Malignant Pleural Mesothelioma: State-of-the-Art on Current Therapies and Promises for the Future

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer of the pleural surface associated with asbestos exposure. The median survival of MPM patients is a mere 8–14 months, and there are few biomarkers and no cure available. It is hoped that, eventually, the incidence of MPM will drop and remain low and constant, given that most nations have banned the use of asbestos, but in the meantime, the incidence in Europe is still growing. The exact molecular mechanisms that explain the carcinogenicity of asbestos are not known. Standard therapeutic strategies for MPM include surgery, often coupled with chemotherapy and/or radiotherapy, in a small percentage of eligible patients and chemotherapy in tumors considered unresectable with or without adjuvant radiotherapy. In recent years, several new therapeutic avenues are being explored. These include angiogenesis inhibitors, synthetic lethal treatment, miRNA replacement, oncoviral therapies, and the fast-growing field of immunotherapy alone or in combination with chemotherapy. Of particular promise are the multiple options offered by immunotherapy: immune checkpoint inhibitors, tumor vaccines, and therapies taking advantage of tumor-specific antigens, such as specific therapeutic antibodies or advanced cell-based therapies exemplified by the CAR-T cells. This review comprehensively presents both old and new therapeutic options in MPM, focusing on the results of the numerous recent and on-going clinical trials in the field, including the latest data presented at international meetings (AACR, ASCO, and ESMO) this year, and concludes that more work has to be done in the framework of tailored therapies to identify reliable targets and novel biomarkers to impact MPM management.


INTRODUCTION
Malignant pleural mesothelioma (MPM) is a rare, incurable, aggressive cancer of the pleural surface associated with asbestos exposure with a median survival of 8-14 months (1,2). Although the incidence in some countries, e.g., the USA (3,200 cases/year) (3), is fairly constant, in Europe, it is growing and is expected to peak between 2020 and 2025 (1). Moreover, the migratory phenomena toward western countries from nations lacking legislation on asbestos use will render MPM even more frequent. At present, no actionable driver mutations have been identified in MPM. However, MPM carcinogenesis and outcome are influenced by many factors: BRCA-associated protein 1 (BAP1) expression status, CDKN2A and neurofibromatosis type 2 (NF2) tumor suppressor inactivation, overexpression of growth factors such as vascular endothelial growth factor (VEGF), mesothelin (MSLN) promoter methylation, and Ras/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/mTOR pathway activation (4,5).

Angiogenesis Inhibitors
The angiogenic process plays an important role in MPM maintenance. VEGF receptor tyrosine kinase inhibitor (TKI) monotherapy yielded modest results (31)(32)(33)(34)(35)(36)(37). The addition of bevacizumab, a humanized monoclonal antibody against VEGF, to cisplatin-pemetrexed CT increases the median overall survival (OS) from 16.1 to 18.8 months and progression-free survival (PFS) from 7.3 to 9.2 months, as shown in the phase III MAPS study (NCT00651456) (38). Since this therapeutic regimen showed manageable toxicities, it has been included in the National Comprehensive Cancer Network guidelines (category 2A) (39), although it is not yet approved by the FDA or EMA. The SWOG S0905 phase I study evaluated the combination of cisplatin-pemetrexed CT with cediranib, a VEGF/PDGF receptor inhibitor, demonstrating a preliminary promising efficacy and reasonable toxicity profile (40) but, when compared to placebo, this combination failed to significantly increase OS and PFS in the following randomized phase II trial (41). Nintedanib is an inhibitor of three (triplet regimen) different growth factor receptors (VEGFR, PDFGR, and FGFR) and its administration in combination with CT improved the objective response rate (ORR) from 44 to 57% and the median PFS (9.7 vs. 5.7 months) compared to placebo in the LUME-Meso trial (42). Data from the phase III LUME-Meso trial (NCT01907100) have recently been published, and the primary PFS endpoint failed, not confirming the previous phase II trial results (43). Other TKIs, such as the anti-VEGFR axitinib (44) or the multi-target inhibitor of VEGFR1/2/3, FGFR-1, PDGFR-β, and RAF/cKit pathway sorafenib failed to improve median OS and PFS in chemonaive or CT-pretreated MPM patients (45,46). The limited success of anti-angiogenic drugs is due to the lack of good predictive biomarkers to guide the selection of suitable patients for this therapy. Recently, blocking of FGF signaling has been pursued through the sequestration of FGFs with the GSK3052230 ligand trap molecule to avoid toxicities associated with FGFR inhibitors. A phase Ib study indicates that a combination of GSK3052230 plus cisplatin-pemetrexed-CT leads to an ORR of 44% and to a median PFS of 7.4 months with limited adverse events (47).

Synthetic Lethal Therapies
Some MPM tumors cannot synthesize arginine due to the loss of argininosuccinate synthetase 1 (ASS1) gene expression. ASS1 deficiency is twice more frequent in the biphasic/sarcomatoid histotypes than in the epithelioid subtype. In vitro experiments suggest that depletion of arginine through exposure to a specific deaminase leads to synthetic lethality (48). The TRAP phase I trial (NCT02029690) demonstrated a positive effect of treatment with pegylated arginine deaminase (ADI-PEG 20) combined with CT in ASS1-deficient MPM patients (49). The ATOMIC-Meso phase III trial (NCT02709512) is recruiting patients with ASS1 gene loss. Genomic studies on MPM cells reported a reduced or absent expression of an enzyme involved in DNA repair and Ca 2+ -dependent apoptosis BAP1 in ∼50% of sporadic MPMs. In vitro studies demonstrated that BAP1-mutated cells are less sensitive to ionizing radiation causing DNA doublestrand breaks (50,51) or to the DNA synthesis inhibitor gemcitabine (52), highlighting the contribution of BAP1 in DNA damage signaling and repair and a possible role as a predictive biomarker (53). Inherited loss-of-function mutations in BAP1 predispose to multiple carcinomas, including mesothelioma (54)(55)(56). Interestingly, MPM patients with germline mutated BAP1 or with genetic alterations in other DNA repair genes and treated with platinum CT showed a significantly longer median OS than patients devoid of the same mutations (57). Hence the BAP1 mutational status at diagnosis could be an important factor in predicting MPM patients' response to CT and may sensitize patients to synthetic lethality therapies that hit other components of the DNA repair machinery. Accordingly, as already suggested by Srinivasan et al. (58), the homologous repair (HR) component PARP-1 would be an excellent target for a synthetic lethality approach, given that MPM cells are frequently characterized by HR deficiency and unrepaired DNA damage accumulation due to the aforementioned BAP1 mutations. PARP-1 inhibitors, such as niraparib and olaparib, clearly decreased MPM cell survival, albeit regardless of BAP1 status. BAP1 loss also up-regulates the expression of EZH2, a Polycomb Repressive Complex-2 (PRC2) component involved in epigenetic silencing (59) and oncogenic pathways (60), suggesting sensitivity of BAP1deficient MPM tumors to EZH2 inhibition. A phase II clinical trial (NCT02860286) is ongoing to evaluate the efficacy of the EZH2 inhibitor tazemetostat in MPM patients (61).
Finally, the synthetic lethality of inhibition of the Focal Adhesion Kinase (FAK) tyrosine kinase with loss of Merlin protein, the first involved in the survival, proliferation, and migration of tumor cells (62) and the second, a tumor suppressor encoded by the NF2 gene frequently mutated in MPM (5), has been proposed. Despite an encouraging positive trend observed in phase I trial in which FAK inhibitor GSK2256098 was tested in MERLIN-negative patients (63), a second large phase II trial (COMMAND, NCT01870609) demonstrated that neither PFS nor OS was improved by the FAK TKI defactinib as compared to placebo when administered as a maintenance treatment after frontline CT (64).

Immunotherapies
Multiple lines of evidence point to the involvement of the immune system in the pathogenesis and sensitivity to therapy of MPM (65,66). Spontaneous regressions in some patients are attributable to an activation of the immune system (67,68). Moreover, B cells are essential for a good prognosis (69) in murine preclinical models of mesothelioma treated with immunotherapy, indicating that antibodies are generated and contribute to the therapeutic effect. Also, the presence of cytotoxic CD8 + tumor-infiltrating lymphocytes (TILs) is a good prognostic marker in MPM (70,71).
MPM can be immunogenic but develops mechanisms to evade immune eradication. PD-L1 is the ligand for PD-1, a receptor expressed by activated T and B cells. Binding of PD-L1 to PD-1 affects effector T-cell and B-cell function and ultimately leads to exhaustion and apoptosis (72). Recently PD-L1 was shown to be expressed in 40% of MPMs, almost all of the sarcomatoid subtype, and was associated with a significantly poorer outcome, with a median survival of 5 months for PD-L1 + MPM patients vs. 14.5 months for PD-L1 − tumors (p < 0.0001) (73). However, PD-L1 expression is heterogeneous among MPM cells and could vary during treatment, limiting the efficacy of anti-PD-(L)1 therapy (74,75).
Despite early enthusiasm for the results of tremelimumab, an anti-CTLA-4 ICI, as first-line therapy (79), its use as a second-or third-line treatment demonstrated no benefit of CTLA-4 inhibition over placebo (DETERMINE, NCT01843374) (80). Nivolumab efficacy was tested as a second-or thirdline treatment alone vs. placebo in MPM patients in two recently completed phase II studies (NivoMes, NCT02497508, and MERIT, JapicCTI-163247) with ORRs of 24.0 and 29.4% and disease control rates (DCRs) of 50.0 and 67.6%, respectively (81,82). A clear correlation between response and PD-L1 expression was reported (81). An ongoing randomized, placebo-controlled phase III trial is testing the efficacy of nivolumab in relapsed mesothelioma (CONFIRM, NCT03063450) (83).
The anti-PD-1 ICI pembrolizumab has been evaluated in different phase I (KEYNOTE-028, NCT02054806) and II (NCT02399371) studies as a second-or third-line treatment, showing promising DCR and prolonged disease stability (84)(85)(86). The results from the randomized phase III trial PROMISE-meso (NCT02991482) were instead disappointing, with relapsed MPM patients receiving pembrolizumab or single-agent CT failing to show an improved median OS and PFS despite a superior ORR for pembrolizumab compared to a CT regimen (22 vs. 6%) (87). Popat and colleagues suggest that ICI treatment should be tested at earlier stages and on patients that are better stratified to benefit from longer periods of immunotherapy.
Other ICIs, like the Inducible T-cell COStimulator (ICOS) agonist GSK3359609, alone or in combination with pembrolizumab, are being evaluated in advanced solid tumors including MPM (INDUCE-I, NCT02723955) (88).

Combination Strategy
Two ICIs against different targets can be combined. An ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) combination was tested in the phase-II MAPS2 trial (89) in relapsed MPM patients. The results indicated that the primary endpoint, DCR after 12 weeks, was reached by combined therapy (50%) and not by nivolumab alone (44%). An ORR of 25.9 vs. 18.5% and a modest increase of median response duration (7.4 vs. 7.9 months) were achieved in the combination and nivolumab groups, respectively. Severe treatment-related side effects were registered in 17% of patients. The same combination is being investigated in a randomized phase III trial (Checkmate 743, NCT02899299) in the front-line setting (90). Similarly, the combined therapy of tremelimumab plus durvalumab, an anti-PD-L1 antibody, tested in the phase II NIBIT-MESO-1 trial (NCT02588131), resulted in grade 3-4 treatment-related side effects in 17.5% of patients (91). A phase III study is evaluating the combination of pembrolizumab with pemetrexed and platinum-based CT vs. pembrolizumab or CT alone as first-line treatment for MPM patients (NCT02784171).

INNOVATIVE THERAPEUTIC APPROACHES FOR MALIGNANT MESOTHELIOMA
miRNA Replacement miRNA replacement is an innovative anti-cancer approach that restores miRNA expression by delivering miRNAs or miRNA mimics. Restored miRNAs can interfere with the expression of proteins endowed with oncogenic activity (92)(93)(94) thereby inhibiting proliferation or inducing apoptosis of tumor cells (95). miR-16 is often downregulated in MPM, while its expression in in vitro and in murine xenografts results in decreased cell proliferation, decreased glucose uptake, and increased mortality (95). The feasibility of miR-16 exploitation by delivering its mimic encapsulated into an anti-EGFR-coated bacteriallyderived shell termed EnGeneIC Dream Vector (TargomiR) (96) was shown in the NCT02369198 trial, which reported efficacy and good tolerability in patients with relapsed MPM (97). TargomiR therapy was associated with a drop in glucose uptake in 60% of patients as measured by PET-CT, while 73% of patients achieved disease control.

Tumor Treating Fields
Recently, the FDA approved an innovative first-line treatment for MPM patients as a humanitarian use device, called NovoTTF-100L, that is based on the delivery of specific electric frequencies (Tumor Treating Fields, TTF) in combination with CT, to interfere with cancer cell proliferation. In vitro and in vivo data (98)

Oncoviral Therapies
In the wake of successful phase I and II studies (100, 101), a phase III clinical trial (INFINITE, NCT03710876) is evaluating the efficacy of an Adenovirus-Delivered Interferon Alpha-2b (rAd-IFN) in combination with celecoxib and gemcitabine in MPM patients who failed previous regimens. A phase II study (NCT04013334) is testing the efficacy of Ad-SGE-REIC/MTG201, an adenoviral vector for the expression of Reduced Expression in Immortalized Cell (REIC)/Dickkopf-3 (Dkk-3) gene in combination with nivolumab. The Dkk-3 protein is a Wnt signaling pathway antagonist that induces cancer cell death and antitumor immune response. A previous phase I/II study showed that intrapleural virus administration was safe and well-tolerated and that Dkk-3 gene expression allowed durable disease control (102). Preclinical studies evaluated the replication-competent neuroattenuated Herpex Simplex Virus (HSV-1716) as oncolytic virotherapy for mesothelioma, showing cytotoxicity in combination with CT or RT in vitro and reduced tumor growth also at low doses in vivo in MPM murine models (103). The results of a phase I/IIa trial (NCT01721018) testing the intrapleural administration of HSV-1716 demonstrated virus replication, pleural Th1 cytokine response, and anti-tumor immunoglobulin production (104). The use of other viral vectors [reviewed in (105)], such as attenuated versions of vaccinia or measles virus genetically engineered to produce human thyroidal sodium iodine symporter (NIS), is being investigated in different phase I clinical trials (NCT02714374, NCT01503177).

Dendritic Cell Vaccination
Cancer vaccines aim at inducing tumor-specific effector T cells that reduce tumor mass and induce tumor-specific memory T cells to curtail tumor relapse (106). Autologous dendritic cell vaccination (DCV) has shown efficacy in MPM treatment. The PMR-MM-002 clinical trial (NCT01241682) demonstrated the safety and feasibility of tumor lysate-pulsed dendritic cells as therapeutic adjuvants in MPM patients (107). The DENIM phase II/III randomized clinical trial (NCT03610360) will treat MPM patients with dendritic cell immunotherapy plus best supportive care (BSC) and compare the results with BSC alone (108). Other vaccination-based therapies currently under investigation are autologous DC loaded with Wilms' Tumor Antigen (WT1) (109) combined with CT (MESODEC, NCT02649829) and autologous TILs plus IL-2 (110). Based on the results obtained by PMR-MM-002 and by ICIs, a phase Ib MESOVAX clinical trial (NCT03546426) is recruiting MPM patients to test the efficacy of a tandem combination of autologous DCV and pembrolizumab at our institute.

Mesothelioma Targeting Antigens
MSLN is a glycoprotein expressed more on the cell surface of several tumors, including MPM cells, than in normal tissues (111). A phase II clinical trial (NCT00738582) testing amatuximab, a chimeric anti-MSLN mAb, plus standard CT compared to CT alone showed a promising OS of 14.8 months (112) that was not confirmed in the ARTEMIS trial (NCT02357147). Anetumab ravtansine (AR), an anti-MSLN antibody conjugated with the cytotoxic anti-tubulin drug ravtansine, showed a 50% ORR and 90% DCR in pretreated patients (113). A second phase II randomized clinical trial showed that AR did not improve survival compared to the anti-mitotic chemotherapeutic, vinorelbine, as a single agent (114). The combined regimen of pembrolizumab plus AR will be evaluated in a phase 1/2 trial (NCT03126630) that is recruiting only MSLN-positive patients. A phase I study (NCT02798536) is currently active to assess a novel low-immunogenic anti-MSLN recombinant immunotoxin, RG778/LMB-100 (115), composed of a human singlechain variable fragment (scFv)-targeting moiety directed against MSLN linked to Pseudomonas exotoxin A (PE). The phase I trial NCT01675765 evaluated the sequential administration of the cancer vaccine CRS-207, an attenuated form of Listeria monocytogenes expressing MSLN, with or without cyclophosphamide followed by consolidation CT, to stimulate an innate and adaptive immunity against MSLN-expressing cells. The cyclophosphamide arm showed acceptable toxicity and a DCR of 89%, a PR of 54%, an SD of 29%, and a median PFS and OS of 7.5 and 14.7 months, respectively (116).
The success of advanced cell-based therapies, e.g., Chimeric Antigen Receptor-transduced T cells (CAR-T) in hematological tumors, awoke interest as well for MPM (117). CAR-T-cell receptors directed against MSLN are being investigated in several phase I clinical trials. The critical issues in Adoptive Cell Therapy (ACT) and CAR-T treatment are the safety profile and the degree of off-tumor toxicity. Intravenous or intra-tumor administration of MSLN-CAR-T cells (NCT01355965) (118) obtained by Tcell electroporation with encoding mRNA to achieve transient expression resulted in moderate responses and low toxicity (119). A phase I study (NCT02414269) drawing on preclinical results in orthotopic mouse MPM models (120,121) is ongoing to test the MSLN-CAR-T cells in multi-treated MPM patients. Preliminary results presented at the AACR [Abstract CT036, (122)] and ASCO [Abstract 2511, (123)] meetings this year have shown an ORR and DCR of 36.8 and 57.8%, respectively, in a cohort treated off protocol in combination with pembrolizumab.
Fibroblast activation protein (FAP) is another interesting target expressed by all MPM subtypes and by cancer-associated fibroblasts (CAFs) and exploited by FAP-targeted CAR-T cells in an ongoing phase I trial (NCT01722149) (124) Preliminary results presented at the ESMO congress this year showed   Frontiers in Oncology | www.frontiersin.org a good tolerance of treatment and persistence of CAR-T cells (125). CD26 is a receptor overexpressed by all MPM histotypes and involved in immune regulation, T-cell activation, and the malignant potential of several cancers (126,127). YS110 is a humanized mAb targeting CD26 that is currently under investigation in a phase I clinical trial (NCT03177668) in MPM patients. Preliminary results show that 50% (13/26) of patients achieved SD, with a median PFS of 43 days (128,129).

DISCUSSION
Despite amazing efforts devoted to understanding and treating MPM better (Figure 1 and Table 1), clinical practice has not changed over the past decades, and CT remains the only standard option. Anti-angiogenic therapies and also ICIs that showed impressive clinical responses in other solid malignancies have little impact on survival in MPM as single agents, while ICI combination efficiency comes at the cost of relevant toxicities. The hopes for patients with MPM are, therefore, innovative therapies such as oncoviral, TTFields, TargomiRs, and CAR therapies in combination with anti-PD-1 ICIs that have shown good preliminary efficacy, although the results need confirmation in larger trials.

AUTHOR CONTRIBUTIONS
FN prepared the manuscript and figure. MM prepared part of the manuscript, provided guidance to FN in preparing the manuscript, and proofread and edited the manuscript. MB, GB, AD, MG, and LC helped with the review and made vital modifications along with suggestions to improve the content. All authors contributed to manuscript revision, read, and approved the submitted version.

FUNDING
Istituto Oncologico Romagnolo (IOR) and Becon AG through AFeVA, Associazione Familiari e Vittime Amianto, are funding our projects on malignant pleural mesothelioma. Neither of the funders contributed to the writing of this review.