Early detection of malignant pleural mesothelioma

Introduction Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the serosal lining of the pleural cavity arising from the mesothelial cells (i.e. from undiff erentiated cells representing the adult remnants of the surface coelomic mesoderm) [1]. MPM is a rare tumor, but epidemiologic studies show a sharply rising incidence [2]. Although rare, its incidence is increasing, principally as a result of the long latency period of the disease [3]. Early diagnosis is a potential key factor to achieve signifi cant progress in MPM management [4]. However, this is hampered by nonspecifi c presenting symptoms and diagnostic diffi culties, including diff erentiation from reactive mesothelium, benign pleural lesions, and adenocarcinoma [5]. Early detection is limited by the long latency period, an inability of imaging to detect the disease at an early stage even when it is used as a screening strategy, and the lack of sensitive and specifi c blood-based markers [6].


Introduction
Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the serosal lining of the pleural cavity arising from the mesothelial cells (i.e. from undiff erentiated cells representing the adult remnants of the surface coelomic mesoderm) [1]. MPM is a rare tumor, but epidemiologic studies show a sharply rising incidence [2]. Although rare, its incidence is increasing, principally as a result of the long latency period of the disease [3]. Early diagnosis is a potential key factor to achieve signifi cant progress in MPM management [4]. However, this is hampered by nonspecifi c presenting symptoms and diagnostic diffi culties, including diff erentiation from reactive mesothelium, benign pleural lesions, and adenocarcinoma [5]. Early detection is limited by the long latency period, an inability of imaging to detect the disease at an early stage even when it is used as a screening strategy, and the lack of sensitive and specifi c blood-based markers [6].

Global burden of malignant pleural mesothelioma
Th e global mesothelioma burden is unclear [7]. Driscoll et al. [8] estimated that as many as 43 000 people worldwide die from the disease each year. Park et al. [9] suggested that one mesothelioma patient may be overlooked for every four to fi ve recorded . However, to date, there is no established global baseline that can be used to evaluate trends in disease occurrence [10].

In industrialized countries
Mesothelioma incidence rates have been increasing throughout the industrialized world [11] (Figs 1 and 2).
Th is refl ects industrial exposure to asbestos, which was common up to the 1980s, combined with a latent period between exposure to asbestos and development of mesothelioma averaging 30-40 years [11]. Th e incidence is predicted to peak around 2020 [12].

In developing countries
Because of scant regulations of asbestos in the developing world, there is an emerging concern about the potential for a signifi cant increase in asbestosrelated disease [13]. Developing countries are still in the early stages of diagnosing mesothelioma and may be prone to misdiagnosis and reporting errors [14].

In Egypt
MPM is an increasing disaster in Egypt, which is underestimated and neglected [15]. Mesothelioma in Egypt is mainly attributed to an environmental origin -that is, exposure to asbestos, with a high incidence in women and young adults. Epidemiological data of 635 malignant mesothelioma patients over 4 years were collected from the National Cancer Institute, Cairo University and Abbassia Chest Hospital. Th is number is more than four times the number of patients that were diagnosed in the previous 11 years at National Cancer Institute [16].
Data obtained from the information network of the General Organization for Industrialization in Egypt showed that 14 asbestos factories were present in Egypt in the year 2004 [17]. Th ese factories aff ect an area of ∼5-7 km in radius, which explains the high Early detection of malignant pleural mesothelioma Hussein F. Mahmoud Malignant pleural mesothelioma (MPM) is a rare tumour. Exposure to asbestos is a well-established aetiological factor for MPM. Patients typically present with shortness of breath due to pleural effusion or chest pain in a more advanced stage. The diagnosis is usually suggested by imaging studies (unilateral pleural thickening; pleural effusion). An occupational history must be obtained. Cytological examination of the effusion can be diagnostic, but often shows equivocal results. Therefore, histology, including immunohistochemistry, is the gold standard. Thoracoscopy, a video-assisted surgical procedure or open pleural biopsy in a fused pleural space may be necessary to provide suffi cient material for accurate histological diagnosis. There are three main histological types (epithelial, sarcomatous and mixed) with ∼60% being epithelial.
incidence of mesothelioma in the neighborhood of these factories. Workers employed since 1948 by the Egyptian asbestos company Sigwart at the mills in greater Cairo (El-Maasara and Shubra El-Kheima) had an increased risk for mesothelioma ( Fig. 3). In Egypt, the ministerial council decided to ban asbestos imports in 2004 and the Sigwart plants were closed in November 2004. Th erefore, the predicted incidence of Mesothelioma in Egypt will reach its peak around 2040 [18].

Diagnosis of malignant pleural mesothelioma
Th e diagnosis of MPM is based on clinical presentation, history of asbestos exposure, radiological fi nding as well as pathological diagnosis [19].

History
Occupational asbestos exposure accounts for the majority of mesothelioma patients [20]. Th us, the diagnosis of mesothelioma in a worker should be viewed as a sentinel event, pointing to the need to obtain a comprehensive occupational history to elucidate possible occupational and nonoccupational asbestos exposures in the past [21].

Clinical picture
Early in the course of the disease, dyspnea is the commonest symptom and is due to the presence of eff usion. Chest pain may occur as the tumor locally invades the intercostal nerves. Eventually, the lung becomes encased by the tumor leading to worsening of dyspnea and chest tightness [22].

Radiology
Th e radiological diagnosis of pleural mesothelioma requires a high degree of clinical suspicion [23].

Chest radiography
Th e radiological manifestations tend to be those of pleural eff usion and/or pleural thickening [24] (Fig. 4).
In late stages, ipsilateral shift of the mediastinum and retraction of the involved hemithorax are characteristic unless the tumor volume becomes very large [25].

Computed tomography scan
Pleural thickening or small focal pleural masses may be seen on computed tomography (CT). CT of the chest is useful in diff erentiating pleural fl uid from pleural thickening [22] (Fig. 5).
Th e CT scan provides much greater sensitivity than the usual chest radiograph for identifying fl uid and visualizing pleural-based masses [26].

Spiral computed tomography
Th e introduction of spiral CT into the fi eld has yielded advantages in the diagnosis of MPM and its staging. Spiral CT combines the advantage of single breathhold acquisition and three-dimensional reconstruction capabilities between bronchus, artery, vein, pleura, and lobular septum [27].
It is also better in visualization of the prediaphragmatic region in a single breath-hold, minimizing motion artifacts and thus better visualization of the diaphragmatic extension of MPM [28].

Magnetic resonance imaging
MRI showed tumor spread, tumor invasion of and through the diaphragm, and invasion of bony structures better than CT. Invasion of the chest wall and mediastinal soft tissue and tumor growth into the lung emphysema were equally well seen on both imaging methods [29] (Fig. 6).

Magnetic resonance imaging versus computed tomography scan
Recent studies comparing CT and MRI have shown that MRI is not signifi cantly better than CT in defi ning local extent of the tumor. Th erefore, CT remains the standard imaging study [30].

Position emission tomography
PET is a nuclear medicine imaging technique. PET scanning detects γ rays emitted by a tracer that is delivered into the body by a biologically active molecule, typically 18 F-FDG, a form of glucose. Th e eff ectiveness of the scan develops in part on how much 18 F-FDG is absorbed by the cancer cells, and hence can be seen on imaging [31] (Fig. 7).

Position emission tomography-computed tomography
PET-CT seems to be superior to other imaging modalities in detecting more extensive disease involvement and in identifying unsuspected occult distant metastases [32].

Gallium scan
Mesothelioma is reported to take up gallium 76. Gallium 76 scans in seven patients obtained before resection were compared with pathology, where no defi nite correlation was found between gallium 76 uptakes and the histological type [33] (Fig. 7).
In another study, uptake of gallium citrate by mesothelioma was positive in 43 of the 49 patients (88%) with pleural mesothelioma [25] (Fig. 8).

Thallium 201
Planar thallium 201 scintigraphy in a single mesothelioma patient revealed diff use pleural accumulation. It can demonstrate the exact tumor location [34]. Map of Greater Cairo. fx1 Industrial areas highly polluted with asbestos.

Fig. 4
Chest radiograph showing left-sided pleural mesothelioma. Appearance on chest computed tomography of malignant pleural mesothelioma. Red bars measure the thickness of pleural disease.

Additional imaging studies
Bone scans are indicated to evaluate specific symptoms (i.e. localized bone pain or laboratory abnormalities such as an elevated alkaline phosphatase) [25].

Thoracocentesis
Th oracocentesis is often the initial diagnostic intervention. Cytological diagnosis of MPM from pleural fl uid is, however, unreliable, as reactive mesothelial cells and cells from other malignant tumors such as sarcomas and adenocarcinomas are often very diffi cult to distinguish from malignant mesothelioma cells [35].

Pleural biopsy
Tissue sampling must be carried out to diagnose MPM correctly [23].

Closed pleural biopsy
Percutaneous pleural biopsy yields a diagnosis of malignancy in up to one-third of patients [30].
Th e relatively low yield of blind pleural biopsies is owing to several factors that may be relevant to the stage of the disease when pleural biopsy is performed, to the absence of visualization of the area being sampled, and to operator inexperience [36].

Computed tomography-guided cutting needle biopsy
CT-guided cutting needle biopsy of the pleural tissue associated with a pleural eff usion is a relatively new technique compared with Abram's biopsy [37]. Results of observational series suggest that this technique might improve the diagnostic sensitivity to about 80% for pleural malignancy [38].

Thoracoscopy
Th oracoscopy is an excellent tool to diagnose pleural disease defi nitively and yields almost a 100% success rate [39]. Video-assisted thoracoscopy has allowed improved visualization of the pleura and off ers the ability to perform biopsies from multiple sites [40] (Fig. 9).

Open (surgical) pleural biopsy
Surgical biopsy would be an ultimate possibility to explore the pleural space, providing both excellent visualization of the target areas and the largest possible biopsy size. In addition, it may be combined with a potentially curative therapeutic approach by partial pleurectomy and/or partial chest wall resection. Open biopsy has a sensitivity of 97% and specifi city of 56% for identifying epithelial MPM [39].

Pathological diagnosis
Diagnosis is usually achieved by the application of a combination of techniques, including histopathology, histochemistry, immunohistochemistry, ultrastructural examination, and mesothelial markers [41]. PET scan for a patient with malignant pleural mesothelioma.

Fig. 8
Anterior and posterior scan images show diffuse Ga-67 uptake in the left middle and lower lung fi elds.
Th e epithelial subtype is the most common and carries the most favorable prognosis [42].

Histochemical techniques
Th e most frequently used biochemical stain is the Periodic acid Schiff stain with diastase. Th e presence of strong Periodic acid Schiff positivity in tumor cells is indicative of epithelial mucin, which strongly argues the diagnosis of malignant mesothelioma. Alcian blue stains hyaluronic acid positively in mesothelioma [41].

Immunohistochemistry
Immunohistochemistry is currently the most widely applied technique in establishing the diagnosis of mesothelioma. Epithelial mesothelioma typically stains positive for calretinin, cytokeratins, and vimentin, and negative for CEA and thyroid transcription factor-1 [43] (Fig. 11).
A pathology panel was convened at the International Mesothelioma Interest Group biennial meeting (October 2006) to develop the practical guidelines for the pathologic diagnosis of malignant mesothelioma. Th e International Mesothelioma Interest Group recommends that markers have either sensitivity or specifi city greater than 80% of the lesions in questions. Interpretation of positivity generally should take into account the localization of the stain (e.g. nuclear vs. cytoplasmic) and the percentage of cells staining (>10% is suggested for cytoplasmic membranes markers). Th ese guidelines are meant to be a practical reference for the pathologist [44].  Stains used to diagnose mesothelioma. CK5, cytokeratin 5/6.

Ultrastructural examination
Th e epithelial variant is composed of polygonal cells with numerous long slender branching surface microvilli, desmosomes, abundant tonofi laments, and intracellular lumen formation. In sarcomatoid variant, elongated nuclei and abundant rough endoplasmic reticulum are found. Th e biphasic nature of mesothelioma is characterized by stromal cells separated by matrix containing collagen fi bers, which appear spindle or ovoid [45] (Fig. 12).

Microarray
A microarray or gene chip measures the expression level of a gene by determining the amount of messenger RNA ( mRNA) that is present. A microarray allows the simultaneous analysis of the expression levels of hundreds, thousands, or even tens of thousands of genes in a single experiment. mRNA is copied into labeled cDNA with reverse transcriptase so that the relative abundance of individual mRNAs is refl ected in the cDNA product. Th is method has proven to provide excellent specifi city and reproducibility using this technique, based on the expression levels of a small number of genes, and can be useful in the early and accurate diagnosis of MPM [46].

Biomarkers
Improved detection methods for the diagnosis of asymptomatic MPM are essential for an early and reliable detection and treatment of this type of neoplastic diseases. Th us, focus has been on fi nding tumor markers in the blood that can be used for noninvasive detection of malignant mesothelioma [47].

Mesothelin
Mesothelin is a 40-κD cell surface glycosylated phosphatidylinositol-anchored glycoprotein, which functions in cell-to-cell adhesion [48]. Mesothelin is expressed by normal mesothelial cells; however, it is highly overexpressed in cancers such as MPM, pancreatic, or ovarian carcinoma [49].
Soluble mesothelin-related peptide ( SMRP) is another protein product of the human mesothelin gene [50]. SMRP can be detected in blood, and has been found highly increased in blood of patients with mesothelioma [51]. Th e fi rst report of SMRP as a marker of mesothelioma suggested excellent values for sensitivity (84%) and specifi city (virtually 100%) [51]. Evidence has been published indicating that an enzyme-linked immunosorbant assay using two antibodies to SMRP, OV569 and 4HS, holds promise for detection of MPM [52].

Megakaryocyte potentiating factor (MPF)
MPF is a soluble protein produced by proteolytic cleavage of the mesothelin precursor protein and is secreted by the mesothelioma cell lines [52]. Holleroet et al. found that MPF can be used as a serum biomarker of malignant mesothelioma. At 95% specifi city, MPF had a sensitivity of 68% [53].

Osteopontin
Osteopontin, also known as early T-lymphocyte activation 1 (Eta-1), is a secreted multifunctional glycoprotein. Its putative functions include roles in bone metabolism, urine regulation, wound healing, cell survival, and tumor progression [54].
Serum osteopontin levels are higher in patients with mesothelioma than in those with asbestos-related, nonmalignant pleural diseases [54]. Osteopontin levels are elevated in patients with both the epithelioid and sarcomatoid subtypes as well as in patients with several other malignancies and nonmalignant conditions [55]. Osteopontin has been seen as a promising biomarker because of its expression on gene expression arrays to predict survival and recurrence patterns in patients with pleural mesothelioma [56], and it seems especially interesting as a potential early diagnostic marker because it has been shown to diff erentiate asbestos-exposed patients from stage I mesothelioma patients [57].
Tables 1 and 2 summarize the data on the previous biomarkers.
Tabata et al. [60] demonstrated that Ang-1 stimulated the cell growth and migration of MPM cells in invitro studies. Th ey also demonstrated that patients with MPM had signifi cantly higher serum levels of Ang-1 in comparison with a population with a history of asbestos exposure that did not develop MPM. Th e authors suggest that Ang-1 could be a novel useful serum prognostic factor.  Transmission electron micrograph of an epithelial mesothelioma showing long microvilli.

Fibulin-3
Fibulin-3 is a highly conserved member of the extracellular glycoprotein fi bulin family encoded by the gene, epidermal growth factor-containing fi bulinlike extracellular matrix protein ( EFEMP1), on chromosome 2p 16 [61]. Plasma fi bulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with eff usion fi bulin-3 levels, plasma fi bulin-3 levels can further diff erentiate mesothelioma eff usions from other malignant and benign eff usions [3].

Fibronectin
Fibronection is a high-molecular-weight glycoprotein, which binds to receptors known as integrins. It exists in two forms, an insoluble form in the extracellular matrix and a soluble form found in the plasma [62]. Its role as a tumor marker in malignant mesothelioma has been investigated by Emri et al. [63].
Th e level of fi bronectin in the pleural fl uid and the plasma was elevated in patients with malignant mesothelioma but was not statistically signifi cant in comparison with tuberculous and other nonmesotheliomatous eff usions. Th us, measurement of serum fi bronectin may not add much to the existing diagnostic aids.

CYFRA21-1
CYFRA21-1 is a soluble cytokeratin 19 fragment and can be used as a tumor marker in many cancers [62].
Paganuzzi et al. [ [65]. Th e sensitivity of serum CYFRA21 levels for the diagnosis of MPM has been stated to be 40% [65].

Platelet derivative growth factor (PDGF)
PDGF is a protein secreted from the alpha granules of the platelets and mesenchymal cells. It regulates cell division and growth and also has a role in migration and angiogenesis. It exists as a dimer with the isoforms: AA, AB, and BB. PDGF AB was found to be signifi cantly higher in patients with mesothelioma compared with their healthy controls [66].
Th e levels showed a correlation with the histological subtypes, higher levels seen in the epitheloid subtype. Serum PDGF level can be used as a marker to assess the prognosis in patients diagnosed with malignant mesothelioma [62].

Tissue polypeptide antigen
Tissue polypeptide antigen is a growth and proliferative cell marker. It is a polypeptide composed of cytokeratin 8, 18, and 19. It is found to be elevated in many cancers including squamous cell carcinoma of the head and neck and thyroid cancer. Its role in malignant mesothelioma has been investigated in few studies [62]. It was found to be a useful marker in predicting disease progression and survival [67].

Serum hyaluronan
Hyaluronan is a glycosaminoglycan distributed widely in the body. It is found in the synovial fl uid connective tissue and vitreous humor of the eye. Serum and pleural fl uid hyaluronan levels have been found to be increased in patients with malignant mesothelioma. It can serve as a potential tumor marker or as a marker of progression of the disease [68].
Th e literature reports the sensitivity and specifi city of serum hyaluronan to be 65% and 85%, respectively, as an indicator of disease progression [69].

Conclusion
Th e diagnosis of mesothelioma can be challenging. If the diagnosis of mesothelioma is suspected, a careful occupational history must be obtained. Improved detection methods for the diagnosis of asymptomatic MPM are essential for an early and reliable detection and for treatment of the disease. MPM should be considered in any patient with either pleural fl uid or pleural thickening, especially if chest pain is present.
Th oracoscopy and open surgical biopsy remain the best diagnostic procedures in suspected MPM. Identifi cation of tumor markers and development of assay to measure them are important goals in oncology. Th e proposed markers have insuffi cient accuracy to replace cytohistology and cannot be presently proposed as screening tools. Th e usefulness of biological markers should be further evaluated in selected highly exposed population including the involuntary surveillance protocol. Th erefore, new mesothelioma-specifi c biomarkers are needed to detect MPM at an earlier stage.