[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.

Pleural mesothelioma (PMe): The evolving molecular knowledge of a rare and aggressive cancer.

Mesothelioma is a type of late-onset cancer that develops in cells covering the outer surface of organs. Although it can affect the peritoneum, heart, or testicles, it mainly targets the lining of the lungs, making pleural mesothelioma (PMe) the most common and widely studied mesothelioma type. PMe is caused by exposure to fibres of asbestos, which when inhaled leads to inflammation and scarring of the pleura. Despite the ban on asbestos by most Western countries, the incidence of PMe is on the rise, also facilitated by a lack of specific symptomatology and diagnostic methods. Therapeutic options are also limited to mainly palliative care, making this disease untreatable. Here we present an overview of biological aspects underlying PMe by listing genetic and molecular mechanisms behind its onset, aggressive nature, and fast-paced progression. To this end, we report on the role of deubiquitinase BRCA1-associated protein-1 (BAP1), a tumour suppressor gene with a widely acknowledged role in the corrupted signalling and metabolism of PMe. This review aims to enhance our understanding of this devastating malignancy and propel efforts for its investigation.

[Research progress on pathogenesis of malignant mesothelioma].

The occurrence of malignant mesothelioma is related to exposure of asbestos. And many researchers have conducted in-depth analysis of the molecular changes of mesothelioma, showed that its molecular characteristics were chromosome changes, including chromosome rearrangement, gene mutation and gene deletion. Recent studies have strengthened our understanding of molecular characterization of mesothelioma, such as targeted mutations of tumor suppressor genes, differential gene expression, changes of miRNA and signal pathways. It is of great significance for the early diagnosis, clinical treatment and prognosis of malignant mesothelioma to explore the pathogenesis and development of malignant mesothelioma. This article reviews the research progress on the pathogenesis and carcinogenesis-related molecules of malignant mesothelioma.

POLD1 Is Required for Cell Cycle Progression by Overcoming DNA Damage in Malignant Pleural Mesothelioma.

BACKGROUND/AIM: The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways. MATERIALS AND METHODS: The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines. RESULTS: High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G1/S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines. CONCLUSION: POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G1/S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM.

Single-cell transcriptomic analysis of human pleura reveals stromal heterogeneity and informs in vitro models of mesothelioma.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant, including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here, we present the first comprehensive single-cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiple in vitro models of benign and malignant mesothelial cells, and characterise these through comparison with in vivo transcriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at the single-cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. It has already enabled us to shed light on the transdifferentiation of mesothelial cells, allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

Comparison of Immunohistochemistry, Next-generation Sequencing and Fluorescence In Situ Hybridization for Detection of MTAP Loss in Pleural Mesothelioma.

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2A fluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P < .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.

p53 dry gene powder enhances anti-cancer effects of chemotherapy against malignant pleural mesothelioma.

Dry gene powder is a novel non-viral gene-delivery system, which is inhalable with high gene expression. Previously, we showed that the transfection of p16INK4a or TP53 by dry gene powder resulted in growth inhibitions of lung cancer and malignant pleural mesothelioma (MPM) in vitro and in vivo. Here, we report that dry gene powder containing p53- expression-plasmid DNA enhanced the therapeutic effects of cisplatin (CDDP) against MPM even in the presence of endogenous p53. Furthermore, our results indicated that the safe transfection with a higher plasmid DNA (pDNA) concentration suppressed MPM growth independently of chemotherapeutic agents. To develop a new therapeutic alternative for MPM patients without safety concerns over "vector doses", our in vitro data provide basic understandings for dry gene powder.

Genes and Pathways Involved in the Progression of Malignant Pleural Mesothelioma: A Meta-analysis of Genome-Wide Expression Studies.

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm of the pleural tissue that lines the lungs and is mainly associated with long latency from asbestos exposure. This tumor has no effective therapeutic opportunities nowadays and has a very low five-year survival rate. In this sense, identifying molecular events that trigger the development and progression of this tumor is highly important to establish new and potentially effective treatments. We conducted a meta-analysis of genome-wide expression studies publicly available at the Gene Expression Omnibus (GEO) and ArrayExpress databases. The differentially expressed genes (DEGs) were identified, and we performed functional enrichment analysis and protein-protein interaction networks (PPINs) to gain insight into the biological mechanisms underlying these genes. Additionally, we constructed survival prediction models for selected DEGs and predicted the minimum drug inhibition concentration of anticancer drugs for MPM. In total, 115 MPM tumor transcriptomes and 26 pleural tissue controls were analyzed. We identified 1046 upregulated DEGs in the MPM samples. Cellular signaling categories in tumor samples were associated with the TNF, PI3K-Akt, and AMPK pathways. The inflammatory response, regulation of cell migration, and regulation of angiogenesis were overrepresented biological processes. Expression of SOX17 and TACC1 were associated with reduced survival rates. This meta-analysis identified a list of DEGs in MPM tumors, cancer-related signaling pathways, and biological processes that were overrepresented in MPM samples. Some therapeutic targets to treat MPM are suggested, and the prognostic potential of key genes is shown.

Genomic profiling of tissue and blood predicts survival outcomes in patients with resected pleural mesothelioma.

PURPOSE: Pleural mesothelioma (PM) is an aggressive tumor still considered incurable, in part due to the lack of predictive biomarkers. Little is known about the clinical implications of molecular alterations in resectable PM tissues and blood. Here, we characterized genetic alterations to identify prognostic and predictive biomarkers in patients with resected PM. EXPERIMENTAL DESIGN: Targeted next-generation sequencing was performed in retrospective pleural tumor tissue and paired plasma samples from stage IB-IIIB resected PM. Association between prognosis and presence of specific mutations was validated in silico. RESULTS: Thirty PM tissues and paired blood samples from 12 patients were analyzed. High tissue tumor mutational burden (TMB) (>10 mutations/Mb), tissue median minor allele frequency (MAF) (>9 mutations/Mb), and blood TMB (>6 mutations/Mb), tissue KMT2C, PBRM1, PKHD1,EPHB1 and blood LIFR mutations correlated with longer disease-free survival and/or overall survival. High concordance (>80%) between tissue and blood was found for some mutations. CONCLUSIONS: Tissue TMB and MAF, blood TMB, and specific mutations correlated with outcomes in patients with resected PM and should be further studied to validate their role as prognostic biomarkers and potentially predictive factors for combinations with immune-checkpoint inhibitors. This suggest that molecular profiling could identify longer survivors in patients with resected PM.

LAG3 is an independent prognostic biomarker and potential target for immune checkpoint inhibitors in malignant pleural mesothelioma: a retrospective study.

BACKGROUND: Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor; novel LAG3 immune checkpoint inhibitors (ICIs) exhibit therapeutic activity in melanoma. The role of LAG3and ICIs of LAG3 are unknown in malignant pleural mesothelioma (MPM). This study aimed to uncover the prognostic landscape of LAG3 in multiple cancers and investigate the potential of using LAG3 as an ICIs target in patients with MPM. METHODS: We used The Cancer Genome Atlas (TCGA) cohort for assessing mRNA expression and our cohort for immunohistochemical expression. TCGA cohort were analyzed using the Wilcoxon rank-sum test to compare mRNA expression between normal and tumor tissues in multiple cancers. We used 86 MPM cases from TCGA and 38 MPM cases from our cohort to analyze the expression of LAG3 in tumor-infiltrating lymphocytes. The mean LAG3 mRNA expression was set as the cut-off and samples were classified as positive/negative for immunohistochemical expression. Overall survival (OS) of patients with MPM was determined using the Kaplan-Meier method based on LAG3 mRNA and immunohistochemical expression. OS analysis was performed using the multivariate Cox proportional hazards model. The correlation of LAG3 expression and mRNA expression of tumor immune infiltration cells (TIICs) gene markers were estimated using Spearman correlation. To identify factors affecting the correlation of LAG3 mRNA expression, a multivariate linear regression model was performed. RESULTS: LAG3 mRNA was associated with prognosis in multiple cancers. Elevated LAG3 mRNA expression was correlated with a better prognosis in MPM. LAG3 expression was detected immunohistochemically in the membrane of infiltrating lymphocytes in MPM. LAG3 immunohistochemical expression was correlated with a better prognosis in MPM. The multivariate Cox proportional hazards model revealed that elevated LAG3 immunohistochemical expression indicated a better prognosis. In addition, LAG3 mRNA expression was correlated with the expression of various gene markers of TIICs, the most relevant to programmed cell death 1 (PD-1) with the multivariate linear regression model in MPM. CONCLUSIONS: LAG3 expression was correlated with prognosis in multiple cancers, particularly MPM; LAG3 is an independent prognostic biomarker of MPM. LAG3 regulates cancer immunity and is a potential target for ICIs therapy. PD-1 and LAG3 inhibitors may contribute to a better prognosis in MPM. TRIAL REGISTRATION: This study was registered with UMIN000049240 (registration day: August 19, 2022) and approved by the Institutional Review Board (approval date: August 22, 2022; approval number: 2022-0048) at Tokyo Women's Medical University.

Genomic Landscape of Pleural Mesothelioma and Therapeutic Aftermaths.

PURPOSE OF REVIEW: In this article, we provide a comprehensive analysis of recent progress in the genetic characterisation of pleural mesothelioma, and the translation of these findings to clinical practice. RECENT FINDINGS: Advancements in sequencing technology have allowed the identification of driver mutations and improved our understanding of how these mutations may shape the mesothelioma tumour microenvironment. However, the identification of frequently mutated regions including CDKN2A, BAP1 and NF2 have, to date, not yet yielded targeted therapy options that outperform standard chemo- and immunotherapies. Similarly, the association between mutational profile and the immune microenvironment or immunotherapy response is not well characterised. Further research into the link between tumour mutational profile and response to therapy is critical for identifying targetable vulnerabilities and stratifying patients for therapy.

Sarcomatoid mesothelioma diagnosed in a patient with mesothelioma in situ: a case report on morphologic differences after 9-month interval with details analysis of cytology in early-stage mesothelioma.

BACKGROUND: Overlapping morphological features of mesothelial cells have been rendered it difficult to distinguish between reactive and malignant conditions. The development of methods based on detecting genomic abnormalities using immunohistochemistry and fluorescence in situ hybridization have contributed markedly to solving this problem. It is important to identify bland mesothelioma cells on cytological screening, perform efficient genomic-based testing, and diagnose mesothelioma, because the first clinical manifestation of pleural mesothelioma is pleural effusion, which is the first sample available for pathological diagnosis. However, certain diagnostic aspects remain challenging even for experts. CASE PRESENTATION: This report describes a case of a 72-year-old man with a history of asbestos exposure who presented with pleural effusion as the first symptom and was eventually diagnosed as mesothelioma. Mesothelioma was suspected owing to prominent cell-in-cell engulfment in mesothelial cells on the first cytological sample, and the diagnosis of mesothelioma in situ was confirmed by histology. Unexpectedly, sarcomatoid morphology of mesothelioma was found in the second pathology samples 9 months after the first pathological examination. Both the mesothelioma in situ and invasive lesion showed immunohistochemical loss of methylthioadenosine phosphorylase (MTAP) and homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) on fluorescence in situ hybridization. The patient received medication therapy but died of disease progression 12 months after the diagnosis of the sarcomatoid morphology of mesothelioma. CONCLUSION: Our case suggests that cell-in-cell engulfment can be conspicuous in early-stage mesothelioma with inconspicuous nuclear atypia and few multinucleated cells. In addition, the presence of MTAP loss and CDKN2A homozygous deletion are suspected to be involved in early formation to invasive lesions and/or sarcomatoid morphology. We believe that it is important to consider genetic abnormalities when deciding on individual patient management. Furthermore, cases of mesothelioma, even those of an in situ lesion, with MTAP loss and/or CDKN2A deletion should be carefully followed up or subjected to early treatment.

[Effects of microRNA-106b on migration and invasion of human malignant pleural mesothelioma cell NCI-H2452].

Objective: To investigate the inhibitory effect of microRNA-106b in the process of migration and invasion of human malignant pleural mesothelioma cell NCI-H2452. Methods: In April 2017, the expression level of miRNA-106b in malignant pleural mesothelioma cells (NCI-H2452, MSTO-211H, NCI-H2052) and normal mesothelial cells MeT-5A was detected and analyzed. Using NCI-H2452 cells as a model, the NCI-H2452 cell model with miRNA-106b overexpression was established by transfecting miRNA-106b mimics. The expression level of miRNA-106b in the cells was detected by real-time fluorescent quantitative PCR. The effect of miRNA-106b on the migration and invasion ability of NCI-H2452 cells was analyzed. The gene expression data of malignant mesothelioma and the downstream target gene data of miRNA-106b in public databases were analyzed to screen the downstream target genes of miRNA-106b in mesothelioma cells that affect cell migration and invasion ability, and to verify the expression of this gene in NCI-H2452 cells with miRNA-106b overexpression. Results: The expression of miRNA-106b in three MPM cells was decreased compared with MeT-5A cells (P<0.001) . The expression level of miRNA-106b was significantly increased after transfection of miRNA-106b mimics (P<0.001) . The scratch migration levels of the experimental group were 28.45%±4.37%, 38.12%±4.82% and 50.06%±8.92% at 24h, 31h and 48h, respectively. Compared with the control group, the migration level decreased by 37.48%±2.65%, 49.21%±3.45% and 68.14%±3.81% (P<0.01) . The number of cell migration and invasion decreased in the experimental group compared with the control group (P<0.001) . Public databases were used to screen and analyze the possibility that TCF21 gene, as a downstream target gene, could affect the migration and invasion ability of MPM cells. The expression level of TCF21 gene was increased after transfection of miRNA-106b mimics in NCI-H2452 cells (P=0.009) . Conclusion: MiRNA-106b can inhibit the migration and invasion of NCI-H2452 cells and increase the expression of TCF21 gene.

Cullin 4B Ubiquitin Ligase Is Important for Cell Survival and Regulates TGF-ß1 Expression in Pleural Mesothelioma.

We previously demonstrated that cullin 4B (CUL4B) upregulation was associated with worse outcomes of pleural mesothelioma (PM) patients, while the overexpression of its paralog CUL4A was not associated with clinical outcomes. Here, we aimed to identify the distinct roles of CUL4B and CUL4A in PM using an siRNA approach in PM cell lines (ACC Meso-1 and Mero82) and primary culture. The knockdown of CUL4B and CUL4A resulted in significantly reduced colony formation, increased cell death, and delayed cell proliferation. Furthermore, similar to the effect of CUL4A knockdown, downregulation of CUL4B led to reduced expression of Hippo pathway genes including YAP1, CTGF, and survivin. Interestingly, CUL4B and not CUL4A knockdown reduced TGF-ß1 and MMP2 expression, suggesting a unique association of CUL4B with this pathway. However, the treatment of PM cells with exogenous TGF-ß1 following CUL4B knockdown did not rescue PM cell growth. We further analyzed ACC Meso-1 xenograft tumor tissues treated with the cullin inhibitor, pevonedistat, which targets protein neddylation, and observed the downregulation of human TGF-ß1 and MMP2. In summary, our data suggest that CUL4B overexpression is important for tumor cell growth and survival and may drive PM aggressiveness via the regulation of TGF-ß1 expression and, furthermore, reveal a new mechanism of action of pevonedistat.

Primary and hTERT-Transduced Mesothelioma-Associated Fibroblasts but Not Primary or hTERT-Transduced Mesothelial Cells Stimulate Growth of Human Mesothelioma Cells.

Pleural mesothelioma (PM) is an aggressive malignancy that develops in a unique tumor microenvironment (TME). However, cell models for studying the TME in PM are still limited. Here, we have generated and characterized novel human telomerase reverse transcriptase (hTERT)-transduced mesothelial cell and mesothelioma-associated fibroblast (Meso-CAF) models and investigated their impact on PM cell growth. Pleural mesothelial cells and Meso-CAFs were isolated from tissue of pneumothorax and PM patients, respectively. Stable expression of hTERT was induced by retroviral transduction. Primary and hTERT-transduced cells were compared with respect to doubling times, hTERT expression and activity levels, telomere lengths, proteomes, and the impact of conditioned media (CM) on PM cell growth. All transduced derivatives exhibited elevated hTERT expression and activity, and increased mean telomere lengths. Cell morphology remained unchanged, and the proteomes were similar to the corresponding primary cells. Of note, the CM of primary and hTERT-transduced Meso-CAFs stimulated PM cell growth to the same extent, while CM derived from mesothelial cells had no stimulating effect, irrespective of hTERT expression. In conclusion, all new hTERT-transduced cell models closely resemble their primary counterparts and, hence, represent valuable tools to investigate cellular interactions within the TME of PM.

Spatially resolved, high-dimensional transcriptomics sorts out the evolution of biphasic malignant pleural mesothelioma: new paradigms for immunotherapy.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a dreadful disease escaping the classical genetic model of cancer evolution and characterized by wide heterogeneity and transcriptional plasticity. Clinical evolution of MPM is marked by a progressive transdifferentiation that converts well differentiated epithelioid (E) cells into undifferentiated and pleomorphic sarcomatoid (S) phenotypes. Catching the way this transition takes place is necessary to understand how MPM develops and progresses and it is mandatory to improve patients' management and life expectancy. Bulk transcriptomic approaches, while providing a significant overview, failed to resolve the timing of this evolution and to identify the hierarchy of molecular events through which this transition takes place. METHODS: We applied a spatially resolved, high-dimensional transcriptomic approach to study MPM morphological evolution. 139 regions across 8 biphasic MPMs (B-MPMs) were profiled using the GeoMx™Digital Spatial Profiler to reconstruct the positional context of transcriptional activities and the spatial topology of MPM cells interactions. Validation was conducted on an independent large cohort of 84 MPMs by targeted digital barcoding analysis. RESULTS: Our results demonstrated the existence of a complex circular ecosystem in which, within a strong asbestos-driven inflammatory environment, MPM and immune cells affect each other to support S-transdifferentiation. We also showed that TGFB1 polarized M2-Tumor Associated Macrophages foster immune evasion and that TGFB1 expression correlates with reduced survival probability. CONCLUSIONS: Besides providing crucial insights into the multidimensional interactions governing MPM clinical evolution, these results open new perspectives to improve the use of immunotherapy in this disease.

Circulating FGF18 is decreased in pleural mesothelioma but not correlated with disease prognosis.

BACKGROUND: Pleural mesothelioma (PM) is a relatively rare malignancy with limited treatment options and dismal prognosis. We have previously found elevated FGF18 expression in PM tissue specimens compared with normal mesothelium. The objective of the current study was to further explore the role of FGF18 in PM and evaluate its suitability as a circulating biomarker. METHODS: FGF18 mRNA expression was analyzed by real-time PCR in cell lines and in silico in datasets from the Cancer Genome Atlas (TCGA). Cell lines overexpressing FGF18 were generated by retroviral transduction and cell behavior was investigated by clonogenic growth and transwell assays. Plasma was collected from 40 PM patients, six patients with pleural fibrosis, and 40 healthy controls. Circulating FGF18 was measured by ELISA and correlated to clinicopathological parameters. RESULTS: FGF18 showed high mRNA expression in PM and PM-derived cell lines. PM patients with high FGF18 mRNA expression showed a trend toward longer overall survival (OS) in the TCGA dataset. In PM cells with low endogenous FGF18 expression, forced overexpression of FGF18 resulted in reduced growth but increased migration. Surprisingly, despite the high FGF18 mRNA levels observed in PM, circulating FGF18 protein was significantly lower in PM patients and patients with pleural fibrosis than in healthy controls. No significant association of circulating FGF18 with OS or other disease parameters of PM patients was observed. CONCLUSIONS: FGF18 is not a prognostic biomarker in PM. Its role in PM tumor biology and the clinical significance of decreased plasma FGF18 in PM patients warrant further investigation.

Characterizing the distribution of alterations in mesothelioma and their correlation to morphology.

OBJECTIVES: Mesothelioma is a lethal disease that arises from the serosal lining of organ cavities. Several recurrent alterations have been observed in pleural and peritoneal -mesotheliomas, including in BAP1, NF2, and CDKN2A. Although specific histopathologic parameters have been correlated with prognosis, it is not as well known whether genetic alterations correlate with histologic findings. METHODS: We reviewed 131 mesotheliomas that had undergone next-generation sequencing (NGS) at our institutions after pathologic diagnosis. There were 109 epithelioid mesotheliomas, 18 biphasic mesotheliomas, and 4 sarcomatoid mesotheliomas. All our biphasic and sarcomatoid cases arose in the pleura. Of the epithelioid mesotheliomas, 73 were from the pleura and 36 were from the peritoneum. On average, patients were 66 years of age (range, 26-90 years) and predominantly male (92 men, 39 women). RESULTS: The most common alterations identified were in BAP1, CDKN2A, NF2, and TP53. Twelve mesotheliomas did not show a pathogenic alteration on NGS. For epithelioid mesotheliomas in the pleura, the presence of an alteration in BAP1 correlated with low nuclear grade (P = .04), but no correlation was found in the peritoneum (P = .62). Similarly, there was no correlation between the amount of solid architecture in epithelioid mesotheliomas and any alterations in the pleura (P = .55) or peritoneum (P = .13). For biphasic mesotheliomas, cases with either no alteration detected or with an alteration in BAP1 were more likely to be epithelioid predominant (>50% of the tumor, P = .0001), and biphasic mesotheliomas with other alterations detected and no alteration in BAP1 were more likely to be sarcomatoid predominant (>50% of the tumor, P = .0001). CONCLUSIONS: This study demonstrates a significant association between morphologic features associated with a better prognosis and an alteration in BAP1.

Damage-associated molecular patterns and sensing receptors based molecular subtypes in malignant pleural mesothelioma and implications for immunotherapy.

Objectives: Malignant pleural mesothelioma (MPM) is characterized as an incredibly aggressive form of cancer with a dismal diagnosis and a dearth of specific biomarkers and therapeutic options. For MPM patients, the effectiveness of immunotherapy may be influenced by damage-associated molecular pattern (DAMP)-induced immunogenic cell death (ICD).The objective of this work is to create a molecular profile associated with DAMPs to categorize MPM patients and predict their prognosis and response to immunotherapy. Methods: The RNA-seq of 397 patients (263 patients with clinical data, 57.2% male, 73.0% over 60 yrs.) were gathered from eight public datasets as a training cohort to identify the DAMPs-associated subgroups of MPMs using K-means analysis. Three validation cohorts of patients or murine were established from TCGA and GEO databases. Comparisons were made across each subtype's immune status, gene mutations, survival prognosis, and predicted response to therapy. Results: Based on the DAMPs gene expression, MPMs were categorized into two subtypes: the nuclear DAMPs subtype, which is classified by the upregulation of immune-suppressed pathways, and the inflammatory DAMPs subtype, which is distinguished by the enrichment of proinflammatory cytokine signaling. The inflammatory DAMPs subgroup had a better prognosis, while the nuclear DAMPs subgroup exhibited a worse outcome. In validation cohorts, the subtyping system was effectively verified. We further identified the genetic differences between the two DAMPs subtypes. It was projected that the inflammatory DAMPs subtype will respond to immunotherapy more favorably, suggesting that the developed clustering method may be implemented to predict the effectiveness of immunotherapy. Conclusion: We constructed a subtyping model based on ICD-associated DAMPs in MPM, which might serve as a signature to gauge the outcomes of immune checkpoint blockades. Our research may aid in the development of innovative immunomodulators as well as the advancement of precision immunotherapy for MPM.

[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.