REVIEW URRENT C OPINION
Comparison of mesothelin and fibulin-3 in pleural fluid and serum as markers in malignant mesothelioma Jenette Creaney a,b, Ian M. Dick a,b, and Bruce W. Robinson a,b,c
Purpose of review Malignant mesothelioma is an asbestos-induced, aggressive tumour, which frequently presents with pleural effusion. There are over 60 reported causes that can result in the development of a pleural effusion. Currently, there are no tumour biomarkers in widespread clinical use for the differential diagnosis of mesothelioma from other diseases. With the incidence of mesothelioma expected to continue to increase, it is timely to review the current status of effusion-based biomarkers for mesothelioma diagnosis. Recent findings The majority of recent studies have evaluated soluble mesothelin in effusions in a diagnostic setting for mesothelioma. However, at high specificity, the sensitivity of the assay is limited to approximately 60% at the time of diagnosis. There is considerable research effort directed toward the identification of new markers for mesothelioma through a variety of genomic, proteomic and immunomic based platforms. One of the few new biomarkers to be identified through a biomarker discovery pipeline and evaluated in pleural effusions is fibulin-3. Preliminary results on the diagnostic accuracy of fibulin-3 have been inconsistent. Summary To date, soluble mesothelin remains the best available biomarker for mesothelioma and a positive result is clinically useful in patients with pleural effusions in whom the diagnosis is uncertain. Keywords diagnosis, effusions, fibulin-3, mesothelin, mesothelioma
INTRODUCTION Malignant mesothelioma is a highly aggressive tumour predominantly associated with asbestos exposure. Tumours arise in the mesothelial cells of the serosal surfaces with approximately 90% of cases occurring in the pleura [1]. Patients with pleural mesothelioma usually present with symptoms of chest pain, discomfort or dyspnoea, which are usually because of the presence of an effusion. Greater than 90% of patients with pleural mesothelioma will have a malignant pleural effusion [2]. Malignant pleural effusions can also arise from metastatic or invasive pleural cancers, one-third of cases having breast cancer and a quarter with lung cancer. Although malignancy is a concern, nonmalignant causes can account for twice as many pleural effusion cases [3]. Cytological analysis of cells from pleural effusions is the most common method for diagnosis of malignant pleural effusions, though the immunohistological examination of biopsy tissue obtained by www.co-pulmonarymedicine.com
thoracoscopy or other invasive methods is considered the ‘gold standard’ [4]. Examination of soluble factors present in the pleural fluid has been utilized for over 40 years in an attempt to find tests that will help clinicians distinguish between benign and malignant effusions [5]. Despite identification of numerous tumour markers and their evaluation in many studies, there has not been widespread uptake or utilization of any particular markers for the diagnosis of malignant pleural effusions.
a
School of Medicine and Pharmacology, bNational Centre for Asbestos Related Diseases, University of Western Australia, Perth and c Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia Correspondence to Prof Jenette Creaney, School Medicine and Pharmacology, M503, 23 Stirling Hwy, Crawley, WA 6009, Australia. Tel: +61 8 6151 0896; fax: +61 8 6151 1028; e-mail: jenette.creaney@uwa. edu.au Curr Opin Pulm Med 2015, 21:352–356 DOI:10.1097/MCP.0000000000000167 Volume 21 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Mesothelin and fibulin-3 as markers for malignant mesothelioma Creaney et al.
KEY POINTS The incidence of mesothelioma is expected to continue to increase and at the present time, there are no tumour biomarkers in widespread clinical use for the differential diagnosis of mesothelioma from other diseases. Mesothelin is currently the best biomarker available, but it lacks sufficient sensitivity. A considerable research effort to identify new markers for mesothelioma through a variety of genomic, proteomic and immunomic-based platforms is being undertaken to improve the early diagnosis of mesothelioma.
Over the last 20 years, we have been intensely investigating the utility of the malignant pleural effusions as a sample for mesothelioma diagnosis [6 ]. Over the past decade in particular, we have focussed on the use of soluble tumour markers. Our publication in 2003 of soluble mesothelin as a biomarker with high specificity for mesothelioma triggered a wave of interest in this field [7]. Our aim in this article is to provide an update on recent studies into soluble markers in mesothelioma, specifically as related to the evaluation in malignant versus benign pleural effusions. &&
MESOTHELIN Mesothelin is a differentiation antigen expressed on normal mesothelial cells and in various tumours, including mesothelioma [8]. Mesothelin can be released from the cell surface and soluble forms of the protein have been detected in blood and effusions of cancer patients. Early studies showed that soluble mesothelin is elevated in pleural and peritoneal fluid from mesothelioma patients relative to levels in benign effusions. Recent studies have further explored mesothelin as part of the diagnostic workup of patients; confirming that an elevated level of mesothelin in the effusion is highly predictive of mesothelioma even when cytology is nondiagnostic [9,10 ,11 ,12]. As the majority of mesothelioma patients present with effusions, elevated fluid mesothelin levels could indicate the need for active invasive investigations such as thoracoscopy and facilitate earlier diagnosis. Indeed, Canessa et al. confirmed that at the time of medical thoracoscopy, 72% of epithelial mesothelioma cases had elevated mesothelin in the effusion; they found one false-positive case in their series of 104 consecutive thoracoscopies (follow-up 24 months) [13]. Another product encoded by the MSLN gene is the soluble, 31-kDa, megakaryocyte potentiating &&
&
factor (MPF), which is also known as N-mesothelin or N-ERC. There is a tight correlation between mesothelin and MPF concentrations in blood and effusions and the two markers have similar discriminatory performance [14–16]. Various assays have been developed to quantitate levels of MPF, and a recent report suggests good diagnostic performance of a new MPF assay, but results have not as yet been compared head-to-head with mesothelin [17].
COMBINING KNOWN MARKERS WITH MESOTHELIN There has been considerable optimism about the potential of combining multiple tumour markers to improve diagnostic sensitivity in mesothelioma. This has been a successful strategy in ovarian cancer biomarker research [18]; albeit most of these improved ovarian biomarker panels contain CA125, the established ovarian biomarker which was first identified in 1981 [19]. One of the early biomarkers, proposed over 30 years ago, for mesothelioma was hyaluronic acid [20]. However, the technically demanding nature of the hyaluronic acid assay limited follow-up studies. Recently, two independent studies have been reported that find combining hyaluronic acid with mesothelin or MPF improves sensitivity for mesothelioma, while maintaining specificity when measured in effusion supernatants [16,21]. Sensitivity of mesothelin for mesothelioma in a dichotomous differential diagnostic setting has been improved by using additional ‘exclusion markers’, such as carcinoembryonic antigen, has been reported [22]. These findings need to be further evaluated in consecutive series of samples.
NEW BIOMARKERS FOR MESOTHELIOMA Various genomic, proteomic, immunomic, imaging and other tools have been used to identify, quantify and characterize novel biomarkers for many different diseases. Generally, there has been a poor rate of conversion of biomarkers identified in discovery projects to clinically validated bioassays that provide favourable health impacts, in part because of lack of independent validation of the original results. Potentially new biomarkers for mesothelioma have been identified on a variety of discovery platforms, including proteomic [23–25,26 ], immunomic [27] and mRNA expression studies [28 ,29]. Of the newly reported mesothelioma biomarkers, only a few have been examined in independent sample sets largely because of lack of availability of assays and reagents, and few have been evaluated in pleural effusion samples.
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FIBULIN-3 Fibulin-3 was shown to be significantly overexpressed in mesothelioma tumours compared with matched normal peritoneum by mRNA gene expression analysis [30]. Fibulin-3 is a secreted glycoprotein, which is believed to play a role in the regulation of cell proliferation and migration. Fibulin-3 is highly upregulated in glioma where it has been shown to promote tumour growth and invasion [31]. The fibulin-3 protein is encoded by the epidermal growth factor-containing fibulinlike extracellular matrix protein 1 (EFEMP1) gene. In mesothelioma, plasma and pleural effusion concentrations of fibulin-3 were shown to be elevated. In plasma, fibulin-3 had a sensitivity of over 96% for mesothelioma at a specificity of 95% at a threshold value of 52 ng/ml, and in pleural effusions, fibulin-3 had a sensitivity of 84% and specificity of 93% at a threshold value of 346 ng/ml when using a commercial ELISA for the protein (USCN Life Science) [28 ]. Although the overall results from this original sample set were validated in an independent cohort of mesothelioma and control samples, a discrepancy in the diagnostic accuracy between the two cohorts was observed [28 ]. From the data presented, it is estimated that in the validation set of samples, fibulin-3 sensitivity for mesothelioma was less than 40% at 95% specificity [28 ]. The discrepancy was possibly not clear because of the use of different threshold values in the two cohorts. Subsequent independent studies to evaluate fibulin-3 as a diagnostic marker for mesothelioma have reported conflicting data. Using the same fibulin-3 ELISA, a small Italian sub-study demonstrated that fibulin-3 concentrations in mesothelioma patient sera, compared with controls and patients with lung cancer, were significantly higher, although there was no difference in levels between mesothelioma patients and those with asbestosis. From the presented data (assuming that serum and plasma results are consistent), we estimate that fibulin-3 was elevated above 52 ng/ml, the originally reported threshold in one of the 14 mesothelioma patients examined (i.e. 7% positive). The 23 lung cancer, 14 asbestosis and 23 control patients were all below this positive fibulin-3 threshold value [32]. In a study specifically designed and powered to compare the diagnostic accuracy of mesothelin and fibulin-3 in mesothelioma patients at the time of diagnosis using the same ELISA as in previous studies, we found, as previously reported, that fibulin-3 concentrations were significantly higher in the plasma of mesothelioma patients compared with patients with other cancers and benign conditions. However, sensitivity was much lower than that reported in &&
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the original study. At the previously reported 52 ng/ ml threshold plasma, fibulin-3 had a sensitivity of 22% (i.e. 18/82) and specificity of 95% (i.e. 114/ 120). Furthermore,we found fibulin-3 wasnotspecific for mesothelioma when pleural effusion samples were examined, with 63% (i.e. 22/35) of benign effusions having a concentration of fibulin-3 above the 346 ng/ml threshold [33 ]. In a small recent Egyptian study with nine cases with benign pleural effusion used as a comparator group for 25 mesothelioma cases, Agha et al. found that both pleural effusion and serum fibulin-3 were significantly elevated in serum and pleural effusions. Although an ELISA was used to quantitate fibulin-3 levels, it was not reported which specific assay was employed in the study. Using independent, internally determined threshold values, they found that serum fibulin-3 had 100% sensitivity for mesothelioma at 78% specificity relative to nonmalignant effusions, and 88% sensitivity for mesothelioma at 82% specificity relative to malignant effusions owing to metastatic disease. When effusion samples were examined, sensitivity was 88% and 72% for mesothelioma compared with nonmalignant and metastatic cancer controls respectively (at a similar specificity of approximately 80%) [34]. Data for the different sample sets were not combined and results could not be compared with previous studies, although appear similar to the original Pass et al.’s study. Of note, the benign effusion samples used in this study were selected such that patients with transudative pleural effusion, serious uncontrolled diseases (including renal, hepatic, cardiac diseases and coagulopathy), and those that were hemodynamically unstable were excluded. It is not clear whether such preselection of control samples would influence the data; our own preliminary analysis suggests patients with benign effusions and elevated effusion lactate dehydrogenase (LDH) levels tend to have higher fibulin-3 concentrations. These recent examples evaluating fibulin-3 as a biomarker for mesothelioma diagnosis in different patient cohorts highlight a number of important issues for biomarker validation in general, as well as for mesothelioma. That is firstly, the choice of appropriate controls; many patients considered at risk of mesothelioma will be in their 70s or older, will have been exposed to asbestos and may have nonmalignant concurrent pleural disease. It is imperative that appropriate control groups are used and characteristics documented; ideally controls should be collected in the same clinical setting and under the same collection and storage protocols as cases. The notion of insisting on appropriate control groups is supported by the observation that most of the recently published biomarkers for mesothelioma, &&
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Mesothelin and fibulin-3 as markers for malignant mesothelioma Creaney et al.
unlike mesothelin, fall down in the area of specificity – other diseases, including nonmalignant diseases, produce positive results. Secondly, for validation studies of an initial promising candidate, the use of a predefined diagnostic threshold should be seriously considered rather than individual study-driven cutoffs. Thirdly, consideration should be given to determining mesothelin concentrations in the samples studied. Despite sensitivity limitations, mesothelin has proved to have a consistent diagnostic performance in numerous studies and comparison of any candidate new mesothelioma biomarker with mesothelin would be informative. A final consideration for authors, and editors, relates to publication bias; in this era of novel biomarker discovery, both negative and positive results of all properly conducted biomarker studies have value.
CONCLUSION When a patient presents with an undiagnosed pleural effusion, thoracentesis is generally the first intervention performed. If no definite cancer cells are apparent in the fluid and no alternative cause for the effusion can be found then thoracoscopy with biopsy will be considered if malignancy is a significant possibility. However, in some patients with significant underlying comorbidities and/or frailty in whom thoracoscopy is considered too high risk, or where thoracoscopy is not available, or declined by the patient, measurement of serum and pleural effusion mesothelin levels is a useful step – a positive result dramatically increases the likelihood of there being a pleural malignancy present. Although soluble mesothelin has a useful clinical role in diagnosis because of its high specificity, its sensitivity remains limited. That means that a positive result is informative, but a negative one is not. A better biomarker for pleural malignant mesothelioma has yet to be identified. Acknowledgements None. Financial support and sponsorship None. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet 2005; 366:397–408. 2. Lee YC. Hunting for a pleural fluid test for mesothelioma: is soluble mesothelin the answer? Thorax 2007; 62:561–562.
3. Thomas R, Lee YC. Causes and management of common benign pleural effusions. Thorac Surg Clin 2013; 23:25–42; v-vi. 4. Roberts ME, Neville E, Berrisford RG, et al. Management of a malignant pleural effusion: British Thoracic Society Pleural Disease Guideline 2010. Thorax 2010; 65 (Suppl 2):ii32–ii40. 5. Light RW, Macgregor MI, Luchsinger PC, Ball WC Jr. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972; 77:507–513. 6. Segal A, Sterrett GF, Frost FA, et al. A diagnosis of malignant pleural && mesothelioma can be made by effusion cytology: results of a 20 year audit. Pathology 2013; 45:44–48. This study describes how perfusion cytology is sufficient to diagnose pleural mesothelioma with a high sensitivity and specificity. 7. Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins and diagnosis of mesothelioma. Lancet 2003; 362:1612–1616. 8. Chang K, Pai LH, Batra JK, et al. Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. Cancer Res 1992; 52:181–186. 9. Davies HE, Sadler RS, Bielsa S, et al. Clinical impact and reliability of pleural fluid mesothelin in undiagnosed pleural effusions. Am J Respir Crit Care Med 2009; 180:437–444. 10. Creaney J, Segal A, Olsen N, et al. Pleural fluid mesothelin as an adjunct to the && diagnosis of pleural malignant mesothelioma. Disease Markers 2014; 2014:413946. This study demonstrates that a mesothelin-positive pleural effusion indicates the likely presence of malignancy and is an indicator for further investigation. 11. Franceschini MC, Ferro P, Canessa PA, et al. Mesothelin in serum and pleural & effusion in the diagnosis of malignant pleural mesothelioma with nonpositive cytology. Anticancer Res 2014; 34:7425–7429. This study indicates that effusion mesothelin is more sensitive than serum mesothelin for mesothelioma diagnosis. 12. Hooper CE, Morley AJ, Virgo P, et al. A prospective trial evaluating the role of mesothelin in undiagnosed pleural effusions. Eur Respir J 2013; 41:18–24. 13. Canessa PA, Ferro P, Manta C, et al. Clinical value of mesothelin in pleural effusions versus histology by medical thoracoscopy: brief report. Med Oncol 2013; 30:649. 14. Creaney J, Sneddon S, Dick IM, et al. Comparison of the diagnostic accuracy of the MSLN gene products, mesothelin and megakaryocyte potentiating factor, as biomarkers for mesothelioma in pleural effusions and serum. Disease Markers 2013; 35:119–127. 15. Hollevoet K, Nackaerts K, Thimpont J, et al. Diagnostic performance of soluble mesothelin and megakaryocyte potentiating factor in mesothelioma. Am J Respir Crit Care Med 2010; 181:620–625. 16. Mundt F, Nilsonne G, Arslan S, et al. Hyaluronan and N-ERC/mesothelin as key biomarkers in a specific two-step model to predict pleural malignant mesothelioma. PloS One 2013; 8:e72030. 17. Sato T, Suzuki Y, Mori T, et al. Newly established ELISA for N-ERC/mesothelin improves diagnostic accuracy in patients with suspected pleural mesothelioma. Cancer Med 2014; 3:1377–1384. 18. Cohen JG, White M, Cruz A, Farias-Eisner R. In 2014, can we do better than CA125 in the early detection of ovarian cancer? World J Biol Chem 2014; 5:286–300. 19. Bast RC Jr, Feeney M, Lazarus H, et al. Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981; 68:1331–1337. 20. Pettersson T, Froseth B, Riska H, Klockars M. Concentration of hyaluronic acid in pleural fluid as a diagnostic aid for malignant mesothelioma. Chest 1988; 94:1037–1039. 21. Creaney J, Dick IM, Segal A, et al. Pleural effusion hyaluronic acid as a prognostic marker in pleural malignant mesothelioma. Lung Cancer 2013; 82:491–498. 22. Filiberti R, Parodi S, Libener R, et al. Diagnostic value of mesothelin in pleural fluids: comparison with CYFRA 21-1 and CEA. Med Oncol 2013; 30:543. 23. Ostroff RM, Mehan MR, Stewart A, et al. Early detection of malignant pleural mesothelioma in asbestos-exposed individuals with a noninvasive proteomicsbased surveillance tool. PloS One 2012; 7:e46091. 24. Mundt F, Johansson HJ, Forshed J, et al. Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma. Mol Cell Proteomics 2014; 13:701–715. 25. Giusti L, Da Valle Y, Bonotti A, et al. Comparative proteomic analysis of malignant pleural mesothelioma evidences an altered expression of nuclear lamin and filament-related proteins. Proteomics Clin Appl 2014; 8:258– 268. 26. Cerciello F, Choi M, Nicastri A, et al. Identification of a seven glycopeptide & signature for malignant pleural mesothelioma in human serum by selected reaction monitoring. Clin Proteomics 2013; 10:16. This is the first description of a multipanel marker for mesothelioma diagnosis. 27. Zhang X, Shen W, Dong X, et al. Identification of novel autoantibodies for detection of malignant mesothelioma. PloS One 2013; 8:e72458. 28. Pass HI, Levin SM, Harbut MR, et al. Fibulin-3 as a blood and effusion && biomarker for pleural mesothelioma. N Engl J Med 2012; 367:1417–1427. This study describes a new biomarker with potentially a higher sensitivity and specificity than mesothelin.
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Diseases of the pleura 29. Gueugnon F, Leclercq S, Blanquart C, et al. Identification of novel markers for the diagnosis of malignant pleural mesothelioma. Am J Pathol 2011; 178:1033–1042. 30. Pass HI, Liu Z, Wali A, et al. Gene expression profiles predict survival and progression of pleural mesothelioma. Clin Cancer Res 2004; 10:849– 859. 31. Hu B, Thirtamara-Rajamani KK, Sim H, Viapiano MS. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol Cancer Res 2009; 7:1756–1770.
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32. Corradi M, Goldoni M, Alinovi R, et al. YKL-40 and mesothelin in the blood of patients with malignant mesothelioma, lung cancer and asbestosis. Anticancer Res 2013; 33:5517–5524. 33. Creaney J, Dick IM, Meniawy TM, et al. Comparison of fibulin-3 and mesothelin && as markers in malignant mesothelioma. Thorax 2014; 69:895–902. This study demonstrates that mesothelin has a higher sensitivity and specificity than fibulin-3. 34. Agha MA, El-Habashy MM, El-Shazly RA. Role of fibulin-3 in the diagnosis of malignant mesothelioma. Egypt J Chest Dis Tuberc 2014; 63:99–105.
Volume 21 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Comparison of mesothelin and fibulin-3 in pleural fluid and serum as markers in malignant mesothelioma Jenette Creaney a,b, Ian M. Dick a,b, and Bruce W. Robinson a,b,c
Purpose of review Malignant mesothelioma is an asbestos-induced, aggressive tumour, which frequently presents with pleural effusion. There are over 60 reported causes that can result in the development of a pleural effusion. Currently, there are no tumour biomarkers in widespread clinical use for the differential diagnosis of mesothelioma from other diseases. With the incidence of mesothelioma expected to continue to increase, it is timely to review the current status of effusion-based biomarkers for mesothelioma diagnosis. Recent findings The majority of recent studies have evaluated soluble mesothelin in effusions in a diagnostic setting for mesothelioma. However, at high specificity, the sensitivity of the assay is limited to approximately 60% at the time of diagnosis. There is considerable research effort directed toward the identification of new markers for mesothelioma through a variety of genomic, proteomic and immunomic based platforms. One of the few new biomarkers to be identified through a biomarker discovery pipeline and evaluated in pleural effusions is fibulin-3. Preliminary results on the diagnostic accuracy of fibulin-3 have been inconsistent. Summary To date, soluble mesothelin remains the best available biomarker for mesothelioma and a positive result is clinically useful in patients with pleural effusions in whom the diagnosis is uncertain. Keywords diagnosis, effusions, fibulin-3, mesothelin, mesothelioma
INTRODUCTION Malignant mesothelioma is a highly aggressive tumour predominantly associated with asbestos exposure. Tumours arise in the mesothelial cells of the serosal surfaces with approximately 90% of cases occurring in the pleura [1]. Patients with pleural mesothelioma usually present with symptoms of chest pain, discomfort or dyspnoea, which are usually because of the presence of an effusion. Greater than 90% of patients with pleural mesothelioma will have a malignant pleural effusion [2]. Malignant pleural effusions can also arise from metastatic or invasive pleural cancers, one-third of cases having breast cancer and a quarter with lung cancer. Although malignancy is a concern, nonmalignant causes can account for twice as many pleural effusion cases [3]. Cytological analysis of cells from pleural effusions is the most common method for diagnosis of malignant pleural effusions, though the immunohistological examination of biopsy tissue obtained by www.co-pulmonarymedicine.com
thoracoscopy or other invasive methods is considered the ‘gold standard’ [4]. Examination of soluble factors present in the pleural fluid has been utilized for over 40 years in an attempt to find tests that will help clinicians distinguish between benign and malignant effusions [5]. Despite identification of numerous tumour markers and their evaluation in many studies, there has not been widespread uptake or utilization of any particular markers for the diagnosis of malignant pleural effusions.
a
School of Medicine and Pharmacology, bNational Centre for Asbestos Related Diseases, University of Western Australia, Perth and c Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia Correspondence to Prof Jenette Creaney, School Medicine and Pharmacology, M503, 23 Stirling Hwy, Crawley, WA 6009, Australia. Tel: +61 8 6151 0896; fax: +61 8 6151 1028; e-mail: jenette.creaney@uwa. edu.au Curr Opin Pulm Med 2015, 21:352–356 DOI:10.1097/MCP.0000000000000167 Volume 21 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Mesothelin and fibulin-3 as markers for malignant mesothelioma Creaney et al.
KEY POINTS The incidence of mesothelioma is expected to continue to increase and at the present time, there are no tumour biomarkers in widespread clinical use for the differential diagnosis of mesothelioma from other diseases. Mesothelin is currently the best biomarker available, but it lacks sufficient sensitivity. A considerable research effort to identify new markers for mesothelioma through a variety of genomic, proteomic and immunomic-based platforms is being undertaken to improve the early diagnosis of mesothelioma.
Over the last 20 years, we have been intensely investigating the utility of the malignant pleural effusions as a sample for mesothelioma diagnosis [6 ]. Over the past decade in particular, we have focussed on the use of soluble tumour markers. Our publication in 2003 of soluble mesothelin as a biomarker with high specificity for mesothelioma triggered a wave of interest in this field [7]. Our aim in this article is to provide an update on recent studies into soluble markers in mesothelioma, specifically as related to the evaluation in malignant versus benign pleural effusions. &&
MESOTHELIN Mesothelin is a differentiation antigen expressed on normal mesothelial cells and in various tumours, including mesothelioma [8]. Mesothelin can be released from the cell surface and soluble forms of the protein have been detected in blood and effusions of cancer patients. Early studies showed that soluble mesothelin is elevated in pleural and peritoneal fluid from mesothelioma patients relative to levels in benign effusions. Recent studies have further explored mesothelin as part of the diagnostic workup of patients; confirming that an elevated level of mesothelin in the effusion is highly predictive of mesothelioma even when cytology is nondiagnostic [9,10 ,11 ,12]. As the majority of mesothelioma patients present with effusions, elevated fluid mesothelin levels could indicate the need for active invasive investigations such as thoracoscopy and facilitate earlier diagnosis. Indeed, Canessa et al. confirmed that at the time of medical thoracoscopy, 72% of epithelial mesothelioma cases had elevated mesothelin in the effusion; they found one false-positive case in their series of 104 consecutive thoracoscopies (follow-up 24 months) [13]. Another product encoded by the MSLN gene is the soluble, 31-kDa, megakaryocyte potentiating &&
&
factor (MPF), which is also known as N-mesothelin or N-ERC. There is a tight correlation between mesothelin and MPF concentrations in blood and effusions and the two markers have similar discriminatory performance [14–16]. Various assays have been developed to quantitate levels of MPF, and a recent report suggests good diagnostic performance of a new MPF assay, but results have not as yet been compared head-to-head with mesothelin [17].
COMBINING KNOWN MARKERS WITH MESOTHELIN There has been considerable optimism about the potential of combining multiple tumour markers to improve diagnostic sensitivity in mesothelioma. This has been a successful strategy in ovarian cancer biomarker research [18]; albeit most of these improved ovarian biomarker panels contain CA125, the established ovarian biomarker which was first identified in 1981 [19]. One of the early biomarkers, proposed over 30 years ago, for mesothelioma was hyaluronic acid [20]. However, the technically demanding nature of the hyaluronic acid assay limited follow-up studies. Recently, two independent studies have been reported that find combining hyaluronic acid with mesothelin or MPF improves sensitivity for mesothelioma, while maintaining specificity when measured in effusion supernatants [16,21]. Sensitivity of mesothelin for mesothelioma in a dichotomous differential diagnostic setting has been improved by using additional ‘exclusion markers’, such as carcinoembryonic antigen, has been reported [22]. These findings need to be further evaluated in consecutive series of samples.
NEW BIOMARKERS FOR MESOTHELIOMA Various genomic, proteomic, immunomic, imaging and other tools have been used to identify, quantify and characterize novel biomarkers for many different diseases. Generally, there has been a poor rate of conversion of biomarkers identified in discovery projects to clinically validated bioassays that provide favourable health impacts, in part because of lack of independent validation of the original results. Potentially new biomarkers for mesothelioma have been identified on a variety of discovery platforms, including proteomic [23–25,26 ], immunomic [27] and mRNA expression studies [28 ,29]. Of the newly reported mesothelioma biomarkers, only a few have been examined in independent sample sets largely because of lack of availability of assays and reagents, and few have been evaluated in pleural effusion samples.
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&
&&
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FIBULIN-3 Fibulin-3 was shown to be significantly overexpressed in mesothelioma tumours compared with matched normal peritoneum by mRNA gene expression analysis [30]. Fibulin-3 is a secreted glycoprotein, which is believed to play a role in the regulation of cell proliferation and migration. Fibulin-3 is highly upregulated in glioma where it has been shown to promote tumour growth and invasion [31]. The fibulin-3 protein is encoded by the epidermal growth factor-containing fibulinlike extracellular matrix protein 1 (EFEMP1) gene. In mesothelioma, plasma and pleural effusion concentrations of fibulin-3 were shown to be elevated. In plasma, fibulin-3 had a sensitivity of over 96% for mesothelioma at a specificity of 95% at a threshold value of 52 ng/ml, and in pleural effusions, fibulin-3 had a sensitivity of 84% and specificity of 93% at a threshold value of 346 ng/ml when using a commercial ELISA for the protein (USCN Life Science) [28 ]. Although the overall results from this original sample set were validated in an independent cohort of mesothelioma and control samples, a discrepancy in the diagnostic accuracy between the two cohorts was observed [28 ]. From the data presented, it is estimated that in the validation set of samples, fibulin-3 sensitivity for mesothelioma was less than 40% at 95% specificity [28 ]. The discrepancy was possibly not clear because of the use of different threshold values in the two cohorts. Subsequent independent studies to evaluate fibulin-3 as a diagnostic marker for mesothelioma have reported conflicting data. Using the same fibulin-3 ELISA, a small Italian sub-study demonstrated that fibulin-3 concentrations in mesothelioma patient sera, compared with controls and patients with lung cancer, were significantly higher, although there was no difference in levels between mesothelioma patients and those with asbestosis. From the presented data (assuming that serum and plasma results are consistent), we estimate that fibulin-3 was elevated above 52 ng/ml, the originally reported threshold in one of the 14 mesothelioma patients examined (i.e. 7% positive). The 23 lung cancer, 14 asbestosis and 23 control patients were all below this positive fibulin-3 threshold value [32]. In a study specifically designed and powered to compare the diagnostic accuracy of mesothelin and fibulin-3 in mesothelioma patients at the time of diagnosis using the same ELISA as in previous studies, we found, as previously reported, that fibulin-3 concentrations were significantly higher in the plasma of mesothelioma patients compared with patients with other cancers and benign conditions. However, sensitivity was much lower than that reported in &&
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the original study. At the previously reported 52 ng/ ml threshold plasma, fibulin-3 had a sensitivity of 22% (i.e. 18/82) and specificity of 95% (i.e. 114/ 120). Furthermore,we found fibulin-3 wasnotspecific for mesothelioma when pleural effusion samples were examined, with 63% (i.e. 22/35) of benign effusions having a concentration of fibulin-3 above the 346 ng/ml threshold [33 ]. In a small recent Egyptian study with nine cases with benign pleural effusion used as a comparator group for 25 mesothelioma cases, Agha et al. found that both pleural effusion and serum fibulin-3 were significantly elevated in serum and pleural effusions. Although an ELISA was used to quantitate fibulin-3 levels, it was not reported which specific assay was employed in the study. Using independent, internally determined threshold values, they found that serum fibulin-3 had 100% sensitivity for mesothelioma at 78% specificity relative to nonmalignant effusions, and 88% sensitivity for mesothelioma at 82% specificity relative to malignant effusions owing to metastatic disease. When effusion samples were examined, sensitivity was 88% and 72% for mesothelioma compared with nonmalignant and metastatic cancer controls respectively (at a similar specificity of approximately 80%) [34]. Data for the different sample sets were not combined and results could not be compared with previous studies, although appear similar to the original Pass et al.’s study. Of note, the benign effusion samples used in this study were selected such that patients with transudative pleural effusion, serious uncontrolled diseases (including renal, hepatic, cardiac diseases and coagulopathy), and those that were hemodynamically unstable were excluded. It is not clear whether such preselection of control samples would influence the data; our own preliminary analysis suggests patients with benign effusions and elevated effusion lactate dehydrogenase (LDH) levels tend to have higher fibulin-3 concentrations. These recent examples evaluating fibulin-3 as a biomarker for mesothelioma diagnosis in different patient cohorts highlight a number of important issues for biomarker validation in general, as well as for mesothelioma. That is firstly, the choice of appropriate controls; many patients considered at risk of mesothelioma will be in their 70s or older, will have been exposed to asbestos and may have nonmalignant concurrent pleural disease. It is imperative that appropriate control groups are used and characteristics documented; ideally controls should be collected in the same clinical setting and under the same collection and storage protocols as cases. The notion of insisting on appropriate control groups is supported by the observation that most of the recently published biomarkers for mesothelioma, &&
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Mesothelin and fibulin-3 as markers for malignant mesothelioma Creaney et al.
unlike mesothelin, fall down in the area of specificity – other diseases, including nonmalignant diseases, produce positive results. Secondly, for validation studies of an initial promising candidate, the use of a predefined diagnostic threshold should be seriously considered rather than individual study-driven cutoffs. Thirdly, consideration should be given to determining mesothelin concentrations in the samples studied. Despite sensitivity limitations, mesothelin has proved to have a consistent diagnostic performance in numerous studies and comparison of any candidate new mesothelioma biomarker with mesothelin would be informative. A final consideration for authors, and editors, relates to publication bias; in this era of novel biomarker discovery, both negative and positive results of all properly conducted biomarker studies have value.
CONCLUSION When a patient presents with an undiagnosed pleural effusion, thoracentesis is generally the first intervention performed. If no definite cancer cells are apparent in the fluid and no alternative cause for the effusion can be found then thoracoscopy with biopsy will be considered if malignancy is a significant possibility. However, in some patients with significant underlying comorbidities and/or frailty in whom thoracoscopy is considered too high risk, or where thoracoscopy is not available, or declined by the patient, measurement of serum and pleural effusion mesothelin levels is a useful step – a positive result dramatically increases the likelihood of there being a pleural malignancy present. Although soluble mesothelin has a useful clinical role in diagnosis because of its high specificity, its sensitivity remains limited. That means that a positive result is informative, but a negative one is not. A better biomarker for pleural malignant mesothelioma has yet to be identified. Acknowledgements None. Financial support and sponsorship None. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet 2005; 366:397–408. 2. Lee YC. Hunting for a pleural fluid test for mesothelioma: is soluble mesothelin the answer? Thorax 2007; 62:561–562.
3. Thomas R, Lee YC. Causes and management of common benign pleural effusions. Thorac Surg Clin 2013; 23:25–42; v-vi. 4. Roberts ME, Neville E, Berrisford RG, et al. Management of a malignant pleural effusion: British Thoracic Society Pleural Disease Guideline 2010. Thorax 2010; 65 (Suppl 2):ii32–ii40. 5. Light RW, Macgregor MI, Luchsinger PC, Ball WC Jr. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972; 77:507–513. 6. Segal A, Sterrett GF, Frost FA, et al. A diagnosis of malignant pleural && mesothelioma can be made by effusion cytology: results of a 20 year audit. Pathology 2013; 45:44–48. This study describes how perfusion cytology is sufficient to diagnose pleural mesothelioma with a high sensitivity and specificity. 7. Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins and diagnosis of mesothelioma. Lancet 2003; 362:1612–1616. 8. Chang K, Pai LH, Batra JK, et al. Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. Cancer Res 1992; 52:181–186. 9. Davies HE, Sadler RS, Bielsa S, et al. Clinical impact and reliability of pleural fluid mesothelin in undiagnosed pleural effusions. Am J Respir Crit Care Med 2009; 180:437–444. 10. Creaney J, Segal A, Olsen N, et al. Pleural fluid mesothelin as an adjunct to the && diagnosis of pleural malignant mesothelioma. Disease Markers 2014; 2014:413946. This study demonstrates that a mesothelin-positive pleural effusion indicates the likely presence of malignancy and is an indicator for further investigation. 11. Franceschini MC, Ferro P, Canessa PA, et al. Mesothelin in serum and pleural & effusion in the diagnosis of malignant pleural mesothelioma with nonpositive cytology. Anticancer Res 2014; 34:7425–7429. This study indicates that effusion mesothelin is more sensitive than serum mesothelin for mesothelioma diagnosis. 12. Hooper CE, Morley AJ, Virgo P, et al. A prospective trial evaluating the role of mesothelin in undiagnosed pleural effusions. Eur Respir J 2013; 41:18–24. 13. Canessa PA, Ferro P, Manta C, et al. Clinical value of mesothelin in pleural effusions versus histology by medical thoracoscopy: brief report. Med Oncol 2013; 30:649. 14. Creaney J, Sneddon S, Dick IM, et al. Comparison of the diagnostic accuracy of the MSLN gene products, mesothelin and megakaryocyte potentiating factor, as biomarkers for mesothelioma in pleural effusions and serum. Disease Markers 2013; 35:119–127. 15. Hollevoet K, Nackaerts K, Thimpont J, et al. Diagnostic performance of soluble mesothelin and megakaryocyte potentiating factor in mesothelioma. Am J Respir Crit Care Med 2010; 181:620–625. 16. Mundt F, Nilsonne G, Arslan S, et al. Hyaluronan and N-ERC/mesothelin as key biomarkers in a specific two-step model to predict pleural malignant mesothelioma. PloS One 2013; 8:e72030. 17. Sato T, Suzuki Y, Mori T, et al. Newly established ELISA for N-ERC/mesothelin improves diagnostic accuracy in patients with suspected pleural mesothelioma. Cancer Med 2014; 3:1377–1384. 18. Cohen JG, White M, Cruz A, Farias-Eisner R. In 2014, can we do better than CA125 in the early detection of ovarian cancer? World J Biol Chem 2014; 5:286–300. 19. Bast RC Jr, Feeney M, Lazarus H, et al. Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981; 68:1331–1337. 20. Pettersson T, Froseth B, Riska H, Klockars M. Concentration of hyaluronic acid in pleural fluid as a diagnostic aid for malignant mesothelioma. Chest 1988; 94:1037–1039. 21. Creaney J, Dick IM, Segal A, et al. Pleural effusion hyaluronic acid as a prognostic marker in pleural malignant mesothelioma. Lung Cancer 2013; 82:491–498. 22. Filiberti R, Parodi S, Libener R, et al. Diagnostic value of mesothelin in pleural fluids: comparison with CYFRA 21-1 and CEA. Med Oncol 2013; 30:543. 23. Ostroff RM, Mehan MR, Stewart A, et al. Early detection of malignant pleural mesothelioma in asbestos-exposed individuals with a noninvasive proteomicsbased surveillance tool. PloS One 2012; 7:e46091. 24. Mundt F, Johansson HJ, Forshed J, et al. Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma. Mol Cell Proteomics 2014; 13:701–715. 25. Giusti L, Da Valle Y, Bonotti A, et al. Comparative proteomic analysis of malignant pleural mesothelioma evidences an altered expression of nuclear lamin and filament-related proteins. Proteomics Clin Appl 2014; 8:258– 268. 26. Cerciello F, Choi M, Nicastri A, et al. Identification of a seven glycopeptide & signature for malignant pleural mesothelioma in human serum by selected reaction monitoring. Clin Proteomics 2013; 10:16. This is the first description of a multipanel marker for mesothelioma diagnosis. 27. Zhang X, Shen W, Dong X, et al. Identification of novel autoantibodies for detection of malignant mesothelioma. PloS One 2013; 8:e72458. 28. Pass HI, Levin SM, Harbut MR, et al. Fibulin-3 as a blood and effusion && biomarker for pleural mesothelioma. N Engl J Med 2012; 367:1417–1427. This study describes a new biomarker with potentially a higher sensitivity and specificity than mesothelin.
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Diseases of the pleura 29. Gueugnon F, Leclercq S, Blanquart C, et al. Identification of novel markers for the diagnosis of malignant pleural mesothelioma. Am J Pathol 2011; 178:1033–1042. 30. Pass HI, Liu Z, Wali A, et al. Gene expression profiles predict survival and progression of pleural mesothelioma. Clin Cancer Res 2004; 10:849– 859. 31. Hu B, Thirtamara-Rajamani KK, Sim H, Viapiano MS. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol Cancer Res 2009; 7:1756–1770.
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32. Corradi M, Goldoni M, Alinovi R, et al. YKL-40 and mesothelin in the blood of patients with malignant mesothelioma, lung cancer and asbestosis. Anticancer Res 2013; 33:5517–5524. 33. Creaney J, Dick IM, Meniawy TM, et al. Comparison of fibulin-3 and mesothelin && as markers in malignant mesothelioma. Thorax 2014; 69:895–902. This study demonstrates that mesothelin has a higher sensitivity and specificity than fibulin-3. 34. Agha MA, El-Habashy MM, El-Shazly RA. Role of fibulin-3 in the diagnosis of malignant mesothelioma. Egypt J Chest Dis Tuberc 2014; 63:99–105.
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