMRP has been detected in the serum of patients with ovarian cancer and in a few patients with other cancers5
PATIENTS AND METHODS Patients and Controls Serum samples were obtained from patients presenting at the respiratory clinics of either the Sir Charles Gardiner Hospital or the Hollywood Specialist Centre in Perth, Western Australia. Forty-four patients (39 male, 5 female) with
*School of Medicine and Pharmacology and ‡Department of Respiratory Medicine, University of Western Australia, Sir Charles Gairdner Hospital, Wedlands; †Western Australian Institute of Medical Research, Queen Elizabeth II Medical Centre, Nedlands; §Biostatistics and Epidemiology Unit, Telethon Institute for Child Health Research, Subiaco, WA, Australia Address correspondence to: Jenette Creaney, School of Medicine and Pharmacology, University of Western Australia, Sir Charles Gairdner Hospital, Verdun St., Nedlands, Western Australia 6009, Australia. Copyright © 2006 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/06/0102-0172
172
cytologically or histopathologically confirmed mesothelioma were included in this study. Of these patients, 25 had epithelial tumors, four had sarcomatoid, and 15 were unclassified. Serum samples from healthy individuals were included as controls. Samples from patients with other malignancies and with non-malignant lung and pleural effusions were examined to be certain that SMRP levels were not increased in other diseases. To determine whether asbestos exposure per se influenced SMRP levels, serum from individuals with a history of asbestos exposure and follow-up information for approximately 8 years, were also studied. These individuals form part of an on-going surveillance program of people who worked or lived in Wittenoom, Western Australia.7,8
SMRP Assays SMRP concentrations were determined by enzymelinked immunosorbent assay using monoclonal antibodies (OV569 and 4H3) that bind to different SMRP epitopes.5 Recently, Fujirebio Diagnostics Incorporated acquired the rights for the use of the SMRP assay and kindly provided us with kits. Quantitative measurements were made against reference standards prepared by the manufacturer. SMRP levels were determined following the manufacturer’s instructions.
RESULTS SMRP concentrations in the serum of patients with established mesothelioma ranged from 0.35 to 78 nM. For the population of 44 individuals, the mean ⫾ standard deviation concentration was 15.33 ⫾ 20.48 nM. SMRP levels in patients with mesothelioma were significantly higher than those in healthy controls with or without a history of asbestos exposure (0.925 ⫾ 0.831 and 1.455 ⫾ 1.524 respectively; P ⬍ 0.001). Increased levels of serum SMRP were only observed in three of the 169 patients with malignant or benign pulmonary or pleural diseases (Table 1). One of these three SMRPpositive patients had non-small cell lung cancer, another had cryptogenic fibrosing alveolitis-idiopathic pulmonary fibrosis, and the third had asbestosis. None of the patients with lung adenocarcinoma (n ⫽ 7) or with pleural effusion (n ⫽ 20), conditions that frequently exhibit symptoms overlapping with mesothelioma, were SMRP-positive. To determine the clinical value of SMRP concentrations as an aid to mesotheloma diagnosis, we measured levels in 20 people who presented at the clinic and had a serum sample available within the 2-month period of pathologically confirmed mesothelioma diagnosis. SMRP concentrations in
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
TABLE 1. SMRP and Disease SMRP-positive samples (n)
SMRP-positive samples (%)
Mesothelioma Other malignancies Non-malignant lung and pleural disease; asbestos-related
37/40 1/36 2/124 1/40
84 2.8 1.6
Healthy controls, non-asbestos exposed Healthy controls Asbestos-exposed (total) Malignancy-free Pre-malignancy
0/28
0
Condition
Soluble Mesothelin Related Protein in Mesothelioma
SMRP levels depending on the temperature at which serum was stored during an approximate 3-year period (data not shown). We also mimicked a series of freeze-thaw cycles using serum from three individuals with low, medium, or high SMRP levels. Figure 1 shows the relative mean level of the SMRP in samples after one, two, five, and 10 freeze/thaw cycles relative to the value determined for the original sample. There was no statistical difference in the SMRP levels after 10 freeze/thaw cycles.
DISCUSSION 7/40 3/36 4/4
17
SMRP, soluble mesothelin related protein.
these symptomatic individuals were significantly different from the above healthy control populations, with or without asbestos exposure (P ⬍ 0.001). SMRP concentrations at the time of mesothelioma diagnosis ranged from 0.982 to 35.45 nM, with a mean value of 6.9 ⫾ 9.9 nM. Of note, four of the 20 patients with early-stage mesothelioma had tumors with a predominantly sarcomatoid histology; the serum SMRP levels in the sarcomatoid patients were lower than those in the patients with predominantly epithelial tumors (data not shown). As previously noted, in this study, we measured SMRP concentrations in 40 healthy controls with a history of asbestos exposure. These individuals were part of a public health epidemiology survey that annually monitors people who worked or lived in Wittenoom, Western Australia, the site of a large asbestos mining operation.7,8 At the time the 40 serum samples were collected, all the individuals were healthy. Interestingly, follow-up data revealed that three people in this group developed mesothelioma 15, 26, and 69 months later, and one person developed lung carcinoma 4 years later. The level of SMRP was higher in these dour individuals than in the people who had not, at the close of the study, died from malignancy (Table 2). A possible confounding element to this study is the stability of SMRP in serum stored frozen for several years and the effect of thawing and refreezing samples. To first assess the stability of the SMRP protein under different storage conditions, we measured levels in serum samples that had been stored for approximately 36 months at either 4ºC, –20ºC, or – 80ºC for three individuals with low, medium, or high SMRP levels. There was no statistical difference in the
Most patients with mesothelioma have increased serum levels of SMRP, which suggests that SMRP is a sensitive marker for this disease. SMRP also seems to be a specific marker, as only three of 160 patients with other non-mesothelioma malignant or benign lung or pleural disease had increased serum SMRP. As SMRP levels were increased in half the patients at the time they were presenting to the clinic with symptoms and levels were also elevated in pleural effusions (data not shown), measurement of this biomarker could be a useful adjunct to the clinician’s repertoire for mesothelioma diagnosis. Much interest surrounds the use of serum tumor markers for screening healthy populations for malignancy with the aim of identifying early-stage disease and providing early treatment.9 In general, asbestos-exposed individuals are compliant to screening strategies. In this small study, we showed that SMRP levels were significantly increased in three people who were apparently healthy but were diagnosed 1 to 5 years later with mesothelioma and in one individual who died of lung adenocarcinoma 4 years after the positive SMRP serum sample. We are currently extending these studies to include a greater number of asbestos-exposed subjects, but the preliminary results coupled with the apparent stability of the protein we report herein suggests that the test may be valuable in screening programs for which samples may need to be collected under field conditions. However, as only half the mesothelioma patients have increased SMRP levels at the
TABLE 2. SMRP and Patient Condition Condition
n
Mean ⴞ SD
Healthy Mesothelioma Lung cancer
36 3 1
1.016 ⫾ 0.61 3.52 ⫾ 1.38 8.49
SMRP, soluble mesothelin related protein.
FIGURE 1.
Copyright © 2006 by the International Association for the Study of Lung Cancer
173
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
Creaney et al.
time of diagnosis, we are currently investigating whether measuring a combination of biomarkers, possibly including the recently described osteopontin,10 would provide the power necessary for an effective screening program. REFERENCES 1. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2006;353:1591–1603. 2. Sugarbaker DJ, Flores RM, et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: Results in 183 patients. J Thorac Cardiovasc Surg 1999;117:54–63;discussion 63–65. 3. Chang MY, Sugarbaker DJ. Extrapleural pneumonectomy for diffuse malignant pleural mesothelioma: Techniques and complications. Thorac Surg Clin 2004;14:523–530.
174
4. Sturgeon C. Practice guidelines for tumor marker use in the clinic. Clin Chem 2002;48:1151–1159. 5. Scholler N, Fu N, et al. Soluble member(s) of the mesothelin/ megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci USA 1999;96: 11531–11536. 6. Hassan R, Bera T, et al. Mesothelin: A new target for immunotherapy. Clin Cancer Res 2004;10(12 Pt 1):3937–3942. 7. Hansen J, de Klerk NH, et al. Environmental exposure to crocidolite and mesothelioma: Exposure-response relationships. Am J Respir Crit Care Med 1998;157:69–75. 8. Musk AW, de Klerk NH, et al. Vitamin A and cancer prevention I: Observations in workers previously exposed to asbestos at Wittenoom, Western Australia. Int J Cancer 1998;75:355–361. 9. Meissner HI, Smith RA, et al. Promoting cancer screening: Learning from experience. Cancer 2004;101(5 Suppl):1107–1117. 10. Pass HI, Lott D, et al. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 2006;353:1564–1573.
Copyright © 2006 by the International Association for the Study of Lung Cancer
Soluble Mesothelin Related Protein in Mesothelioma Jenette Creaney, PhD,* Hanne Christansen, PhD,* Richard Lake, PhD,*† A. Bill Musk, MBBS,‡ Nick de Klerk, PhD,§ and Bruce W.S. Robinson, MD*†
(J Thorac Oncol. 2006;1: 172–174)
T
he worldwide incidence of malignant mesothelioma is increasing. This is a highly aggressive asbestos-related tumor with a median survival of less than a year.1 Treatment options have been limited and have done little to extend the median survival, although some strategies, such as complete tumor resection in patients with early disease, seem promising.2,3 Serum markers have been useful for the clinical management of different cancers.4 Currently no serum markers are in routine use for the clinical management of patients with mesothelioma. We have therefore evaluated a newly described tumor marker, soluble mesothelin related protein (SMRP),5 in patients with mesothelioma. SMRP is a member of the mesothelin family of proteins. Little is known of the function of this family of proteins. Mesothelin itself is a 40kDa cell surface protein that is present on normal mesothelial cells and on some cancers including mesothelioma, ovarian, and pancreatic cancer.6 It is believed that alternative splicing of the mesothelin gene results in the production of SMRP, a putatively soluble protein with an N-terminal region identical to that of mesothelin but with a different C-terminal. Using a doubledeterminant enzyme-linked immunosorbent assay, SMRP has been detected in the serum of patients with ovarian cancer and in a few patients with other cancers5
PATIENTS AND METHODS Patients and Controls Serum samples were obtained from patients presenting at the respiratory clinics of either the Sir Charles Gardiner Hospital or the Hollywood Specialist Centre in Perth, Western Australia. Forty-four patients (39 male, 5 female) with
*School of Medicine and Pharmacology and ‡Department of Respiratory Medicine, University of Western Australia, Sir Charles Gairdner Hospital, Wedlands; †Western Australian Institute of Medical Research, Queen Elizabeth II Medical Centre, Nedlands; §Biostatistics and Epidemiology Unit, Telethon Institute for Child Health Research, Subiaco, WA, Australia Address correspondence to: Jenette Creaney, School of Medicine and Pharmacology, University of Western Australia, Sir Charles Gairdner Hospital, Verdun St., Nedlands, Western Australia 6009, Australia. Copyright © 2006 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/06/0102-0172
172
cytologically or histopathologically confirmed mesothelioma were included in this study. Of these patients, 25 had epithelial tumors, four had sarcomatoid, and 15 were unclassified. Serum samples from healthy individuals were included as controls. Samples from patients with other malignancies and with non-malignant lung and pleural effusions were examined to be certain that SMRP levels were not increased in other diseases. To determine whether asbestos exposure per se influenced SMRP levels, serum from individuals with a history of asbestos exposure and follow-up information for approximately 8 years, were also studied. These individuals form part of an on-going surveillance program of people who worked or lived in Wittenoom, Western Australia.7,8
SMRP Assays SMRP concentrations were determined by enzymelinked immunosorbent assay using monoclonal antibodies (OV569 and 4H3) that bind to different SMRP epitopes.5 Recently, Fujirebio Diagnostics Incorporated acquired the rights for the use of the SMRP assay and kindly provided us with kits. Quantitative measurements were made against reference standards prepared by the manufacturer. SMRP levels were determined following the manufacturer’s instructions.
RESULTS SMRP concentrations in the serum of patients with established mesothelioma ranged from 0.35 to 78 nM. For the population of 44 individuals, the mean ⫾ standard deviation concentration was 15.33 ⫾ 20.48 nM. SMRP levels in patients with mesothelioma were significantly higher than those in healthy controls with or without a history of asbestos exposure (0.925 ⫾ 0.831 and 1.455 ⫾ 1.524 respectively; P ⬍ 0.001). Increased levels of serum SMRP were only observed in three of the 169 patients with malignant or benign pulmonary or pleural diseases (Table 1). One of these three SMRPpositive patients had non-small cell lung cancer, another had cryptogenic fibrosing alveolitis-idiopathic pulmonary fibrosis, and the third had asbestosis. None of the patients with lung adenocarcinoma (n ⫽ 7) or with pleural effusion (n ⫽ 20), conditions that frequently exhibit symptoms overlapping with mesothelioma, were SMRP-positive. To determine the clinical value of SMRP concentrations as an aid to mesotheloma diagnosis, we measured levels in 20 people who presented at the clinic and had a serum sample available within the 2-month period of pathologically confirmed mesothelioma diagnosis. SMRP concentrations in
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
TABLE 1. SMRP and Disease SMRP-positive samples (n)
SMRP-positive samples (%)
Mesothelioma Other malignancies Non-malignant lung and pleural disease; asbestos-related
37/40 1/36 2/124 1/40
84 2.8 1.6
Healthy controls, non-asbestos exposed Healthy controls Asbestos-exposed (total) Malignancy-free Pre-malignancy
0/28
0
Condition
Soluble Mesothelin Related Protein in Mesothelioma
SMRP levels depending on the temperature at which serum was stored during an approximate 3-year period (data not shown). We also mimicked a series of freeze-thaw cycles using serum from three individuals with low, medium, or high SMRP levels. Figure 1 shows the relative mean level of the SMRP in samples after one, two, five, and 10 freeze/thaw cycles relative to the value determined for the original sample. There was no statistical difference in the SMRP levels after 10 freeze/thaw cycles.
DISCUSSION 7/40 3/36 4/4
17
SMRP, soluble mesothelin related protein.
these symptomatic individuals were significantly different from the above healthy control populations, with or without asbestos exposure (P ⬍ 0.001). SMRP concentrations at the time of mesothelioma diagnosis ranged from 0.982 to 35.45 nM, with a mean value of 6.9 ⫾ 9.9 nM. Of note, four of the 20 patients with early-stage mesothelioma had tumors with a predominantly sarcomatoid histology; the serum SMRP levels in the sarcomatoid patients were lower than those in the patients with predominantly epithelial tumors (data not shown). As previously noted, in this study, we measured SMRP concentrations in 40 healthy controls with a history of asbestos exposure. These individuals were part of a public health epidemiology survey that annually monitors people who worked or lived in Wittenoom, Western Australia, the site of a large asbestos mining operation.7,8 At the time the 40 serum samples were collected, all the individuals were healthy. Interestingly, follow-up data revealed that three people in this group developed mesothelioma 15, 26, and 69 months later, and one person developed lung carcinoma 4 years later. The level of SMRP was higher in these dour individuals than in the people who had not, at the close of the study, died from malignancy (Table 2). A possible confounding element to this study is the stability of SMRP in serum stored frozen for several years and the effect of thawing and refreezing samples. To first assess the stability of the SMRP protein under different storage conditions, we measured levels in serum samples that had been stored for approximately 36 months at either 4ºC, –20ºC, or – 80ºC for three individuals with low, medium, or high SMRP levels. There was no statistical difference in the
Most patients with mesothelioma have increased serum levels of SMRP, which suggests that SMRP is a sensitive marker for this disease. SMRP also seems to be a specific marker, as only three of 160 patients with other non-mesothelioma malignant or benign lung or pleural disease had increased serum SMRP. As SMRP levels were increased in half the patients at the time they were presenting to the clinic with symptoms and levels were also elevated in pleural effusions (data not shown), measurement of this biomarker could be a useful adjunct to the clinician’s repertoire for mesothelioma diagnosis. Much interest surrounds the use of serum tumor markers for screening healthy populations for malignancy with the aim of identifying early-stage disease and providing early treatment.9 In general, asbestos-exposed individuals are compliant to screening strategies. In this small study, we showed that SMRP levels were significantly increased in three people who were apparently healthy but were diagnosed 1 to 5 years later with mesothelioma and in one individual who died of lung adenocarcinoma 4 years after the positive SMRP serum sample. We are currently extending these studies to include a greater number of asbestos-exposed subjects, but the preliminary results coupled with the apparent stability of the protein we report herein suggests that the test may be valuable in screening programs for which samples may need to be collected under field conditions. However, as only half the mesothelioma patients have increased SMRP levels at the
TABLE 2. SMRP and Patient Condition Condition
n
Mean ⴞ SD
Healthy Mesothelioma Lung cancer
36 3 1
1.016 ⫾ 0.61 3.52 ⫾ 1.38 8.49
SMRP, soluble mesothelin related protein.
FIGURE 1.
Copyright © 2006 by the International Association for the Study of Lung Cancer
173
Journal of Thoracic Oncology • Volume 1, Number 2, February 2006
Creaney et al.
time of diagnosis, we are currently investigating whether measuring a combination of biomarkers, possibly including the recently described osteopontin,10 would provide the power necessary for an effective screening program. REFERENCES 1. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2006;353:1591–1603. 2. Sugarbaker DJ, Flores RM, et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: Results in 183 patients. J Thorac Cardiovasc Surg 1999;117:54–63;discussion 63–65. 3. Chang MY, Sugarbaker DJ. Extrapleural pneumonectomy for diffuse malignant pleural mesothelioma: Techniques and complications. Thorac Surg Clin 2004;14:523–530.
174
4. Sturgeon C. Practice guidelines for tumor marker use in the clinic. Clin Chem 2002;48:1151–1159. 5. Scholler N, Fu N, et al. Soluble member(s) of the mesothelin/ megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci USA 1999;96: 11531–11536. 6. Hassan R, Bera T, et al. Mesothelin: A new target for immunotherapy. Clin Cancer Res 2004;10(12 Pt 1):3937–3942. 7. Hansen J, de Klerk NH, et al. Environmental exposure to crocidolite and mesothelioma: Exposure-response relationships. Am J Respir Crit Care Med 1998;157:69–75. 8. Musk AW, de Klerk NH, et al. Vitamin A and cancer prevention I: Observations in workers previously exposed to asbestos at Wittenoom, Western Australia. Int J Cancer 1998;75:355–361. 9. Meissner HI, Smith RA, et al. Promoting cancer screening: Learning from experience. Cancer 2004;101(5 Suppl):1107–1117. 10. Pass HI, Lott D, et al. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 2006;353:1564–1573.
Copyright © 2006 by the International Association for the Study of Lung Cancer