Prognostic risk assessment model for alternative splicing events and splicing factors in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and highly malignant thoracic tumor. Although alternative splicing (AS) is associated with tumor prognosis, the prognostic significance of AS in MPM is unknown. METHODS: Transcriptomic data, clinical information, and splicing percentage values for MPM were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox analyses were performed to establish a model affecting the prognosis of MPM. Survival and ROC analyses were used to test the effects of the prognostic model. LASSO/multivariate Cox analysis was used to construct the MPM prognostic splicing factor (SF) model. The SF-AS interaction network was analyzed using Spearman correlation and visualized using Cytoscape. The association between the MPM prognostic SF model and drug sensitivity to chemotherapeutic agents such as cisplatin was analyzed using pRRophetic.R. RESULTS: The LASSO/multivariate Cox analysis identified 41 AS events and 2 SFs that were mostly associated with survival. Nine prognostic prediction models (i.e., seven types of AS model, total AS model, and SF model) were developed. An MPM prognostic SF-AS regulatory network was subsequently constructed with decreased drug sensitivity in the SF model high-risk group (p = 0.025). CONCLUSION: This study provides the first comprehensive analysis of the prognostic value of AS events and SFs in MPM. The SF-AS regulatory network established in this study and our drug sensitivity analysis using the SF model may provide novel targets for pharmacological studies of MPM.

Assessment of miR-103a-3p in leukocytes-No diagnostic benefit in combination with the blood-based biomarkers mesothelin and calretinin for malignant pleural mesothelioma diagnosis.

Malignant pleural mesothelioma (MPM) is a cancer associated with asbestos exposure and its diagnosis is challenging due to the moderate sensitivities of the available methods. In this regard, miR-103a-3p was considered to increase the sensitivity of established biomarkers to detect MPM. Its behavior and diagnostic value in the Mexican population has not been previously evaluated. In 108 confirmed MPM cases and 218 controls, almost all formerly exposed to asbestos, we quantified miR-103-3a-3p levels in leukocytes using quantitative Real-Time PCR, together with mesothelin and calretinin measured in plasma by ELISA. Sensitivity and specificity of miR-103-3a-3p alone and in combination with mesothelin and calretinin were determined. Bivariate analysis was performed using Mann-Whitney U test and Spearman correlation. Non-conditional logistic regression models were used to calculate the area under curve (AUC), sensitivity, and specificity for the combination of biomarkers. Mesothelin and calretinin levels were higher among cases, remaining as well among males and participants ≤60 years old (only mesothelin). Significant differences for miR-103a-3p were observed between male cases and controls, whereas significant differences between cases and controls for mesothelin and calretinin were observed in men and women. At 95.5% specificity the individual sensitivity of miR-103a-3p was 4.4% in men, whereas the sensitivity of mesothelin and calretinin was 72.2% and 80.9%, respectively. Positive correlations for miR-103a-3p were observed with age, environmental asbestos exposure, years with diabetes mellitus, and glucose levels, while negative correlations were observed with years of occupational asbestos exposure, creatinine, erythrocytes, direct bilirubin, and leukocytes. The addition of miR-103a-3p to mesothelin and calretinin did not increase the diagnostic performance for MPM diagnosis. However, miR-103a-3p levels were correlated with several characteristics in the Mexican population.

Assessment of Risk of Hereditary Predisposition in Patients With Melanoma and/or Mesothelioma and Renal Neoplasia.

Importance: In BAP1 tumor predisposition syndrome, clear cell renal cell carcinoma (RCC) is frequently associated with melanoma and/or mesothelioma, while germline MITF p.E318K alterations are being increasingly reported in melanoma/RCC. Limited data exist on the co-occurrence of melanoma and/or mesothelioma with renal neoplasia and the prevalence of associated germline alterations. Objective: To assess the frequency of melanoma and/or mesothelioma co-occurring with renal neoplasia using our institutional nephrectomy registry and to determine the prevalence of BAP1 and MITF alterations within this cohort. Design, Setting, and Participants: In this genetic association study, medical records from 8295 patients from 1970 to 2018, renal neoplasia co-occurring with melanoma and/or mesothelioma within a single institutional nephrectomy registry was reevaluated based on contemporary histopathologic criteria and the medical records were reviewed. Data were analyzed from September 2019 to May 2021. Main Outcomes and Measures: Identified cases were screened for BAP1 loss using immunohistochemistry; while patients with melanoma and clear cell RCC were screened for MITF p.E318K alterations. Tumors from patients with potential germline alterations were analyzed with comprehensive molecular profiling using a 514-gene next generation sequencing panel. Results: Of a total of 8295 patients, 93 (1.1%; 95% CI, 0.9%-1.4%) had melanoma and/or mesothelioma co-occurring with renal neoplasia (cutaneous melanoma, n = 76; uveal melanoma, n = 11; mesothelioma, n = 6). A total of 69 (74.2%) were male; 24 (25.8%) were female; median age at diagnosis of renal neoplasia was 63 years (IQR, 58-70 years) and the median duration of follow-up was 8.5 years (IQR, 5.0-14.6 years). Two patients with clear cell RCC had germline BAP1 alterations in the setting of cutaneous melanoma and mesothelioma. Two patients with hybrid oncocytic tumors had biallelic inactivation of FLCN in a setting of Birt-Hogg-Dubé (BHD) syndrome associated with uveal melanoma and mesothelioma. Tumor-only screening of clear cell RCC associated with cutaneous (n = 53) and uveal melanoma (n = 6) led to the identification of 1 patient with a likely germline MITF p.E318K alteration. After excluding benign renal neoplasia (such as oncocytoma and angiomyolipoma), alterations of BAP1, FLCN, and MITF were identified in 5 of 81 patients (6.2%) with melanoma and/or mesothelioma and renal neoplasia. In contrast to hybrid oncocytic tumors in BHD, no unique genotype-phenotype correlations were seen for clear cell RCC with pathogenic BAP1/ MITF alterations and VHL loss of function variants. Four of 5 cases (80%) met current National Comprehensive Cancer Network criteria for germline testing based on a combination of age, multifocality, histologic findings, and family history. Conclusions and Relevance: In this genetic association study, findings support the continued use of these National Comprehensive Cancer Network criteria and suggest more stringent screening may be warranted in this patient population.

Assessment of signaling pathway inhibitors and identification of predictive biomarkers in malignant pleural mesothelioma.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive tumor with limited therapeutic options, requiring the development of efficient targeted therapies based on molecular phenotype of the tumor and to identify predictive biomarkers of the response. MATERIALS AND METHODS: The effect of inhibitors was investigated by cell viability assessment on primary MPM cell lines established in our laboratory from patient tumors, well characterized at the molecular level. Effects on apoptosis, cell proliferation and viability on MPM growing in multicellular spheroid were also assessed for verteporfin. Gene and protein expression, and gene knockdown by RNA interference were used to define mechanism of inhibition and specific predictive biomarkers. RESULTS: Anti-tumor effect of eight major signaling pathways inhibitors involved in mesothelial carcinogenesis was investigated. Three inhibitors were more efficient than cisplatin, the drug used as first-line chemotherapy in patients with MPM: verteporfin, a putative YAP inhibitor, defactinib, a FAK inhibitor and NSC668394, an Ezrin inhibitor. Verteporfin, the most efficient inhibitor, induced cell proliferation arrest and cell death, and is effective on 3D spheroid multicellular model. Verteporfin sensitivity was YAP-independent and related to molecular classification of the tumors. Biomarkers based on gene expression were identified to predict accurately sensitivity to these three inhibitors. CONCLUSION: Our study shows that drug screening on well-characterized MPM cells allows for the identification of novel potential therapeutic strategies and defining specific biomarkers predictive of the drug response.

Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine. Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PD-L1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi. Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response.

Possible repurposing of pyrvinium pamoate for the treatment of mesothelioma: A pre-clinical assessment.

Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, whose incidence is increasing worldwide. Unfortunately, no effective therapies are currently available and the prognosis is extremely poor. Recently, the anti-helminthic drug pyrvinium pamoate has attracted a strong interest for its anti-cancer activity, which has been demonstrated in many cancer models. Considering the previously established inhibitory effect of pyrvinium pamoate on the Wnt/ß-catenin pathway and given the important role of this pathway in MM, we investigated the potential anti-tumor activity of this drug in MM cell lines. We observed that pyrvinium pamoate significantly impairs MM cell proliferation, cloning efficiency, migration, and tumor spheroid formation. At the molecular level, our data show that pyrvinium pamoate down-regulates the expression of ß-catenin and Wnt-regulates genes. Overall, our study suggests that the repurposing of pyrvinium pamoate for MM treatment could represent a new promising therapeutic approach.

Malignant pleural mesothelioma and mesothelial hyperplasia: A new molecular tool for the differential diagnosis.

Malignant pleural mesothelioma (MPM) is a rare asbestos related cancer, aggressive and unresponsive to therapies. Histological examination of pleural lesions is the gold standard of MPM diagnosis, although it is sometimes hard to discriminate the epithelioid type of MPM from benign mesothelial hyperplasia (MH).This work aims to define a new molecular tool for the differential diagnosis of MPM, using the expression profile of 117 genes deregulated in this tumour.The gene expression analysis was performed by nanoString System on tumour tissues from 36 epithelioid MPM and 17 MH patients, and on 14 mesothelial pleural samples analysed in a blind way. Data analysis included raw nanoString data normalization, unsupervised cluster analysis by Pearson correlation, non-parametric Mann Whitney U-test and molecular classification by the Uncorrelated Shrunken Centroid (USC) Algorithm.The Mann-Whitney U-test found 35 genes upregulated and 31 downregulated in MPM. The unsupervised cluster analysis revealed two clusters, one composed only of MPM and one only of MH samples, thus revealing class-specific gene profiles. The Uncorrelated Shrunken Centroid algorithm identified two classifiers, one including 22 genes and the other 40 genes, able to properly classify all the samples as benign or malignant using gene expression data; both classifiers were also able to correctly determine, in a blind analysis, the diagnostic categories of all the 14 unknown samples.In conclusion we delineated a diagnostic tool combining molecular data (gene expression) and computational analysis (USC algorithm), which can be applied in the clinical practice for the differential diagnosis of MPM.

Sarcomatoid mesothelioma: future advances in diagnosis, biomolecular assessment, and therapeutic options in a poor-outcome disease.

Malignant pleural mesothelioma (MPM) is the most frequent pleural neoplasm, with asbestos exposure as one of the recognized carcinogen agents, causative in 80% of cases. The prognosis is poor; median survival of untreated cases is 6-9 months, with fewer than 5% of patients surviving 5 years. Sarcomatoid mesothelioma (SM) represents the subtype with the worst outcome and median survival ranging from 3.5 to 8 months. In the last few years, an accurate differentiation between the subtypes of MPM has become a crucial issue, due to differences in chemosensitivity and clinical outcome, and several studies have evaluated different immunohistochemical markers to better define the diagnosis. The different and worse outcome of patients with SM and, in general, nonepithelioid subtypes makes it intriguing to select these cases to better study the biomolecular profile in order to find factors linked to prognosis and/or predictive of therapeutic response. Considering recent studies on miRNA and genetic mapping, further investigation of this rare subtype might represent a field for basic and clinical-translational research providing for more tailored therapies.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. METHODS: We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. RESULTS: Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. CONCLUSIONS: We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

New strategies in pleural mesothelioma: BAP1 and NF2 as novel targets for therapeutic development and risk assessment.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer with limited therapeutic options. Recent work has focused on the frequent somatic inactivation of two tumor suppressor genes in MPM-NF2 (Neurofibromatosis type 2) and the recently identified BAP1 (BRCA associated protein 1). In addition, germline mutations in BAP1 have been identified that define a new familial cancer syndrome, which includes MPM, ocular melanoma, and other cancers. These recent advances may allow screening of high-risk individuals and the development of new therapies that target key pathways in MPM.

[Epidemiology, molecular biology, diagnostic and therapeutic strategy of malignant pleural mesothelioma in 2007 - an update]. / Prise en charge dignostique et thérapeutique du mésothéliome pleural malin en 2007.

Malignant pleural mesothelioma (MPM) is a rare tumour due to occupational asbestos exposure. The incidence of MPM will continue to increase until 2020-2030. The incidence reaches 100 cases/million/year in occupationally exposed populations as opposed to 1 case/million/year in the general population, leading to 800 to 1,000 cases per year in France. The molecular carcinogenesis of MPM is incompletely understood but alterations to genes NF2, c-met, WT1 RASSF and p16 have been described. These genes are involved in cell invasion and motility, cell division and apoptosis control. Histological diagnosis remains difficult and depends on immunohistochemical analysis as described by the French Mesopath group. Clinical diagnosis relies on thoracoscopy and large pleural biopsies, with increasing use of CT-PET for the evaluation of disease extent. Therapeutic strategy includes prophylactic irradiation following drainage or thoracoscopy to prevent tumour nodule development along drainage channels and puncture sites. In selected patients, extensive extra-pleural pneumonectomy can be performed with curative intent. First line chemotherapy is based on a combination of pemetrexed and cisplatin that has demonstrated an improvement in overall survival and quality of life in phase 3 trials. Antiangiogenic agents such as bevacizumab (Avastatin) may be of interest but need to be tested in phase 3 trials. The Mesothelioma Avastatin Pemetrexed Study (MAPS) is ongoing, coordinated by the French Thoracic Cancer Intergroup (IFCT).

Assessment of the mutations of p53 suppressor gene and Ha- and Ki-ras oncogenes in malignant mesothelioma in relation to asbestos exposure: a study of 12 American patients.

In our previous study, we found no genetic alteration in exons 1 and 2 of Ha- and Ki-ras oncogenes nor in exons 5 to 9 of the p53 suppressor gene in seven Japanese malignant mesothelioma patients exposed to asbestos. To examine further whether malignant mesothelioma due to asbestos has genetic alterations in the p53 suppressor gene and in Ha- and Ki-ras oncogenes, we analyzed point mutations of these genes in paraffin embedded operative open biopsied samples of the primary tumor of malignant mesothelioma in twelve American patients. The genetic analysis was conducted by the PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) method in all patients and by sequencing analysis of DNA bases in the two patients with suspected gene mutation. The analysis of the p53 suppressor gene showed an amino acid converting mutation of exon 7 in one patient and a polymorphism of exon 6 in another patient; the former patient was a heavy smoker with a biphasic cell type. No genetic alteration was found in exons 1 and 2 of Ha- and Ki-ras oncogenes in any of the patients. The results suggest that the effects of asbestos on the p53 suppressor gene and Ha- and Ki-ras oncogenes in malignant mesothelioma are negligible. Further studies are needed to examine whether the observed mutation of the p53 suppressor gene is due to the combined effects of asbestos and smoking or to other unknown factors.

Assessment of mutations of Ha- and Ki-ras oncogenes and the p53 suppressor gene in seven malignant mesothelioma patients exposed to asbestos--PCR-SSCP and sequencing analyses of paraffin-embedded primary tumors.

To examine whether malignant mesothelioma due to asbestos has genetic alterations in the Ha- and Ki-ras oncogenes or in the p53 suppressor gene, we analyzed the point mutations of these genes in paraffin-embedded autopsy samples of the primary tumors of malignant mesothelioma in seven asbestos patients who died from malignant mesothelioma. The genetic analysis was conducted by the polymerase chain reaction-single strand comformation polymorphysms (PCR-SSCP) method in all patients, and through the sequencing of deoxyribonucleic acid (DNA) bases in one patient. No genetic alterations were found in exons 1 or 2 of Ha- and Ki-ras oncogenes, or in exons 5 to 9 of the p53 gene, in any of the patients. Further studies on a larger number of patients are required to reach a definite conclusion concerning the genetic effects of asbestos on malignant mesothelioma.

Potential for interferon-alpha-based therapy in mesothelioma: assessment in a murine model.

Malignant mesothelioma is an aggressive tumor, usually induced by asbestos exposure, that has a poor prognosis and is unresponsive to conventional therapy. The present study was aimed at assessing the potential for interferon-alpha (IFN-alpha)-based therapies in a murine model for malignant mesothelioma. The effect of recombinant human IFN-alpha B/D on tumor growth, alone and in combination with either of two immunomodulatory and antiproliferative agents beta-carotene or alpha-difluoromethylornithine (DFMO), was assessed. The data suggest that IFN-alpha treatment is most efficacious when commenced early in tumor development. Combination of IFN-alpha with either DFMO or dietary beta-carotene supplementation improved the effect of an otherwise suboptimal IFN-alpha therapy regimen. Both IFN-alpha and beta-carotene had in vivo stimulatory effects on immune cells, perhaps indirectly by inhibiting TGF-beta generation. The immunomodulatory effects may contribute, at least in part, to the positive antitumor and clinical activities of the treatments in this model.