Malignant Pleural Mesothelioma Tissue Research Articles

Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Prognostic significance and immune cell infiltration analysis of differentially expressed genes in malignant pleural mesothelioma

Objective: To explore and analyze differential expressed genes in malignant pleural mesothelioma (MPM) by bioinformatics method, and to study their prognostic value in MPM and their potential role in immunotherapy. Methods: In January 2022, the dataset GSE51024 was downloaded from the GEO database, and MPM (55 cases) and normal tissue (41 cases) samples were obtained. Using R software and HMDD and miRNet database, MPM-related differential genes were screened and co-expressed genes were identified. Co-expressed genes were enriched and functionally annotated, and protein-protein interaction (PPI) networks were constructed and key genes were identified using the STRING database and Cytoscape software. TRRUST and GEPIA databases were used to predict transcription factors of key genes and to analyze prognosis and survival. The correlation between key genes and the degree of infiltration of immune cells was analyzed using TIMER. Results: A total of 435 co-expressed genes were obtained, which were mainly concentrated in the extracellular matrix tissue and the signaling pathways of cell adhesion molecules. Combined with PPI and TRRUST database, seven key MPM prognostic genes were identified. Among them, cyclin 20 (CDC20) , cell cycle checkpoint kinase 1 (CHEK1) , enhancer of Zeste homolog 2 (EZH2) , ribonucleotide reductase subunit M2 (RRM2) , topoisomerase 2A (TOP2A) , ubiquitin like plant homeodomain and ring finger domain 1 (UHRF1) were significantly up-regulated in MPM, while cyclin A1 (CCNA1) was significantly down-regulated. The expressions of CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 genes were significantly associated with MPM overall survival (P<0.05) . The expressions of CDC20, CHEK1, EZH2, RRM2 and TOP2A genes were positively correlated with B cells and dendritic cells (P<0.05) , and negatively correlated with neutrophils (P<0.05) . Conclusion: CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 may be potential prognostic markers in MPM patients, and their expressions may be related to MPM tumor immunity.

Expression of CD24 gene in human malignant pleural mesothelioma and its relationship with prognosis

Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.

Analysis of Primary Cilium Expression and Hedgehog Pathway Activation in Mesothelioma Throws Back Its Complex Biology

The primary cilium (PC) is a sensory organelle present on the cell surface, modulating the activity of many pathways. Dysfunctions in the PC lead to different pathologic conditions including cancer. Hedgehog signaling (Hh) is regulated by PC and the loss of its control has been observed in many cancers, including mesothelioma. Malignant pleural mesothelioma (MPM) is a fatal cancer of the pleural membranes with poor therapeutic options. Recently, overexpression of the Hh transcriptional activator GL1 has been demonstrated to be associated with poor overall survival (OS) in MPM. However, unlike other cancers, the response to G-protein-coupled receptor smoothened (SMO)/Hh inhibitors is poor, mainly attributable to the lack of markers for patient stratification. For all these reasons, and in particular for the role of PC in the regulation of Hh, we investigated for the first time the status of PC in MPM tissues, demonstrating intra- and inter-heterogeneity in its expression. We also correlated the presence of PC with the activation of the Hh pathway, providing uncovered evidence of a PC-independent regulation of the Hh signaling in MPM. Our study contributes to the understanding MPM heterogeneity, thus helping to identify patients who might benefit from Hh inhibitors.

The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

EP07.02-001 Evaluation of FGF18 as a Contributing Factor in Malignant Pleural Mesothelioma Growth and Its Role as a Potential Biomarker

Malignant pleural mesothelioma (MPM) is an aggressive malignancy originating from the mesothelial lining of the pleural cavity. Despite the numerous advances in treatment approaches in recent years, the prognosis remains poor with a median overall survival time (OS) ranging from 10 to 22 months. Therefore, the identification of novel non-invasive biomarkers is urgently needed in order to identify patients with a better prognosis and to make the best personalized therapeutic decision. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens.

Expression and clinical significance of COL1A1 and COL1A2 genes in malignant pleural mesothelioma tissues

Objective: To investigate the expression levels and clinical significance of collagen typeⅠ α1 chain (COL1A1) and collagen type Ⅰ α2 chain (COL1A2) in malignant pleural mesothelioma (MPM) tissues. Methods: In January 2020, MPM tissues and adjacent normal pleural tissues were collected from 26 MPM patients, and the expression levels of COL1A1 and COL1A2 genes in the tissues were determined by quantitative reverse transcription PCR, and the efficacy of both levels in diagnosing MPM was assessed using receiver operating characteristic (ROC) curves. The relationship between COL1A1 and COL1A2 gene expression and clinicopathological features was analyzed by the Cancer Genome Atlas (TCGA) database, and the relationship between the expression levels of both and overall survival (OS) and disease-free progression survival (DFS) of MPM patients was dynamically analyzed by gene expression profiling, and the factors affecting the prognosis of MPM patients were explored by Cox proportional risk regression model. The TIMER 2.0 platform was used to explore the relationship between COL1A1 and COL1A2 gene expression in MPM and tumor immune infiltrative cells. Results: Compared with normal pleural tissues, the expression of COL1A1 and COL1A2 genes was significantly increased in MPM tissues (P<0.01) , and their expression was positively correlated (P<0.001) . The ROC curves showed that the area under the curve for COL1A1 and COL1A2 expression levels diagnostic of MPM was 0.900 and 0.897, respectively. The expression of COL1A1 gene was correlated with tumor type in MPM patients (P<0.05) , and COL1A2 gene expression was correlated with T stage in MPM patients (P<0.05) . Both COL1A1 and COL1A2 gene expression were associated with OS in MPM patients (Logrank P<0.05) , but there was no significant correlation with DFS (Logrank P>0.05) . Cox multivariate analysis showed that patients with high COL1A1 and COL1A2 gene expression and biphasic mixed MPM had a higher risk of death (P<0.05) . TIMER 2.0 platform analysis showed that COL1A1 and COL1A2 gene expression in MPM patients was positively correlated with macrophages, COL1A2 gene expression in MPM was negatively correlated with neutrophils (P<0.05) . Conclusion: High expression of COL1A1 and COL1A2 genes in MPM tissues is valuable for diagnosis, disease prediction and prognostic assessment of MPM, and both may jointly contribute to the development of MPM.

NFE2L3 as a Novel Biomarker Associated With IL-2/STAT5/NLRP3 Signaling Pathway in Malignant Pleural Mesothelioma and Other Cancers.

Background: Malignant pleural mesothelioma (MPM) is a malignant tumor originating from pleural mesothelial cells and has a high mortality rate worldwide. With the advent of immunotherapy in MPM treatment, there is an urgent need to elucidate the immune-related mechanisms in this caner. Methods: Single-sample gene set enrichment analysis (ssGSEA) was used to score the immunocytes infiltration of data from different database sources. Identification of immunocyte-related genes was performed with weighted gene co-expression network analysis (WGCNA), differentially expressed genes (DEGs) analysis, and correlation analysis. Pan-caner analysis was performed using “DiffExp” and “Correlation” modules in TIMER. Results: T-helper 2 (Th2) cell was found to be a poor prognostic factor for patients with MPM. Then a transcription factor, NFE2L3, was identified as a biomarker that showed a strong positive correlation with Th2 cell infiltration, and was highly expressed in MPM tissues and was related to the poor prognosis of these patients. At the same time, multiple NFE2L3 methylation sites were negatively correlated with Th2 cell infiltration, and patients with a high degree of methylation enjoy a better prognosis. Pan-caner analysis indicated that NFE2L3 might promote the differentiation of Th2 cells through the IL-2/STAT5/NLRP3 signaling pathway in MPM and many other cancers. Conclusion: We believe that NFE2L3 can serve as a potential biomarker related to the diagnosis and prognosis of patients with MPM, and speculate that NFE2L3 could promote Th2 cell differentiation via IL-2/STAT5/NLRP3 signaling pathway in MPM and many other cancers.

Sulfated Glycans Recognized by S1 Monoclonal Antibody can Serve as a Diagnostic Marker for Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a malignant neoplasm of the pleura caused by asbestos exposure. For diagnosis of MPM, immunohistochemistry using multiple markers is recommended to rule out differential diagnoses, such as pulmonary adenocarcinoma. However, the specificity of currently used markers is not fully satisfactory. We previously developed a monoclonal antibody named S1, which recognizes 6-sulfo sialyl Lewis x, an L-selectin ligand expressed on high endothelial venules. During the screening process, we discovered that this antibody stained normal pleural mesothelium. This finding prompted us to hypothesize that the epitope recognized by S1 might serve as a new diagnostic marker for MPM. To test this hypothesis, we immunostained human MPM (n = 22) and lung adenocarcinoma (n = 25) tissues using S1 antibody. 77.3% of MPM were S1 positive, and if limited to epithelioid type, the positivity rate was 100%, while that of lung adenocarcinoma was only 36.0%. Statistical analysis revealed a significant difference in the S1 positivity rate between each disease. Furthermore, immunohistochemistry using a series of anti-carbohydrate antibodies combined with glycosidase digestion revealed the structure of sulfated glycans expressed in MPM to be 6-sulfo sialyl N-acetyllactosamine attached to core 2-branched O-glycans. We propose that the S1 glycoepitope could serve as a new diagnostic marker for MPM.

Elemental Mapping of Human Malignant Mesothelioma Tissue Samples Using High-Speed LA-ICP-TOFMS Imaging.

This is the first report of the use of laser ablation–inductively coupled plasma time-of-flight mass spectrometry (LA–ICP–TOFMS) to analyze human malignant pleural mesothelioma (MPM) samples at the cellular level. MPM is an aggressive, incurable cancer associated with asbestos exposure, with a long latency and poor overall survival. Following careful optimization of the laser fluence, the simultaneous ablation of soft biological tissue and hard mineral fibers was possible, allowing the spatial detection of elements such as Si, Mg, Ca, and Fe, which are also present in the glass substrate. A low-dispersion LA setup was employed, which provided the high spatial resolution necessary to identify the asbestos fibers and fiber fragments in the tissue and to characterize the metallome at the cellular level (a pixel size of 2 μm), with a high speed (at 250 Hz). The multielement LA–ICP–TOFMS imaging approach enabled (i) the detection of asbestos fibers/mineral impurities within the MPM tissue samples of patients, (ii) the visualization of the tissue structure with the endogenous elemental pattern at high spatial resolution, and (iii) obtaining insights into the metallome of MPM patients with different pathologies in a single analysis run. Asbestos and other mineral fibers were detected in the lung and pleura tissue of MPM patients, respectively, based on their multielement pattern (Si, Mg, Ca, Fe, and Sr). Interestingly, strontium was detected in asbestos fibers, suggesting a link between this potential toxic element and MPM pathogenesis. Furthermore, monitoring the metallome around the talc deposit regions (characterized by elevated levels of Al, Mg, and Si) revealed significant tissue damage and inflammation caused by talc pleurodesis. LA–ICP–TOFMS results correlated to Perls’ Prussian blue and histological staining of the corresponding serial sections. Ultimately, the ultra-high-speed and high-spatial-resolution capabilities of this novel LA–ICP–TOFMS setup may become an important clinical tool for simultaneous asbestos detection, metallome monitoring, and biomarker identification.

ATG7 immunohistochemical expression in malignant pleural mesothelioma. A preliminary report.

Literature evidence has demonstrated a high incidence of asbestos-related malignant pleural mesothelioma (MPM) in a Sicilian town (Biancavilla, Italy), where fluoro-edenite (FE) fibers were discovered some decades ago. As ATG7 immunohistochemical analysis has been ascribed as a prognostic tool of improved survival, we decided to investigate, in MPM patients, exposed and not exposed to FE fibers, the immunohistochemical expression of this autophagy-related protein named ATG7. We analyzed the correlation between ATG7 immunohistochemical level and clinicopathological parameters. Twenty MPM tissue samples, from patients with available clinical and follow-up data, were included in paraffin and processed for immunohistochemistry. The immunohistochemical results confirmed activation of the autophagic process in MPM. Densitometric and morphometric expressions of ATG7 were significantly increased in MPMs when compared to the control tissues. A significant association of a high level of ATG7 with increased survival was demonstrated, with a mean overall survival (OS) of 12.5 months for patients with high expression vs. a mean OS of 4.5 months for patients with low ATG7 expression. In addition, a significant correlation between ATG7 expression and the survival time of MPM patients was observed. This study represents a starting point to hypothesize the prognostic role of ATG7 which could be a reliable prognostic indicator in MPM.

P35.01 YB-1 is a Key Player in Aggressive Behaviour and Chemoresistance in Mesothelioma

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome. YB-1 expression was analysed by qPCR or western blot in cell lines, and in patient material by immunohistochemistry. YB-1 overexpression was achieved by a doxycycline-inducible, retroviral construct, which was stably introduced into MPM cell lines. YB-1 knockdown was performed using YB-1 specific siRNA. For inhibition of YB-1 phosphorylation, the RSK inhibitor BI-D1870 was used. Effects of drug interaction (CI values) were calculated using the CompuSyn software. Cell migration was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. For the zebrafish model, RFP-expressing MPM cells were injected into 48 hour old larvae, imaged after 1 and 2 days and the number of cells in the tail was manually counted. We previously reported that YB-1 is up-regulated in MPM cell lines compared to non-malignant controls. In this study we analysed MPM tissue (n>120) and normal pleura (n=3) specimens for YB-1 expression. While all control samples were negative, tumours showed a heterogeneous YB-1 expression. YB-1 knockdown decreases MPM cell migration and invasion, hence we evaluated the impact of YB-1 overexpression on these phenotypes. MPM cell lines (n=6) which overexpress YB-1 in a doxycycline-inducible manner showed significantly increased cell scattering and migration. Furthermore, YB-1 overexpression lead to significantly higher migratory capacity in vivo using a zebrafish xenograft model. Additionally, when tumour spheroids were co-cultured with endothelial cells, YB-1 overexpression led to a significantly more extensive formation of gaps in the endothelial layer. Finally, since we found that YB-1 knockdown not only decreases cell growth in vitro and in vivo but also decreases the expression of LRP1 and ABCC1, two genes involved in drug resistance, we combined YB-1 knockdown as well as inhibition of YB-1 phosphorylation via an RSK inhibitor with cisplatin chemotherapy. Our data showed highly synergistic combination effects (CI values: 0.1 – 0.5) for several cisplatin doses in combination with either YB-1 siRNA or the RSK inhibitor. In this study we report a deregulated YB-1 expression in MPM tumor specimens compared to non-malignant controls. Our data show an important role of YB-1 in the regulation of cell migration and invasion in vitro and in vivo, which are key characteristics of MPM. Additionally, YB-1 knock down as well as pharmacological inhibition of YB-1 phosphorylation not only reduce MPM cell growth but also sensitise cells to cisplatin chemotherapy. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

The Role of E-Cadherin and microRNA on FAK Inhibitor Response in Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited effective treatment options. Focal adhesion kinase (FAK) inhibitors have been shown to efficiently suppress MPM cell growth initially, with limited utility in the current clinical setting. In this study, we utilised a large collection of MPM cell lines and MPM tissue samples to study the role of E-cadherin (CDH1) and microRNA on the efficacy of FAK inhibitors in MPM. The immunohistochemistry (IHC) results showed that the majority of MPM FFPE samples exhibited either the absence of, or very low, E-cadherin protein expression in MPM tissue. We showed that MPM cells with high CDH1 mRNA levels exhibited resistance to the FAK inhibitor PND-1186. In summary, MPM cells that did not express CDH1 mRNA were sensitive to PND-1186, and MPM cells that retained CDH1 mRNA were resistant. A cell cycle analysis showed that PND-1186 induced cell cycle disruption by inducing the G2/M arrest of MPM cells. A protein−protein interaction study showed that EGFR is linked to the FAK pathway, and a target scan of the microRNAs revealed that microRNAs (miR-17, miR221, miR-222, miR137, and miR148) interact with EGFR 3′UTR. Transfection of MPM cells with these microRNAs sensitised the CHD1-expressing FAK-inhibitor-resistant MPM cells to the FAK inhibitor.

MicroRNA-206 suppresses mesothelioma progression via the Ras signaling axis

Malignant pleural mesothelioma (MPM) is an incurable surface neoplasm with peculiar pathobiology. MPM proliferates by using the tyrosine-kinase-Ras pathway. Despite representing an attractive therapeutic target, there are no standard agent(s) specifically inhibiting Ras signaling adopted in clinical settings. We posited that biologic effects of microRNA (miRNA) can disrupt this molecular network. Using patient samples, cell lines, and murine tumor xenograft models, we confirmed specific genes in the Ras pathway are targeted by an MPM-associated miRNA and then examined its therapeutic effects. We verified significant and consistent downregulation of miR-206 in MPM tissues. When miR-206 is ectopically re-expressed in MPM cells and delivered to tumor xenografts in mice, it exerted significant cell killing by suppressing multiple components of the receptor-tyrosine-kinase-Ras-cell-cycle-signaling network; some of which were prognostic when overexpressed and/or have not been druggable. Of note, we validated CDK6 as a novel target of miR-206. Overall, this miR-206-targeting mechanism manifested as induced G1/S cell cycle arrest. In addition, we identified a novel MPM therapeutic combination by adding systemic-route abemaciclib with local-route miR-206, which showed additive efficacy translating to improved survival. Our pre-clinical study suggests a potential pathophysiologic role for, and therapeutic relevance of, miR-206 in MPM.

Metastasis-associated gene 1 (MTA1) enhances cisplatin resistance of malignant pleural mesothelioma by ATR-Chk1-mediated DNA repair.

BackgroundMalignant pleural mesothelioma (MPM) chemoresistance remains a challenge to oncologists. In our previous study, we demonstrated that the aberrant expression of metastasis-associated gene 1 (MTA1) is associated with carcinogenesis and metastasis in MPM. The aim of the present study was to investigate the mechanism of MTA1 and chemo-resistance in MPM.MethodsWestern blotting and real-time polymerase chain reaction were used to analyze the protein and mRNA levels. A stable clone with a knockdown of MTA1 was generated with shRNA via lentivirus technology in MPM cell lines. Cell Counting Kit-8 assay and crystal violet assay were used to measure cell viability. Immunochemical staining was employed to detect MTA1 expression in MPM tissues. The cell cycle of MPM cells was determined by phosphohistone H3 staining and flow cytometric analysis.ResultsThe MTA1 protein was upregulated and enhanced cisplatin resistance in MPM. Cisplatin stabilized the expression of the MTA1 protein by inhibiting its ubiquitination, and MTA1 enhanced G2/M cell cycle delay and regulated and protected the tumor genome from chemotherapeutic drugs via participating in the phosphorylation of the ataxia telangiectasia mutated and rad3 related-checkpoint kinase 1 (ATR-Chk1) pathway.ConclusionsThese data suggest that MTA1 enhances cisplatin resistance by ATR-Chk1-mediated DNA damage repairment and cisplatin stabilizes MTA1 expression via affecting on the ubiquitination pathway of MTA1 in MPM. Our findings indicate that MTA1 could serve as a novel therapeutic target to overcome chemoresistance in MPM.

Alterations in BAP1 Are Associated with Cisplatin Resistance through Inhibition of Apoptosis in Malignant Pleural Mesothelioma.

The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

CDKN2A and MTAP Are Useful Biomarkers Detectable by Droplet Digital PCR in Malignant Pleural Mesothelioma: A Potential Alternative Method in Diagnosis Compared to Fluorescence In Situ Hybridisation.

BackgroundThe diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH). The tumor suppressor gene, CDKN2A, is frequently silenced by epigenetic mechanisms in many cancers; in the case of MPM it is mostly silenced via genomic deletion. Co-deletion of the CDKN2A and methylthioadenosine phosphorylase (MTAP) genes has been researched extensively and discovered to be a highly specific characteristic of MPM. Most studies have used FISH to detect the deletion of CDKN2A and IHC for MTAP as a surrogate for this. In this study, we aim to investigate and validate droplet digital PCR (ddPCR) as an emerging alternative and efficient testing method in diagnosing MPM, by particularly emphasizing on the loss of MTAP and CDKN2A.MethodsThis study included 75 formalin fixed paraffin embedded (FFPE) MPM tissue, and 12 normal pleural tissue and 10 RMH as control. Additionally, primary MPM cell lines and normal pleural samples were used as biomarker detection controls, as established in our previous publication. All FFPE specimens were processed to isolate the DNA, that was subsequently used for ddPCR detection of CDKN2A and MTAP. FFPE samples were also analyzed by fluorescence in situ hybridization (FISH) for CDKN2A and MTAP deletion, and for MTAP IHC expression. Concordance of IHC and ddPCR with FISH were studied in these samples.Results95% and 82% of cases showed co-deletion of both MTAP and CDKN2A when determined by FISH and ddPCR respectively. ddPCR has a sensitivity of 72% and specificity of 100% in detecting CDKN2A homozygous loss in MPM. ddPCR also has a concordance rate of 92% with FISH in detecting homozygous loss of CDKN2A. MTAP IHC was 68% sensitive and 100% specific for detecting CDKN2A homozygous loss in MPM when these losses were determined by ddPCR.ConclusionOur study confirms that MTAP is often co-deleted with CDKN2A in MPM. Our in-house designed ddPCR assays for MTAP and CDKN2A are useful in differentiating MPM from RMH, and is highly concordant with FISH that is currently used in diagnosing MPM. ddPCR detection of these genetic losses can potentially be utilized as an alternative method in the diagnosis of MPM and for the future development of a less-invasive MPM-specific detection technique on MPM tumor tissue DNA.

EIF4G1 and RAN as Possible Drivers for Malignant Pleural Mesothelioma.

For malignant pleural mesothelioma (MPM) novel therapeutic strategies are urgently needed. In a previous study, we identified 51 putative cancer genes over-expressed in MPM tissues and cell lines. Here, we deepened the study on nine of them (ASS1, EIF4G1, GALNT7, GLUT1, IGF2BP3 (IMP3), ITGA4, RAN, SOD1, and THBS2) to ascertain whether they are truly mesothelial cancer driver genes (CDGs) or genes overexpressed in an adaptive response to the tumoral progression (“passenger genes”). Through a fast siRNA-based screening, we evaluated the consequences of gene depletion on migration, proliferation, colony formation capabilities, and caspase activities of four MPM (Mero-14, Mero-25, IST-Mes2, and NCI-H28) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model. The depletion of EIF4G1 and RAN significantly reduced cell proliferation and colony formation and increased caspase activity. In particular, the findings for RAN resemble those observed for other types of cancer. Thus, we evaluated the in vitro effects of importazole (IPZ), a small molecule inhibitor of the interaction between RAN and importin-β. We showed that IPZ could have effects similar to those observed following RAN gene silencing. We also found that primary cell lines from one out of three MPM patients were sensitive to IPZ. As EIF4G1 and RAN deserve further investigation with additional in vitro and in vivo studies, they emerged as promising CDGs, suggesting that their upregulation could play a role in mesothelial tumorigenesis and aggressiveness. Furthermore, present data propose the molecular pathways dependent on RAN as a putative pharmacological target for MPM patients in the view of a future personalized medicine.

Delayed-Phase Enhancement for Evaluation of Malignant Pleural Mesothelioma on Computed Tomography: A Prospective Cohort Study

BackgroundRadiologic assessment of malignant pleural mesothelioma (MPM) on computed tomography (CT) imaging can be limited by similar attenuations of MPM and adjacent tissues. This can result in inaccuracies in defining the presence and extent of pleural tumor burden. We hypothesized that increasing the time delay for pleural enhancement will optimize discrimination between MPM and noncancerous tissues on CT. Here we conduct a prospective observational study to determine the optimal time delay for imaging MPM on CT. Patients and MethodsAdult MPM patients (n = 15) were enrolled in this prospective exploratory imaging trial. Patients with < 1 cm MPM thickness, prior pleurectomy, pleurodesis, pleural radiotherapy, or antiangiogenic therapy were excluded. All patients underwent a dynamically-enhanced CT with multiple time delays (0 - 10 minutes) after intravenous contrast administration. Tumor tissue attenuation was measured at each phase of enhancement. A qualitative assessment of tumor enhancement kinetics was also performed. The optimal phase of enhancement based on qualitative lesion conspicuity and quantitative tumor enhancement was then compared. ResultsMPM tumor enhancement was quantitatively and qualitatively increased at time delays beyond the conventional time delay for thoracic CT imaging (40-60 seconds). Patient tumor enhancement kinetics, displayed as the fraction of maximal tumor tissue attenuation as a function of time, revealed an optimal time delay of 230 to 300 seconds after intravenous contrast administration. There was an association between degree of tumor enhancement and subjective lesion conspicuity. ConclusionOptimal MPM contrast enhancement occurs at a later phase than typically acquired with conventional thoracic CT imaging.

Prognostic and clinicopathological utility of programmed death-ligand 1 in malignant pleural mesothelioma: A meta-analysis

ObjectiveProgrammed death ligand 1 (PD-L1) has been reported to be connected to prognosis in individuals with malignant pleural mesothelioma (MPM), although there is no consensus based on data from previous studies. Accordingly, this quantitative meta-analysis investigated prognostic and clinicopathological utility of PD-L1 in patients with MPM. MethodsA comprehensive search of the PubMed, Web of Science, Embase, and Cochrane Library databases for articles published up to October 4, 2019 was performed. Studies using immunohistochemical techniques to detect/quantify the expression of PD-L1 in MPM tissue were enrolled in the analysis. The combined hazard ratio (HR) and corresponding 95% confidence interval (CI) was applied to assess the association between PD-L1 expression and overall survival (OS). ResultsA total of 11 studies comprising 1606 patients was included in the present meta-analysis. For OS, pooled data revealed an HR of 1.50 (95% CI 1.32–1.70; p < 0.001), suggesting that patients with PD-L1 overexpression experience inferior OS. Subgroup analysis revealed that elevated PD-L1 remained a significant prognostic indicator for worse OS, irrespective of sample size, cut-off value, ethnicity, and Newcastle-Ottawa Scale score. Moreover, PD-L1 overexpression was associated with non-epithelioid histology (odds ratio 4.30 [95% CI 1.89–9.74]; p < 0.001). ConclusionsResults of this meta-analysis show that elevated expression of PD-L1 could be a factor predicting poorer survival in patients with MPM.