Mesothelioma Progression Research Articles

Differential expression of DMD transcripts as a novel prognostic biomarker in histologically diverse mesotheliomas.

The identification of prognostic biomarkers is crucial for guiding treatment strategies in mesothelioma patients. The Duchenne muscular dystrophy (DMD) gene and its specific transcripts have been associated with patient survival in various tumours. In this study, we aimed to investigate the prognostic potential of DMD gene expression and its transcripts in mesothelioma patients. We analysed The Cancer Genome Atlas (TCGA) mesothelioma RNAseq, mutation, and clinical data to assess the association between DMD gene expression and its transcripts (Dp427, Dp71 splice variants) and mesothelioma survival. We also evaluated the specific Dp71 transcript as a unique prognostic biomarker across mesothelioma subtypes. Additionally, we performed differential gene expression analysis between high and low DMD gene/transcript expression groups. The analysis included 57 epithelioid, 23 biphasic, two sarcomatoid, and five not otherwise specified (NOS) histological subtypes of mesothelioma samples. Univariate analysis revealed that high expression of the DMD gene and its Dp71 transcript was significantly associated with shorter survival in mesothelioma patients (P=0.003 and P<0.001, respectively). In a multivariate analysis, the association between Dp71 expression and survival remained significant [hazard ratio (HR) 2.29, 95% confidence interval (CI): 1.24-4.23, P=0.008] across all mesothelioma patients, and also among patients with mesotheliomas without deep CDKN2A deletions (HR 3.58, 95% CI: 1.31-9.80, P=0.01). Pathway analysis revealed enrichment of cell cycle (P=3.01×10-4) and homologous recombination (P=0.01) pathways in differentially expressed genes (DEGs) between high and low Dp71 groups. Furthermore, there were correlations between Dp71 transcript expression and tumour microenvironment (TME) cells, including a weak positive correlation with macrophages (R=0.32, P=0.002) specifically M2 macrophages (R=0.34, P=0.001). Our findings indicate that the differential expression of specific DMD transcripts is associated with poor survival in mesothelioma patients. The specific Dp71 transcript can serve as a potential biomarker for predicting patient survival in diverse histological subtypes of mesothelioma. Further studies are needed to understand the role of specific dystrophin transcripts in cancer and TME cells, and their implications in the pathogenesis and progression of mesothelioma. Identifying patients at risk of poor survival based on DMD transcript expression can guide treatment strategies in mesothelioma, informing decisions regarding treatment intensity, follow-up schedules, eligibility for clinical trials, and ultimately, end-of-life care planning.

Abstract 4716: First-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 triggers mesenchymal-to-epithelial transition in mesothelioma

Abstract Mesothelioma, a lethal cancer associated with asbestos exposure, lacks effective drug therapy particularly in the relapsed treatment setting. Epithelial-to-mesenchymal transition (EMT) confers an aggressive sarcomatoid phenotype capable of invasion, metastasis, and drug resistance. Pharmacological targeting of EMT could favorably alter the progression and treatment of mesothelioma. We identified a geospatial transcriptomic gradient between adjacent epithelioid and sarcomatoid regions in patient-derived biphasic mesotheliomas involving hypoxia tolerance, TGF-β, mitochondrial oxidative phosphorylation, and EMT. Mesothelioma cells rely on buffering of mitochondrial oxidative stress through the expression and activity of mitochondrial peroxiredoxin 3 (PRX3), which is covalently inactivated by the first-in-class inhibitor RSO-021, now under investigation in the phase 1/2 MITOPE clinical trial (NCT05278975). To better understand the pharmacodynamics of RSO-021, transcriptomic profiles of drug sensitive and resistant biphasic mesothelioma cell lines were generated. Differential analysis revealed that RSO-021 induced up-regulation of ROS response and apoptosis signaling, while down-regulating TGF-β and EMT markers, WNT signaling, and TEAD target genes. Additionally, we observed altered expression of the mesothelioma specific EMT genes Col5A2, mesothelin and VISTA. Resistance to RSO-021 conferred significant proliferative defects and was reversible upon RSO-021 removal, however the down-regulation of EMT genes was conserved, suggesting an irreversible mesenchymal-to-epithelial transition. In summary PRX3 is a clinically actionable drug target, inhibition of which correlates with EMT modulation. This activity suggests a mechanism to alter mesothelioma progression and prevent the emergence of the most aggressive mesothelioma phenotype. Citation Format: Terri Messier, Victoria Gibson, David J. Seward, Essa Y. Baitei, Peter Wells Jordan, Charlotte Poile, Joanna Dzialo, Aleksandra Bzura, Kudzayi Kutywayo, Apostolos Nakas, George N. Naumov, Dean A. Fennell, Brian Cunniff. First-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 triggers mesenchymal-to-epithelial transition in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4716.

MTH1 Inhibition Alleviates Immune Suppression and Enhances the Efficacy of Anti-PD-L1 Immunotherapy in Experimental Mesothelioma.

MTH1 protects tumor cells and their supporting endothelium from lethal DNA damage triggered by oxidative stress in the tumor microenvironment, thus promoting tumor growth. The impact of MTH1 on the tumor-related immune compartment remains unknown. We hypothesized that MTH1 regulates immune fitness and therefore enhances the activity of currently used immunotherapeutic regimens. Our hypotheses were validated in two syngeneic murine mesothelioma models using the clinically relevant MTH1 inhibitor, karonudib. We also examined the effect of combined MTH1 and PD-L1 blockade in mesothelioma progression, focusing on the main immune players. Karonudib administration enhances M1 macrophage polarization, stimulates CD8 expansion and promotes the activation of DC and T cells. Combined administration of PD-L1 and MTH1 inhibitors impairs mesothelioma tumor growth and mesothelioma-associated pleural effusion accumulation more effectively compared to each monotherapy. Combined MTH1 and PD-L1 inhibition holds promise for the successful clinical management of mesothelioma.

La eficacia de [18F]FDG PET/TC en el seguimiento de la progresión de la enfermedad en el mesotelioma pleural maligno

ObjectiveIn the event of suspicion of malignant pleural mesothelioma (MPM) progression, imaging plays an important role. We aimed to evaluate the efficacy of [18F]FDG PET/CT in monitoring disease progression by comparing it with CT, and estimate median overall survival (OS) according to progression status with CT and [18F]FDG PET/CT. Materials and MethodsThis was an observational, retrospective, single-institution study with MPM patients who had both [18F]FDG PET/CT and CT for monitoring disease progression from March 2009 to February 2020. Clinical features, radiological findings, and progression status according to CT [radiologic progression negative (RPN), radiologic progression positive (RPP)] and [18F]FDG PET/CT [metabolic progression negative (MPN), metabolic progression positive (MPP)] were recorded. The discrepancies and concordance between two methods were evaluated. The OS was estimated using the Kaplan–Meier method. ResultsA total of 56 patients were included. There were thirty-one (55.3%) RPN and 25 (44.7%) RPP, while there were 26 (46.5%) MPN and 30 (53.5%) MPP. All RPP patients were also found to be MPP, however, among RPN, 5 patients (8.9% of all patients) were evaluated as MPP. The concordance between two methods in monitoring disease progression was very good (K=.423; P<.01). The OS was 26 ± 2.6 months in all patients. Kaplan-Meier curves between RPN and RPP, and between MPN and MPP did not show statistically significant differences (P=.56 and P=.25, respectively). ConclusionsBoth methods are equally acceptable in monitoring disease progression in MPM, even though [18F]FDG PET/CT detected more progression than CT did.

The efficacy of 18F-FDG PET/CT in monitoring disease progression in malignant pleural mesothelioma

ObjectiveIn the event of suspicion of malignant pleural mesothelioma (MPM) progression, imaging plays an important role. We aimed to evaluate the efficacy of 18F-FDG PET/CT in monitoring disease progression by comparing it with CT, and estimate median overall survival (OS) according to progression status with CT and 18F-FDG PET/CT. Materials and MethodsThis was an observational, retrospective, single-institution study with MPM patients who had both 18F-FDG PET/CT and CT for monitoring disease progression from March 2009 to February 2020. Clinical features, radiological findings, and progression status according to CT [radiologic progression negative (RPN), radiologic progression positive (RPP)] and 18F-FDG PET/CT [metabolic progression negative (MPN), metabolic progression positive (MPP)] were recorded. The discrepancies and concordance between two methods were evaluated. The OS was estimated using the Kaplan–Meier method. ResultsA total of 56 patients were included. There were thirty-one (55.3%) RPN and 25 (44.7%) RPP, while there were 26 (46.5%) MPN and 30 (53.5%) MPP. All RPP patients were also found to be MPP, however, among RPN, 5 patients (8.9% of all patients) were evaluated as MPP. The concordance between two methods in monitoring disease progression was very good (K = 0.423; p < 0.01). The OS was 26 ± 2.6 months in all patients. Kaplan-Meier curves between RPN and RPP, and between MPN and MPP did not show statistically significant differences (p = 0.56 and p = 0.25, respectively). ConclusionsBoth methods are equally acceptable in monitoring disease progression in MPM, even though 18F-FDG PET/CT detected more progression than CT did.

Oral metastasis of pleural sarcomatoid mesothelioma, a rare aggressive variant of mesothelioma and a diagnostic challenge

<h3>Introduction</h3> Malignant mesothelioma is a rare neoplasm that most commonly develops after exposure to asbestos and arises from the serosal cells of the pleura or rarely peritoneum. Various histological subtypes have been recog- nized: epithelioid, sarcomatoid, biphasic-epithelioid, and biphasic-sarcomatoid, with the sarcomatoid variant being a less common, but more aggressive variant. Numerous genetic alterations have been identified in the development and progression of malignant mesothelioma, most notably mutations in tumor suppressor genes, <i>neurofibromatosis type 2 (NF2), BRCA1-associated protein-1 (BAP1),</i>and <i>cyclin-dependent kinase inhibitor 2A/alternative reading frame (CDKN2A/ARF)</i>. Mesothelioma metastasis to the oral cavity is infrequent, with only nineteen cases reported in the literature. Soft tissue involvement of the oral cavity has been reported in the tongue, followed by the mandibu- lar gingiva, and alveolar mucosa. Prognosis for malignant mesothelioma is poor as it is typically unresponsive to conventional chemotherapeutic agents. New targeted therapies are currently in ongoing clinical trials. <h3>Case Findings</h3> An 81-year-old male, undergoing treatment for malignant pleural mesothelioma and with a history of asbestos exposure, presented for evaluation of a raised, necrotic mass on the left posterior mandibular buccal and lingual gingiva. Panoramic radiograph revealed extensive bone loss. The histopathology was that of a sarco- matoid neoplasm. Immunohistochemistry demonstrated positive staining for WT1, cyclin D1, and P53, supporting the diagnosis of metastatic malignant mesothelioma, sarcomatoid variant. Other stains that help to define mesothe- lioma such as CK5/6, calretinin, and D2-40 were negative. Despite equivocal immunostaining results, the diagnosis was further supported by comprehensive genomic analysis with identification of mutated <i>NF2</i>and <i>BAP1</i>tumor sup- pressor genes. <h3>Conclusion</h3> The diagnosis of malignant pleural mesothelioma relies on morphology supplemented with immuno- histochemistry. The diagnosis is challenging, especially so for the sarcomatoid variant, with immunohistochemical staining profiles often being inconsistent. The current case demonstrates the utility of comprehensive genomic analysis in firmly establishing the diagnosis.

Reactive oxygen species in the progression and treatment of malignant mesothelioma

Malignant mesothelioma (MM) is an aggressive cancer that affects the pleural and peritoneal mesothelial lining of the lungs and abdomen. Survival rates for patients with MM remain extremely low and effective treatments are limited. MM tumors harbor both genotypic and phenotypic features that indicate MM tumor cells are under increased oxidative stress, similar to other aggressive cancers. This increased oxidative stress in MM cells supports aggressive growth while providing a therapeutic vulnerability exploitable by redox-modulating compounds. MM tumor cells also exhibit altered mitochondrial structure and function that contribute to the disease through perturbations in metabolism and reactive oxygen species (ROS) production and metabolism. Targeting the altered redox status in cancer through increasing cellular ROS levels directly or inhibiting cellular antioxidant pathways and disrupting ROS scavenging mechanisms has become an exciting area for therapeutic intervention. This review discusses ROS sources and signaling, mitochondrial structure and function and targeting mitochondria ROS as a therapeutic approach for the treatment of MM.

CSF1/CSF1R Axis Blockade Limits Mesothelioma and Enhances Efficiency of Anti-PDL1 Immunotherapy.

Simple SummaryCSF1/CSF1R signaling mediates tumor-associated macrophages recruitment and M2 polarization. M2 TAMs are dominant immune populations infiltrating mesothelioma tumors. We evaluated the role of CSF1/CSF1R axis blockade in tumor-infiltrating immune subsets. We also examined the effect of combined anti-CSF1R and anti-PDL1 treatment in mesothelioma progression. We show that CSF1R inhibition impedes mesothelioma progression, abrogates infiltration of TAMs, facilitates an M1 anti-tumor phenotype and activates tumor dendritic and CD8+ T cells. We also show that this inhibitor was able to significantly improve the effectiveness of anti-PDL1 immunotherapy.Colony-Stimulating Factor 1 (CSF1)/Colony-Stimulating Factor Receptor 1 (CSF1R) signaling orchestrates tumor-associated macrophage (TAM) recruitment and polarization towards a pro-tumor M2 phenotype, the dominant phenotype of TAMs infiltrating mesothelioma tumors. We hypothesized that CSF1/CSF1R inhibition would halt mesothelioma growth by targeting immunosuppressive M2 macrophages and unleashing efficient T cell responses. We also hypothesized that CSF1/CSF1R blockade would enhance the efficacy of a PDL1 inhibitor which directly activates CD8+ cells. We tested a clinically relevant CSF1R inhibitor (BLZ945) in mesothelioma treatment using syngeneic murine models. We evaluated the role of CSF1/CSF1R axis blockade in tumor-infiltrating immune subsets. We examined the effect of combined anti-CSF1R and anti-PDL1 treatment in mesothelioma progression. CSF1R inhibition impedes mesothelioma progression, abrogates infiltration of TAMs, facilitates an M1 anti-tumor phenotype and activates tumor dendritic and CD8+ T cells. CSF1R inhibition triggers a compensatory PD-1/PDL1 upregulation in tumor and immune cells. Combined CSF1R inhibitor with an anti-PDL1 agent was more effective in retarding mesothelioma growth compared to each monotherapy. In experimental mesotheliomas, CSF1R inhibition abrogates tumor progression by limiting suppressive myeloid populations and enhancing CD8+ cell activation and acts synergistically with anti-PDL1.

Syndecan-1 Overexpressing Mesothelioma Cells Inhibit Proliferation, Wound Healing, and Tube Formation of Endothelial Cells.

Simple SummaryThe transmembrane proteoglycan syndecan-1 (SDC-1) is an important mediator of cell-matrix interactions. The heparan sulfate side-chains of SDC-1 can bind to a multitude of growth factors, cytokines, and chemokines, thereby regulating a plethora of physiological and pathological processes, including angiogenesis. The extracellular region of SDC-1 can be released from the cell surface by the action of sheddases including matrix metalloproteinase-7 and 9, resulting in a soluble protein that is still active and can act as a competitive activator or inhibitor of the cell surface receptor. Accelerated shedding and loss of cell surface SDC-1 is associated with epithelial to mesenchymal transition (EMT) and achievement of a more invasive phenotype in malignant mesothelioma (MM). Transfection with SDC-1 reverts the morphology in epithelioid direction and inhibits the proliferation and migration of MM cells. This study aimed to investigate the role of SDC-1 in angiogenesis. We demonstrate that overexpression and silencing of SDC-1 alters the secretion of angiogenic proteins in MM cells. Upon SDC-1 overexpression, several factors collectively inhibit the proliferation, wound closure, and tube formation of endothelial cells, whereas SDC-1 silencing only affects wound healing.Malignant mesothelioma (MM) is an aggressive tumor of the serosal cavities. Angiogenesis is important for mesothelioma progression, but so far, anti-angiogenic agents have not improved patient survival. Our hypothesis is that better understanding of the regulation of angiogenesis in this tumor would largely improve the success of such a therapy. Syndecan-1 (SDC-1) is a transmembrane heparan sulfate proteoglycan that acts as a co-receptor in various cellular processes including angiogenesis. In MM, the expression of SDC-1 is generally low but when present, SDC-1 associates to epithelioid differentiation, inhibition of tumor cell migration and favorable prognosis, meanwhile SDC-1 decrease deteriorates the prognosis. In the present study, we studied the effect of SDC-1 overexpression and silencing on MM cells ability to secrete angiogenic factors and monitored the downstream effect of SDC-1 modulation on endothelial cells proliferation, wound healing, and tube formation. This was done by adding conditioned medium from SDC-1 transfected and SDC-1 silenced mesothelioma cells to endothelial cells. Moreover, we investigated the interplay and molecular functional changes in angiogenesis in a co-culture system and characterized the soluble angiogenesis-related factors secreted to the conditioned media. We demonstrated that SDC-1 over-expression inhibited the proliferation, wound healing, and tube formation of endothelial cells. This effect was mediated by a multitude of angiogenic factors comprising angiopoietin-1 (Fold change ± SD: 0.65 ± 0.07), FGF-4 (1.45 ± 0.04), HGF (1.33 ± 0.07), NRG1-β1 (1.35 ± 0.08), TSP-1 (0.8 ± 0.02), TIMP-1 (0.89 ± 0.01) and TGF-β1 (1.35 ± 0.01). SDC-1 silencing increased IL8 (1.33 ± 0.06), promoted wound closure, but did not influence the tube formation of endothelial cells. Pleural effusions from mesothelioma patients showed that Vascular Endothelial Growth Factor (VEGF) levels correlate to soluble SDC-1 levels and have prognostic value. In conclusion, SDC-1 over-expression affects the angiogenic factor secretion of mesothelioma cells and thereby inhibits endothelial cells proliferation, tube formation, and wound healing. VEGF could be used in prognostic evaluation of mesothelioma patients together with SDC-1.

Proteasomal degradation of polycomb-group protein CBX6 confers MMP-2 expression essential for mesothelioma invasion

The aggressive invasiveness of malignant mesothelioma limits cancer therapy, however, the molecular mechanisms underlying the invasiveness remain largely unknown. Here we found that the matrix metalloproteinase-2 (MMP-2) was required for the invasion of mesothelioma cells in the collagen matrix and the gene expression of MMP-2 was correlated with the invasive phenotype. The MMP-2 gene expression was regulated by DNA and histone methylation around the transcription start site, implicating the involvement of the polycomb repressive complex (PRC). Knockdown of PRC component chromobox 6 (CBX6) promoted MMP-2 expression and invasion of mesothelioma cells. Transcriptome analysis suggested that CBX6 regulates sets of genes involved in cancer cell migration and metastasis. In invasive but not non-invasive cells, CBX6 was constantly unstable owing to ubiquitination and protein degradation. In human tissues, CBX6 localized in the nuclei of normal mesothelium and benign mesothelioma, but the nuclear staining of CBX6 was lost in malignant mesothelioma. These results suggest involvement of proteasomal degradation of CBX6 in mesothelioma progression.

SARCOID-LIKE REACTION TO IMMUNOTHERAPY MIMICS PROGRESSIVE MALIGNANCY ON IMAGING

SESSION TITLE: Medical Student/Resident Lung Cancer Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Recurrence or progression of thoracic malignancies can often be mimicked by numerous processes discovered on imaging including infection and inflammation. There is increasing evidence that immunotherapy can be associated with a sarcoid-like reaction causing enlarged, PET-avid lymph nodes that can resemble recurrence or progression of malignancy.1,2 We present a case in which progression of mesothelioma was suspected based on surveillance imaging, however, pathology was consistent with a sarcoid-like reaction in the setting of recent immunotherapy. CASE PRESENTATION: A 75-year-old female former smoker with a history of asbestos exposure subsequently diagnosed with epithelioid mesothelioma presents for evaluation of recurrence of her mesothelioma. She was initially diagnosed in 2018 when she presented with cough, pleuritic chest pain, weight loss and a pleural effusion. Diagnosis was made by thoracentesis and CT-guided biopsy of a left lower lobe nodule. Her initial stage was T2N0. She was treated with neoadjuvant Atezolizumab, Pemetrexed Disodium, Cisplatin followed by surgery (left pleurectomy, decortication, mediastinal lymph node dissection, and wedge resections for peripheral pleural nodules) and postoperative adjuvant Atezolizumab. Six months later, PET CT showed enlarged hypermetabolic mediastinal lymph nodes, a perifissural nodule, enlarging pleural nodules, and increased pleural thickening, suspicious for disease progression. The patient was asymptomatic and evaluated with bronchoscopy and endobronchial ultrasound with transbronchial needle aspiration of multiple mediastinal lymph nodes. Pathology revealed granulomatous inflammation. Given her asymptomatic presentation, enlarged and PET avid mediastinal lymphadenopathy and recent immunotherapy exposure, she was diagnosed with a sarcoid-like reaction as a cause for her mediastinal lymphadenopathy. Adenopathy later resolved after withdrawal of immunotherapy. DISCUSSION: We present a case in which a sarcoid-like reaction to immunotherapy mimics metastatic progression of mesothelioma. In this case, tissue biopsy indicated a lower disease stage compared to imaging and allowed for local rather than systemic treatment options. CONCLUSIONS: Recognition of sarcoid-like reactions is important during surveillance and restaging of malignancy in patients undergoing treatment with immunotherapy. This case illustrates the importance of direct tissue sampling in diagnosing mimickers of thoracic malignancies. Reference #1: Nishino M, Sholl LM, Awad MM, Hatabu H, Armand P, Hodi FS. Sarcoid-Like Granulomatosis of the Lung Related to Immune-Checkpoint Inhibitors: Distinct Clinical and Imaging Features of a Unique Immune-Related Adverse Event. Cancer Immunol Res. 2018;6(6):630-635. Reference #2: Tetzlaff MT, Nelson KC, Diab A, et al. Granulomatous/sarcoid-like lesions associated with checkpoint inhibitors: a marker of therapy response in a subset of melanoma patients. J Immunother Cancer. 2018;6(1):14. DISCLOSURES: stockholder relationship with Merck Please note: $20001 - $100000 Added 03/09/2020 by Adam Fox, source=Web Response, value=Ownership interest stockholder relationship with MMM Please note: $20001 - $100000 Added 03/09/2020 by Adam Fox, source=Web Response, value=Ownership interest No relevant relationships by Courtney Linkous, source=Web Response No relevant relationships by Gerard Silvestri, source=Web Response

AXL Inactivation Inhibits Mesothelioma Growth and Migration via Regulation of p53 Expression.

Simple SummaryPrevious studies have shown that AXL is a crucial protein that is activated in mesothelioma to cause growth and invasiveness. p53 is the tumor suppressor protein that is most frequently inactivated by mutations in human cancers, but these p53 inactivating mutations are not common in mesothelioma. In the studies reported herein we demonstrate that AXL activation causes p53 functional inactivation in mesothelioma. Specifically, we show that AXL suppresses p53 expression by binding to DNA sequences upstream from the p53 gene, thereby blocking transcription of p53 DNA into RNA. We also show that AXL inhibition by a selective drug inhibits mesothelioma cell viability, migration, and invasion. p53 inactivation attenuates the impact of the AXL inhibitor, providing further support for interplay between AXL and p53 in mesothelioma oncogenesis. These studies demonstrate a novel feedback regulation loop between AXL and p53, and provide a rationale for mesothelioma therapies targeting AXL/p53 signaling.Malignant mesothelioma is a locally aggressive and highly lethal neoplasm. Dysregulation and activation of Gas6/AXL tyrosine kinase signaling are associated with mesothelioma progression, but the mechanisms of these AXL tumorigenic roles are poorly understood. p53 mutants in lung carcinoma upregulate AXL expression by binding and acetylating the AXL promoter. Although TP53 mutations are uncommon in mesothelioma, we hypothesized that these tumors might have alternative feedback mechanisms between AXL and p53. In the current report, we investigated AXL regulation of TP53 transcription, expression, and biological function in mesothelioma. AXL expression was stronger in mesothelioma than most of the other tumor types from the TCGA gene expression profile dataset. AXL knockdown by shRNA induced wild-type and mutant p53 expression in mesothelioma cell lines, suggesting that AXL pro-tumorigenic roles result in part from the suppression of p53 function. Likewise, induced AXL inhibited expression of wild type p53 in COS-7 cells and 293T cells. Immunofluorescence staining showed nuclear colocalization of AXL and p53; however, association of AXL and p53 was not demonstrated in immunoprecipitation complexes. The AXL effects on p53 expression resulted from the inhibition of TP53 transcription, as demonstrated by qRT-PCR after AXL silencing and TP53 promotor dual luciferase activity assays. Chromatin immunoprecipitation-qPCR and sequencing showed that AXL bound to the initial 600 bp sequence at the 5′ end of the TP53 promoter. AXL inhibition (shRNA or R428) reduced mesothelioma cell viability, migration, and invasion, whereas TP53 shRNA knockdown attenuated antiproliferative, migration, and invasive effects of AXL silencing or AXL inactivation in these cells. These studies demonstrate a novel feedback regulation loop between AXL and p53, and provide a rationale for mesothelioma therapies targeting AXL/p53 signaling.

Malignant mesothelioma in situ: diagnostic and clinical considerations

In situ stages of malignancy have been characterised in various neoplasms. Mesothelioma in situ (MIS) has been a controversial diagnosis, lacking clear diagnostic criteria and understanding as to whether it is truly a premalignant lesion in the progression of malignant mesothelioma (MM). Originally understood as a concept and defined as atypical mesothelial proliferation in the presence of invasion, it has now been suggested that loss of nuclear labelling for BRCA1-associated protein-1 (BAP1) in flat, non-invasive mesothelial lesions can define MIS. This study aimed to characterise BAP1 expression in a cohort of 19 patients diagnosed with MIS (either pure MIS, n=3, or MIS-predominant invasive MM, n=16) and to compare survival between MIS, MIS-predominant MM and MM (n=114) in order to gain insight into the characteristics of MIS. We defined pure MIS as any architectural pattern of surface mesothelial cells with loss of BAP1 in the absence of invasion, but in specimens with superficial stromal invasion we also accepted the original definition of cytologically and architecturally atypical mesothelial proliferation, in the absence of inflammatory features, with or without loss of BAP1. We observed that MIS associated with minimal invasion was associated with significantly improved survival compared to MM (8 months vs 22 months). This suggests that MIS is indeed a precursor to MM and that these cases represent earlier stage disease. Loss of BAP1 was present in 60% of mesotheliomas with invasion, so not all early cases can be detected by BAP1 loss, but our study provides evidence that BAP1 loss may be an early molecular alteration in MM pathogenesis in patients that have loss of BAP1. We confirm that BAP1 loss can be useful for diagnosis of pure MIS in surgical specimens, permitting earlier diagnosis. However, identification of a predominant MIS component with minimal invasion has prognostic and conceptual implications. Whilst no approved therapy is available for MIS, close follow up of patients with BAP1 mutation in mesothelial cells and/or diagnosis of MIS is required to monitor for disease progression and potentially investigate earlier treatment interventions.

Connective tissue growth factor produced by cancer‑associated fibroblasts correlates with poor prognosis in epithelioid malignant pleural mesothelioma.

Malignant mesothelioma is an aggressive neoplasm for which effective treatments are lacking. We often encounter mesothelioma cases with a profound desmoplastic reaction, suggesting the involvement of cancer-associated fibroblasts (CAFs) in mesothelioma progression. While the roles of CAFs have been extensively studied in other tumors and have led to the view that the cancer stroma contains heterogeneous populations of CAFs, their roles in mesothelioma remain unknown. We previously showed that connective tissue growth factor (CTGF), a secreted protein, is produced by both mesothelioma cells and fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of α-smooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and Elastica-Masson staining. We also analyzed the expression of mesenchymal stromal cell- and fibroblast-expressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancer-restraining CAFs and differ from αSMA-positive CAFs, by in situ hybridization. The extent of fibrosis and CTGF expression in mesothelioma cells did not correlate with patient prognosis. However, the expression of αSMA and CTGF, but not Meflin, in CAFs correlated with poor prognosis. The data suggest that CTGF+ CAFs are involved in mesothelioma progression and represent a potential molecular target for mesothelioma therapy.

MTH1 favors mesothelioma progression and mediates paracrine rescue of bystander endothelium from oxidative damage.

Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor's response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.

Data mining analysis of the PP2A cell cycle axis in mesothelioma patients.

Mesothelioma is an aggressive tumor that affects thousands of people every year. The therapeutic options for patients are limited; hence, a better understanding of mesothelioma biology is crucial to improve patient survival. To find new molecular targets and therapeutic strategies related to the protein phosphatase 2A (PP2A) network, we analyzed the gene expression of known PP2A inhibitors in mesothelioma patient samples. Our analysis disclosed a general overexpression of all PP2A-negative regulators in mesothelioma patients. Moreover, the expression of ANP32E and CIP2A genes, increased in 16% and 11% of cases, positively correlates with the ones of all the other PP2A regulators and the ones of the main cyclins and CDKs, suggesting the existence of a feed-forward loop that might contribute to the mesothelioma progression via PP2A inactivation. Overall, our study indicates the existence of a strategic and targetable axis between PP2A inhibitors (ANP32E and CIP2A) and cell cycle regulators (cyclin B2/CDK1) and provides a valuable rationale for using a personalized combinational therapy approach to improve mesothelioma patient survival.

Versican modulates tumor-associated macrophage properties to stimulate mesothelioma growth

ABSTRACTVersican promotes experimental tumor growth through cell- and non cell-autonomous mechanisms. Its role in mesothelioma progression has not been investigated so far. In this study we investigated the impact of tumor-derived versican in mesothelioma progression and the underlying mechanism of its action. For this purpose, versican-silenced or control ΑΕ17 and ΑΒ1 murine mesothelioma cells were intrapleuraly injected into syngeneic mice, in order to create pleural mesotheliomas and pleural effusions. Intratumoral and pleural immune subsets were assessed using flow cytometry. Mesothelioma cells were co-cultured with syngeneic macrophages to examine versican’s impact on their interaction and endothelial cells to assess the effect of versican in endothelial permeability. Versican expression was assessed in human mesotheliomas and mesothelioma-related pleural effusions and benign pleural tissue and effusions. We observed that, versican silencing reduced mesothelioma mass and pleural fluid volume by affecting tumor cell proliferation and apoptosis in vivo, while tumor cell growth remained intact in vitro, and limited pleural vascular permeability. Mice harboring versican-deficient tumors presented fewer tumor/pleural macrophages and neutrophils, and fewer pleural T-regulatory cells, compared to the control animals. Macrophages co-cultured with versican-deficient mesothelioma cells were polarized towards M1 anti-tumor phenotype and demonstrated increased tumor cell phagocytic capacity, compared to macrophages co-cultured with control tumor cells. In co-culture, endothelial monolayer permeability was less effectively stimulated by versican-deficient cells than control cells. Versican was over-expressed in human mesothelioma tissue and mesothelioma-associated effusion. In conclusion, tumor cell-derived versican stimulates mesothelioma progression by shaping a tumor friendly inflammatory milieu, mainly by blunting macrophage anti-tumor activities.

Genomic changes of chromosomes 8p23.1 and 1q21: Novel mutations in malignant mesothelioma

IntroductionMalignant mesothelioma is an aggressive malignancy of the thoracic cavity caused by prior asbestos exposure. In the peritoneum the mesothelioma is an extremely rare condition. In the present preliminary study, high-resolution array-comparative genomic hybridization (a-CGH) was performed to identify genetic imbalances in a series of malignant peritoneal mesothelioma cases. Materials and methodsBetween 1990 and 2008, among the cases recorded in the Apulia Mesothelioma Register, we found 22 peritoneal mesothelioma cases. CGH-array was performed on samples from all patients. ResultsThe CGH-array analysis revealed multiple chromosomal imbalances. Interestingly, deletion at 8p23.1 was observed in 12 cases. Furthermore, another novel deletion at 1q21 was present in 11. Often, 1q21 and 8p23.1 losses were present in the same patient (7 cases). Losses of BAP1 and CDKN2A loci were not detected. DiscussionThe region at 8p23.1 contains the beta-defensin gene cluster (DEF) and 1q21 contains ubiquitin conjugating enzyme E2 (UBE2Q1). We hypotesized that the loss of function of ubiquitination, as well as of the defensins, could play an important role in the initial development and subsequent progression of mesothelioma.

Role of angiopoietins in mesothelioma progression

Background and objectiveAnti-angiogenic treatment has been recently shown to be clinically beneficial for mesothelioma patients. Angiopoietins-1 and -2 are key regulators of tumor angiogenesis. Ang-1 is mainly known to promote angiogenesis and vessel stability, while Ang-2 could serve as an antagonist of Ang-1 causing vessel regression and destabilization or enhance angiogenesis in a context-dependent manner. We hypothesized that Ang-1 would promote and Ang2 would halt experimental mesothelioma by affecting tumor angiogenesis. MethodsTo examine the effects of angiopoietins in mesothelioma angiogenesis and in vivo growth we constructed Ang-1 or Ang-2 overexpressing AE17 and AB1 mesothelioma cells and implanted them in the respective syngeneic animals. We also explored the clinical relevance of our observations using the human tumoral mRNAseq data available in the TCGA database. Results and conclusionsAng-1 promotes mesothelioma angiogenesis and growth while the effect of Ang-2 is context-dependent. Low Ang-1 levels in human mesotheliomas are associated with the epitheloid subtype. Tumors of high Ang-1, or concurrent high Ang-2 and VEGF expression present high PECAM-1 and CDH5 expression, markers of vascularity and vascular stability, respectively. Our results highlight the importance of angiopoietins in mesothelioma pathophysiology and pave the way for the clinical development of novel anti-angiogenic strategies.

Abstract 2769: Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3

Abstract Malignant mesothelioma (MM) is an aggressive tumor of mesothelial tissues that has poor prognosis and limited therapeutic options. Pass et al. (N Engl J Med 2012) identified the extracellular matrix protein fibulin-3 as an accurate biomarker of MM progression, which can be detected in the tumor parenchyma and in pleural effusions. Here, we report a systematic study of the mechanisms of fibulin-3 in MM and its value as a molecular target. Fibulin-3 activated canonical NFkB signaling and regulated the expression of NFkB-downstream genes, including the proteases MMP9 and MMP13, and the proinvasive proteins TNC and MLCK. Fibulin-3 knockdown downregulated NFkB signaling, reduced MM cell viability and potentiated the cytotoxicity of cisplatin in vitro. We next developed a function-blocking antibody ("mAb428.2") against an epitope of fibulin-3 required for NFkB activation (Nandhu et al., Clin Cancer Res 2017). This antibody blocked fibulin-3 signaling in vitro and reduced the expression of this protein, as well as NFkB-regulated MMP9, in subcutaneous (sc) MM tumors implanted in nude mice. Next, we treated mice carrying sc or intrapleural models of MM with mAb428.2 or control IgG1 (8x 30 mg/kg, q24h) delivered IV. Our anti-fibulin-3 antibody caused a significant reduction of tumor burden and extension of overall survival in both models. Overall, downregulation and inhibition of fibulin-3 proved a successful approach to enhance MM therapy. Given the value of this protein as a MM biomarker, we expect that detection of fibulin-3 may serve to identify patients with the highest chance to respond to combination therapies including anti-fibulin-3 approaches, achieving maximum therapeutic against these aggressive tumors. Citation Format: Mohan Sobhana Nandhu, Chandra Goparaju, Sharon L. Longo, Harvey I. Pass, Mariano S. Viapiano. Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2769.