**3. Pathogenesis and biology**

#### **3.1. How asbestos causes cancer**

Since most mesothelioma is associated with asbestos exposure, a lot of research has focused on how this inorganic fibre causes cancer. The physical properties of asbestos are more important for its carcinogenicity than its chemical composition. Many studies have confirmed that long (≥8 μm), thin (≤0.25 μm) fibres are most strongly associated with mesothelioma. Fibres of that size have aerodynamic features that are fine enough to allow them on the one hand to be deposited beyond the ciliated airways where they escape mechanical clearance, and on the other, large enough to frustrate phagocytosis by macrophages. Having been deposited, they migrate through still poorly understood pathways to the parietal pleura where they promote oncogenesis on the mesothelium. The chemical properties of the fibre are important insofar as they determine the biopersistence of the fibre. Chrysotile fibres, for example, are weak and fracture easily into shorter fibres which are easier to clear. Their apparent weak association with mesothelioma has been attributed to the fibre's lower biopersistence [33].

Asbestos is specifically cytotoxic to mesothelial cells in culture but not to fibroblasts [34], [35], therefore other processes must be involved *in vivo* to prevent cell death, promote cell survival and drive malignant transformation. The most widely accepted hypothesis invokes a chronic and genotoxic inflammatory response which over time drives tumourigenesis. As the long fibres become deposited and transported to the parietal pleura, they are progressively taken up by macrophages. However, because of their sheer size and biopersistence, they could not be cleared effectively, markedly lengthening their dwell time at the mesothelium compared to other particulates. Furthermore, macrophages are unable to engulf the entirety of these large fibres. Such frustrated phagocytosis is a potent stimulation of the macrophage's inflammatory response which results in respiratory bursts and secretion of toxic metabolites such as reactive oxygen species, growth factors and cytokines. The asbestos fibres have also themselves been implicated in carcinogenesis through direct interference with the mitotic apparatus, and direct generation of free radicals through its interaction with mobilisable iron on the fibre surface [36]. Over a long period of time, it could be envisaged the unrelenting exposure to these mutagens and growth signals could drive neoplastic transformation.

#### **3.2. Molecular pathogenesis**

*2.3.2. SV40*

treatment for SV40 infection.

132 Principles and Practice of Cardiothoracic Surgery

**3. Pathogenesis and biology**

**3.1. How asbestos causes cancer**

*2.3.3. Irradiation*

[31] [32].

SV40 is a polyoma virus which has long been studied as a carcinogen. The concerns over SV40 arose from the widespread administration of SV40-contaminated polio vaccines which were distributed worldwide in the 1960's. Certainly in the laboratory, SV40 virus has the ability to transform human cells and induce mesothelioma in experimental animals both directly, and as a co-carcinogen acting synergistically with asbestos [26] [27]. However, its relevance to clinical mesothelioma is less clear and the evidence is largely circumstantial. SV40 DNA fragments have been identified in 40 to 60% of mesothelioma samples [28] [29], but the copy numbers were exceedingly low (less than one per cell) [29]. Furthermore, there is no current

Irradiation causes cancer and mesothelioma is no exception, Thorotrast is an alpha-emitting thorium dioxide radiocontrast used between 1930s and 1950s. It has a physical half-life of 1010 years and a biological half-life of several hundred years, and so it is retained lifelong, constantly exposing tissue including mesothelium to irradiation. Thorotrast is associated with many malignancies including mesothelioma [30]. Patients with lymphoma who underwent mantle radiotherapy represent another cohort with irradiation to the pleura. They also have a higher risk of mesothelioma compared to population with similar levels of asbestos exposure

Since most mesothelioma is associated with asbestos exposure, a lot of research has focused on how this inorganic fibre causes cancer. The physical properties of asbestos are more important for its carcinogenicity than its chemical composition. Many studies have confirmed that long (≥8 μm), thin (≤0.25 μm) fibres are most strongly associated with mesothelioma. Fibres of that size have aerodynamic features that are fine enough to allow them on the one hand to be deposited beyond the ciliated airways where they escape mechanical clearance, and on the other, large enough to frustrate phagocytosis by macrophages. Having been deposited, they migrate through still poorly understood pathways to the parietal pleura where they promote oncogenesis on the mesothelium. The chemical properties of the fibre are important insofar as they determine the biopersistence of the fibre. Chrysotile fibres, for example, are weak and fracture easily into shorter fibres which are easier to clear. Their apparent weak association

Asbestos is specifically cytotoxic to mesothelial cells in culture but not to fibroblasts [34], [35], therefore other processes must be involved *in vivo* to prevent cell death, promote cell survival and drive malignant transformation. The most widely accepted hypothesis invokes a chronic and genotoxic inflammatory response which over time drives tumourigenesis. As the long

with mesothelioma has been attributed to the fibre's lower biopersistence [33].

A central player which promotes mesothelial transformation is believed to be tumour necrosis factor-alpha (TNF-α). Asbestos stimulates both macrophages and human mesothelial cells to express TNF-α. TNF-α has been shown to promote mesothelial cell survival in the face of asbestos exposure *in vitro*, and the effect appears to be mediated through Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells (NF-κB) [35]. NF-κB activation results in release of a p16 subunit which translocates to the nucleus to induce expression of antiapoptotic genes. Meanwhile, activated macrophages also secrete a host of other cytokines and growth factors including interleukins, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), as well as reactive oxygen and nitrogen species which directly causes DNA and chromosomal damage. Together, this sustained insult of mutagens and growth stimulators causes the first genetic alterations behind malignant mesothelioma.

Whilst most cancers have inactivation of the p53 and pRb tumour suppressor genes, mutation of these genes in mesothelioma is surprisingly rare [37] [38]. In contrast, over 70% of meso‐ theliomas have deletions of 9p21 and about 40% mesothelioma have loss of heterozygosity at the 22q12 locus [39] [40] [41] [42] [43]. 9p21 contains the INKa/ARF locus which encodes two proteins p16INK4a and p14ARF alternatively spliced from the same mRNA. Functionally, p14ARF stabilises p53 whilst p16INK4a inhibits the inactivation of pRb, thereby restricting progression through the cell cycle G1 checkpoint. Thus, mutations in the INKa/ARF locus effectively lead to loss of both tumour suppressor pathways. Positional cloning also identified the neurofibromatosis NF2 gene within the 22q12 locus, which encodes for the protein merlin. Merlin integrates signals from various adhesion molecules and cytoskeletal components and promotes cell adhesion, establishes apical polarity and mediates contact inhibition. It also has the capacity to migrate to the nucleus to modulate gene expression.

The precise mechanism of NF2 tumour suppression remains unclear [44] but it is likely to have a salient role. Whilst only 40% mesothelioma have truncations of NF2 or merlin, in the remaining cases, merlin is functionally inactivated through increased phosphorylation at Ser518 [45] and changes in microRNA expression [46]. Simultaneous loss of both INKa/ARF and NF2 appears to be important in the pathogenesis of mesothelioma. In experimental models of asbestos-induced mesothelioma, loss of the remaining NF2 allele is accompanied by the concomitant loss of INKa/ARF [47]. Indeed functionally, loss of INKa/ARF appears to be permissive for NF2 tumourigenesis [48]. Thus, the evidence points to mutations in INKa/ARF and NF2/merlin as driver mutations central to the pathogenesis of mesothelioma. As a result of the genetic instability conferred by these tumour suppressor gene mutations, a large number of genetic lesions appear causing dysregulation of growth factor expression and signalling, angiogenesis and apoptosis, conferring on the cell the phenotype of malignancy.

The quality of tissue available to the pathologist greatly influences the ability to make a diagnosis. Fluid from a pleural effusion is helpful at narrowing the differential diagnosis but it has poor sensitivity (about 50-60%) for the diagnosis of malignancy, and in most studies the negative predictive value is around 70% [55]. However, a diagnosis may be more forthcoming when aspiration cytology is repeated [56]. For the specific diagnosis of mesothelioma, cytology has an overall sensitivity of about 30-50%, and is almost useless at diagnosing sarcomatoid

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Tissue for histopathology can be obtained by percutaneous or thoracoscopic means. Of the percutaneous methods, the Abram's needle has the highest yield, but is only similar to cytological diagnosis [58] [59]. Although pneumothorax can be expected in 15% of patients, few require intervention and the overall complication rate is low in safe hands [60] [61]. The addition of image guidance to target areas with >5mm pleural thickening significantly

Thoracoscopy allows direct visual inspection and target selection, at the same time enabling greater amounts of tissue to be obtained, thereby improving the diagnostic yield for malig‐ nancy to about 95%. Video-assisted thoracoscopy requires a general anaesthetic and single lung ventilation and may be less suited to frail patients, however, it offers the opportunity to proceed to other procedures such as opening up loculations, pleurodesis or insertion of an

Thoracotomy and pleural excision remains the gold-standard for diagnosis of mesothelioma. Whilst pleural biopsy, both open and close, have a high sensitivity for the diagnosis of mesothelioma, the sensitivity for the determination of tissue subtype is approximately 80-86%

The separation of benign from malignant mesothelial proliferation can be extremely difficult. Most processes that affect the pleural space, from pneumothorax to thoracic surgery, pulmo‐ nary diseases to systemic diseases cause a degree of pleuritis with a degree of reactive mesothelial hyperplasia. This hyperplasia can be accompanied by quite florid cytological atypia, sometimes more florid than seen in some mesothelioma. Therefore, cytological features

Whilst invasion necessarily implies malignancy, benign processes in the pleura can also produce features that mimic invasion. To demonstrate invasion requires surrounding fat and stroma within the biopsy specimen, that is a reason why the diagnostic yield is higher with larger surgical specimens which contain the full thickness of the pleura and the deeper surrounding tissue. To illustrate the difficulty in clinching a diagnosis, even expert members of the US-Canadian Mesothelioma Reference Panel disagree 22% of the time on selected cases

Some benign histological features can appear ominous, one such is entrapment, where organising pleuritis within the pleural space overlying a pleural surface gives the deceptive appearance of mesothelial invasion. This can be complicated by the concomitant appearance

improves the diagnostic yield to >80% and reduces the rate of complications [61].

mesothelioma (20% sensitivity) [57].

indwelling drain in case of trapped lung.

referred to them [65] [66].

and is less accurate for non-epithelioid subtypes [62] [63].

**4.3. Differentiating benign from malignant mesothelial proliferation**

of a specimen are not helpful in the diagnosis of malignant mesothelioma [64].
