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Pleural Microparticles and Malignant mesothelioma

INTRODUCTION 

Malignant mesothelioma (MM) is a devastating cancer mesothelial cells caused mainly by asbestos exposure.

Limited knowledge regarding the detection of asbestos exposure and the early diagnosis of MM, as well as a lack of effective treatment options for this deadly cancer, underscores an immediate need to understand the mechanisms of MM development.

Much research is currently underway on the study of nanovesicles and their enormous potential to contain signature molecules representative of different diseases as well as to communicate with distant targets.

In this area of research, many scientists have been particularly interested the role of exosomes in the biology of MM.

In this bibliography review, we have summarized the most noteworthy topics from the published literature in the field.

We hope that the ongoing research in MM will help advance the field by revealing the mechanisms of MM development and survival so that we can develop new treatment strategies for this disease.

Microparticles

Extracellular vesicles are cell-derived membrane-bound vesicles in response to cell activation or apoptosis.

They are very heterogeneous both in content and size, with a diameter ranging between 0.1 to 1 μm.

Microvesicles are miniature versions of the parent cell and can reflect it by expressing parental antigens.

"Microvesiculation" is a biological process and, as such, can occur in all types of cells and every fluid in our bodies.

There is very little data on microparticles and pleural fluid, although initial research has been published since the early 2000s.

The pleural fluid is theoretically an ideal biological fluid for studying microparticles: sampling is minimally invasive, there is abundant material available for analysis, there is little "background noise" because it usually has a low cell count and very little cellular debris.

Microparticles deriving from cancer cells are of great interest because, by definition, they reflect the cancer cell that they derive from and can therefore provide a lot of information about it.

They are membrane-bound heterogeneous sacs that are released from the surface of cancer cells into the extracellular environment.

Tumor cells can constitutively produce extracellular microvesicles apparently without the need for stimulation.

Although several features of these tumor microparticles have been described, they all show the great potential of tumor cells for the survival and growth of the cancer.

Exosomes

Exosomes are specific extracellular microparticles and are in fact nanospheres with a diameter of less than 150 nm.

Although they were first discovered in the 1980s, this research was not particularly appealing because at that time, exosomes were simply considered as cellular residues, or waste. This was probably because it was not possible to demonstrate the interactions ongoing between exosomes and nearby cells.

The enormous potential of these particles, whose surface is composed of a lipid membrane that can contain surface proteins, DNA, miRNA, RNA, lipids, etc., has only recently emerged.

Due to their supply of surface proteins, loading capacity and stability, exosomes are potential extracellular messengers that can reach very distant cells within our body. Exosomes are therefore essential for intercellular communication: these nanovesicles are in fact transported from producer cells to target cells is important to normal physiology as well as disease states such as cancer.

Exosomal communication is implicated in multiple biological systems such as immune function, tissue repair, nervous system signaling, cardiac health, etc.

The wave of studies in the field of exosomes has brought valuable information about basic biology and diseases into the scientific realm.

We now know that exosomes are more than simple waste receptacles used by cells to rid themselves of unwanted material, but are sophisticated molecular messaging systems that can act locally or distally from where vesicles are secreted.

This special ability opens up avenues of important research from a diagnostic standpoint: researchers are studying the association between the amount of exosomes in biological fluids and the presence of tumor cells, which show a strong "impetus to communicate", i.e., produce a high number of these messenger particles.

Exosomes are also potentially interesting from a therapeutic standpoint: cells could be targeted by targeted therapies based on specific exosomes, transformed into transporters of agents.

Because of the pivotal role exosomes play in disease, they could be used for biomarker identification for diagnostic and prognostic means and developing new therapeutic strategies.

Cancer is the most studied field where the roles of exosomes have been explored in various processes such as diagnosis, prognosis, metastasis and therapy.

Malignant mesothelioma and exosomes

The earliest research on exosomes and MM was focused on identifying exosomes and their protein cargo from the pleural effusions of cancer patients.

Exosomes were isolated by sucrose-gradient ultracentrifugation from the pleural fluid of patients with MM, lung cancer, breast cancer or ovarian cancer. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis indicated large amounts of peptides originating from immunoglobulins and various complement factors, as well as previously undescribed exosomal proteins such as the sorting nexing protein (SNX25), B-cell translocation protein gene 1 (BTG1), and pigment epithelium-derived factor (PEDF). Both BTG1 and PEDF were in increased abundance in exosomes from malignant processes and likely involved in the tumor exosome biogenesis. Moreover, Western blot analysis verified the presence of the MHC class II molecule, HSP90, and immunoglobulin G and M.

Research has also been conducted on the protein composition of exosomes that are secreted from MM tumor cells. Knowledge about tumor antigens in MM is limited and so research in this area has been useful to delineate the proteomic load of MM exosomes. MM tumor cell lines were created; exosomes were isolated using ultracentrifugation and were characterized by TEM for their morphology and size. The exosomal proteins were subjected to MALDI-TOF analysis, and of these identified proteins, four were also confirmed by Western blot analysis, namely: fascin, -tubulin, HSC70 and HSP90.

In addition, as reported in in vivo systems, these tumor exosomes were also enriched with MHC class I molecules, and the researchers also indicated high levels of annexins which may be involved in membrane-cytoskeleton dynamics. This report revealed several proteins that had not yet been indicated on tumor exosomes or in MM cell lines, thus providing novel information on MM and tumor exosomes as a whole that could advance our understanding of the disease.

In 2005, researchers published their work on the immunological functions of exosomes secreted by cancer cells (breast cancer and mesothelioma), and how these tumor exosomes altered the expression of the NKG2D receptor on target blood leukocytes. The exosomes secreted from these MM cancer cells were positive for their expression of NKG2D ligands, which was directly related to the ability of MM exosomes to decrease the capacity of effector T cells to kill target cells. This research demonstrated that the two MM cell lines used had a high expression of NKG2D ligands and appeared to correlate with the aptitude of MM exosomes to more effectively suppress NKG2D expression on target cells. Overall, this report indicates a role of MM exosomes in phenotypically altering immune cells that can aide cancer cells in immune evasion through the presence of exosome ligands to NKG2D.

A promising field of therapeutic cancer research is focused on the use of tumor-associated antigens (TAAs) present on tumor exosomes as a mode of dendritic cell-based immunotherapy immunotherapy. The concept being that tumor exosomes bearing TAAs, mostly secreted from immunogenic cancers, are adept at inducing antitumor responses in mouse cancer models through the activation of dendritic cells. This is an exciting advance in this field because MM is regarded as a non-immunogenic cancer with very few TAAs known. This research investigated whether MM exosomes were potential antigen sources for dendritic cell-based immunotherapy. Initially, a lethal dose of MM tumor cells was injected into BALB/c mice. After seven days of tumor formation in the mice, a single bolus dose of dendritic cells was injected for immunotherapy. These dendritic cells, however, had been loaded with either MM exosomes or MM cell lysate to test whether exosomes had an immunogenic priming capacity on the dendritic cells. The median overall survival of the tumor-bearing mice was significantly increased in the dendritic cell immunotherapy loaded with MM tumor exosomes compared to the cell lysate, suggesting that the exosomes could be a potential immunotherapeutic approach for MM as well as other non-immunogenic tumors.

Subsequent research on exosomes in MM has focused on the formation of tunneling nanotubes (TnT), which are actin-based cell extensions involved in intercellular cargo transport. The relationship between TnT formation and their communicatory effects with MM tumorigenesis is unknown, which led to research on exosomes as possible mediators for TnT formation in MM. MM exosomes were purified and added to MM cells cultured separately, showing that in these conditions, MM cancer cells produced significantly more TnTs than the cells cultured without the addition of exogenous exosomes. The researchers suggested that the added tumor exosomes were enriched with TnT, a process that could be involved in how exosomes interact with target cells. It has also been shown that exosomes can localize and "surf" on filipodia (cellular protrusions similar to actin filaments) before internalization. The uptake of MM exosomes by MM cells apparently facilitated more TnT connections between the tumor cells, and the connected cells had nearly twice as many lipopods. Overall, MM exosomes may act as an induction agent of TnT formation between MM tumor cells and perhaps this connection could be an important conduit of cellular information vital for MM progression.

To increase our understanding of the MM secretome, researchers published a study on MM-derived exosomal proteomic cargo. Using quantitative proteomics, they delineated the protein composition of exosomes from four human MM cell lines and identified a total of 2178 proteins from all cells, with 631 common exosomal proteins between the groups. Of these MM exosome proteins, the researchers delineated candidate biomarkers based on clinical relevance, including tubulin isotypes TUBB4A, Q8IWP6 and B3KPS3; galectin-3-binding and LGB3P; alpha enolase, annexin 1 and G6PD. Furthermore, it was also shown that exosomes contained mesothelin, calreticulin, vimentin and superoxide dismutase, all highly expressed in MM. This research also uncovered the presence of 26 immunoregulatory components in MM exosomes (such as oncostatin-M oncostatin receptor (OSMR), the drug resistance-associated protein 1 (ABCC1), and the SUMO-1 activating receptor SUMO-1 (SAE1)), as well as 16 tumor-derived antigens, including glycan-1, which has been identified in many tumor-derived exosomes and considered a potentially valuable biomarker for pancreatic cancer. Importantly, this study also provided valuable information showing that MM exosomes regulate the cells in the tumor microenvironment by increasing the migratory capacity of fibroblasts and endothelial cells in vitro. Overall, this research suggests that MM exosomes contain many proteins relevant to cancer, angiogenesis, metastasis, migration and immune regulation.

The known complexity of the MM secretome was also elucidated using iTRAQ proteomic analysis. Using six MM cell lines in comparison with three primary mesothelial cell cultures, it was seen that MM cell secretomes contained higher abundances of exosomal proteins.

Another study analyzed a small number of patient samples demonstrated the potential utility of extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) in diagnosing benign or malignant MM. The ratios of mesothelin, galectin-1, osteopontin, and VEGF were higher in MM samples compared with benign effusion, whereas exosomal angiopoietin-1 was higher in MPM samples. These findings are encouraging and need to be validated with larger sample populations.

Although more emphasis has been placed on the exosomal proteomic signature, a study suggests that a specific exosomal microRNA signature can discriminate malignant pleural mesothelioma (MPM) from past asbestos exposure (PAE) subjects. This study was conducted in a small number of subjects and needs to be verified in larger cohorts.

Later, researchers using the MM tumor stromal pattern demonstrated that endothelial cell-derived exosomes enriched in miR-126 were differentially distributed within the stroma. These findings suggest an important role regarding the exosomal transfer of miR-126 in its antitumor response in MM. The same researchers further demonstrated that MPM-derived spheroids, when treated with the miR-126-enriched exosome, showed antitumor effect initially. However, this effect later vanished due to the loss of miR-126 from the cells that could be restored by inhibition of exosome secretion.

Researchers investigated the proteomic cargo and gene modulatory effects of exosomes from asbestos-exposed cells. First, epithelial lung cells (BEAS2B) or macrophages (THP1) (the first known cells to encounter asbestos during inhalation) were cultured with asbestos and their exosomes isolated. These asbestos exosomes were subjected to tandem mass spectrometry for protein identification. 145 proteins were identified in the epithelial cell exosomes, of which 55 were significantly different in abundance in the asbestos-exposed group, including plasminogen activator inhibitor 1, vimentin, thrombospondin and glycan-1, and glycan-1.

Exosomes from asbestos-exposed epithelial cells were also found to lead to genetic changes in the target primary pleural human mesothelial cells (HPM3), which were reminiscent of epithelial to mesenchymal (EMT) transition: down-regulation of E-cadherin, desmoplakin and the IL1 receptor antagonist. 785 proteins were identified upon proteomic analysis of the macrophage exosomes, of which 32 had significantly different abundances between the exosomes in the asbestos-exposed group and the control. Fifteen of these exosomal proteins were in greater abundance in the asbestos group compared with the control, and interestingly, vimentin and SOD were among those that showed an increase in exosomes from the macrophages after asbestos exposure. In response to exposure of asbestos exosomes from macrophages to target primary mesothelial cells, it was shown that significant genetic alterations occurred in the mesothelial cells: 498 genetic changes in total, with 241 up-regulated and 257 down-regulated. As a positive control, the group used asbestos fibers on mesothelial cells, and found that 206 genes were mutually altered in the asbestos-exposed exosomes or asbestos-exposed group of mesothelial cells.  This exciting discovery suggests that exosomes from asbestos-exposed cells are able to modify the genetics of mesothelial cells in similar ways to how asbestos fibers would change on their own.

As an initial step towards an in vivo study, researchers began to define the proteomic signature of mouse serum exosomes in an asbestos-exposure model. C57/Bl6 mice were exposed to asbestos via oropharyngeal aspiration, and 56 days later, the serum exosomes were isolated for proteomic analysis. Tandem mass spectrometry for protein identification again showed that there were 376 quantifiable proteins present in the mouse serum exosomes, with the majority of proteins being more abundant in the asbestos-exposed group. Of these more abundant proteins in the asbestos-exposed group, three were validated by Western blot analysis, all of which were acute-phase proteins: haptoglobin; ceruloplasmin, the copper carrying glycoprotein previously seen to be increased in the blood of MM patients and asbestos-exposed individuals, and fibulin-1, which is implicated in asbestos exposure and MM. The findings on the secreted exosomes of mesothelioma cells compared with healthy mesothelial cells showed that the tumor cells secreted  significantly different patterns of miRNA compared with their healthy counterparts. In particular, it was shown that miR-16-5p expression was significantly increased within the exosomes released by the cancer cells. The hypothesis was that the mesothelioma cells developed a preferential secretion mechanism to rid themselves of miR-16-5p due to its well-established tumor suppressor functions. Many studies have indicated the functionality of this secretion and the possibility of targeting this pro-tumor phenotype.

Another study looked at a number of different human cancers by analyzing the vesicles and extracellular particles (EVPs) via a comprehensive proteomic analysis. This research demonstrated that EVP proteins can be used for cancer-type detection. Focusing on the mesothelioma data, the paper showed that immunoglobulins were the main family of proteins found in EVPs at a high frequency in mesothelioma. The study suggested that plasma-derived EVP protein signatures could be beneficial for cancer-type detection in patients. While encouraging, these findings need to be further validated and tested in a larger cohort of patients to confirm the results.

Besides the above-mentioned published studies, numerous studies have been performed with human mesothelioma cells, plasma from asbestos-exposed samples and mesothelioma patient samples. Studies have also been performed on plasma exosomes isolated from healthy volunteers, from the asbestos-exposed non-cancer group and asbestos-exposed mesothelioma group. Although the number of exosomes per ml of plasma did not differ in the various groups, there was a greater quantity of exosomal proteins in the various disease groups compared with the controls. The proteomic analysis performed on these samples showed the presence of coagulation-related proteins in the exosomes from the disease groups (mesothelioma and asbestos-exposed) compared with the controls. The control group plasma exosomes presented a signature comprising immunoglobulins, lipoproteins, and platelet-bound proteins. These data indicate an altered immune surveillance in MM samples concomitant with the increase of coagulation factors.

Conclusions

The studies reviewed above provide an initial framework for understanding potential biomarkers and the underlying biology of MM and asbestos exposure. Based on these findings, researchers can try to further identify means for early detection of asbestos exposure or the development of asbestos-related diseases, as well as discovering much-needed therapeutic targets.

Understanding the mechanism of how MM develops and progresses is an important area that can be used for the treatment of MM patients.

Ultimately, we hope that exosome research in MM will continue along this path and that more significant discoveries will be made toward understanding how asbestos causes cancer and finding ways to identify dangerous exposure to asbestos and the early detection of cancer before a fatal diagnosis.

Although the field of exosome research is very prolific and has offered many opportunities for advancement in the medical field, it is not without challenges and limitations. Areas for improvement include methods for isolating exosomes, understanding the mechanisms of biogenesis, and the characterization of exosome cargo. We have achieved remarkable progress in this area, which gives us hope for potential breakthrough treatments and / or technologies.

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New therapeutic strategies for malignant pleural mesothelioma: CAR T cells

INTRODUCTION 

As we all know, malignant pleural mesothelioma (MPM) is an aggressive neoplasia that does not respond well to the standard treatment currently being used in clinical practice and that has a bleak prognosis.
Nevertheless, new immunotherapeutic approaches are currently being tested and, specifically, there is some data regarding the possibility of developing therapies aimed at specific targets. One of these is the tumor- associated antigen mesothelin.

Within this framework, new therapeutic strategies are being developed such as the possibility of using laboratory-designed CAR T cells that are able to target mesothelin.
The following bibliographic review aims at compiling literature data concerning the application of the CAR T therapeutic approach to malignant pleural mesothelioma, considering this treatment strategy as a future perspective for improving the prognosis and outcomes of patients suffering from this neoplasia.

We will therefore attempt to provide some answers to simple questions that the reader may have when dealing with these complex issues but aimed at therapeutic innovation with regard to Malignant Pleural Mesothelioma.

MESOTHELIN

Mesothelin (MSLN) was discovered in 1992 in an effort to find new surface targets for immunotherapy through the application of monoclonal antibodies. 

It is expressed at low levels in healthy mesothelial cells of the pleura, pericardium, and peritoneum, while ideally any neoplastic tissue of MPM might show a notable expression of MSLN.

The physiological role of MSLN in healthy tissues is currently not fully understood. MSLN is initially expressed as a 71-kDa protein, which is then cleaved by furin, causing the release of a 31-kDa protein, called Megakaryocyte Potentiation Factor (MPF), while the remaining 40-kDa fragment stays attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. 

Mesothelin (MSLN) maturation. 

Image taken from an article by Castelletti et al. Biomarker Research (2021)

Surface MSLN may also be released by creating Soluble Mesothelin-Related Peptide (SMRP), which can likewise be detected in the blood of patients affected by MPM.

MSLN has been the focus of immunotherapy research ever since it was discovered. The characteristics that make MSLN an ideal immunotherapeutic target in MPM are many, but they can be summed up as follows:
- a high level of MSLN expression in the cancer tissue and low or no expression in healthy tissue, thus minimizing possible toxicities;
- 85 to 90% of cases in the MPM epithelioid subtype have high MSLN expression [12]; 
- its expression at high levels has been linked to increased aggressiveness and invasiveness.

MSLN is made up as follows.
The extracellular domain of MSLN includes three adjacent elements: 
1) region I (residues 296-390), 
2) region II (391-486), 
3) region III (487-598). 

Region I is the distal part of the membrane and can bind to MUC16 mucin (also known as CA125), which is also expressed by most MPM cells and is linked to neoplastic aggressiveness characteristics. This MSLN-MUC16 interaction has proven to be of great significance for tumor cell adhesion and metastasis and is the primary target of current immunotherapies, including CAR T cell therapy.
 

A model of protein structure of human mesothelin

CAR T CELLS

What are cellular therapies?
Cell therapy uses hematological cells (obtained from blood) genetically modified in the laboratory through specific molecular engineering methods. To proceed with these genetic approaches, highly complex instruments as well dedicated and well-equipped laboratories are needed.The cells, suitably engineered, can be injected into the diseased organism, where they can perform the desired and intended therapeutic action.

What does CAR stand for?
CAR is an acronym used to indicate chimeric antigen receptors, also referred to as chimeric immunoreceptors or chimeric T-cell receptors or engineered T-cell receptors. Essentially, these are receptor proteins designed to give T lymphocytes the new ability to detect a specific protein target. The receptors are called "chimeric" because they combine both antigen-binding and T-cell activation functions in a single receptor.

What are CAR-T cells?
Hence, CAR T cells chimeric antigen receptor cells and are genetically modified T lymphocytes. The main objective of engineering these cells is to produce an engineered T-cell receptor, which is used in the treatment of certain hematological neoplasia. This is, therefore, a powerful example of the clinical efficacy of cell therapies, which produce CAR-T cells "genetically trained" to search for, recognize and eliminate neoplastic cells.

How are CAR-T cells produced?
The production of CAR-T cells is extremely complex.
The first step consists in taking a blood sample from the patient, which is then processed in a lab in order to separate the cellular material (blood cells) from the plasma, using a technique called apheresis. This procedure makes it possible to collect and isolate the patient's lymphocytes. These cells are then sent to laboratories specialized in engineering according to well-defined scientific protocols.

Once the T cells have been isolated, the CAR (Chimeric Antigen Receptor), which is capable of recognizing cancer cells, is introduced. In fact, these T lymphocytes, defined at this point CAR-Ts, express a surface receptor that detects specific antigens expressed by neoplastic cells.

What does CAR-T therapy consist of?
Generally speaking, we can say that CAR-T therapy uses T cells engineered with CAR, the therapeutic approach for some neoplasia. The rationale for this treatment application is the ability of CAR T cells to modify T cells so that they can recognize the tumor and, consequently, attack and fight it in the most effective way possible.

This treatment is completed starting from the patient's blood, which is drawn and processed in the laboratory, leading to the extraction of T cells. These cells, after being suitably isolated, are then processed using a vector, which usually consists of a modified lentivirus, so that they can express a defined CAR that guides them towards a specific tumor antigen. It is crucial that the CAR T cells be designed as being specific for antigens that are typical of neoplastic cells and that are not as present in healthy tissues, in order to develop a treatment that is as effective as possible, but also as non-toxic as possible. After such treatment, the cells thus modified can be reinfused into patients with neoplasia. In particular, CAR T cells can be defined as:

- autologous, if obtained from the patient's own T cells,

- allogenic, if obtained from a healthy donor.

CAR-T cells destroy neoplastic cells through a variety of mechanisms, among these they are able to increase the degree of toxicity (cytotoxicity), they can contribute to increased secretion of factors affecting cytokines, interleukins and growth factors. Precisely because of this action, one the side effects of the therapeutic application of CAR-T cells is the so-called "cytokines storm", which can create serious damage, related to cytokine activation and other factors such as tumor volume and the patient’s specific pathophysiological state. This adverse reaction usually takes place in the first days after therapeutic administration and is often treated with corticosteroids and IL6 inhibitors (tocilizumab).

Image taken from an article by Castelletti et al. Biomarker Research (2021)

CAR-T THERAPY AND MALIGNANT PLEURAL MESOTHELIOMA

CAR-T cells used against malignant pleural mesothelioma are T lymphocytes engineered to target mesothelin.
As we know, CAR-T cell therapy is effective in the treatment of hematologic neoplastic diseases, whilst applications known to date for solid tumors are limited.

In 2019, during the conference held by the American Association for Cancer Research (ASCR), a number of scientists presented encouraging results against this type of thoracic cancer, targeting mesothelin and using CAR-Ts specific for this target. 
Initial results from a phase I study (trial) were presented in March 2019 at the annual meeting of the American Association for Cancer Research (Abstract CT036) and again in the Journal of the American Society of Clinical Oncology (ASCO – Abstract 2511).
Briefly, these were the results of this preliminary study: 21 patients with malignant pleural disease (19 MPM, 1 lung cancer, 1 breast cancer) were treated (40% had received ≥3 lines of prior therapy). 18 patients received preconditioning with cyclophosphamide; the first cohort did not receive cyclophosphamide. CAR T cells  were administered to twelve patients using an interventional radiology procedure. One patient had grade 3 cyclophosphamide-related febrile neutropenia, whereas no CAR T-cell-related toxicities above grade 2 were observed. The last cohort of patients was hospitalized 2 weeks after infusion with a temperature of >38°C and fatigue. Intensive monitoring of toxicity carried out through clinical evaluation (chest or abdominal pain), radiological evaluation (CT/PET or echocardiogram for pericardial effusion, ascites), laboratory evaluation (troponin elevation), and other evaluation (electrocardiogram) documented no toxicity. One patient underwent successful surgical resection for treatment purposes 6 weeks after infusion of CAR T-cells. CAR T cells were detected in the peripheral blood of 13 patients (from day 1 to week 38). T-cell persistence was associated with decreased serum levels of serial soluble MSLN-related peptide (>50% compared with pretreatment) and evidence of tumor regression on imaging studies. Once lack of toxicity was confirmed (6-17 weeks after CAR T-cell infusion), 14 patients were treated with immunotherapy and received anti-PD1 checkpoint blocking agents (1-21 cycles) without toxicity. The best response among the 19 MPM patients (13 patients received an anti-PD1 agent; PD-L1 <10% in all but 1) was achieved by 2 patients who had a complete metabolic response at PET (60 and 32 weeks ongoing); 5 with partial response; and 4 with stable disease.
A few months later, at the European Congress of Medical Oncology (ESMO - Barcelona), the first results of a phase I study using CAR-T cells in three patients with malignant pleural mesothelioma were presented. It was a study limited to very few patients, in fact, conducted on just three patients with malignant pleural mesothelioma, but it represented a new possible path to take against this disease.

Alessandra Curioni Fontecredo, Italian researcher and Head of the Thoracic Tumor Unit at the Department of Oncology and Hematology at the University Hospital of Zurich, talks about her research in this regard and in an interview comments on her study saying: "...we administered CAR-T, developed in partnership with the University of Zurich, to three patients with pleural mesothelioma in a phase I study. The chimeric receptor used, " assembled" on T lymphocytes and able to recognize tumor cells, targets a molecule known as FAP, an acronym for Fibroblast Activating Protein. This molecule is expressed in many epithelial tumors, such as those of the colon or ovary, and is often present in particular in mesotheliomas: it is found in about 80% of cases. The idea of starting precisely from mesotheliomas is that, in this case, researchers can proceed with a local therapy, with the injection of CAR-T cells directly in the thoracic cavity. We can't say anything about the efficacy of the therapy, also because the patients involved in the study underwent chemotherapy before and after the administration of CAR-T cells. However, from a safety standpoint we found no serious side effects or toxicities related to the infused cells. Of the three patients treated, one is alive at one year after the treatment and another at two years. At the moment this study, the first in Europe as regards solid tumors, was stopped, but we are working to optimize the chimeric receptor, for example through the addition of other stimulation molecules, and we hope to start a new trial next year.".

A new therapeutic scenario for malignant pleural mesothelioma has opened up from these insights, and several studies have been proposed in the scientific field in an attempt to confirm this data and elaborate on these findings. Should the reader be interested, please refer to the bibliography provided at the end of this review.

In this bibliographic review we like to mention in particular one of the latest researches on this topic.

The administration was carried out by injecting the therapy through a pleural catheter already in place, or by means of interventional radiology methods.
CAR T cells were detected in peripheral blood for >100 days in 39% of patients.
Previous studies had documented the possibility to combine CAR-T therapy with immunotherapy, already known and widely applied to thoracic neoplasia. More in detail, these experimental studies, conducted on mice, had shown that PD-1 blockade improves the function of CAR T cells in mice. Starting from this preliminary data, a human study was designed to associate pembrolizumab (immunotherapeutic monoclonal antibody) to CAR-T therapy. This trial took place in 18 patients with mesothelioma: among these patients, median overall survival from CAR T cell infusion was 23.9 months (overall survival at 1 year, 83%).

The graph represents the survival of patients with malignant pleural mesothelioma treated in this study.
Stable disease was maintained for ≥6 months in 8 patients; 2 patients showed a complete metabolic response on PET. Therefore, the researchers emphasized in this study how the data supports the study of immunotherapy combined with CAR T cells and PD-1 blocking agents in solid tumors.

The graph represents the outcomes from patients affected by MPM treated in this study
(PR = Partial Response, SD = Stable Disease, PD = Progression Disease.)
The following photographs show practical examples of treatment response in patients with mesothelioma.

Researchers concluded that the local administration CAR T-cell therapy targeting mesothelin followed by the administration of pembrolizumab is feasible, safe, and shows evidence of antitumor efficacy in patients with malignant pleural disease.

CONCLUSIONS

The study of CAR T cells made it possible to analyze their application not only for hematologic diseases, but for solid tumors as well.
Among these, malignant pleural mesothelioma has become a potential target of these treatments.
Specific cells of the immune system suitably engineered, become effective against specific targets and, if these targets are found on the neoplastic cells of malignant pleural mesothelioma, then it is possible to develop well-defined therapeutic strategies.
A new therapeutic approach in this innovative scenario for malignant pleural mesothelioma is represented by the application of CAR T cells. Preliminary results in this regard show interesting transversal implications and present encouraging future prospects.
Further dedicated studies will be able to confirm these researches and eventually bring into clinical practice new therapies for this pleural pathology.

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Is DNA repair a potential target for effective therapies against malignant mesothelioma?

AUTHORS:
Ilaria Fuso Nerini, Elisa Roca, Laura Mannarino, Federica Grosso, Roberta Frapolli, Maurizio D'Incalci

JOURNAL:
Cancer Treat Rev 2020 Nov; 90:102101. doi: 10.1016/j.ctrv.2020.102101. Epub 2020 Aug 25.

BIBLIOGRAPHY:
PMID: 32892058, DOI: 10.1016/j.ctrv.2020.102101

INTRODUCTION

The bibliography review for the latter half of 2020 looks at DNA repair mechanisms as a new potential target for treating malignant pleural mesothelioma (MPM).
DNA damage can be induced by physical and chemical factors, but our bodies can activate repair mechanisms that can overcome and resolve this damage.
Studying and understanding how these mechanisms are activated is of vital importance for us to be able to develop new therapeutic strategies that are more targeted and effective.  
This premise has led to an interest in investigating this topic for a disease whose prognosis remains poor to this day.

This bibliography review was recently published as an overview in a scientific journal, which can be accessed at the following link: https://pubmed.ncbi.nlm.nih.gov/32892058/

Is DNA repair a potential target for effective therapies against malignant mesothelioma?

Malignant pleural mesothelioma (MPM) is a rare malignancy mainly caused by asbestos exposure.

Germinal and acquired mutations in genes of DNA repair pathways, in particular of homologous recombination repair, are frequent in MPM.

As we know, germinal mutations are alterations that occur in the cells that give rise to a new life (germ cells); conversely, acquired mutations occur in any other cell of the body (somatic cells). It is important to remember that only germline mutations can be inherited. There are many ways of repairing DNA damage, and homologous recombination in particular is considered one of the main mechanisms for repairing double-strand breaks. In practice, homologous recombination consists of exchanging the double-strand DNA strands, or segments with an identical or very similar sequence. This exchange allows one double-strand DNA segment to act as a template for repairing lost or damaged information in another double-strand DNA segment. This mechanism repairs these alterations which may frequently occur in DNA replication cycles, and therefore constitutes an essential repair system for all proliferating cells.

The purpose of this review is to explore and report on the experimental data available, which suggest how an altered DNA repair system can affect the pathogenesis of MPM.

Studies about DNA molecule repair systems are particularly important. In 2015, the Nobel Chemistry Prize was awarded to three scientists for their work on these repair mechanisms, namely Sweden’s Tomas Lindahl, Turkish-born Aziz Sancar, and American Paul Modrich. They are very important methods and pathways for cells and are active 24 hours per day! DNA can be subjected to continuous stress from external sources, such as chemical or physical agents, or from the body itself, for example in the case of specific process errors such as DNA duplication. However, cells are equipped with specific systems to repair this damage to prevent dangerous consequences to the cell and the whole body.

Alterations in the DNA repair mechanisms may mean that there are genetic alterations that are in some way involved in this neoplasm.
Specifically, DNA repair defects appear to be a vulnerability or a limitation of MPM cells.

A potential therapeutic strategy for MPM in the future could be the use of targeted drugs to specifically target these DNA repair deficiencies.  

Specifically, these findings may be useful in the future for developing innovative therapies. PARP inhibitors are an example of a therapeutic approach that leverages this hypothesis. These therapies act against tumor cells characterized by defects in the homologous repair process.  PARP inhibitors block the action of Parp enzymes, which are molecules involved in the repair of single-strand DNA breaks. These drugs make it impossible for the neoplastic cells to repair DNA damage, with the resulting increase in double-strand breaks. This results in a continuous loop system because all these alterations that have accumulated in the tumor cells cannot be corrected since the homologous repair system in these cells has been “deactivated”, so to speak, by the drugs. This does not affect healthy cells, in which the mechanism is still active and which therefore survive.

This review confirms how vital it is to continue research to further understand the most deeply-seated mechanisms of MPM in order to find treatments that are more targeted and effective.

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1. Are patients with malignant pleural mesothelioma (MPM) more at risk for contracting COVID 19?

MPM patients are not at greater risk of contracting COVID 19, but like all cancer patients, if they become infected they are at greater risk of serious complications.

2. Which symptoms of MPM could mimic those of COVID 19?

The most common COVID 19 symptoms presenting in patients with thoracic tumors were analyzed in a recently published international study called TERAVOLT¹. The symptoms were fever, dyspnea (difficulty breathing) and cough, all of which are very common symptoms of MPM, particularly the latter two. Pain is not a common symptom of COVID 19, although myalgias (muscle pain) may occur at the onset, which can also affect the chest. The possibility of COVID 19 infection should not be ruled out, particularly if fever is present.

3. Are there data available regarding the prognosis and course of COVID 19 in MPM patients?

The number of MPM patients (8 of 200 total) was very low in the original TERAVOLT study publication, and the data presented referred to the overall population of patients with thoracic cancers so mainly patients with lung cancers. Most of these patients were on active cancer treatment and had concomitant illnesses (comorbidities) besides cancer, mainly hypertension, diabetes, chronic pulmonary disease, cardiovascular disease.  78% of patients in the study required hospitalization, with 8% of cases requiring intensive care. The death rate from COVID 19 in these patients was very high (31%).

Another analysis from the TERAVOLT study was recently presented and included a larger number of patients with less frequent thoracic tumors (microcytoma of the lung, MPM, thymus tumors and even rarer tumors)². This analysis included 22 MPM patients from a total of 581; the mortality rate from COVID 19 in MPM patients was 36%.   

4. What should patients with MPM be aware of with respect to COVID 19?

The data reported from the TERAVOLT study should be interpreted with caution. There were very few MPM patients, and only those patients with symptomatic and severe COVID 19 infection were included. 91% of these patients were in fact hospitalized, including 9% of whom were in intensive care.  Similar to the general population, MPM patients may contract COVID 19 in a milder or asymptomatic form; however, there is a high possibility of complications so this situation must not be underestimated and should be closely monitored together with timely treatments prescribed by the attending physicians (oncologists and general practitioners).

5. Are there any special precautions that MPM patients should take to prevent COVID 19 compared to the general population?

MPM patients must strictly observe all the regulations for preventing the spread of COVID 19 (social distancing, use of masks in closed spaces and in open spaces if an adequate distance cannot be maintained, frequent handwashing). Patients should consult their doctors for evaluation if their symptoms worsen, particularly if they have a fever, and very importantly if they know they have been in contact with anyone infected with COVID 19.

6. Can treatments for MPM change if patients become infected with COVID 19?

The results from the TERAVOLT study on thoracic tumors and other similar studies on cancer in general (COVID19 and Cancer Consortium – CCC19³ and UK coronavirus Cancer Monitoring Project Team⁴) have not shown a negative impact from COVID 19 on the prognosis of patients if they have been administered oncology treatments during the previous weeks. Obviously, treatments should be suspended until the infection has been resolved in the case of full-blown COVID 19.  In other words, the available data thus far suggest that there is no reason to suspend or discontinue cancer therapy for fear of contracting COVID 19, but it is obviously a different matter if there is active COVID 19 infection.

7. Could there be a delay in cancer diagnosis, such as MPM, due to the COVID 19 pandemic?

Unfortunately, the pandemic has resulted in the slowdown or halting of diagnostic procedures (such as invasive and/or surgical procedures, and also other procedures) for several months, especially in the most affected areas of Italy. Moreover, the fear of contracting COVID 19 in a hospital setting has probably led to many patients postponing their appointments at diagnostic procedure facilities. These two factors together may have led to delays in diagnosis at that stage.  Given the current situation of Italian hospitals, these delays are no longer warranted and patients should not be afraid of consulting qualified hospitals for a timely diagnosis of MPM or other cancers.

8. What advice can be given to MPM patients who are afraid of contracting COVID 19 or whom we suspect may have become infected?

As already mentioned above, MPM patients and their family members and contacts must carefully observe all the regulations for preventing the spread of COVID 19 (social distancing, use of masks in closed spaces and in open spaces if an adequate distance cannot be maintained, frequent handwashing). Patients should immediately consult their doctors for an evaluation if their symptoms worsen, particularly if they have a fever or other typical symptoms (for example, changes in taste and smell, muscle pain, intestinal pain and diarrhea), and very importantly if they know they have been in contact with anyone infected with COVID 19.

ESSENTIAL BIBLIOGRAPHY

1. Garassino et al., Lancet Oncol 2020;

2. Cortellini et al., presentation at AACR Virtual Meeting, COVID19 and Cancer, July 2020;

3. Kuderer et al., Lancet 2020; 4. Lee et al., Lancet 2020.

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DEFINITION

HMGB1 is an acronym that stands for High Mobility Group Box 1. It is a protein that belongs to the high mobility group family, in other words, high electrophoretic mobility proteins.
("Electrophoretic mobility" can be defined as a measurement of the ability of a chemical substance to move when subjected to an electric field, and usually depends on various parameters such as the charge, size, conformation characteristics, the voltage applied to the field and the concentration of
the electrophoretic medium).
This protein is also known as amphoterin or HMG1, and is a non-histone scaffold protein of chromatin.
HMGB1 also belongs to a subfamily of proteins that contain a domain involved in DNA binding, namely HMG-box.
Below is a three-dimensional image of the structure of this protein.

(from PDB: Protein Data Bank. https://www.rcsb.org/structure/1aab)

The HMGB1 gene is located on the long arm of chromasome 13 13q12.
The figure below shows the 5 exons of the HMGB1 gene in the form of small parallelepipedshollow for translated regions and solid for non-translated regions).
Part (B) of the figure below shows the 215 amino acid residues and is composed of three domains: A box, B box and an acidic C-terminal tail. There are three cysteine residues at positions 23, 45 and 106, which regulate HMGB1 function in response to oxidative stress.
Part C of the figure shows HMGB1, which is loosely and transiently associated with nucleosomes. HMGB1 is important for spatial segregation and nuclear homeostatis.

(from He SJ, et al. Oncotarget. 2017)

In general, HMGB1 is ubiquitously expressed (only 10 times less than core histones). However, HMGB1 expression and subcellular localization varies depending on cell types and tissues and are developmentally regulated to cues from the environment.

(from He SJ, et al. Oncotarget. 2017)

MAIN FUNCTIONS

HMGB1 is found in large quantities within the nucleus of all eukaryotic cells and its main role is to remodel chromatin.
It has also recently been discovered that this protein is an important mediator in the inflammation process, especially in the case of cellular necrosis. As such, HMGB plays an important role in triggering inflammation but it also appears to be involved in innate and adaptive responses and in
repairing tissue damage.
Cells undergoing stress actively secrete HMGB1, which is relocated from the nucelus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space.

(from Bianchi ME, et al. Immunol Rev. 2017)

The HMGB1 protein can be passively released from dead cells, as shown on the right in the figure.
In other cases, it is actively secreted as a result of cellular stress, as shown on the left.
Under normal conditions, this protein is located in the nucleus in a reduced and unacetylated form.
Following tissue damage, this unmodified protein is released from dead cells and subsequently converted into the disulfide-HMGB1 form by spontaneous oxidation, or through reactive oxygen species (ROS), which are produced in abundance by inflammatory cells.
Leukocytes can also secrete HMGB1, which is first released into the cytoplasm and subsequently acetylated or phosphorylated, and then passes into the extracellular space after being transformed.
This often occurs after it has been loaded into secretory lysosomes or through a little-known mechanism into non-hematopoietic cells.
Secreted HMGB1 can be distinguished from passively released HMGB1 (in yellow), due to the acetylated state, shown in green in the figure. The secreted form is also oxidized.
The figure at left shows the pathway of LPS- and interferon-induced HMGB1 secretions after a bacterial or viral infection.
Immune cells are first recruited to the damaged tissue site and are then activitated after arriving at the site.
The HMGB1 protein supports tissue repair by coordinating the switch of macrophages to a tissuehealing phenotype, activation and profileration of stem cells, and neoangiogenesis. Unfortunately,
this protein similarly helps repair tissue of all damaged cells, including tumor cells.
 

(from Bianchi ME, et al. Immunol Rev. 2017)

The figure above shows the role of HMGB in tissue repair.
As shown, this protein plays an important role during muscle injury.
HMGB1 is released from damaged or necrotic muscle cells. Under these conditions, this protein can also promote the recruitment of immune system cells such as leukocytes by forming heterocomplexes with CXCL12. Inflammation occurs when the leukocytes arrive at the damaged tissue site. HMGB1 is then oxidized to disulfide through free oxygen radicals, which are formed after infiltration by the leukocytes. HMGB1 also activates the leukocytes to promote the release of a series of pro-inflammatory cytokines and chemokines, but loses its ability to form heterocomplexes
with CXCL12.
After resolving the inflammatory state, the tissue-healing phenotype macrophages release HMGB1, which can activate stem cells and promote angiogenesis as well as coordinate muscle injury repair.

HMGB1 AND CANCER

The HMGB1 protein appears to play an important role in cancer progression.
The figure below shows how HMGB1 can interact biologically to promote carcinogenesis.
In the extracellular space, as shown in part A, HMGB1 signals through receptors such as RAGE, TLRs, TIM3 and CXCR4, driving cell proliferation, invasion and angiogenesis, metastasis, apoptosis evasion, inflammation and immunity. The interaction between HMGB1 and CXCR4 is
dependent on CXCL12. TLR9 is initially localized on the endoplasmic reticulum (ER) and then redistributes to endosomes upon stimulation with CpG-DNA via an HMGB1-dependent pathway.
In part B, HMGB1 is present at the cell surface and promotes cell migration and tumor-cell metastasis.
In the cytoplasm, as shown in part C of the figure, HMGB1 regulates autophagy and promotes cell proliferation.
In the nucleus, as shown in part D, HMGB1 acts as a DNA chaperone participating in DNA repair and transcription. HMGB1 can also interact with transcription factors such as p53, p73 and RB and enhance their activities. Nuclear HMGB1 enhances telomerase activity and modulates telomere
homeostatis.

(from He SJ, et al. Oncotarget. 2017)

HMGB1 AND MALIGNANT PLEURAL MESOTHELIOMA

HMGB1 is highly involved in tumor biology.
HMGB1 appears to be associated with mesothelioma. In this neoplasm, asbestos causes inflammation of the mesothelium but the biomolecular pathways underlying this inflammatoryneoplastic process are not completely known.
However, it was recently discovered that asbestos induces the death of mesothelial cells by necrosis,
with the resulting release of HMGB1 into the extracellular space and recall of inflammatory cells.
The persistence of the asbestos fibers is one of the main causes of the perpetuation of inflammation in the pleura and often the lungs of subjects who have been exposed to this carcinogen and then develop MPM. High levels of HMGB1 have been seen in the blood of subjects exposed to asbestos
as well as those with MPM, demonstrating that this protein could be involved in these carcinogenesis and inflammation processes. Here too, it is not fully understood how persistent inflammation can induce carcinogenesis but some studies suggest that macrophages play a role in cell survival, demonstrated by the fact that macrophages have been found in abundance in neoplastic tissue.

The figure below shows the pro- or anti-tumoral activity of HMGB1.
In part A, mesothelial cells are damaged by exposure to asbestos and induce programmed necrotic cell death, resulting in the release of HMGB1.
Part B shows the pro-tumoral activity of HMGB1, which bends TLR4 and produces a state of chronic inflammation leading to malignant transformation. Macrophages are present in the mesothelial tissue and HMGB1 is constitutively secreted by the mesothelioma cells.
Part C shows the anti-tumoral characteristics of this protein. The question mark shown in the figure means that the mechanisms involving HMGB1 and the pathogenesis of mesothelioma have never beeen fully investigated. However, the activity of this protein against various tumors has been widely documented. HMGB1 is secreted by the cells and probably involved in the mechanisms that lead to the responses of B- and T-cells to immunological memory.

(from Bianchi ME, et al. Immunol Rev. 2017)

CONCLUSIONS

Further research into this protein will lead to a greater understanding of the pathogenesis of cancer.
Many researchers are specifically trying to determine the actual rol of HMGB1 in MPM to better define the carcinogenesis of this disease.
Future research also aims to design therapeutic approaches that may involve this protein or pathways activated by it.

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INTRODUCTION

Like most neoplastic diseases, malignant pleural mesothelioma (MPM) requires a great deal of radiological evaluation both at the time of diagnosis and for monitoring patient response during therapy.
For this reason, the FBU also wished to be involved in this area of diagnosis and treatment of MPM and has initiated a project to fund a radiologist specializing in this field, currently under way at the UFIM of Alessandria / Casale Monferrato.
This month's bibliography review provides an informative and simplified overview of the use of radiology in this disease.
(Please see the bibliography for further information or for the "experts in the field".)

USE OF RADIOLOGY

Chest X-rays

Radiological techniques allow us to determine pleural alterations and their characteristics, such as the presence of thickening and pleural plaques, their pattern of distribution, and the eventual presence of pleural effusion. Radiological analysis can be used to arrive at the diagnosis as well as for staging the disease (1).
The initial examination is usually a standard chest X-ray, but this is not always conclusive, especially if the presence of pleural lesions is suspected.

Computed tomography
Because a chest X-Ray does not allow for a detailed examination of suspected pleural lesions, the first real in-depth examination for this disease should be computed tomography (CT). According to the AIOM national guidelines, the data obtained by CT analysis have demonstrated a specificity of 78% (95% CI, 72%-84%), but a sensitivity of only 68% (CI 95%, 62%-75%). This is necessary particularly in the differential diagnosis of pleural effusion with a negative CT scan that is negative for pleural lesions, in case you want to exclude the diagnosis of malignant disease.
Consequently, this often means subjecting the patient to an invasive diagnostic procedure, such as thoracentesis or a pleural biopsy. In these cases, the decision should be based on the clinical data rather than the negative CT scan (2).
If the thoracic CT scan shows evidence of MPM, the examination should be extended to the abdomen to exclude any secondary disease in the abdominal organs and the peritoneum in particular.

Ultrasonography

A simple ultrasound is one of the possible ultrasonography approaches to MPM, allowing us to analyze both the presence of pleural fluid and any parietal lesions. Ultrasound may also be used together with color Doppler or contrast media (CEUS). As such, ultrasonography allows us to easily identify pleurisy and pleural thickening and also determine any suspicious lesions due to malignancy based on their vascularization (3).

Magnetic resonance

Several studies have shown that nuclear magnetic resonance imaging (MRI) appears to be superior to CT in differentiating between benign and malignant pleural thickening, and particularly in assessing the possible infiltration of the chest wall and diaphragm (4). However, it is important to point out that the introduction of new generation and increasingly sophisticated TC equipment has greatly reduced this discrepancy. MRI could therefore be useful mainly to further the CT findings, particularly as an additional examination before performing an intervention. Preliminary studies also suggest the possibility of using MRI with special techniques, such as diffusion-weighted imaging (DWI), to assess the histology of patients with pleural mesothelioma using the apparent diffusion coefficient (ADC) (5). However, although the results are promising these methods are currently experimental.

Positron emission tomography

18-FDG PET-CT has been studied because it is a technique that helps to distinguish between benign and malignant pleural lesions (6). It is also used in the clinic for staging, in other words to identify metastatic sites not shown by other radiological procedures.
This metabolic method has demonstrated greater sensitivity, specificity and accuracy in lymph node staging (7). However, the reliability of the method is limited due to the possibility of false negatives (especially in the presence of micrometastases <4 mm) and false positives (very often related to non-necrotizing granulomatous reactions) (8). However, the gold standard for the most accurate pleural staging remains the thoracoscopy, as suggested by at least one study which compared metabolic imaging with this procedure (9).
A total body 18-FDG PET-CT is recommended for the staging of patients eligible for multimodal treatment due to its greater accuracy in extra-thoracic and lymphatic staging compared to a CT scan. The optimal timing for performing this procedure is before conducting any invasive procedures such as pleurodesis due to the risk of subsequent false positive results due to the procedure (10, 11). Precisely because of the above limitations, the use of this method for evaluating response to treatment is still being studied and it is not recommended for routine use (12).
Metabolic assessments could be used not only in the diagnosis and staging of the disease but also for monitoring the malignant lesions during antiblastic therapy. In fact, a recent study suggests that there is a possible role for metabolic imaging to identify the non-responders among patients with stable disease according to mRECIST criteria. In this subgroup of patients, a ≥ 25% increase of SUVmax compared to baseline was associated with a statistically significant reduction in median time to progression (10.0 vs 13.7 months, p <0.001). (13)

RECIST criteria

The radiological criteria usually evaluated are known as RECIST (Response Evaluation Criteria in Solid Tumor), which were updated and published in 2004 ("modified RECIST"). However, the use of these criteria for evaluating response in mesothelioma is rather complex. The modified RECIST1 system published in 2004 allows for more accurate measurements. Even though this has led to an improvement over the initial RECIST criteria, the rate of variability and inaccuracy of the measures remains very high. It is important to point out that compliance of the radiology specialists with the correct method greatly influences the evaluation of disease response to treatment.
Currently the modified RECIST criteria are based on the CT measurement of the thickness of the neoplasm perpendicular to the chest wall or the mediastinum at three different levels, so as to take into account the irregularity of the tumor (Tables 1 and 2) (14). These criteria are the diagnostic standard, since the response evaluated with these tools has shown a statistically significant correlation with overall survival and respiratory function.

Studying volumetric variation using CT is a promising approach in this area, considering also the potential correlation with survival, if analyzed together with several clinical parameters (15).
An article was also recently published proposing further modifications to the modified RECIST version 1.0, with a recommendation to adopt a new RECIST version 1.1 (16). Specifically, the continuous updating of the RECIST criteria evaluates different approaches that are reflected in clinical practice. The main ones are as follows:

• Definition of measurable lesions
• Evaluation of non-pleural lesions
• Characterization of non-measurable pleural disease
• Definition of pathological lymph nodes
• Definition of disease progression

CONCLUSIONS

Radiology plays a fundamental role for malignant pleural mesothelioma and is useful for diagnosing, staging and, more importantly, for monitoring the disease during specific antiblastic treatment.
However, continuous updates are needed specifically in this area and the role of the radiology specialist in this field is increasingly necessary.
The FBU, which has always been involved in the diagnosis and treatment of MPM, also wished to contribute to this sector by funding a radiology specialist dedicated to this neoplasm.

BIBLIOGRAPHY

1. Surea B, Thukral BB, Mittal MK, Mittal A, Sinha M. Radiological review of pleural tumors. Indina J Radiol Imaging. 2013;23:313-20
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3. Sartori S, Postorivo S, Vede FD, Ermili F, Tassinari D, Tombesi P. Contrast-enhanced ultrasonography in peripheral lung consolidations: what’s its actual role? World J radiol. 2013;5:372-80
4. Gill RR, Gerbaudo VH, Jacobson FL, Trotman-Dickenson B, Matsuoka S, Hunsaker A, Sugarbaker DJ, Hatabu H. MR imaging of benign and malignant pleural disease. Magn Reson Imaging Clin N Am; 16(2008) 319-339
5. Gill RR, Umeoka S, Mamata H, Tilleman TR, Stanwell P, Woodhams R, Padera RF, Sugarbaker Dj, Habau H. Diffusion-weighted MRI of malignant pleural mesothelioma: preliminary assessment of apparent diffusion coefficient in histologic subtypes. AJR Am J Roentgenol 2010;195(2):W125-30
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7. Zahid I, Sharif S, Routledge T, Scarci M. What is the best way to diagnose and stage malignant pleural mesothelioma? Interact Cardiovasc Thorac Surg. 2011;12:254-9
Sørensen JB1, Ravn J, Loft A, Brenøe J, Berthelsen AK for the Nordic Mesothelioma Group. Preoperative staging of mesothelioma by 18F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography fused imaging and mediastinoscopy compared to pathological findings after extrapleural pneumonectomy. Eur. J. Cardiothorac. Surg. 2008;34: 1090-6
9. Pinelli V, Roca E, Lucchini S, et al. Positron emission tomography/computed tomography for the pleural staging of malignant pleural mesothelioma: how accurate is it?. Respiration 2015;89:558-64
10. Murray JG, Erasmus JJ, Bahtiarian EA, Goodman PC. Talc pleurodesis simulating pleural metastases on 18Ffluorodeoxyglucose positron emission tomography. AJR Am J Roentgenol 1997; 168:359-60
11. Nguyen NC, Tran I, Hueser CN, et al. F-18 FDG PET/CT characterization of talc pleurodesis induced pleural changes over time: a retrospective study. Clin Nucl Med 2009;34:886-90
12. Schaefer NG, Veit-Heibach P, Soyka JD, et al. Continued pemetrexed and platin-based chemotherapy in patients with malignant pleural mesothelioma (MPM): value of 18F-FDG.PET/CT.Eur J Radiol 2012;81:e19-25
13. Kanemura S, Kuribayashi K, Funaguchi N, et al. Metabolic response assessment with 18F-FDG PET/CT is superior to modified RECIST for the evaluation of response to platinum-based doublet chemotherapy in malignant pleural mesothelioma. Eur J Radiol 2017;86:92-98
14. Byrne M.J., Nowak A.K.. Modified RECIST criteria for assessment of response inmalignant pleural mesothelioma, Ann. Oncol. 15 (2004) 257–260
15. Labby ZE, Nowak KA, Dignam JJ, Straus C, Kindler HL, Armato III SG. Disease volumes as a marker for patient response in malignant pleural mesothelioma. Ann Oncol 2013;24(4):999-1005
16. Armato SG 3rd, Nowak AK. Revised modified response evaluation criteria in solid tumors for assessment of response in malignant pleural mesothelioma (version 1.1). J Thorac Oncol. 2018;13:1012–1021.

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Neoplastic pathologies are treated using different approaches depending on the stage of the disease; hence, there are early-stage tumours attacked by radical therapies, such as, for example, surgery and radiotherapy, and there are advanced-stage or metastatic tumours which are usually treated with palliative therapies, such as chemotherapy, immunotherapy or radiotherapy.
Malignant Pleural Mesothelioma (MPM) generally has an unlucky prognosis, and it is usually diagnosed at a late phase, when the disease is already in an advanced stage and cannot be subjected to radical treatment through a surgical approach. Therefore, most patients affected by MPM are treated with systemic chemotherapy or experimental protocols, which have nevertheless a merely palliative purpose, in addition to aiming at making the disease a chronic one for as long as possible.
However, radiotherapy also plays a role in the treatment of MPM, and can be used in different settings and even for opposite purposes.
This bibliographic review aims to evaluate the last studies published in scientific reviews which investigate the role of radiotherapy in the treatment of MPM.

Introduction

MPM is considered to be a radiotherapy-resistant neoplasia. Indeed, several studies have shown minimum benefits of this approach combined with the other standard treatments usually implemented in this area(1) . Moreover, very often the use of radiotherapy is postponed due to the significant toxicity which this treatment can have on the lung itself.
In fact, a common collateral effect is represented by post-actinic pneumonias(2). Already back in 1991, studies were published highlighting the pulmonary toxicity caused by radiotherapy applied at the level of the entire hemi-thorax: this data underscores how this treatment entails a moderate-severe and irreversible pulmonary damage in most patients subjected to radiotherapy(3).
Therefore, radiotherapy has been mainly considered within the setting of palliative treatments, such as, for example, pain management(4).
However, recent publications have determined that this recommendation should be changed as it has been observed that patients subjected to radiotherapy for palliative purposes also showed a good response depending on the administration of specific dosages(5).
In vitro studies were also conducted assessing the usefulness of radiotherapy on the cell lines of malignant mesothelioma(6). This data described the cellular sensitivity of mesothelioma to radiotherapy treatments, and it was shown that after two dosages of 2 Gy of radiation were administered, about 60-80% of the mesothelioma cells suffered a damage resulting in the inability to generate new colonies(7).
With regards to this, experiments were also carried out on cell lines of murine models, which have demonstrated cellular survival in only 10% of the cases after 5 Gy of radiotherapy were administered(8).
It is interesting to observe that some authors also proved the existence of a systemic activation and an increase in the proliferation of immune system cells (T-cells), in murine models in which cells previously irradiated in vitro with 5-15 Gy were injected under the skin9 .
If this research conducted on murine cells is applied to human cells, we can observe that sensitivity to radiotherapy is closely dependent upon the histological sub-type of the MPM. For instance, administering 25 Gy in vitro determines an increase in the release of damage signals, such as, for example, HMGB1 (High Mobility Group box 1), especially in the case of epithelioid MPA cells and not of sarcomatoid MPA cells. Since the release of HMGB1 is considered a pro-inflammatory factor able to activate dendritic cells and induce an immunotherapy response against the tumour, the absence of HMGB1 in sarcomatoid MPM might explain the different sensitivity of the two histological subtypes to radiotherapy(10).
Medicines that act on the cell cycle could increase sensibility to radiotherapy, and this was tested on MPM cell lines MPM(11 12 13). Indeed, the radiotherapy treatment causes damage at the DNA level through the generation of oxygen free radicals, consequently causing the cells to die(14 15). Treatments that inhibit DNA repair or increase death of the cells therefore work in synergy with the radiotherapy.
In this case as well, however, the data is inconsistent since this was observed above all in the epithelioid MPM cell lines and not in sarcomatoid MPM cell lines. This difference may be tied to the fact that sarcomatoid tumours have such a resilience that allows them to better defend themselves against the DNA damage due to the sarcomatoid histotypes. Another explanation for the resistance of the sarcomatoid phenotype seems to be tied to the influence of the expression and of the release of fibroblasts growth factor(16).

Prophylactic radiotherapy

In the case of surgery or surgical diagnostic procedures, there may be a dissemination of tumour cells at the level of the site where said approach was implemented.
The risk of local dissemination during a biopsy increases with the size of the procedure and ranges from 10% in case of transparietal biopsy, 13% in case of pleuroscopy, up to 26% in case a thoracotomy is performed(17).
The metastasised tract may cause serious problems not only related to the worsening of the stage and of the prognosis, but also in terms of quality of life, since it can be the cause of several symptoms, the most frequent of which is pain.
For this reason, prophylactic therapy plays a role in these patients, since it reduces the risk of metastasis resulting from these diagnostic or therapeutic procedures.
The initial data pertaining to these findings was published in 1995; the authors proved that a reduction in this dissemination could be tied to the reduced angiogenesis and to the diminished release in tumour growth factors which occur thanks to the use of radiotherapy treatment(18).
Nevertheless, this data is inconsistent, since other studies have not confirmed this research and, on the contrary, have shown a difference in terms of reduction of the risk of metastasis after an invasive procedure, between the group of patients treated with radiotherapy and the control group(19 20 21 22 23 34 25 26).

Palliative radiotherapy

We speak of palliative radiotherapy for patients suffering from MPM when the treatment is aimed, for example, at the management of pain, of dysphagia, of obstruction of the upper respiratory tract and at relief of vena cava compression(27). In fact, the radiotherapy treatment is able to bring about an improvement in severe symptoms for the patient and, although this setting does not entail an increase in survival, it is nevertheless an effective method for improving the quality of life.
Actually, radiotherapy can be used for palliative purposes also for the remote treatment of metastasis, such as, for example, bone or encephalic lesions. In this case, radiotherapy aims at blocking the secondary lesion, improving any symptoms that may be present, as wells as at reducing any symptoms and signs that may develop in the future due to these metastases, and that may lead to a further worsening of the patient’s quality of life(28 29 30 31).
In fact, it has been demonstrated that, especially for non-sarcomatoid histology, palliative radiotherapy was useful and that sensitivity to radiotherapy was correlated to an improvement of the patient’s overall conditions and, consequently, a clear clinical benefit(32 33 34).

Adjuvant radiotherapy

Adjuvant radiotherapy is the treatment administered after surgery as an “adjuvant” to the surgical approach in order to obtain the best outcome.
The Memorial Sloan Kettering Cancer Center (MSKCC, New York City, NY, USA) was a pioneer in the study of this technique(35 36 37 38 39 40).
The radiotherapy approach may also be used intra-surgery. However, it was demonstrated that this option is not an effective treatment and, moreover may cause severe complications such as pleural empyema(41 42 43).
The use of hemithoracic adjuvant radiotherapy at high dosages seems to entail a reduction in the risk of relapse; in fact, some authors claim that with this treatment, the risk of locoregional relapse is approximately 15%(44 45 46).
Intensity-modulated radiotherapy (IMRT) has also been used as hemithoracic therapy at high dosages, and it has shown to provide a benefit in terms of relapse reduction, especially when applied at the level of the base of the ribcage, in areas with a higher risk of a relapse of the locoregional disease(47 48 49).
It is important to remember that these treatments are not risk-free. For example, the literature includes cases of lethal contralateral pneumonia, especially after use of radiotherapy after an extrapleural pneumonectomy(50 51 52). For this reason, a crucial factor is the proper dosage of the radiotherapy in addition to a careful evaluation of the patient’s respiratory functionality(53 54 55).
Other authors, on the other hand, have demonstrated how radiotherapy, and in particular the modulated-intensity type, may be an approach that is easily tolerated by patient and featuring a not-so-high risk of post-actinic pneumonias(56 57 58 59 60 61 62).
Moreover, a more innovative technique would allow a further reduction in the risk of complications: this innovative technique is called VMAT (Volumetric-Modulated Arc Therapy)(63 64 65).
Increased control over the disease has been described in patients subjected to adjuvant radiotherapy(66 67).
With these purposes, new randomised and prospective studies on the association between chemotherapy and adjuvant radiotherapy may possibly provide data demonstrating greater efficacy(68 69 70 71 72 73 74). Indeed, chemotherapy may reduce the risk of a relapse while radiotherapy may increase local control(75 76 77 78 79 80 81 82 83 84 85 86).

Induction radiotherapy

Trimodal therapy consists of the application of chemotherapy, followed by extrapleural pneumonectomy and by hemithoracic adjuvant radiotherapy at high dosages.
Use of the therapy in this setting resulted in better survival and local control of the disease(87). On the basis of this research, we can hypothesise that radiotherapy can also be applied at the early stages of MPM, rather than using chemotherapy: this therapy setting is known as "induction" radiotherapy(88 89).
In studies on the application of radiotherapy in this setting, the surgical complications which occurred after the induction radiotherapy treatment were similar to those recorded after the adjuvant chemotherapy treatment(90).
In this case too, the patients with epithelioid MPM had a better outcome compared to those affected by the other histological type(91).
Several studies have underscored how the tumour volume is correlated with the outcome and how it could also be a guiding factor in the choice of whether or not to subject a patient to induction therapy(92 93 94 95). On the contrary, the same data cannot be applied to the response to chemotherapy, which instead does not depend on the volume of the neoplasias, as it happens with radiotherapy and surgery(96). This data indicates that patients with localised disease may be good candidates for treatment with induction radiotherapy, whilst those with a large-sized tumour would benefit instead from chemotherapy(97).

Conclusions

Future prospects include the possibility to combine standard treatments for MPM, such as radiotherapy, and immune-system treatments(98 99 100). In the past fifteen years, the role of radiotherapy in the treatment of MPM has grown, also supported by scientific evidence demonstrated in various treatment settings.
However, the real benefit of this method has yet to be completely defined for this type of neoplasia, but new and innovative approaches, such as IMRT at the pleural level and induction-accelerated hemithoracic radiotherapy performed after surgery may very well be feasible and well-tolerated.
New studies will make it possible to provide an answer these still unanswered questions.

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INTRODUCTION

A major change has been brought to thoracic oncology by the advent of biological therapies, and by “targeted” treatments in particular. These new approaches have changed the natural course of disease in patients with lung cancer, particularly those whose lung cancer is characterized by specific genetic mutations and rearrangements who may benefit from therapies that target these biomolecular alterations.
Among these drugs, molecules acting against multiple molecular targets have also been investigated, particularly Vargatef, which is a mult-target TKI that inhibits the VEGFR, FGFR and PDGFR receptors. It is currently approved in combination with docetaxel for advanced/metastatic lung adenocarcinoma after front-line chemotherapy. The scientific data for this treatment in lung cancer has shown benefit compared to standard chemotherapy alone in terms of progression-free survival (PFS) (4,0 vs 2,8) and overall survival (OS) (12,6 vs 10,3), with a reduction of 17% in the risk of death and a disease control rate of 60,2% (vs 44,0%). An analysis of the European population of the Lume-Lung 1 study showed a further increase in survival (13,4 vs 8,7).
This treatment was therefore considered as potentially useful also for MPM, an aggressive disease with a poor prognosis.
The purpose of this bibliographic review is to provide preliminary data on the use of nintedanib in mesothelioma.

Malignant pleural mesothelioma (MPM) is a cancerous disease that is no longer rare and unfortunately is very aggressive: if it is not treated, the average survival is 6-9 months.(1-4)
The standard first-line treatment for unresectable MPM is cisplatin and pemetrexed chemotherapy.(5-9) Although this therapy helps increase median survival, it is still only reaches approximately one year so it is essential that new first-line treatments are developed.(10-13)
One approach to fulfil this goal is the investigation of antiangiogenics.(14-22)

Specifically, the effectiveness of bevacizumab in combination with standard treatment as demonstrated in the MAPS study (Mesothelioma Avastin Cisplatin Pemetrexed Study), has led to renewed interest in inhibiting VEGF (vascular endothelial growth factor) as a therapeutic approach. In fact, many signaling molecules, which are involved in the angiogenesis regulation processes, are involved in both the pathogenesis and the prognosis of MPM.(23,24) The VEGF pathway therefore plays a key role in regulating angiogenesis and consequently tumor growth, and is an important factor that can induce the proliferation of MPM cells.(25) MPM patients also have high blood levels of VEGF, which is considered a negative prognostic factor.25
In the MAPS study, survival was shown to be significantly greater in MPM patients treated with cisplatin and pemetrexed plus bevacizumab, compared to those who received chemotherapy alone (18.8 months [confidence interval 95% (CI), 15.9-22.6] vs 16.1 months [95% CI 14.0-17.9]; hazard ratio [HR], 0.77 [95% CI 0.62-0.95]; P =.0167).(26) Up until then, no therapy had ever been shown to increase survival in MPM since the approval of pemetrexed by the US Food and Drug Administration in 2004. These results show that VEGF could be an effective approach as a therapeutic target.
One such drug is nintedanib, which acts on different signal transduction pathways that are involved in the pathogenesis of MPM. Specifically, the VEGF receptor is one of the targets of nintedanib and so this molecule is also considered an angiogenesis inhibitor.
The Phase III portion of the global Phase II/III LUME-Meso is evaluating the safety and efficacy of nintedanib in combination with pemetrexed and cisplatin in patients with unresectable epithelioid MPM.
Initially, this was a randomized, double-blind exploratory Phase II study, which was amended to include a confirmatory Phase III study, following an evaluation by an Internal Data Monitoring Committee after they had reviewed the Phase II results.
The Phase III study will enroll 450 patients not previously treated with chemotherapy, who will be randomized to receive pemetrexed/cisplatin on Day 1 and nintedanib or placebo from Day 2 to 21 for up to 6 cycles. Patients without progressive disease who are eligible to continue treatment in the study may receive maintenance treatment with nintedanib or placebo until disease progression or excessive toxicity. The primary endpoint of the study is progression-free survival (PFS); the key secondary endpoint is overall survival (OS). The study will also include interim analyses to ensure it is adequately powered for the statistical survival analyses. The study is currently enrolling patients.

THE DRUG

Nintedanib is a tyrosine kinase inhibitor (TKI) that targets three growth factor receptors: the vascular endothelial growth factor receptor (VEGFR), the fibroblast growth factor receptor (FGFR), and the platelet-derived growth factor receptor (PDGFR), as well as other targets such as FLT3, RET, Abl and Src tyrosine-protein kinase signaling.(27,28)

This drug is also indicated for idiopathic pulmonary fibrosis (IPF), which is a non-cancerous disease characterized by slow progression and fatal.(29-31)
It is associated with dyspnea, cough and reduced quality of life. Currently, the objectives of patient care include improving outcomes, obtained by slowing disease progression, increasing life expectancy and improving the quality of life.(32-34) Timely and accurate diagnosis is important so that patients can be treated in the early stages of the disease and also so they can be considered for a lung transplant.(35)
The conditional recommendation of nintedanib for IPF is based on the results of the TOMORROW and INPULSIS studies, which showed that nintedanib 150 mg administered twice daily lowered the annual rate of change in FVC compared to patients who received placebo.(36-41) The INPULSIS study results showed that nintedanib was consistently effective across the various subgroups defined by different characteristics such as age (<65 vs ≥65 years), race (Caucasian vs Asian), predicted FVC % (≤70% vs >70%; ≤80% vs >80%, <90% vs >90%), predicted DLCO % (>40% vs ≤40%), and several other diagnostic criteria (such as the presence of honeycombing on high-resolution CT and or confirmation of the presence of UIP by biopsy vs the potential presence of UIP with traction bronchiectasis detected by high-resolution CT, without biopsy evaluation).(42-46)
The TOMORROW and INPULSIS studies also showed that nintedanib reduced exacerbations and mortality due to IPF vs. placebo.(47)
Regarding the tolerability profile, adverse events occurred in more than 10% of patients treated with nintedanib. These adverse events included diarrhea, nausea, abdominal pain, vomiting, and raised liver enzymes, which occurred more frequently in patients receiving nintedanib than placebo.(48,49) For the most part, the adverse events were managed by reducing the dose of the drug or discontinuing treatment.(50) Results from INPULSIS_ON, the ongoing extension of the INPULSIS study, confirm the good tolerability profile of nintedanib and its effectiveness in lowering FVC for over three years.(51)
In oncology, nintedanib (BIBF 1120) in combination with docetaxel was approved by the European Medicines Agency (EMA) for use in the European Union and several other countries for the treatment of locally advanced, metastatic or locally recurrent NSCLC after first-line chemotherapy.(52)
It can also be given in combination with various anti-neoplastic treatments due to the efficacy and good safety profile it has shown when used for the treatment of different types of tumors.(27)
The ability of nintedanib to act on the three major pro-antiangiogenic signaling pathways (VEGF, PDGF and FGF) may offer greater clinical benefit to patients with unresectable MPM than that obtained with agents that target other known anti-angiogenic targets. Nintedanib also inhibits Src,(27) a molecule that plays an important role in several neoplastic pathways and is involved in the pathogenesis of mesothelioma. Inhibition of Src has also been proposed as a therapeutic target for MPM.(53) In preclinical studies, nintedanib reduced the growth and ability of MPM cell lines to metastasize and increased survival in an orthotopic xenograft model of MPM. It is therefore considered a valid candidate for the treatment of unresectable MPM.(54)

THE STUDY

LUME-Meso is a randomized, double-blind, placebo-controlled Phase II/III study.
The study compares the efficacy of nintedanib in combination with backbone pemetrexed+cisplatin chemotherapy followed by maintenance treatment with nintedanib, vs. placebo in combination with pemetrexed+cisplatin followed by placebo monotherapy in patients with unresectable MPM.
The study was initially an exploratory randomized, double-blind Phase II study only, but it was enlarged to include a confirmatory Phase III following the recommendation of an Internal Data Monitoring Committee after a review of the Phase II results.
After completing enrollment in the Phase II study, the results for the primary endpoint of PFS were presented.(55) This led to nintedanib being granted Orphan Drug Designation by the US FDA on 12 December 2016. The Phase III study is currently enrolling and the data will be reviewed by an independent committee.

Study Purpose and Design and Treatment Regimen

The purpose of the study is to evaluate the tolerability and efficacy of backbone chemotherapy in combination with nintedanib followed by maintenance therapy with nintedanib, vs. backbone chemotherapy of cisplatin+pemetrexed in combination with placebo, followed by placebo monotherapy, as first-line treatment of unresectable MPM.

Patients are randomized 1:1 to receive pemetrexed (500 mg/m2)/cisplatin (75 mg/m2) on Day 1 for up to 6 cycles, in combination with nintedanib (200 mg twice per day) or placebo (twice per day) from Day 2 to Day 21. Patients will subsequently receive maintenance therapy with nintedanib or placebo until evidence of progressive disease (PD), the development of severe toxicity, withdrawal of consent or death. Patients who, in the opinion of the investigator, may derive a clinical benefit from continuing treatment after disease progression may continue treatment with nintedanib/placebo.
Based on the results of the Phase II study, which included patients with epithelioid and biphasic MPM histologies, the Phase III will enroll 450 patients with epithelioid MPM only.
Figure 1. Design of the Phase III LUME-Meso trial∗Nintedanib is administered from Day 2 to 21; ¶Cisplatin 75 mg/m2 intravenously for 2 hours on Day 1 of each cycle, for a duration of 21 days, for up to 6 cycles; §Pemetrexed 500 mg/m2 intravenously for 10 minutes on Day 1 of each cycle, for a duration of 21 days, for up to 6 cycles; &lowest;∗Treatment after progression is permitted if clinical benefit is anticipated.
Abbreviations: b.i.d. = twice daily; I.V. = intravenous; MPM = malignant pleural mesothelioma; OS = overall survival; PD = Progressive disease; PFS = progression-free survival.

Inclusion criteria

The study is conducted in accordance with the Helsinki Declaration and following approval by the Ethics Committee.
All patients must provide their informed consent in writing. The Phase III study is currently enrolling patients with histologically confirmed unresectable epithelioid MPM. Although patients eligible for radical resection or elective surgery (e.g., pleurectomy) are not eligible for enrollment, prior surgery, if performed at least 4 weeks before randomization, is permitted if the patient is fully healed and still has measurable disease.

Study Endpoint

The primary endpoint is PFS, while OS is the main secondary endpoint. Other secondary endpoints include overall response rate (ORR) and the percentage of patients with a complete or partial response, or stable disease control rate (DCR), as measured by modified RECIST Criteria.(56) Another objective of the Phase III study includes evaluating the quality of life associated with health status as measured by the EuroQoL-5 self-evaluation questionnaire and the Lung Cancer Symptom Scale (LCSS-Meso) for mesothelioma.(57,58)
Biological samples will be tested by immunohistochemical or molecular genetic analysis to determine the value of markers such as mesothelin, merlin protein (produced by the NF2 gene) and protein 1 associated with BRCA1, as predictive or prognostic factors.
During the study, tolerability of the treatment will be monitored by evaluating variations in the laboratory parameters and the frequency and severity of adverse events in accordance with the CTCAE criteria (Common Terminology Criteria for Adverse Events version 4.03), established by National Cancer Institute (NCI). .(59)

CONCLUSION

As always, the main objective of the study is to increase survival and improve the quality of life.
The study of the new therapy aims to achieve these objectives, although the research requires time in order to validate the data before the results can be applied in clinical practice.
The Phase II/III LUME-Meso study will determine whether nintedanib in combination with the backbone of cisplatin+pemetrexed can provide clinical benefit to patients.
The Phase III study is currently underway, and eligible patients with unresectable MPM are being enrolled at sites in North and South America, Europe, Africa, Australia and Asia.

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Foreword

New findings about the interaction between the immune system and the immune microenvironment have led to the development of important checkpoint inhibitors drugs for cancer. More specifically, these drugs target the PD-1/PD-L1 pathway.

One of these drugs, pembrolizumab, has been the subject of many articles and press releases.
It has even been hailed by some as the “syrup that dissolves tumors”, but this assertion needs to be clarified!
The press often goes beyond its own expertise by describing some treatments as miraculous but without having any knowledge of the drugs in question and without any actual rigorous documentation about the scientific studies published.
By doing this, the concerns of patients and their loved ones are not taken into account and we become caught in a vortex of words that do not contain any actual knowledge.

In view of this, we thought it would be useful to describe the main studies involving this therapy in our bi-annual review of the literature.
We will attempt to evaluate how this drug could be useful for the treatment of malignant pleural mesothelioma (MPM), and will describe as simply and graphically as possible some of the other immunotherapies that have been investigated or are being studied for MPM.
For the "insiders" who are involved in the study of MPM and for further information, please see the Reference section at the end of this revision for a list of publications.

Introduction

Immune checkpoint inhibitors

To prevent autoimmune phenomena, the activation and functions of T lymphocytes are finely regulated to different levels through “control points”, known as immune checkpoints.
Cytotoxic T lymphocytes associated with the antigen 4 (CTLA-4) and "programmed death 1" (PD-1) play an essential role in this process. Specifically, CTLA-4 (CD152) is an immunosuppressive receptor and a member of the superfamily of immunoglobulin CD28/B7, which is expressed mainly in the CD4 T lymphocytes and to a lesser extent in the antigen-presenting cells (APC) and granulocytes(1) . The recruitment of CTLA-4 decreases the amplitude of the T cell response: it competes with its costimulatory CD28 receptor by inhibiting B7-1 (CD80) and B7-2 (CD86). These interactions are critically important for the initial activation of naïve T cells, by inhibiting the function of the T cells and preventing an inappropriate immune response against the “self” (or the body’s own) antigens in the secondary lymph organs and by limiting the extent and duration of the immune response(2) .

On the other hand, the PD-1 pathway regulates the effector T cells in the later stages of the immune response in peripheral tissues(3) .
PD-1 is expressed primarily in activated T and B cells, but also in monocytes, natural killer cells and tumor-infiltrating lymphocytes (TILS) (4) .
PD-1 binds two molecules, PD-L1 and PD-L2, which are members of the B7 family. PD-1 is expressed in leukocytes, while PD-2 only in dendritic cells and monocytes. PD-1 directly inhibits the functions of the TRC-mediated effector, acting as a negative regulator of the immune response. Additionally, PD-L1 is highly expressed in many malignancies, including MPM (5). PD-L1 expression in tumor cells appears to attenuate the immune response against tumors by inhibiting the activation of T cells and increasing the apoptosis of antigen-specific cell clones (6 7) .
CTLA-4 plays an important role in regulating the suppressive function of regulatory T lymphocytes (Treg), which are usually found in tumor tissue and are thought to locally inhibit antitumor immunity by inhibiting the response of effector T cells (8 9) .
In this context, by using a model of human suppressor T cell lines (MT-2), it has recently been shown that exposure to asbestos increases the function of Tregs with an increase in the production of suppressive cytokines such as IL-10 and TGFb (10).
In some tumors, the CTLA-4 and PD-1 inhibitory pathways are activated much more than in physiological conditions, so they are therefore believed to be involved in the suppression of the immune response by the tumor and in the ability to escape recognition by the immune system.
Various therapeutic approaches targeting CTLA-4 and PD-1/PD-L1 have therefore been designed over the last few years.

Anti CTLA-4

The efficacy of inhibiting CTLA-4 in combination with chemotherapy or radiotherapy in MPM has been shown in several in vivo and animal model studies (11 12 13).
In fact, several mouse models of mesothelioma treated with an anti-CTLA-4 monoclonal antibody have demonstrated a significant inhibition of tumor growth, inhibition of repopulation by cancer cells, and an increase in T infiltrating lymphocytes (14).

Anti PD-1

PD-L1 expression has been reported in 40% of patients with mesothelioma and appears to be associated with a poor prognosis (15 16 17) . However, it is important to remember that guidelines and standard methods for investigating these targets and determining the cut-off, i.e, the thresholds indicating the positivity or negativity of this alteration, have not yet been defined 18. Another aspect still being investigated is clarifying which cell types should be evaluated for PD-L1 expression (19 20) .

Pembrolizumab

Pembrolizumab (MK-3475, lambrolizumab, Keytruda®) is a highly selective humanized IgG4 monoclonal antibody against PD-1. The amplified regulation of PD-1 occurs in some tumors and the pathway that is activated through this molecule helps to inhibit the active T cells as immune surveillance against tumors.
Pembrolizumab is able to destroy the bond that occurs between PD-1 and its PD-L1 and PD-L2 ligands, thus blocking the inhibitory signal to the T cells. By so doing, the cancer cells can be recognized by the cytotoxic T cells(21) .
This drug was recently approved for solid tumors such as non-small cell lung cancer (NSCLC) expressing PD-L1, and advanced unresectable melanoma (22 23).
The diagram below shows the immune system activation against tumor cells and regulation of the signal occurring between PD-1 and its PD-L1-PD-L2 ligands.

(From: S. Karim, et al. Future Oncol. 2016)

Efficacy of pembrolizumab

The efficacy of this drug has been recently documented in various clinical studies in patients with solid tumors of different histologies.
The Phase I KEYNOTE-001 study is evaluating the efficacy and safety of pembrolizumab in patients with NSCLC. The results of this study showed an objective response rate (ORR) of 19.4% with a median duration of response of 12.5 months, progression-free survival (PFS) of about 3.7 months, and a median overall survival (OS) of 12 months . An analysis of the results showed an association between the efficacy of pembrolizumab and the presence of PD-L1 expression. Specifically, in patients with expression of PD-L1>/= 50%, the ORR was 45.2%, together with greater PFS and OS rates versus those patients with lower PD -L1 expression(25) .
Other studies have been initiated to assess the efficacy of the pembrolizumab, which for the sake of simplicity are listed in the following table:

(From: S. Karim, et al. Future Oncol. 2016)

(From: S. Karim, et al. Future Oncol. 2016)

(From: S. Karim, et al. Future Oncol. 2016)

Safety of pembrolizumab

The safety and side effects of pembrolizumab were also evaluated in the above-mentioned KEYNOTE-001 study. We can say that pembrolizumab is generally well tolerated, without any clear evidence of adverse effects associated with it.
However, the most frequently reported side effects were asthenia, pruritus and decreased appetite.
The most serious grade 3 adverse events reported in a small percentage of patients (9.5%) were mainly dyspnea (3.8%), pneumonia (1.8%), decreased appetite (1%) and asthenia (1%) (26).
Some side effects involving the immune-mediated response were also reported. More specifically, there were cases of hypothyroidism (6.9%), pneumonia (3.6%) and drug infusion reactions (3%) (27).

Pembrolizumab and mesothelioma

The first data from the KEYNOTE-028 study of pembrolizumab in MPM were presented at the American Association for Cancer Research (AACR) conference.
Data from the study entitled "Single-agent pembrolizumab for patients with malignant pleural mesothelioma” were presented at the International Association for the Study of Lung Cancer (IASLC), World Conference on Lung Cancer (WCLC), in September 2015.
For completeness, the original abstract is included below:

“Single-agent pembrolizumab for patients with malignant pleural mesothelioma”
EW Alley, JH Schellens, A Santoro, K Beckey, SS Yuan, J Cheng, B Piperdi, LR Molife

Summary: Researchers presented updated safety and efficacy data for pembrolizumab (MK-3475), currently approved for the the treatment of advanced melanoma that progressed after ipilimumab, and BRAF inhibitor therapy if BRFV600 mutant in patients with PD-L1-positive advanced solid tumors with malignant pleural mesothelioma (MPM) enrolled in the KEYNOTE-028 study. In patients with PD-L1—positive MPM, pembrolizumab had significant clinical activity, they concluded. Further study of the durability of responses and the 49.4% 6-month progression-free survival rate is warranted.

Methods:

• For this nonrandomized, multicohort phase 1b study, researchers included patients with measurable disease who had failed standard therapy, had ECOG PS 0-1, adequate organ function, and no autoimmune or interstitial lung disease.
• They defined PD-L1 positivity as expression in ≥1% of tumor cells by IHC at a central laboratory.
• Patients were treated with pembrolizumab (10 mg/kg) every 2 weeks for up to 2 years, or until confirmed progression or unacceptable toxicity.
• Every 8 weeks for the first 6 months and every 12 weeks thereafter, researchers assessed response using RECIST v1.1.
• The primary endpoint was overall response rate (ORR) and secondary endpoints included safety, tolerability, and progression free survival (PFS).

Results:

• In all, 84 patients with MPM were screened for PD-L1 expression; of these, 38 (45%) had PD-L1—positive tumors; and of these, 25 were included in the study.
• As of March 20, 2015, the ORR was 28% (n=7), and 12 patients (48%) had stable disease, for a disease control rate of 76%.
• In 15 patients with only one previous line of therapy, ORR was 20% and DCR was 73%.
• Researchers observed durable responses, with 10 (40%) of patients remaining on treatment.
• After a median follow-up of 8.6 months, median PFS is 5.5 months (95% CI, 3.4-NR) and the 6-month PFS was 49.4%.
• Researchers observed no new safety signals.
• Drug-related adverse events (DRAE) occurred in 15 patients (60%), including 3 (12%) who had grade 3-4 DRAEs.
• Only two patients required dose interruption due to immune-related adverse events, which included transaminitis and uveitis (1 each).
• No treatment-related mortality occurred, and no patients discontinued treatment due to DRAEs.

The study design displayed below shows that pembrolizumab was administered to patients with advanced MPM who had progressed after prior treatment or who could not be treated with standard chemotherapy.
These patients had to be in overall good condition (ECOG PS 0-1) and PD-L1 positive. Patients with autoimmune diseases or interstitial lung disease were excluded.
Other baseline characteristics of the patients are summarized in the figure below:

(From WCLC, September 7, 2015, Denver, Colorado, USA)

Patients received pembrolizumab intravenously at a dose of 10 mg/kg bi-weekly.
Response to treatment was assessed every eight weeks for the first six months and then every 12 weeks thereafter. If patients achieved a partial or complete response, they were treated for 24 months or until disease progression or the emergence of toxicity. Treatment with pembrolizumab was discontinued if patients experienced disease progression or unacceptable toxicity

(From WCLC, September 7, 2015, Denver, Colorado, USA)

Below is an example of microscope images showing the negative or positive PD-L1 expression of this molecule.

(From WCLC, September 7, 2015, Denver, Colorado, USA)

Evaluated by immunohistochemistry, the PD-L1 levels do not appear to be correlated with the ability to respond better to treatment with pembrolizumab, as shown in this graph:

(From WCLC, September 7, 2015, Denver, Colorado, USA)

The drug was generally well tolerated, as evidenced by the low rate of serious side effects.
The most common adverse events were asthenia (24%) and nausea (24%); however, these side effects were reported in less than 20% of patients. Grade 3 side effects included an increase in transaminases (ALT) and thrombocytopenia. There were no patient discontinuations in the study due to pembrolizumab-related side effects, nor were there any drug-related deaths.
Below is a summary of the drug-related side effects:

(From WCLC, September 7, 2015, Denver, Colorado, USA)

The anti-tumor activity of this treatment was as follows:

(From WCLC, September 7, 2015, Denver, Colorado, USA)

The PFS was approximately 5.8 months. Additionally, the PFS after six months of treatment was approximately 50%.

(From WCLC, September 7, 2015, Denver, Colorado, USA)

The results of this study are encouraging, but more studies are needed to confirm whether this treatment can actually increase the survival of these patients.

Other immunotherapy drugs

The Phase II study MEST-TREM-2008 (NCT01649024) is investigating tremelimumab, an anti-CTLA4 human monoclonal antibody, as monotherapy in patients with advanced MPM that has progressed after standard first-line chemotherapy. The results of this study can be summarized as follows: no patients achieved a complete response, while 31% had stable disease; the median overall survival was 10.7 months(28) .
The Phase II study MEST-TREM-2012 (NCT01655888) evaluated a higher dose of tremelimumab, which led to the following results: a disease control rate of 52% was achieved, with a median overall survival of 10.9 months (29 30) .
An international, randomized, double-blind, placebo-controlled Phase IIb study is currently underway, DETERMINE (NCT01843374), in patients with advanced pleural or peritoneal mesothelioma who have progressed after a standard first-line therapy (31) .
Emerging immunotherapy findings have led to other study protocols, such as the Phase II study NIBIT-mESO-1 (NCT02588131), which is evaluating the efficacy of tremelimumab in combination with anti-PD-L1 durvalumab (32) .
The KEYNOTE-028 study (NCT02054806) investigated the efficacy of PD-1 antibody pembrolizumab in PD-L1-positive patients (33 34) .
The NCT02399371 study is also evaluating pembrolizumab in patients with MPM and the role of PD-L1 positivity (35) .
Additionally, pembrolizumab was recently investigated in combination with gemcitabine and defactinib (NCT02546531).
Another drug, nivolumab, was recently tested in MPM in the NivoMes study (NCT02497508), which enrolled patients with recurrent mesothelioma, and in a study investigating the combination of ipilimumab and nivolumab (NCT02716272), in patients with unresectable MPM (36) .
Avelumab is another PD-L1 inhibitor that was investigated for this disease, which resulted in a partial response of 15%, disease control rate of 60% and PFS of approximately 16.3 months (37) .

Future prospects

There are still many unresolved issues, and many aspects need to be clarified in order to better understand the results of these clinical trials. Of particular importance is the definition of a specific, sensitive biomarker to serve as a predictor of response because the association between PD-L1 expression and the response to immunotherapy remains controversial (39 40) .
It is important to remember, however, that there is no standardized method to test for this biomarker and a cut-off threshold to precisely determine positivity has not yet been defined (41).
In some MPM cases, the mutation burden was associated with an increase in the neo-antigen burden and inhibition of PD-1 (42 43) .
Consequently, mutation burden and gene instability are aspects of the pathogenesis of MPM that need to be studied further in order to fully understand this disease and define the most effective drugs.
Although these immunotherapies appear to be very well tolerated, it should also be remembered that the toxicity of these drugs, particularly in the long-term, is not yet well known. Although rare, there may also be autoimmune side effects that could even be lethal (44).

Conclusions

In conclusion, the immune checkpoint blockade appears to be an attractive and certainly promising therapeutic strategy for the treatment of malignant pleural mesothelioma.
However, the results obtained to date are preliminary and many studies are still underway; as such, we are looking forward to the final results.
Additionally, drug combinations acting on different pathogenetic pathways related to immunotherapy must still be investigated.
The discovery of new, more sensitive markers that are predictive of response to these treatments will certainly be useful to identify patients who will truly benefit from these therapies.

References

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Introduction

Here we are once again to discuss scientific information and to disclose news about Malignant Pleural Mesothelioma (MPM).
At this time, we deem it useful to involve the readers in a new consensus conference, in other words in a meeting of experts which resulted in the drafting an update of the state-of-the-art of this topic and in the collection of recommendations on how to deal with this disease.
We are talking about the Third Italian Consensus Conference that took place in Bari back in January 2015, with the support of AIOM (Italian Association of Medical Oncology).
The resulting publication, the tangible output of the work carried out by experts on MPM, is available online at the following link: http://www.ncbi.nlm.nih.gov/pubmed/?term=Mezzapelle+et+al%2C+Sci+Rep+6%3A+22850.
This article is organised into nine different chapters: Introduction, Methods, Epidemiology, Diagnosis, Radiological Check-ups, Surgery, Radiotherapy, Chemotherapy, Psycho-social and legal aspects and future prospects.
This review contains a collection of the main references and an outline summary of the main concepts of this Consensus Conference. For more in-depth information and more specific details, please refer to the full text.

State-of-the-art and recommendations for Malignant Pleural Mesothelioma, according to Italian experts

Epidemiological data

In 2011, the incidence of MPM in Italy was 3.49 and 1.25 cases every 100,000 people/year, respectively, for men and women. A total of 1428 cases were reported: 1035 men and 393 women (Anon, 2015). The national incidence and mortality are currently stable, and there seems to be a one-plateau trend. However, it is believed that a peak will take place in 2020-2025, especially in industrialised countries.
As everyone knows, exposure to asbestos is strictly related to the incidence of MPM, and there is a veritable dose-response relationship (Prince, 2005; Mastrangelo, 2014).
Cumulative exposure to asbestos is an indicator that takes into account the sum of the exposure and is used in various research fields. However, it fails to consider important data such as the duration or intensity of the exposure itself (Thomas, 2013; Lubin, 2006; Vlandereen, 2013; Richardson, 2012).
Nevertheless, we can safely say that, with regards to cumulative exposure to asbestos, there does not seem to be a large difference compared to the previously published Consensus Conferences (Pinto, 2011; Pinto, 2013).
Exposure to asbestos can be of the occupational type, in other words tied to the work which the patient carries out or has carried out in the past, or non-occupational, tied above all to atmospheric and domestic pollution. In Italy, it is estimated that non-occupational exposure takes place in about 10.2% of the cases (National Mesothelioma Register 2012).
Asbestos can also spread through the air under the form of fibres. The WHO (World Health Organisation) has estimated that continuous exposure to 0.4-1 fibre/l can lead to the risk of becoming ill with MPM in 0.4-0.5 cases out of 100,000 people (World Health Organisation Regional Office for Europe, 2000).
Moreover, asbestos can also be found in water; however, there is no evidence of cases of mesothelioma caused by the ingestion of fibres.
There are documented cases of asbestos-containing talc, although this has never happened on Italian soil (Finkelstein, 2012).
Other carcinogenic substances tied to asbestos that have been the cause of MPM in Italy are fluoro-edenite, as in the area of Biancavilla (CT). These cases are similar to those described in Japan, in volcanic areas.
Although there is data of cancerogenesis only in test animals and no cases of MPM were ever described in man, silica carbide is also a carcinogenic agent that can potential cause this disease (Grosse, 2014).
There are also hereditary cases of MPM, associated with genetic alterations such as, for example, the mutations of BAP1 (Klerk, 2013; Betti, 2015. Some of these have also been described in Italy (Ascoli, 2007; Ascoli, 2014).

Diagnosis

Oftentimes, MPM manifests itself with a pleural effusion; however, this collection of fluid in the pleural cavity can also be a secondary effect of different medical conditions. Therefore, it is important to first of all proceed with a differential diagnosis between primitive tumour of the pleura, MPM, and other neoplasias that may metastasize at the pleural level, the most common ones being lung, breast and kidney neoplasias (Smith, 2014). Moreover, it is important to remember that many non-neoplastic pathologies are included within the differential diagnosis which may however cause pleural effusion: for example, infectious or inflammatory pleurisies, cardiac failure, parapneumonic effusion.
The diagnosis of MPM is carried out above all through the analysis of a pleural biopsy, which is often obtained by means of a thoracoscopy or, more rarely, by means of an eco or TC guided percutaneous biopsy (Pinto, 2013; Scherpereel, 2010; van Zandwijk, 2013).
In addition to the histological analysis carried out on the tissue obtained through a biopsy, it is also possibly to conduct a cytological analysis, evaluating the cells found in the pleural fluid. In some cases, this may allow the diagnosis to be made; however, this is exam is not as sensitive as histology (Kawai, 2014; Paintal, 2013; Hjerpe, 2015, Henderson, 2013).
In accordance with WHO, a histological classification of MPM has been defined, according to which tMPM can be subdivided into the epithelioid, sarcomatoid and biphasic histotype (Larsen, 2013).
Different markers are used to better define these tumour characteristics and, in particular to differentiate pleural metastasis from adenocarcinoma and primitive lesions caused by MPM (Ordonez, 2013; Betta, 2012).
The following markers are more commonly used to differentiate epithelioid MPM from adenocarcinomas: calretinin, D2-40 (anti-podoplanin antibody), the protein of Wilms-1 tumour, cytokeratin 5 and 6, mesothelin and thrombomodulin. Markers which are considered negative are; CEA, BerEP4, MOC-31, claudin-4 and CD155 (Henderson, 2013b; Lonardi, 2011, Jo, 2014). Napsin A, TTF1, CDX2, PAX-8, apocrine markers and hormonal receptors are instead useful for differentiating MPM from other localised metastasis at the pleural level. The marker BAP1 has been recently tested to differentiate benign mesothelial lesions from malignant ones (Cigognetti, 2015).
Sarcomatoid MPM expresses above all markers such as pan-cytokeratin, vimentin, smooth muscle differentiation markers, D2-40, calretinin (Pinto, 2013; Ordonez, 2013; Scherpereel, 2010; Churg, 2015; Henderson, 2013b).
Other useful markers for the diagnosis of MPM are mesothelin-related peptides (SMRP), osteopontin, and fibulin-3 (Lao, 2014; Creaney, 2011; Hollevoet 2011; Hollevoet, 2010; Luo, 2010; Wheatley-Price, 2010; Creaney 2014a; Franceschini, 2014).

Radiological tests

There are different radiological methods mainly used to diagnose MPM (Hallifax, 2015). The first approach usually takes place through a chest X-ray, which normally allows to identify the presence of a pleural or pericardial effusion or even very extended pleural lesions.
The chest TC, on the other hand, is considered a second-choice exam that allows one to obtain much more detailed morphological information compared to the chest X-ray.
The ultrasound scan can be useful to visualise some specific pleural anomalies, in addition to being used as a guide for conducting a thoracentecis procedure and, if necessary, as a guide for pleural biopsies.
The PET scan can be applied above all to evaluate the metabolism of some lesions; no changes have occurred since the previous Consensus Conference (Pinto, 2013).
As regards invasive diagnostic procedures, there are no changes in indications and recommendations compared to the ones described in the previous experts’ review (Pinto, 2013).
Thoracentesis is still the first minimally invasive diagnostic approach, and cytological analysis can be useful to diagnose the presence of malignant cells in about 60% of the cases. The thoracentesis procedure applied under ultrasound guidance can be useful to minimise any complications that may arise (Hooper, 2010).
Ultrasound and CT-guided biopsy have permanently replaced biopsies done blindly, and they are useful for performing accurate biopsies of lesions, irregularities or pleural thickening (Maskell, 2003; Qureshi, 2006; Adamset, 2001; Metintas, 2010a).
Thoracoscopy is the “gold standard” invasive diagnostic technique, and allows to obtain a diagnosis in 90% of the cases (Churg, 2014; Boutin, 1993; Hansen, 1998; Galbis, 2011; Brimset, 2012).
The Endo-Bronchial UltraSound (EBUS) technique, used to analyses lymph nodes that drain neoplastic cells deriving from MPM, can offer certain advantages with respect to the mediastinoscopy (Rice, 2009; Tournoy, 2008; Zielinski, 2010; Richards, 2010).
All of the radiological techniques used for diagnostic purposes also play a crucial role in establishing the stage the disease is in, thus allowing, in addition to the determination of the prognosis, a definition of the therapeutic approaches, which obviously change depending on the stage of the disease.
The methods mostly used for this purpose are still the CT scan and the PET scan (Truong, 2013a; Nickellet, 2014; Basu, 2011; Erasmus, 2005; Rice, 2009; Flores, 2003; Sørensen, 2008; Truong, 2013b; Armato, 2013; Frauenfelder, 2011; Labby, 2013a; Labby, 2013b; Byrne, 2004).

Therapeutic approaches

Surgery
Surgery plays a role in the diagnostic approach since, through invasive methods, including the ones described above, it can be extremely useful for obtaining histological material (Greillier, 2007; Buenoet, 2004; Attanoos, 2008 ).
Surgery is also employed in the treatment of malignant pleural effusion. In fact, in addition to being used for diagnostic purposes, thoracoscopy can also be for medical purposes since it allows the intrapleural administration of talc for the purpose of obtaining a pleurodesis. In the same way, specific surgical drainage methods can be applied for each clinical case (Waller, 1995; Halstead 2005; Martin-Ucar, 2001; Nakas, 2008).
Of course, the role of surgery in the treatment of MPM aims at the complete resection of the disease. It is important to remember that this is possible only in those cases where the MPM is resectable and, consequently, it can only be used in the earlier stages (Rice, 2011 Aug; Gomez, 2014; Treasure, 2014; Flores Pass, 2008; Lang-Lazdunski, 2012; Taioli, 2015; (Cao, 2014; Sugarbaker, 2014; Nakas, 2012 ).
(For specific recommendations and detailed indications of surgery in MPM, please refer to the full text of the Consensus Conference.)

Radiotherapy
In the past, radiotherapy was used to treat the progress of the section used for access of thoracoscopy or pleural drainages. In fact, it was believed that irradiating this tract would decrease the possibility of developing metastasis along the anatomical course of the optic instrument or of the drainage used during invasive procedures. However, studies are contradictory and, as of today, there is no evidence such as to suggest stopping the use of radiotherapy (Boutin, 1995; Bydder, 2004; O’Rourke, 2007).
There is no random data in support of the usefulness of adjuvant therapy for MPM. Nevertheless, it is believed that a total dosage of 54 Gy may be associated to a reduced failure of the local treatment (Rusch, 2001). Different studies have compared radiotherapy technique with modulated intensity with standard radiotherapy. However, effective radiotherapy would seem to be the one applied to the entire hemi-thorax concerned by the disease (Forster, 2003; Rice, 2007; Stahel, 2014). At present, there is some preliminary data available on the potential use of radiotherapy with modulated intensity, used to spare the lung contained in the hemi-thorax affected by MPM (Rosenzweig, 2012; Minatel, 2014; Chance, 2015).
Palliative radiotherapy is certainly crucial for controlling the symptoms and, in particular, for pain management (Bissett, 1991; Lindén, 1996; MacLeod, 2015).
(For specific recommendations and detailed indications of radiotherapy in MPM, please refer to the full text of the Consensus Conference.)

Chemotherapy
Standard chemotherapy indications were widely described in the previous Consensus Conference (Pinto, 2013).
Nevertheless, please remember that the first therapeutic line of this disease is based on the administration of a combination of platinum salts and third-generation antifolates (Fennell, 2008; Muers, 2008; Vogelzang, 2003; Van Meerbeeck, 2005; Santoro, 2008; van den Bogaert, 2006; Buikhuisen, 2013, Anon, 2016; Ceresoli, 2008; Ceresoli 2014).
New knowledge aimed at understanding the pathogenic ways of this disease have allowed the identification of new therapeutic targets, including of the biological type (Kindler, 2012; Zalcman, 2010, Zalcman, 2015; Hassan, 2014).
The second-line therapy has not allowed a clear improvement in terms of survival compared to the support therapy only, although certain standard drugs, such as Pemetrexed, have contributed favourable data in terms of better objective response and control of the disease rate (Jassem, 2008; Ceresoli, 2014). There are no pharmaceutical agents approved for second-line therapy, consequently the possibility to enrol patients in clinical studies may be considered a good treatment opportunity.
There is still no confirmed scientific evidence concerning the use of second-line biological drugs (Ceresoli, 2014; Krug, 2015; Calabrò, 2013; Anon, 2016; Alley, 2015; Ceresoli, 2011; Bearz, 2012; Zucali, 2012).

Conclusions

Unfortunately, the efficacy of current therapies for MPM is still quite limited, and the prognosis of this disease remains regrettably negative.
New therapeutic approaches are needed, and the research conducted in this area is offering interesting results that require confirming, randomised, multicentric and reproducible studies.
In the meantime, it is useful to continue with a strict surveillance of the subjects at risks and, consequently, there is some advice that can be easily applied.
In fact, the surveillance programmes being implemented are aimed at different objectives, such as:

• informing subjects exposed to asbestos of the possible risks associated with it, both in terms of present exposure and past exposure;
• informing the relatives of subjects exposed to asbestos and the possible risk for these individuals, even though they may have not been directly exposed;
• carefully reconstructing the patient’s work history, especially entering into details of exposure to carcinogenic substances, its duration and intensity;
• arranging for spreading information concerning diagnostic and therapeutic instruments and the medical prospects available abroad as well;
• providing support for claims in order to obtain payments and compensations;
• conduct proper counselling aimed at getting people to stop smoking cigarettes and follow a proper and healthy lifestyle.

Future therapeutic prospects are just around the corner, and a series of research projects underway offer new hope for the treatment of this pathology.

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Introduction

Tumors have developed multiple mechanisms to avoid destruction by the immune system(1).
There are, in fact, various inhibitory pathways in the immune system that allow this complex system to tolerate cells and antigens physiologically present in the body, and not to mount an excessive immune response. These inhibitory pathways are essential because they allow the immune T-cells to block the growth of cancer cells; however, tumors use mechanisms to escape the control of the immune system, preventing the T-cells from using their cytotoxic activity against the tumor. Both the innate and adaptive immune responses can act against tumors.
A deficiency in cytotoxic T lymphocytes and natural killer cells can, in fact, increase tumor incidence.
One of the mechanisms by which tumors can escape the immune system is the expression of ligands that inhibit T-cell expression, such as CTLA-4 (cytotoxic T-lymphocyte antigen 4), PD-L1 (programmed death-ligand 1) and PD-1 (programmed death-1)(2). Malignant Pleural Mesothelioma (MPM) is considered an “inflammatory” tumor because it is often characterized by a prominent infiltration of lymphocytes, macrophages and T-cells(3 4).
The continuous, chronic inflammation of the mesothelial cells helps at first to promote, progress and transform these healthy cells into cancer cells. Similarly, the escape mechanisms from the immune system allow the tumor to evade the immune response from the host.
The role of the immune system in the biogenesis of MPM is also complex and multifaceted and appears to involve both the innate and adaptive immune responses.(5 6) This is why research is being conducted in immune mechanisms that allow tumors to grow by escaping the body’s control, with the goal of identifying suitable targets for an effective immunotherapy for MPM.
PD-L1 expression in MPM appears to be associated with a greater extent of disease at the time of presentation and with a greater incidence of sarcomatoid histology.(7) This could be a plausible explanation for the poor prognosis seen in these cases.
Immunotherapy represents a new frontier in the treatment of cancer. The significant progress made in our understanding of the immune system has led to the development of new molecules that can increase the immune response of patients. Regardless of their genetic or metabolic disorders, many cancer patients may benefit from treatment because it is the immune response itself of the patient that is targeted and not the cancer cell.

Immune Check-Points

The term immune-checkpoints refers to a series of inhibitory pathways in the immune system that are crucial for maintaining self-tolerance and preventing the excessive, prolonged and potentially harmful activity of T-cells in peripheral tissues.(8)
It is now clear that tumors can use these immune-checkpoints to evade the antitumor immune response, such as through the loss of expression of tumor-associated antigens (TAA) and/or major histocompatibility complex (MHC) antigens, or through the production of cytokines and the expression of new, inhibitory membrane molecules.
This continuous molecular remodeling phenomenon is known as “cancer immunoediting”, and consists of three main, consecutive phases:
- Elimination (complete destruction of the tumor cells by the host’s immune system)
- Equilibrium (tumor cells selected by the T-cells become resistant to the immune system)
- Escape (the tumor cells give rise to clinically detectable lesions)(9)
The immune-checkpoints currently known to be involved in the development of lung cancer are the cytotoxic T-lymphocyte antigen-4 receptor (CTLA-4) and the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1).

Cytotoxic T lymphocyte antigen-4

Because immune-checkpoints are activated in most cases by a ligand-receptor interaction, they can be inhibited by antagonist antibodies or recombinant forms of ligands/receptors.
The CTLA-4 receptor, known also as CD152, is a member of the immunoglobulin (Ig) superfamily that is expressed on cytotoxic T lymphocytes.(10 11 12) After binding with one of its ligands, B7-1 or B7-2 expressed on the antigen-presenting cells (APC), it transmits an inhibitory signal to the lymphocyte, thus helping to homeostatically regulate the immune response.(13)
CTLA-4 plays a vital role in the maintenance of immune tolerance to the tumor.(14) Specifically, the CTLA-4 receptor acts on the CD80 and CD86 costimulatory immune signals activated by the antigen-presenting cells, so they increase the activation threshold of the T lymphocytes. The systemic administration of anti-CTLA-4 inhibitors as monotherapy or in combination with other therapeutic cancer vaccines has been shown to induce a regression of melanoma and colon cancer in murine models.(15 16)
In a Phase II study evaluating the activity of an anti-CTLA-4 antibody (tremelimumab) that enrolled 29 patients with chemotherapy-resistant MPM (28 pleural and 1 peritoneal)(17 18 19), objective clinical responses were observed only in 29 patients, however, stable disease was seen in 9 patients, or approximately 31% of patients with epithelial histology. The median overall survival rate at one year was 48% and 37% at two years.

Programmed death-1 receptor

PD-1 is a cell surface receptor belonging to the Ig superfamily that is expressed on T-cells and pro-B cells and binds two ligands, PD-L1 and PD-L2. PD-L1 is a transmembrane protein that binds to its PD-1 and B7.1 receptors on the surface of T-cells, deactivating them.(20)
The PD-1 receptor stimulates the cells into inactivation by allowing the tumor cell to escape the surveillance of the immune system. (21)
This receptor is activated by binding with its ligand: programmed death-ligand 1 (PD-L1), which is usually found in the tumor microenvironment on the surface of cancer cells. (22)
Researchers have shown that PD-L1 is present on rat murine cells in vivo.(23) PD-L1 expression increases in response to an increase in the concentration of interferon y (IFN)-? and the draining of T-cells into the tumor-draining lymph nodes, supporting the hypothesis that this is an important local immunosuppressive pathway.
The inhibitory action of PD-L1 on the different subpopulations of T-cells produces opposite effects on tumor progression and suggests that the immune suppression of the tumor is mediated by a specific subclass of T-cells.
Researchers have demonstrated that PD-L1 was expressed in approximately 40% of 106 samples of mesothelioma analyzed, all of which were tumors with sarcomatoid histology with a poor prognosis (5.0 months versus 14.5).

Treatments

Targeted immunotherapies

The first immunopotentiation study dates back to 1975, investigating the intrapleural administration of a vaccine consisting of irradiated and sterilized BCG, which led to a reduction in tumor growth, secondary to the activation of the immune system.(25 26) Other studies followed the same line of research using Mycobacterium vaccae in combination with chemotherapy, for example.(27 28 29) Another attempt at potentiating the immune response is the use of cytokines, such as interferon.(30 31 32 33( Viral vectors have also been used to increase the efficacy of therapy.34 35 36 Interleukin-2 has also been tested in this disease to activate the immune system against the tumor. (37 38 39 40) Another cytokine used in immunopotentiation studies is GM-CSF (granulocyte/macrophage colony-stimulating factor)(41 42 43 44) Several studies have evaluated the possibility of increasing the expression of major histocompatibility molecules (MHCs) to increase T-cell clones with antitumor effect. Other studies have evaluated coadjuvant therapies such as CpGm oligodeoxynucleotide, CpG ODNs, Toll-like receptor 3 agonist, Toll-like receptor-7 agonist. (47 48 49) Immunomodulation involves modifying the lack of cell immunity within tumor sites. (50 51)
The first immunomodulation studies were conducted in the 1990s, evaluating therapies such as autologous and allogeneic LAK cells to reduce pleural effusions.(52) Other studies demonstrated the cytotoxic action of CTLs against MPM cells.53 More recently, research has been conducted on the activity of dendritic cells and their ability to protect antitumor immunity. (54) Other studies have investigated CD40-activated follicular B-cells and autologous T-cells expressing a chimeric antigen receptor (CAR).(55 56)
Immunodepletion, often known as lymphodepletion, is another area of immunotherapy that involves eliminating the cells that have infiltrated the tumor foci.57 Promising studies have investigated the depletion of tumor-associated macrophages (TAMs) from the tumor mass and the possibility of reversing these cells from TAMs to M1 status.(58 59) Similarly, other studies have demonstrated the utility of activating the TAMs infiltrating MPM tissue, promoting the release of cytokines and chemokines such as TNF-a, IFN-inducible protein 10 and IL6.(60 61)

Vaccines

The discovery of mesothelin in the 1990s was very important because researchers hoped it could be used as a specific marker for MPM.(62) Indeed, since this protein is also expressed in other tumor types, promising vaccines against these cancers were developed. (63 64)
Notwithstanding the controversy surrounding the possible role of the SV40 virus in the biogenesis of MPM, SV40 antigens have been tested as potential immunological targets for the disease.(65 66 67 68)
Wilms’ tumor 1 protein (WT1) has also been investigated as a potential diagnostic marker specifically for MPM.(69 70 71 72)
One of the treatment strategies for this disease is developing cellular vaccines and a number of studies have evaluated the effects of cancer cells transfected with IL-4, IL-2, GM-CSF, B7-1, as vaccine cells.(73 74 75 76) Dendritic-cell based vaccines have also been investigated to activate both Th1 cells and CTLs by activating phagocytosis and apoptosis.(77 78) Researchers have also reported that MPM cells in apoptosis could be used as potent inducers of anticancer CTLS.79

Monoclonal Antibodies

Monoclonal antibodies have long been used in immunohistochemical techniques to obtain a differential diagnosis between MPM and other tumors.(80)
The first approach in this area was to test a monoclonal antibody and in combination with a toxin as a potential immunotherapy. (81) Other studies evaluated monoclonal antibody reagents in combination with the 7D3 transferrin receptor, ricin A or doxorubicin. (82 83)
The monoclonal antibody K1 targeting mesothelin has been investigated as a potential immunotherapy for MPM.(84)
Various biomedical engineering methods have been used to design monoclonal antibodies combined with toxins to construct chimeric agents. (85 86 87 88)
Fusion proteins that can target cancer cells when subjected to radiotherapy have also been developed, and whose results suggest greater cytotoxic effects with fewer side effects. (89)
Researchers have developed an anti-MPM immunotoxin by combining Pseudomonas exotoxin with a fragment of the anti-mesothelin antibody. (90) Human mesothelin has also been combined with an antibody targeting a dendritic cell receptor, increasing the potential for vaccination with mesothelin.(91)
Wingless-type (Wnt) protein, which is involved in various cancers, is another important antigen against which monoclonal antibodies have been developed.(92 93 94)
Antibodies against the surface antigen CD26, which is involved in tumor growth, have also been developed.(95)
Other examples of monoclonal antibodies described above include CTLA-4 inhibitors (ipilimumab and tremelimumab), and PD-L1 (pembrolizumab) and PD-1 receptor (nivolumab) inhibitors. (96 97 98 99)
Tremelimumab was investigated in a single-arm Phase II study of pretreated patients but the primary endpoint of objective response rate was not achieved. (100) The disease control rate of patients treated with tremelimumab was around 31%, progression-free survival (PFS) was 6.2 months (95% CI 1.3–11.1), and the mean survival time (mST) was 10.7 months (0.0–21.9).(101) Studies of anti-PD-L1 and PD-1 monoclonal antibodies are also currently underway.(102)

Recent clinical trials

Below is a short list of recent clinical studies that have been completed or currently ongoing. Please see www.clinicaltrials.gov for further details about these studies.

Completed studies:

- Dendritic Cell-based Immunotherapy in Mesothelioma (tumor lysate-loaded autologous dendritic cells).
- Dendritic Cell-based Immunotherapy Combined With Low-dose Cyclophosphamide in Patients With Malignant Mesothelioma (DC+CTX)(103)
(Allogeneic Tumor Cell Vaccine (K562); Drug: Celecoxib; Drug: cyclophosphamide)(104 105 106).
- Pilot Study of Allogeneic Tumor Cell Vaccine With Metronomic Oral Cyclophosphamide and Celecoxib in Patients Undergoing Resection of Lung and Esophageal Cancers, Thymic Neoplasms, and Malignant Pleural Mesotheliomas (Allogeneic Tumor Cell Vaccine (K562); Drug: Celecoxib; Drug: cyclophosphamide).(104 105 106).
- Cyclophosphamide Plus Vaccine Therapy in Treating Patients With Advanced Cancer (allogeneic tumor cell vaccine; Biological:autologous tumor cell vaccine; Biological: recombinant interferon alfa; Biological: recombinant interferon gamma; Biological:sargramostim; Drug:cyclophosphamide).
- Study of Safety and Tolerability of Intravenous CRS-207 in Adults With Selected Advanced Solid Tumors Who Have Failed or Who Are Not Candidates for Standard Treatment (Biological: CRS-207, Live-attenuated Listeria monocytogenes expressing human Mesothelin).
- Safety and Immune Response to a Multi-component Immune Based Therapy (MKC1106-PP) for Patients With Advanced Cancer (PSMA/PRAME).

Ongoing studies:

- Safety and Efficacy of Listeria in Combination With Chemotherapy as Front-line Treatment for Malignant Pleural Mesothelioma (Immunotherapy plus chemotherapy; Biological: Immunotherapy with cyclophosphamide plus chemotherapy).
- Dendritic Cells Loaded With Allogeneous Cell Lysate in Mesothelioma Patients (MesoCancerVac).
- CAR T Cell Receptor Immunotherapy Targeting Mesothelin for Patients With Metastatic Cancer (Fludarabine; Biological:Anti-mesothelin CAR; Drug:Cycolphosphamide; Drug:Aldesleukin).
- Genetically Modified T Cells in Treating Patients With Stage III-IV Non-small Cell Lung Cancer or Mesothelioma (Aldesleukin; Biological: Autologous WT1-TCRc4 Gene-transduced CD8-positive Tcm/TnLymphocytes; Drug:Cyclophosphamide; Other: Laboratory Biomarker Analysis; Procedure: Therapeutic Conventional Surgery).
- The Anti-CTLA-4 Monoclonal Antibody Tremelimumab in Malignant Mesothelioma (Tremelimumab).
- Adjuvant Tumor Lysate Vaccine and Iscomatrix With or Without Metronomic Oral Cyclophosphamide and Celecoxib in Patients With Malignancies Involving Lungs, Esophagus, Pleura, or Mediastinum (H1299 Lysate Vaccine; Drug:Cyclophosphamide; Drug:Celecoxib).
- A Study of Tremelimumab Combined With the Anti-PD-L1 MEDI4736 Antibody in Malignant Mesothelioma (NIBIT-MESO-1) (tremelimumab plus MEDI4736).
- A Clinical Study With Tremelimumab as Monotherapy in Malignant Mesothelioma (Tremelimumab).
- Phase II Study of Adjuvant WT-1 Analog Peptide Vaccine in MPM Patients After MSK10-134 (WT-1-vaccine Montanide+GM-CSF; Biological:Montanide adjuvant + GM-CSF).
- Randomized Study of Adjuvant WT-1 Analog Peptide Vaccine in Patients With Malignant Pleural Mesothelioma (MPM) After Completion of Combined Modality Therapy (WT-1-vaccine Montanide +GM-CSF; Biological: Montanide adjuvant + GM-CSF (This arm is closed))
- Nivolumab in Patients With Recurrent Malignant Mesothelioma.
- Gene Therapy for Pleural Malignancies (Adenoviral-mediated Interferon-beta; Biological:SCH721015).
- Four Versus Six Cycles of Pemetrexed/Platinum for MPM (Pemetrexed/platinum chemotherapy).
- Intrapleural AdV-tk Therapy in Patients With Malignant Pleural Effusion (AdV-tk+ valacyclovir).

Response to Immunotherapy

Immunotherapy has changed the way in which we measure objective responses in both clinical studies as well as clinical practice. Melanoma immunotherapy studies have shown that the antitumor response is not seen until weeks or months after the start of treatment, with a survival gain seen after several months. This is because immunotherapy drugs activate the immune system, which in turn elicits a cell-mediated response.
The response to treatment is measured by using RECIST or WHO criteria. During immunotherapy, these conventional criteria are not capable of adequately measuring the presence of peritumoral inflammatory infiltrate which may mimic a pseudoprogression, a phenomenon typically encountered during this type of treatment. To avoid this problem, immune-related response criteria (irRC) have been developed, in which an initial radiographic progression, in other words the appearance of new lesions and/or an increase in the size of existing lesions, in the absence of clinical progression, must be confirmed by another evaluation.(107) The correct use of irRC can identify long-term survivors, including patients who would be considered by coventional criteria to be progressing and so may not continue to benefit from targeted treatment.(108) Another aspect evidenced by immunotherapy is the need to understand whether this treatment is suitable for everyone or only certain patients who are most likely to benefit from immunotherapy.

Conclusions

The association between the immune system and MPM is complex and multifaceted. Immunity certainly plays a key role in inducing damage to the DNA of mesothelial cells, which is strongly and pathogenically linked to exposure to asbestos.
Dividing the immune system into innate response and adaptive response also helps balance the inhibition and activation of the cells involved in this complex mechanism.
Although the results from immunotherapy studies have not yet been earth-shattering, they are nevertheless extremely promising and provide a new view of this disease in anticipation that it will lead to new therapeutic approaches.

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Introduction

Tumours are characterised by a series of genetic anomalies with a proliferative potential that leads to cellular transformation.

Nevertheless, it is believed that the micro-environment that surrounds the tumour (tumour micro-environment or TME) may also have an important role in the fate of tumour cells, acting on the cellular progression or regression.

The correlations which exist between cancer and inflammation have been documented since 1863, when Virchow observed that tumour tissue is often surrounded by inflammatory cells which are discovered in the analysis of bioptic samples(i).

In immunodeficient mouse models, it has been demonstrated that inflammation may precede the development of malignant mesothelioma(ii). Moreover, epidemiological studies have revealed that chronic inflammation caused by chemical or physical agents and the inflammatory and autoimmune reactions of uncertain origin predispose people to certain types of tumours(iii iv).

Growing evidence shows that the «inflammation-cancer» connection is not only limited to initial processes of tumour development; in fact, all types of cancers would seem to have an active inflammatory component in their micro-environment. These experimental and clinical observations lead to a greater confirmation that inflation related to cancer may be one of the main typical characteristics of neoplasias itself(v).

Inflammation

One of the main factors that characterise the tumour micro-environment is persistent chronic inflammation(vi).

Tumour-related inflammation is mainly triggered by innate immunity cells (especially macrophages), which are present in large quantities in the tumour micro-environment, but it is also maintained by stromal cells, such as fibroblasts, by blood vessel cells or by the same tumour cellsvii.

Two pathogenic ways correlate cancer and inflammation.

The intrinsic way is guided by genetic alterations that cause neoplasias. For example, these genetic modifications may lead to the triggering of various types of oncogenes, to mutation, to rearrangement or to chromosomal amplification, or they may make oncosuppressor genes inactive, which initiate an inflammatory process inside the neoplastic cell.

The extrinsic way, on the other hand, is mediated by inflammatory cells of the innate immunity; we are talking mainly of macrophages.

These two inflammatory paths join the activation of transcription factors, such as the NF-.B, of signal transducers, of transcriptional activators 3 (STAT3) and of the hypoxia-inducible factor 1 (HIF1).

In the past ten years, the mechanisms through which chronic inflammation supports tumour growth have been further delved into. Different soluble inflammatory mediators, either produced by macrophages or by tumour cells, act as growth factors that directly stimulate the proliferation of tumour cells and increase their resistance to apoptotic stimuli. These include, for example, the primary inflammatory cytokines IL-1 and TNF, which activate NF-kB, the key regulator of the inflammatory response.

In tumour cells, NF-kB activates the expression of anti-apoptotic genes (for example, c-IAP, BCL2, c-FLIP) and of genes that regulate cellular proliferation (for example, Cyclin, c-Myc).

In the macrophages, NF-kB activates different genes that encode for cytokines (Egil-1, TNF, IL-6), chemokines (i.e., CCL2, CCL5, CXCL8) and reactive enzymes (for example, COX-2), which further stimulate the inflammatory response, thus amplifying the recruitment of new inflammatory cells in the tumour.

Cytokine IL-6 activates transcription factor STAT3, another important inflammation and tumour development regulator. In tumour cells, STAT3 stimulates cellular survival and proliferation, whilst in the macrophages its persistent activation leads to immune suppression.

Besides, little is known about the mechanisms that lead to tumour initiation within the context of chronic inflammation. There is evidence that inflammatory mediators such as cytokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS) lead to epigenetic alterations in pre-cancerous cells, cause the silencing of onco-suppressor genes and the inhibition of DNA repairing mechanismsviii. Certain inflammatory cytokines and other mediators increase the survival of tumour cells, the motility and invasiveness, also encouraging the angiogenic capacity, which is crucial for allowing oxygen, nutrients and growth factors to reach tumour cells(ix x).

In this way, chronic inflammation favours the accumulation of DNA mutations and increases the proliferation potential of the cells. It is believed that this cancerogenesis process induced by inflammation may require several years, as a consequence of a lack of balance between continuous casual mutations and DNA repair, cellular death and cellular proliferation, recognition or escape from control of the immune system.

While in the past 15 years incredible progress has been made in terms of understanding the mechanisms through which cancer-related inflammation might have a negative impact on tumour progression, little is known to this day with regards to the effects of chronic inflammation on cancerogenesis. It is believed that long-term exposure to inflammatory mediators (cytokines, reactive oxygen and nitrogen species) causes genotoxic damage to the DNA, constantly putting pressure on the DNA repair system. Cells where the DNA repair response is inhibited or is less efficient are at high risk of genomic instability and are more predisposed to malignant transformation. At present, little is known about the mechanisms underlying these processes.

Macrophages associated with tumours

Macrophages associated with tumours (TAM) are innate immunity cells that are abundantly present in tumours. They are key initiators of the persistent inflammation present in the tumour micro-environment (TME), since they are the main producers of reactive mediators that perpetuate and amplify the inflammatory cascade(xi xii).

A typical characteristic of macrophages is their functional plasticity. In fact, the acquisition of their various functions is precisely dictated by specific local stimuli that activate separate functional processes: actually, they are not only able to fight the onset and progression of the tumour but also lead to the start of the tumour as well(xiii).

Macrophages can be classified, in a simplistic way, as M1 or classic macrophages, having the tumour-suppressor phenotype and capable of product large quantities of inflammatory cytokines and M2, or alternative macrophages that have the immune-suppressor phenotype and control the trophic activity of tissues as well as the angiogenesis(xiv xv xvi).

Macrophages perform many actions aimed at encouraging tumour progression: they produce growth and survival factors for tumour cells and the vascularisation (neoangiogenesis), they contribute to the deterioration of the extracellular matrix and to the remodelling, they facilitate the invasion of tumour cells and the metastasis, and they produce immunity mediators that suppress anti-tumour activity(xvii xviii).

Consequently, the quantity of TAM in most solid and haematological tumours has been associated with an unlucky prognosis and resistance to therapies (xix).

The TAMs have a limited cytotoxic action against neoplastic cells and, according to certain studies, it appears that they are in fact capable of encouraging tumour proliferation, the deterioration of the extra-cellular matrix and the ability to elude the control of the immune system(xx xxi xxii xxiii xxiv).

Moreover, the presence of TAMs in tumour tissue is associated with the quick rate of progression (xxv xxvi). Hence, macrophages constitute a source of inflammatory mediators at the tumour level. This also occurs for MPM, although it has been reported in literature that the mesothelial cells of the pleura are also capable of producing reactive mediators in response to asbestos fibres.

On the basis of functional activities and gene expression profiles, some researchers have demonstrated that TAMS are polarised macrophages M2xxvii. Moreover, TAMs have been characterised in various mouse tumour models, and the inflammatory paths that are involved the most in the pro-tumour activity have been defined(xxvii xxix).

In recent years, great emphasis has been placed in identifying macrophages at the tumour site for therapeutic purposes. Moreover, it has been demonstrated that inhibiting these cells in experimental contexts would limit tumour growth and metastatic spreading(xxx). Inhibiting the recruitment of monocytes at the tumour sites, in combination with chemotherapy, appears to significantly increase the efficacy of the therapeutic treatment in mice with tumours. This is probably due to the fact that the presence of TAMs and myeloid cells is also strongly implicated in the ineffectiveness of anti-tumour therapies(xxxi xxxii xxxiii). Recent clinical studies have also provided interesting results, through the use of inhibitors that limit the action of the chemokines(xxxiv).

pleural mesothelioma

Pleural mesothelioma is a pathological condition characterised by chronic persistent inflammation. It is a very aggressive tumour caused by the neoplastic transformation of the mesothelial cells that line the body’s serous cavities and internal organs; in 80% of the cases it is of pleural origin and it is defined as malignant pleural mesothelioma (MPM)(xxxv). MPM is usually discovered in the advanced stage, since there are no markers that allow early diagnosis(xxxvi). Malignant mesothelioma is almost insensitive to current chemotherapy, and still has a very limited global survival rate.

It is a highly malignant disease associated with long-term exposure to asbestos or other particulate fibres(xxxvii). In fact, its incidence is strongly linked to exposure to airborne asbestos fibres(xxxviii). Once the asbestos enters the lungs, the macrophages are locally recruited and activated in an attempt to eliminate the fibres, but they are unable to carry out this «clean up» due to the non-degradable nature of asbestos. This failed deterioration of asbestos fibres by the macrophages leads to a state of chronic inflammation and to a fibrogenic response by the fibroblasts, which in the long term facilitates the transformation of healthy pleural cells into tumour cells(xxxix xl xli xlii).

Hence, the inhaled fibres are not degradable, and they cause a persistent local state of inflammation. Due to the volatile nature of particulate fibres, the people who work directly with asbestos are not the only ones at risk, as entire populations who live in areas where asbestos was present may also be affected. Therefore, it is possible to state that malignant mesothelioma is a tumour that is certainly related to chronic inflammations(xliii).

Genetic anomalies tied to MPM have been widely studied. In fact, a wide range of genetic mutations has been identified, including, for example: BAP1, CDKN2A, Ras, Wnt, p16, TP53, SMACB1, NF2, PIK3CA(xliv xlv xlvi).

This wide spectrum of genetic mutation indicates that the anomalous proliferation of the neoplastic cells is not caused by the oncogenic activity of one or of some oncogenes, as it happens in many types of tumours (for example, KRAS and pancreatic or lung cancer, BRCA1 and breast cancer)xlvii xlviii. In this case, we are dealing instead with the result of casual damage to the DNA, due to an upstream condition (for example, long-term inflammation), confirming that inflammation is indeed one of the main causes of carcinogenesis(xlix 1).

It is known that certain polymorphisms of genes related to inflammations cause a predisposition to the disease. For example, SNPs in Toll-like receptors have been found to be related to infections and chronic inflammatory diseasesli. For example, the SNPs of gene NLRP3 appear to be related to susceptibility to the HIV virus, to Crohn’s Disease, to rheumatoid arthritis and to diabeteslii liii liv. Girardelli et all have demonstrated that in patients suffering from MPM, the SNPs in gene NLRP1 are more frequent(lv).

Several studies have reported the expression of inflammatory mediators in MPM(lvi lvii lviii). Hegmans JP et all have demonstrated that the inflammatory cellular infiltrate of MPM is full of macrophages, thus implying that these cells play a crucial role in the biology of the mesotheliomalix.

It is well known that asbestos fibres cause the inflammatory sublayerlx lxi. The recruitment of the macrophages is also induced by the adipocytes involved in the inflammation caused by the presence of asbestos. In fact, some researchers have demonstrated that adipocytes exposed to asbestos fibres are capable of producing inflammatory cytokines (IL6 and CCL2), which in turn draw and recruit macrophages in the inflammatory micro-environment.

However, at present a complete characterisation of the inflammatory paths involved in MPL is still not available.

Conclusions

Inflammation is present in the micro-environment that surrounds the tumour tissue and, probably, it is not simply a cellular characteristic surrounding the neoplasias, but instead appears to be and active component involved in the carcinogenesis.

Several studies aim to study the mechanisms that lead to the neoplastic transformation of various neoplasias and, among these, of mesothelioma, focusing on the inflammatory response.

Researchers are currently attempting to understand which inflammatory paths are most involved in the onset and progression of mesothelioma, and if there are specific characteristics that can explain why the selected individuals develop the disease.

It would be crucial to identify subjects at a high risk of developing mesothelioma, and to find out more about chronic inflammation and its capacity to create a predisposition to carcinogenesis.

This research may lead to the discovery of new molecular targets useful for therapy or for prevention drugslxii.

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INTRODUCTION

Malignant pleural mesothelioma (MPM) is a tumor associated with exposure to asbestos (1, 2). It is an aggressive disease with a poor prognosis whose standard treatment does not greatly improve survival (3-9), and it is the reason why many studies are currently investigating MPM to discover new findings that may help increase our knowledge about the disease and thus optimize treatment goals (10-26).

Emerging scientific evidence has shown that tumor aggressiveness may be associated with the genome and the aberrant expression of some genes. Several studies have therefore focused on the role of microRNAs (miRNAs) in the tumor genesis of MPM.

MiRNAs are small (17–22 nucleotides), single-stranded, non-coding RNAs involved in many cellular processes that regulate gene expression (7).

MiRNA are often aberrantly expressed in tumors. Specifically, multiple miRNA expression profiles have been documented in MPM cells as opposed to healthy mesothelial cells, suggesting a potential role for miRNAs as both oncogenes and tumor suppressors in tumor genesis.

We hope that new diagnostic methods will be found to improve the diagnosis and treatment of MPM (8,10), which is why research has focused on defining new clinical prognostic factors. New biomarkers are also being investigated because they can help predict the evolution of the disease. Specific biomarkers that can diagnose MPM have not yet been validated, although studies are currently being conducted (27-31). Identifying new MPMP tumor markers could certainly help in the early diagnosis and treatment of this disease.

The purpose of this bibliography revision is to communicate the recent findings about the new class of microRNA markers.

DEFINITION OF miRNAs

Biogenesis of miRNAs
MicroRNA (miRNA) are short, single-stranded, non-coding RNAs acting as a new class of regulatory genes (32) that bind to the 3’ untranslated region of their target mRNAs, thus regulating post-transcriptional gene expression.

The translation of miRNA in proteins is inhibited due to the partial complementary binding between the miRNA and its target. Conversely, full complementary binding leads to the degradation of the miRNA.

Function of miRNAs
A single miRNA can regulate hundreds of targets downstream, so these molecules play an important role in various cell processes, including proliferation, development, differentiation, apoptosis, and response to stress. They are also aberrantly expressed in various cancers and so could play a key role in tumor genesis.

Recent studies have shown that miRNAs are non-invasive biomarkers that may offer new pathways for early diagnosis and treatment of various cancers (33-35) due to their tumor-specific expression profiles and presence in peripheral blood (36-37). MiRNAs could be useful for defining profiles that identify different tumor subtypes (50-51).

MiRNAs are tissue-specific and can be investigated to identify cancer tissue origin (36-37).

The presence of miRNAs in bodily fluids such as serum, plasma, saliva and urine might be useful to predict the clinical outcome and response to cancer treatment (38-41).

They could also be used as prognostic markers, which has been documented in various cancer types (43-49).

Analysis of miRNAs
Microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR) are the most commonly used methods of investigating miRNAs.

Microarray profiling is a technique in which microscopic probes are attached to a solid surface such as glass, plastic or silicon chips to form an array (matrix) (52), permitting the simultaneous analysis of many genes within a sample.

Microarrays use a technique known as inverse hybridization, which consists of attaching all the DNA segments (known as probes) onto a medium and marking the nucleic acid that we want to identify (known as the target). This method was developed in the 1990s and allows us to analyze gene expression by monitoring the RNA produced by thousands of genes in a single procedure.

MiRNAs are studied by first extracting them from the cells, converting them into cDNA using an enzyme known as reverse transcriptase and marking them with a fluorescent probe. When the probe in the matrix and the cDNA hybridize, the cDNA target binds to the probe and can be identified simply by looking at the position where it is bound.

The second method is qRT-PCR, which is a more sensitive, quantitative profiling instrument that analyzes the expression of a single cell, which could be applied in real time using primers together with specific samples (or tests?) [53].

These two methods should be used together to confirm the data and make them more credible, even though many studies have obtained results using a single techniqe.

Role of miRNAs

Few studies have actually evaluated the different expression of mRNAs with the aim of improving the diagnosis and treatment of MPM.

Early studies investigated the microarrays used to analyze how miRNAs are expressed differently in cancerous tissue versus normal tissue (54).

Biomarkers for the early identification of MPM
There is a growing desire to discover new biomarkers to identify MPM and a number of studies have been involved in defining the accuracy, feasibility and specificity of miRNAs as clinical biomarkers.

Abnormal increases in miRNA levels in different tumor types have been observed, and researchers suggest that these data could also be used to define the development and progression of MPM (55). Vascular endothelial growth factor (VEGF) has also been described as one of the targets of a specific miRNA, miR-126, and MPM patients have very high VEGF levels in their blood (55).

Several studies have shown that miR-126 expression is reduced in cancerous cells, which are characterized instead by increased VEGF expression, suggesting that this miRNA may play a role in tumor suppression (56).

A correlation between the levels of miR-126 found in serum and SMRP, a specific marker in patients at high risk of developing MPM, has been observed so it could presumably be used as a marker for the early diagnosis of MPM.

Recent research has shown that miR-126 can distinguish patients with MPM from patients with NSCLC (non-small cell lung cancer), and low levels of circulating miR-126 have in fact been found in MPM versus NSCLC samples (57).

This marker is not tumor-specific, however, and is often expressed in low levels in other tumor types so it should be used in combination with other markers such as mesothelin rather than alone (57).

Diagnosis
Up to now, it has been difficult to differentiate between MPM and adenocarcinomas or epithelial metastases of other cancers, and since there are no accurate markers miRNA expression could be an interesting option for obtaining a more differential diagnosis.

Several studies have shown that different patterns of miRNA expression can distinguish between MPM, lung adenocarcinoma or other cancers of the pleura (58-59).

Recent research has shown that some miRNA (miR-17-92) may be up-regulated in MPM cells, while other miRNA are down-regulated as typically occurs in other tumor types (miR-31, miR-221, miR-222) (60-61).

However, some statistical studies have shown that although specific miRNA (miR-17-5p, miR-30, miR-221, miR-222) are more characteristic of some histological tumor types, they cannot be considered as diagnostic markers to differentiate MPM from malignant mesothelial proliferations (58,59,61,63).

MiR-625-3p could be a promising marker, and some studies have shown that it is up-regulated in patients with MPM compared to the controls (65).

The miR-103 marker may be able to differentiate between the diagnosis of MPM and the controls who were exposed to asbestos (66).

Prognostic factors
MiRNAs have also been studied as prognostic factors.

Specifically, some researchers have observed that the patient population can be divided into two groups, namely those with a good prognosis and those with a bad prognosis based on the expression profile of the specific miRNAs (67-69).

MiR-29 is considered a prognostic factor in terms of relapse and survival after cytoinductive surgery, and it does seem to be more highly expressed in patients with epithelial MPM as opposed to those with non-epithelial MPM.

High levels of miR-29 expression may be able to predict a more favorable prognosis compared to patients with lower levels of this miRNA. MiR-29 probably plays a role in inhibiting proliferation, migration, cell invasion and the formation of colonies.

On the other hand, other miRNAs such as miR-31 are associated with a worse prognosis.

Several miRNAs also appear to be specific to some histological subgroups of MPM, such as miR-17-5p, miR-29a, miR-30e-5p, miR-106a and miR-143 (64).

Other miRNAs such as mi-17-5p and miR-30c also have prognostic value, and might be able to identify sarcomatoid MPM with better outcomes.

It is important to remember that besides serving as prognostic markers, miRNAs have also been investigated for their potential role as predictive markers with promising results (54, 55, 58, 59, 61, 63).

Potential targets for anti-tumoral therapies
Several researchers have studied the role of miRNA biomarkers.

Various studies have shown that there is an actual pattern of miRNA that distinguishes between malignant mesothelioma and healthy mesothelial cell cultures (63). The genes involved in regulating the cell cycle could be targets of various mRNA, such as the members of the onco-miR miR-17-92 (miR 17-5p, 18a, 19b, 20a, 25, 92, 106a, 106b) cluster.

There are also specific miRNAs such as miR-31 that could be useful for defining new approaches to treating MPM.

Functional studies have shown that the forced re-expression of this miRNA may lead to the overexpression of the cell cycle and inhibit important factors involved in DNA replication and progression of the cell cycle.

It has been shown that miR-31 has tumor-suppression properties, suggesting the possibility of developing new therapeutic agents for MPM and other tumor types expressing the loss of chromosome 9p21.3 (71).

Another miRNA that has been studied at length is miR-34b/c, which appears to play a role in gene-silencing and suppression of some oncologic characteristics. Here too, the forced expression of this miRNA resulted in a significant anti-cancer effect, secondary to cell cycle arrest, suppression of migration, invasion and cell mobility. MiRNA may also have important therapeutic implications in the near future (72-73).

Cells transfected with miR-34 inhibitors are characterized by increased proliferation, migration and cell invasion (74).

It has been observed that methylation of miR-34b/c circulating in the blood is associated with MPM (75).

Treatment with ranpirnase (Onconase) induces an increase in miR-17 expression, which leads to a down-regulation of miR-30 and NF-kB expression, translating in turn to an increase in apoptosis and a decrease in the aggressiveness of the tumor (77).

MiR-1 appears to have a tumor-suppressive function in MPM treatment (80), and is in fact down-regulated in MPM cell lines versus normal mesothelium (78-79).

A decrease in miR-15 and miR-16 expression has been observed in mesothelioma cell lines. Strong expression of these miRNA is associated with an inhibition of the growth of MPM cells (81-85).

CONCLUSIONS AND FUTURE PROSPECTS

All the studies described in this bibliography revision show the importance of miRNAs in MPM due to their potential role as both diagnostic and prognostic markers and as antineoplastic agents for the treatment of this disease.

The Buzzi Foundation has also funded research in the miRNA field, and a report on the current project is available for consultation: “New targets in mesothelioma cells: hitting translational control and mirnas”. The project researchers have been able to describe a miRNA signature present in mesothelioma tissues. They have analyzed the level of expression of these and other miRNAs in 10 mesothelioma cell lines. The researchers have also demonstrated an extremely heterogeneous situation. In summary, it is very difficult at this stage to define if and which miRNAs have any diagnostic, prognostic or therapeutic significance. The results are in line with the heterogeneity seen in the literature about miRNAs in mesothelioma. However, a review of the data available points to not only heterogeneity in the tumor but also in the analytical methods. In this context, the researchers decided to develop an innovative method to unambiguously define which miRNA are expressed in human mesothelioma tissue, a necessary step in order to understand whether miRNAs are effective markers. This technology could be transferred to diagnostic laboratories in the near future.

Very few studies have actually focused on miRNA expression in MPM, so it is not surprising that there are discordant results among the different histotypes, the various sampling sources used in the in vitro and in vivo research, the control groups, the approaches, the standardization techniques and the qRT-PCR and microarray analyses.

Further standardized, multicenter studies are needed using proven and standardized methods. In conclusion, all the results shown confirm the significance of miRNAs in the diagnosis, prognosis and treatment of MPM. All these data should be validated in a uniform manner to identify miRNAs as potential predictive and prognostic markers.

The recent progress in this field will certainly help define future prospects based on new treatment approaches and new opportunities for experimental treatment protocols.

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INTRODUCTION

The literature review in this section focuses on several key concepts regarding the genetics and pathways involved in the tumorigenesis of malignant mesothelioma (MM).

With this in mind, approximately 250 articles were analyzed and summarized below but we do not claim that this is an in-depth, exhaustive review, so please consult the bibliography at the end of this text for further further information.

MOLECULAR BIOLOGY OF MESOTHELIOMA

Malignant mesothelioma (MM) is caused by the abnormal proliferation of tumors in the pleura, pericardium, peritoneum, tunica vaginale testis or ovarian epithelium  (1,2).

Its incidence rate is increasing, and unfortunately the prognosis is often poor (3,4). A number of diverse pathogenetic hypotheses for this disease have been investigated in detail (5-8).

MM is characterized by a long latency period before the appearance of the initial symptoms that can lead to a diagnosis and during this long period, genetic mutations may occur and characterize the neoplastic changes (9-11).  The purpose of this literature review is to focus on the genetics and pathogenetic pathways associated with this neoplasm.

GENES

The main chromosomes affected by this neoplasm are: 1, 3, 4, 6, 9, 13 and 14 (12).

The genetic abnormalities most commonly associated with malignant pleural mesothelioma (MPM), and which will be analyzed individually, are the following: p16^INK4a /p14^ARF (13,14), NF2 (15,16), p53 (17-20), PTEN (21-23), BAP-1 (24), LATS2 (25), PI3K/AKT/mTOR (22,26), EGFR (27,28), VEGF (29-31), pRb (32,33), BCL-2 (34-36), hippo (37-39) and Wnt (40,41).

p16 INK4a/p14ARF

The p16^INK4a/p14^ARF gene is also known as CDKN2A/ARF and is located on chromosome 9p21.

This is a very important tumor suppressor gene that codifies for two proteins: p16^INK4a and p14^ARF.  (42-43)

Protein p16^INK4a inhibits CDK, which inactivates pRb.

Protein p14^ARF, on the other hand, regulates the function of p53 and inhibits its degradation by interacting with MDM2 (27,44,45).

These modifications play a fundamental role in regulating the control of the cell cycle; these genetic mutations also appear to be associated with more aggressive tumors and a poorer prognosis (13,14).

These genes in particular are implicated in the development of different types of neoplasia (46-48). Similarly, the same type of genetic mutations may also occur in MPM (13, 50-54).

Scientific studies have shown that if this gene is “switched off”, cancerogenesis may be “accelerated” due to exposure to asbestos  (55-59).

Gene therapy studies are aimed at reactivating the p16^INK4a/p14^ARF gene to restore the functions that have been lost if the gene is mutated. The studies have shown that reactivating this gene halts the cell cycle of mesothelioma cells, inhibits the phosphorylation of pRb, and decreases cell growth. All these modifcations may therefore increase survival, increase the levels of protein p53, and boost cell apoptosis (60-63,12).  Gene therapy aimed at restoring the functions altered by the mutation of this gene has shown promising preliminary results.

NF2

NF2, the abbreviation for the type 2 neurofibromatosis gene, is a genetic trait that follows an autosomal dominant inheritance pattern leading to tumor predisposition syndrome, and is characterized by the development of bilateral vestibular schwannomas on the eighth cranial nerve and other brain tumors, including meningiomas and ependymomas.  

This syndrome results from the lack of expression of the NF2 gene, which is a tumor suppressor.

Although known for the above syndrome, this gene is also associated with malignant mesothelioma (64-69).

The lack of protein activity associated with the codification of the mutated gene appears to be associated with a greater possibility of carcinogenesis, as opposed to patients who do not have this genetic mutation, and which is certainly greater for patients who have been exposed to asbestos (22,70).  However, the precise functionality of this gene has not yet been fully defined.

Gene therapy associated with this gene involves trying to “over-express” it through the use of viral vectors. These studies have shown interesting results, such as controlling the cell cycle and proliferation (71-75).

Re-expression of the NF2 gene in patients with MM could certainly be of considerable help in inhibiting cell proliferation and tumor invasion (76).

BAP-1

Several clinical studies have sought to understand how there appears to be a genetic predisposition to MPM in certain localities.  BAP-1 was found among the genes that were mutated and thus deemed to be involved in this disease (77-79).

Recent studies have also shown that BAP-1 is a tumor suppressor located on chromosome 3p21, which appears to play a role in regulating the cell cycle and responding to DNA damage (80-81).

This genetic mutation was found in patients with MM, especially the squamous histotype rather than the epithelial histotype  (82-84).

This pathologic genetic modification seems to be particularly associated with a poorer prognosis (85-86), as well as the development of neoplasia (87).

Gene therapy is being investigated not only to find an effective treatment for patients with a genetic alteration of this gene, but also to eventually prevent MM in subjects with a mutated BAP-1 gene.

LATS2

The Large Tumor Suppressor (LATS) was the first tumor marker identified in Drosophila (88).

In humans, this gene is located in a region of chromosome 13 (13q11-12) and is often mutated in tumors (89-90).

Two forms of LATS have been identified: LATS1 and LATS2. LATS2 in particular is a centrosomal protein which appears to be involved in mitotic subdivision (91), regulating the inhibition of the growth of Hippo (37) and activating p53 (92-93).

This gene has been studied in MM, particularly in cell lines characterized by a deletion of chromosome 13q11-12. Comparative genomic hybridization techniques were used for these analyses, subsequently confirmed by PCR.

These studies have shown the presence of genetic mutations of LATS2 in MM cells (25).

According to these studies, LATS2 appears to play a role in cell proliferation and survival. However, further studies are needed to confirm whether this gene actually plays a causal role in the development of MM.

DNA methylation

Studies investigating DNA methylation in MM have shown promising results.

They have shown that the methylation profile can be a differentiating factor between the physiological pleura and their pathological mutations, especially those that are characteristic of mesothelioma (94).

Some studies maintain that the methylation profile could even be considered a diagnostic marker that can be used to identify primitive and secondary pleural tumors (95).

Other studies investigated the relationship between patient outcomes and their methylation status and have observed interesting differences in survival associated this genetic mutation (96).

Other studies have also analyzed diagnosis and an eventual epigenetic therapeutic approach (15,97).

MicroRNA

miRNA expression is another important mechanism in the development of tumors, which supports their ability to control different biological processes.

For this reason, many researchers have focused their attention on the profile of miRNA to verify if there are any discrepancies/associations between these various genetic expressions and the pleura (98-103).

Other genes

A component of the Hippo cascade, the salvador gene (SAV) was dicovered in the Drosophila 81349 and is considered one of the gene suppresors altered in different neoplastic forms (16, 104-105).  Deletion of the chromosome 14q22 was recently demonstrated in approximately 5% of mesothelioma cell lines; however, the actual role of this gene in the pathogenesis of this disease is still being studied (25).

Deletion of the β-catenin (CTNNB1) gene in MM cell lines was discovered in approximately 10% of cases (106). CTNNB1 appears to be a cell growth stimulation factor in different tumor forms (107), although further studies are needed in this case too to clarify their pathogenetic role.

Recent studies have suggested that the Hedgehog signal pathway is activated in MM cell lines (108). This pathway in fact appears to be regulated by 13 genes in cancer pathogenesis.  However, only three of these genes were mutated in MM cell lines: PTCH1, SMO and SUFU (108-110).

The circadian rhythm is regulated by different genes and proteins that involve different processes: sleep, body temperature, hormones, the immune response and many others (111). Various studies have demonstrated a possible correlation between changes in the circadian rhythm and the development of cancer (112-113). Studies are investigating different genes with respect to MM, including the following: the clock genes PER (period), CRY (cryptochrome) BMAL1 (aryl hydrocarbonreceptor nuclear translocator-like) (114-116).

CONCLUSIONS

Genetic mutations associated with MM are being studied, with many already identified and many others being defined.

All these studies are aimed at increasing our knowledge about the genetics of MM, to understand how genetic mutations are associated with this disease.

Defining their pathogenetic role and cause would open up new avenues of research and certainly to potential experimental therapeutic strategies aimed at restoring the correct genetics whenever possible, which appear to be distorted in this disease.

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A review of the current scientific literature allows us to highlight the latest updates on treating Malignant Pleural Mesothelioma (MPM).
The key points from the world of science were updated in December 2013 and are summarized below to show how MPM is being treated at the dawn of 2014.

Introduction

Mesothelioma, as we know, is a rare neoplasm that usually develops in the mesothelial cells lining the surface of the pleural cavity, less frequently in the peritoneal surface area, and very rarely in the tunica vaginalis or the pericardium.
This neoplasm has a very poor prognosis (1) and the treatments currently used in clinical practice have not yet led to a definitive cure for this disease (2,3).
Many patients with MPM have symptoms that develop gradually and which are often of a respiratory nature (dyspnea, cough, thoracic pain). The presence of symptoms often leads to a diagnosis of extensive intrathoracic disease.
With respect to the diagnosis, we should point out that physicians should always suspect MPM if they see symptoms typically associated with a history of exposure to asbestos. However, a definitive diagnosis can always be made by performing a histological examination of an adequate sample of the neoplastic tissue.
MPM is staged using the same system widely employed by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC), which is known as TNM: T(Tumor), N (lymph nodes), M (metastasis) (4).
The clinical takes a multidiscplinary approach towards treating MPM, based on evaluating the extent of the disease, the overall condition of the patient (including cardiopulmonary function and other comorbidities) and their agreement to undergo treatment that is more or less aggressive. In fact, we must never forget to evaluate the wishes and hopes of patients to ensure that they have the best quality of life possible in accordance with their own personal parameters.
After evaluating these parameters, the patients can be subdivided into groups depending upon the recommended treatment: surgery or chemotherapy.
Different studies have evaluated various clinical and pathological parameters to identify patients with a good or poor prognosis. These characteristics have a prognostic value and are defined as “prognostic factors” .
The Cancer and Leukemia Group B (CALGB) and the European Organization for Research and Treatment of Cancer (EORTC) have identified some very interesting clinical prognostic factors (5-7). The best known prognostic factor is histology; in fact, patients with sarcomatoid mesothelioma as opposed to biphasic seem to have a worse prognosis than patients with epithelial mesothelioma.
Many biomarkers are currently being studied that could be both prognostic factors and predictive factors, in other words they can identify which patients respond to a certain treatment as opposed to another(8,9).
Defining the clinical benefit is essential for evaluating the effectiveness of the treatment:

• Treatment response rate
• Disease control rate
• Progression free survival
• Overall survival 10,11.

There are currently two radiographic methods for measuring the response rate used for the Computed Tomography (CT) evaluation: the RECIST system and the modified RECIST system (12,13,14).
Besides CT, other radiographic methods are also used, such as PET/CT (Computed Tomography with Positron Emission Tomography).
PET can define the metabolic activity of the body by evaluating the consumption of radiolabeled glucose that is injected as a contrast just before the scan (15). However, studies have also shown that the PET responses must be evaluated by experts only because this tool cannot be considered a gold standard diagnostic examination due to the various cases of false positives and negatives (16).
The importance of this nuclear exam is aimed at evaluating the response to the disease, tumoral activity or relapse, but the standard method for confirming the diagnosis is to perform a histological analysis.
There are also very promising biomolecular methods such as measuring the serum mesothelin-related peptide (SMRP) levels (16).

Surgery

Patients who are candidates for surgery
These are patients with resectable disease that is limited to one hemithorax and who are able to withstand surgery.
In these cases, multimodal treatment approaches can be used which involve Maximal Complete Resection (MCR) together with chemotherapy and radiation therapy.
Patients who are not candidates for surgery
These are patients whose disease is such that an MCR cannot be performed, or because they are too old and suffer from insufficient cardiopulmonary function or other comorbidities.
In these cases, chemotherapy and symptomatic treatment are the best approaches and which could actually lead to a clinical benefit.

Chemotherapy

Nowadays, MPM is treated with a combination of drugs rather than a single agent. In fact, the chemotherapy regimen of cisplatin combined with pemetrexed administered together with vitamin B12 and folic acid supplements (19), is now the standard of care for patients with non-resectable disease or who cannot undergo surgery. This decision is based on a study that showed an increase in survival using this combination versus cisplatin alone.
Other platinum-based regimens have been shown to be useful but further studies are not required to determine their efficacy (19).
The combination of Raltitrexed on top of cisplatin improves survival versus cisplatin alone in patients with advanced MPM who have not been treated previously (27,28).
Gemcitabine together with platinum has shown response rates with acceptable toxicity levels (29-35).
Cisplatin has also been studied with other older chemotherapy agents such as doxorubicin or epirubicin, the combination of fluoruracil, mitomycin plus etoposide, and the combination of methotrexate and vinblastine (36-41).
The role of maintenance chemotherapy with pemetrexed after completing four or six cycles of therapy with a platinum-based combination is still controversial (19).
It is important to remember that these treatments are not devoid of toxicities, even though treatment with folic acid and vitamin B12 supplements can alleviate these side effects (20-21).
If the side effects need to be reduced, carboplatin can be used instead of cisplatin with pemetrexed (23-25). The treatment response rates appear to be similar and so carboplatin can be a good alternative, especially for patients who are not in good overall condition and cannot tolerate the side effects from cisplatin.
Although treatment with single agents is considered inferior to combinations, they still have a role as second-line therapy (10).
Agents that have been investigated and which can be used for this purpose are cisplatin (42), carboplatin (43-44), pemetrexed /45-50), methotrexate (51), edatrexate (52), raltitrexed (53), gemcitabine (54-56), anthracycline (57-59) and vinca alkaloids (56,60,61).
There are still no predictive biomarkers for response to chemotherapy, although research is moving forward in this area. For example, the serum levels of thymidylate synthase appear to be associated with a better response to chemotherapy and a better prognosis (22).

Experimental approches

There are many new experimental approaches that are being studied to improve the systemic treatment of MPM.
Among new agents are angiogenesis inhibitors, such as bevacizumab (62) or thalidomide (63).
Tyrosine kinase inhibitors could also be very promising, such as sorafenib (64), sunitinib (65), imatinib (65-67), vatalanib (68) and cediranib (69).
Histone deacetylase inhibitors such as vorinostat (70-71) are also new treatments.
Last but not least, immunotherapy could be very useful for treating this disease either alone or in combination with chemotherapy (72-77).

Conclusions

MPM can no longer be considered a rare disease due to the increased incidence and improved diagnostic capabilities.
It should be pointed out that there are guidelines for physicians to follow because they are considered the best treatment approach since they are based on scientific evidence.
As of now, there is no treatment that can completely cure advanced stage MPM, but there is a variety of therapeutic approaches that will allow this disease to become as chronic as possible.
It is essential that we take into account the decisions and personal wishes of the patient so that we can treat their symptoms as effectively as possible and optimize their quality of life.
New experimental approaches are being studied and are very promising, although they are not yet considered as a standard treatment for MPM.
However, future prospects seem to be opening up at the dawn of 2014 and while research is moving forward at the laboratory bench, we hope that the products quickly become effective tools in clinical practice.

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Introduction

Second revision of the scientific literature on malignant pleural mesothelioma (MPM).
What new discoveries, studies and research protocols have been developed during the last few months?
How is research progressing on mesothelioma, a subject close to our hearts?

PubMed, the free access database containing articles, references, abstracts, revisions, etc., about science and medicine, serves as the definitive starting point for this new revision.
We therefore consulted all the scientific literature published between January 1 and June 30, 2013.
Under the general topic of “Mesothelioma”, we found the following for this period:

• 345 total publications
• By filtering the search to studies conducted in humans only, we narrowed it down to 75 studies during the last 6 months (not bad for a disease considered “rare” by some!)

Our revision does not claim to be a scientific review, but it offers patients, their families and general practitioners a concise idea of the latest scientific research on mesothelioma. This is not a critical analysis of individual articles, rather it is a recent snapshot of the most well-known scientific bibliography in the world. People who would like more detailed information can delve further into the topics by reviewing the references provided at the end of this short revision.

Diagnosis

Various research groups are looking for new biomarkers that can be used to diagnose MPM.
For example, claudin-4 is a protein involved in cellular junctions and is considered a useful immunohistochemical biomarker to distinguish between epithelioid mesothelioma and carcinoma metastases(1).
Another biomarker being studied is BAP1, which is a deubiquitylase involved in the cellular cycle, gluconeogenesis, response to DNA damage, cellular differentiation and cell death. Researchers have discovered that a germline mutation of BAP1 may be associated with a “syndrome” that causes melanoma in young people and may lead to the development of mesothelioma, uveal and cutaneous melanoma, and perhaps other neoplasms in older people(2). Other noteworthy mesothelioma biomarkers currently under investigation include fibulin(4 5 6), PTEN(7), GLUT, MCT-1 and MCT-4(8), IMP3(9).
New technologies and methods are currently being defined for earlier and better identification of this disease(10), others could be developed in addition to those already existing to provide further information(11).
Mesothelioma must be differentiated from other benign diseases such as fibrous pleuritis. Several researchers are focusing on this topic and have evaluated a biomarker that could distinguish between these two diseases(12).
The distinction between mesothelioma and lung cancer is also important, especially because it leads to different treatment approaches; this topic is also being studied(13).
In addition to biomarkers, we must not forget the importance of differential diagnosis(14) and an accurate case history to determine the possibility of environmental exposure(15 16 17 18 19) so we can arrive at the most accurate and early diagnosis possible.

Therapy

Notwithstanding the increased survival rate obtained through multimodal therapy based on a combination of surgery and chemotherapy, we need new treatments to further improve the results.
With this in mind, new therapeutic approaches are appearing on the horizon for the treatment of MPM.
Several researchers have investigated the administration chemotherapy or other agents directly into the thoracic or pleural cavity(20 21 22). Effective results have not yet been obtained, however.
There have been a few case reports of spontaneous remission after intratumoral lymphocytic infiltration, which have increased the median survival rate.
Based on these reports, several researchers are investigating the results further. For example, studies on immunotherapeutic approaches for MPM are underway with the aim of obtaining better results than those offered by standard therapy(23).
Various studies have shown that patients who develop post-operative empyema after pulmonary resection have an improved survival rate.
Based on this data, we can hypothesize about the importance of the immune system against the tumor and the need to find drugs that can increase the immune response against cancer(24 25 26).
Various studies have investigated the intrapleural injection of Calmette-Guerin bacillus as an adjuvant to surgery, but significant clinical benefits have not yet been obtained(27).
Various studies have investigated the systemic administration of immunotherapy such as interleukin and interferon gamma. However, the results thus far have been no more effective than current therapy and it is important to evaluate the side effects to determine the risk versus the benefit of these treatments(28 29).
Several researchers have analyzed the possibility of administering immunostimulant cytokines into the intrapleural cavity to treat MPM. Their research has shown a significant tumoral response using both IL2 and IFN gamma. The treatment seems more effective in patients with early stage MPM and these results could be truly promising(30 31 32 33 34).
The search for an adequate, effective treatment for MPM continues and new methods for novel, more effective and less toxic approaches are under investigation.
Gene therapy and new, emerging technologies in particular are examining the use of “transfer” genes to potentially transport cancer drugs. Gene vectors have been researched in clinical and preclinical studies and are characterized by complex liposomes/DNA or modified viruses, including herpes, vaccinia and adenoviruses(35 36).
The results from these studies have been mixed and need further research, but they are promising and offer much hope for the future.
Several researchers(37) have documented a dose-dependent response to intrapleural administration. The case of two “long surviving” patients whose disease stabilized after 6 months was reported a few years later . Also reported(39) were complete responses to the treatment, partial responses and stable disease evaluations after therapy(40 41 42 43).
There has been much discussion about “suicide gene therapy” and “cytokine gene therapy”.
“Suicide gene therapy” is a treatment characterized by the transduction of tumor cells with a gene codifying for an enzyme that induces sensitivity to the chemotherapy drugs normally used. In other words, a prodrug is transformed into a toxic metabolite by introducing an enzyme into the malignant cells, resulting in the death or suicide of the tumor cells(44 45 46 47 48 49 50).
The rationale behind “cytokine gene therapy” is the fact that activated tumor cells cause the release of many immunostimulatory cytokines, which in turn leads to an immune response against cancer(51 52 53 54).
Local administration of these cytokines could certainly avoid the side effects that have been documented with systemic administration(55 56 57 58 59).
All these new treatments have led to improved patient survival and quality of life than in the past. New studies will certainly help offer patients new hope for the future.

Conclusions

This is the latest news on the scientific research being conducted to find new treatments for MPM. We must emphasize that these clinical studies need further investigation and more data before they can be translated into clinical practice.
Nonetheless, guidelines for the diagnosis and treatment of this disease are being used on a daily basis. Continual congresses and conferences allow physicians to remain up-to-date and exchange information about new scientific achievements. For example, recommendations on total MPM patient care were published following the “Second Italian consensus conference on malignant pleural mesothelioma”(60). We therefore propose that MPM patients consult clinical centers dedicated to this disease in order to receive care that is personalized and focused on the patient rather than just the disease, in the knowledge that there is an answer to the question posed at the beginning of this article: are scholars, scientists and researchers making progress in their work? Are they focusing on a subject that is close to our hearts? Are they studying mesothelioma?

The answer is yes.

References

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Introduction

Malignant pleural mesothelioma (MPM) is a highly aggressive, rare form of cancer with a growing rate of incidence and an often unfavorable prognosis. Optimal management of this disease has not yet been defined due to the lack of an indisputedly effective therapy, although various scientific societies have proposed practical guidelines that are being applied in standard clinical practice. These guidelines emphasize the difficulty of diagnosing MPM and note the poor results of current treatments, thus emphasizing the need for innovative therapies and methods to monitor patients suffering from this disease.

Although the prognosis of MPM is often unfavorable and the prospects pessimistic, recent studies investigating the pathogenesis and biology of this disease have revealed some interesting findings, pointing to promising significant progress for the future treatment of these patients. Translational research in this disease is making great progress and different molecular oncogenic pathways leading to the growth and progression of MPM have been characterized and better defined, leading to exciting pharmaceutical developments. However, more in-depth analysis still needs to be done to further define the processes, including increased early mesothelial cell profileration to the progression of invasive mesothelioma. All this information will help define an effective, more personalized treatment for these cancer patients.

The purpose of this bibliographic review of the scientific literature is to provide an overview of the recent advances in our knowledge of the biology of MPM and their potential therapeutic-diagnostic applications.

This article is written in a non-scientific style to make it more available and accessible to a varied audience. We therefore refer interested readers and experts to the chapter containing the references, which could prove useful to anyone who would like to learn more about the studies analyzed in this review.

New therapeutic approaches

The role of surgery and radiotherapy for the treatment of MPM is still controversial and further studies may eventually provide more information in this regard.

However, medical therapy is considered the standard treatment in clinical practice, and the combination of platinum-based chemotherapy with antimetabolites (pemetrexed/raltitrexed) in particular has been considered the best first-line therapy for patients with MPM (1,2). Results obtained thus far have been limited, however, especially in terms of survival.

The main goal is to increase knowledge about the pathogenesis of MPM to define and improve target therapies and new therapeutic agents currently under investigation. The purpose of this review of the scientific literature is to describe the main therapeutic approaches currently being investigated in preclinical and clinical studies.

Epithelial Growth Factor Receptor

The epithelial growth factor receptor (EGFR) performs a role in the proliferation, differentiation, migration, adhesion and survival of cells (3) and is overexpressed in over 50% of MPM patients (4). The expression of the receptor in mesothelioma cells has led to the hypothesis that a therapy targeted against EGFR will inhibit it and prevent its uncontrolled and often harmful activity. This is why a number of studies have investigated the efficacy of EGFR inhibitors such as gefitinib and erlotinib in chemotherapy- naive patients. These studies have demonstrated that these drugs are not very effective as first-line therapy in MPM when they are administered as monotherapy, so not in conjunction with any standard chemotherapy (5, 6, 7). However, although the EGF receptor is overexpressed in mesothelioma, the reason why EGFR inhibitors are not very effective may be because mutations of this receptor are rare (8).

Some studies disagree about the correlation between the overexpression of EGFR in mesothelioma cells and the response to treatment with inhibitors of this receptor. Some research groups have proven that there is no relationship between the overexpression of EGFR and the clinical outcome of MPM patients (9,10), while others have shown that patients who overexpress the receptor may have a better outcome (11-14). These discrepancies confirm the need for further research to better define the results. In any case, it has been shown that overexpression of EGFR in MPM is more common in the epithelial histological subtype, which is associated with better patient survival but is not an independent prognostic marker (13,14).

Recent results indicate the presence of an important communication network between the EGFR pathways and other cellular signaling pathways. For example, some proteins such as PI3K and AKT which play a role in the EGFR signaling pathway, also act in other cellular growth pathways and interact with other factors such as c- MET and IGF-1 (15,17). The histological overexpression of the c-MET protein has been documented in MPM and also in some normal pleura samples. According to this rationale, c-MET inhibitors have been investigated in mesothelioma cell lines; preliminary results have shown a dose-dependent inhibition of tumor growth (18).

This type of dose- dependent inhibition was also observed in MPM cell lines subjected to IGF receptor inhibitors (19). Moreover, the cytotoxic effect of cisplatin also increased when administered together with these inhibitors (20).

An important biological communication also exists between EGFR and cyclooxygenase 2 (COX-2) (21).

COX2 is overexpressed in many solid tumors and so it is considered a potential therapeutic target (22-24). Immunohistochemical studies of this protein in MPM demonstrated that it was overexpressed in 59-100% of the tumor samples analyzed (25-27). They also showed that treating mesothelioma cell lines with COX2 inhibitors induces cytotoxicity and increases the effect of pemetrexed (28-29).

K-ras, BRAF and PI3KCA mutations

In the search for therapeutic targets, researchers have investigated the presence of genetic mutations associated with neoplastic pathogenesis, leading to studies of K-ras, BRAF and PI3KA gene mutations.

Unfortunately, genetic mutations of K-ras were not seen in the initial studies (30-32), thus changing expectations about this protein as a potential therapeutic target.

Studies of BRAF gene mutations have shown that they are absent in various tissues and tumor cell lines (33); other authors (34) have studied different MPM cell lines to analyze the PI3ka gene, but no mutation has been seen.

PTEN

PTEN is a protein that has been investigated in MPM to evaluate a potential therapy that would interact with this pathogenetic pathway.

Recent studies of various mesothelioma samples have shown that there is a loss of expression of the protein and that the mutation of this protein expression can be considered a negative prognostic value. In fact, patients with a decreased or lack of PTEN expression had a worse prognosis, whereas those with no genetic mutation had a greater survival rate (35).

It was also observed that the loss of PTEN expression might result in increased AKT activity, another important factor associated with the pathogenesis of cancer (36, 34). The loss of PTEN expression, which activates AKT, may induce resistance to various biological treatments such as EGFR inhibitors or anti-EGFR monoclonal antibodies. Therefore, these mutations also have consequences on other pathogenetic pathways, demonstrating the complexity of the pathogenesis and the intersecting network between these biological factors.

VEGF / VEGF Receptors

VEGF receptors have also been studied in mesothelioma cells and preclinical studies have shown they are expressed in both tumor tissue and peripheral blood in MPM patients (37).

The rationale for using drugs that inhibit this biological pathway is because they are expressed in greater levels in MPM patients than in healthy subjects. Also, the increased levels of VEGF are associated with increased microvascular density and appear to be associated with an unfavorable prognosis (38) as well as the likelihood of disease progression (39-41).

Anti- VEGF antibodies that actively inhibit this factor have been investigated. Studies have also tested the efficacy of combined VEGF and EGF inhibitors, in which these combinations achieved disease stabilization in 50% of patients, progression-free survival of 2.2 months and a median survival of 5.8 months (42, 43).

Drugs currently being investigated include vatalanib and cediranib, which are VEGF receptor inhibitors with antitumor activity in a variety of solid tumors (44-47).

Semaxanib is another VEGF-1 receptor inhibitor, but it also acts on the PDGF receptor (PDGFR) and c-kit (48). Another drug is thalidomide, which has been investigated in MPM patients with the following results: no partial or complete responses, 27.5% of patients were progression free after 6 months, and median overall survival of 7.6 months (49).

Sorafenib has shown limited activity in non-resectable MPM patients (50). However, it has also been studied in combination with doxorubicin, which confirmed that this combination is well tolerated thus justifying further clinical studies (51).

Sunitinib has been investigated in a Phase II study in MPM as second-line treatment after chemotherapy with platinum and antimetabolites, with the following results: partial response in 12% of patients; disease stabilization in 65% of patients; mean time to progression of 3.5 months and overall survival of 7 months (Nowak et al., IMIG 2011, unpubl. data).

Various Phase II studies have been conducted to determine the efficacy of imatinib mesylate in MPM patients refractory to chemotherapy or chemotherapy-naive patients (52-54). Combination studies between imatinib, cisplatin and pemetrexed are currently underway (55). New research is investigating the utility of these drugs, which appear to be active in MPM due to their ability to induce apoptosis of the tumor cells and by inhibiting various metabolic pathways such as AKT/PI3K, for example; the efficacy of these drugs has also been demonstrated due their ability to increase the sensitivity of the tumor to chemotherapy with gemcitabine or pemetrexed (56).

PDGF / PDGFR

The discovery that the PDGF receptor is highly expressed in mesothelioma cells has further defined the rationale for targeting treatment against this molecule (57).

The increased secretion of PDGF appears to be associated with thrombocythemia, which is considered a prognostic factor of adverse events that is seen in many MPM patients (58-59). In fact, high serum levels of PDGF in MPM patients appear to be a predictive factor of an unfavorable prognosis.

The expression of c-kit in MPM cells has also been shown in 26% of patients, prompting a number of clinical studies investigating imatinib in this disease (60).

Inhibition of PDGFR using imatinib combined with paclitaxel reduced interstitial fluid pressure, thereby enhancing the effect of the drugs and increasing in vitro efficacy (61). A partial response was seen in a Phase I study of imatinib in combination with gemcitabine (62). Dasatinib has shown cytotoxic effects in preclinical studies, leading to decreased migration and invasion in mesothelioma cells (63-64).

PI3K/AKT/mTOR Pathway

The PI3K/AKT/mTOR biological pathway is often aberrant in MPM, and various in vitro studies have shown that inhibiting this intracellular pathway may induce apoptosis in MPM cell lines (36, 65].

Sirolimus is a drug that has been approved as an immunosuppressant and which is currently used mainly in kidney transplantation and has an anti-proliferative effect on the PI3K/AKT/mTOR pathway.

Temsirolimus, a derivative of rapamycin, was investigated in a Phase I study but did not produce meaningful results (66). Studies investigating the combination of cisplatin and sirolimus are also underway, which have shown synergistic anti-tumor effects in MPM cell lines (67).

 Mesothelin

Mesothelin is highly expressed in a number of cancer types, including ovarian, pancreatic, some squamous carcinomas and the epithelial subtype MPM (68, 69).

Overexpression of the membrane protein mesothelin in MPM and its limited distribution in normal tissue has raised interest in this protein as a potential anti-tumor target (70).

Preliminary studies have not yet produced meaningful results (71); however, a number of drugs with anti-mesothelin activity are currently being investigated (72). Synergistic effects from the combination of these new agents with chemotherapy have been observed (73) offering promising results.

Ribonucleases

Ribonucleases are proteins that act on cellular RNA. Ranpirnase belongs to this group of proteins and has been investigated for its potential to induce apoptosis of tumor cells and inhibit cellular growth and proliferation.

However, various treatment-related adverse events have been observed, such as renal insufficiency, allergic reactions, arthralgia and peripheral edema (73).

Asparagine-Glycine- Arginine-human

TNF has known anti-tumor activity which is activated by inducing apoptosis of tumor cells. Studies of systemic treatment with this drug have shown that it is highly toxic and so it must be administered in such low doses to avoid disabling side effects that it is rendered ineffective (75-76).

Researchers have investigated a molecule consisting of a TNF fused to a peptide (tumor-homing peptide asparagine-glycine-arginine (NGR)) which is capable of selectively binding to mesothelial cells and has shown good tolerability as well as promising responses (77).

HDACi

Histone dacetylase inhibitors (HDACi) have been shown to alter the growth of numerous cancerogenic cell types. These molecules, many of which are derived from natural sources, have shown that they can inhibit proliferation, induce differentiation, and induce apoptosis of tumor cells.

Preliminary data from a Phase I study suggest that vorinostat has clinically significant activity in mesothelioma patients (78).

However, other studies have shown that this drug does not increase survival (79).

Vorinostat has also been investigated in combination with carboplatin and paclitaxel (80) and shown disease stabilization in some cases.

Belinostat is another drug that belongs to this group but has not proved to be superior to the other drugs (81). However, in vitro studies have demonstrated increased efficacy of these inhibitors when they are administered in combination with other agents (82, 83).

CBP501 EIMC- A12

Cells constantly undergo control processes to verify whether they are free of mutations and can continue in their cell cycle and multiply, or whether there are anomalies, in which case their life cycle must stop, lead to apoptosis and destruction of the cells to prevent more extensive damage. There are actually check-points that the cells must pass to obtain “permission” to proceed in their cellular cycle. Although they are anomalous, tumor cells can overcome these controls and can escape from being destroyed.

Drugs that act on these checkpoints have been defined to block the cell cycle of tumor cells which otherwise would continue proliferating and multiplying (84). Studies have also documented partial responses to treatment with these drugs when administered in combination with cisplatin (20), by enhancing the chemotherapy induced by these drugs.

Immunotherapy and Gene Therapy

Immunotherapy is another treatment that has contributed to significant advances and is currently being studied. An example of this treatment is the systemic administration of IL-2, which unfortunately had only limited efficacy and some side effects (85-86). Intrapleural administration of IL-2 has also been investigated and found to be well tolerated with objective responses, although further studies are needed to evaluate whether it offers more benefits than conventional treatment (87). Studies are also investigating systemic therapy with IL-2 by gene transfer of endogenous IL-2 as well as artificial regulation (88). Rapamycin is a natural macrolid that has been approved as an immunosuppressant and appears to have anti-proliferative effects by inhibiting some kinases such as mTOR. Synthetic derivatives of rapamycin known as “rapalogs” have been developed to improve the pharmacological properties of this macrolide; several examples are everolimus, temsirolimus and deforolimus.

Bortezomib is a potent proteasome inhibitor that has shown interesting cytotoxic effects in vitro and in vivo (89-90). Several studies are currently underway based on promising preclinical data (91).

Studies are evaluating the combination of interferon and various standard chemotherapy regimens, which have shown variable response rates to the treatment (92-95).

Vaccine therapies have also been studied, aiming to stimulate immune activity against tumor cells in MPM patients.

Some very interesting studies aimed at activating the immunostimulant ability of dendritic cells have also demonstrated variable but promising results (97-99).

Intrapleural therapy

The pleural cavity provides easy access for therapeutic molecules and intrapleural administration of drugs active in this disease could certainly offer excellent therapeutic prospects (100).

Various studies have evaluated the intracavitary administration of chemotherapy even after surgical resection, with the goal of enhancing local control of the disease (101- 103). Results have shown a 50% relapse of the disease after resection together with intrapleural chemotherapy, but further studies are needed to confirm these results that could probably show better responses.

Studies are also evaluating the intrapleural administration of recombinant viruses to try to sensitize tumor cells to drugs administered later (104-106).

Anti-mesothelin agents have also been injected into the pleural cavity (107-112), with the aim of inducing an immune response that could also act against tumor cells (113).

Conclusion

It is clear that clinicians, pathologists (114) and basic researchers must collaborate constantly to improve the treatment of rare but very aggressive diseases which often have an unfavorable prognosis such as MPM.

Numerous studies have been conducted over the last few years to investigate targeted molecular therapy and the biological pathways involved in the pathogenesis of this disease.

We need to gain a better understanding of the basic mechanisms of the development of this cancer in order to sufficiently understand the biomolecular pathways that play a role in cancerogenesis and so more effectively inhibit them. All these new studies, including those currently underway, have contributed to new advances or encouraging findings. However, further studies and more in-depth analyses carefully conducted and controlled will be able to confirm the results obtained thus far, as well as provide new information in order to achieve an effective therapy.

As MPM experts have already emphasized, all MPM patients must be afforded the opportunity to participate in ongoing clinical studies, not just to contribute to translational research but mainly to to gain access to treatments which, even though they are still being defined and investigated, can contribute to as yet undefined treatment lines regimens (115).

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