Malignant Pleural Mesothelioma Tissue Research Articles

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.

Inhibition of cyclin-dependent kinase 7 down-regulates yes-associated protein expression in mesothelioma cells.

Cyclin‐dependent kinase 7 (CDK7) is a protein kinase that plays a major role in transcription initiation. Yes‐associated protein (YAP) is a main effector of the Hippo/YAP signalling pathway. Here, we investigated the role of CDK7 on YAP regulation in human malignant pleural mesothelioma (MPM). We found that in microarray samples of human MPM tissue, immunohistochemistry staining showed correlation between the expression level of CDK7 and YAP (n = 70, r = .513). In MPM cells, CDK7 expression level was significantly correlated with GTIIC reporter activity (r = .886, P = .019). Inhibition of CDK7 by siRNA decreased the YAP protein level and the GTIIC reporter activity in the MPM cell lines 211H, H290 and H2052. Degradation of the YAP protein was accelerated after CDK7 knockdown in 211H, H290 and H2052 cells. Inhibition of CDK7 reduced tumour cell migration and invasion, as well as tumorsphere formation ability. Restoration of the CDK7 gene rescued the YAP protein level and GTIIC reporter activity after siRNA knockdown in 211H and H2052 cells. Finally, we performed a co‐immunoprecipitation analysis using an anti‐YAP antibody and captured the CDK7 protein in 211H cells. Our results suggest that CDK7 inhibition reduces the YAP protein level by promoting its degradation and suppresses the migration and invasion of MPM cells. Cyclin‐dependent kinase 7 may be a promising therapeutic target for MPM.

Expression of FGFR1-4 in Malignant Pleural Mesothelioma Tissue and Corresponding Cell Lines and its Relationship to Patient Survival and FGFR Inhibitor Sensitivity.

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the current investigation, we determined the expression of all four FGFRs (FGFR1–FGFR4) by immunohistochemistry in tissue samples from 94 MPM patients. From 13 of these patients, we were able to establish stable cell lines, which were subjected to FGFR1–4 staining, transcript analysis by quantitative RT-PCR, and treatment with the FGFR inhibitor infigratinib. While FGFR1 and FGFR2 were widely expressed in MPM tissue and cell lines, FGFR3 and FGFR4 showed more restricted expression. FGFR1 and FGFR2 showed no correlation with clinicopathologic data or patient survival, but presence of FGFR3 in 42% and of FGFR4 in 7% of patients correlated with shorter overall survival. Immunostaining in cell lines was more homogenous than in the corresponding tissue samples. Neither transcript nor protein expression of FGFR1–4 correlated with response to infigratinib treatment in MPM cell lines. We conclude that FGFR3 and FGFR4, but not FGFR1 or FGFR2, have prognostic significance in MPM and that FGFR expression is not sufficient to predict FGFR inhibitor response in MPM cell lines.

MicroRNA-215-5p Treatment Suppresses Mesothelioma Progression via the MDM2-p53-Signaling Axis.

Malignant pleural mesothelioma (MPM) is an incurable, aggressive neoplasm with distinctive features, including preservation of wild-type p53, irrespective of histologic subtype. We posited that this consistent molecular characteristic represents an underexploited therapeutic target that can be approached by leveraging biologic effects of microRNA (miRNA). The Cancer Genome Atlas was surveyed to identify p53-responsive prognostic miRNA(s) in MPM. Using patient samples, invitro MPM cell lines, and murine tumor xenograft models, we verified specific gene pathways targeted by these miRNAs, and we examined their therapeutic effects. miR-215-5p is a poor prognosis miRNA downregulated in MPM tissues, which has not been recognized previously. When miR-215-5p was ectopically re-expressed in MPM cells and delivered invivo to tumor xenografts, it exerted significant cell killing by activating p53 function and inducing apoptosis. The mechanistic basis for this effect is due to combinatorial effects of a positive feedback loop of miR-215-MDM2-p53 signaling, additional mouse double minute 2 (MDM2)-p53 positive feedback loop(s) with other miRNAs such as miR-145-5p, and suppression of diverse gene targets associated with cell cycle dynamics not previously drug treatable in MPM clinical studies. Our results suggest a potential pathophysiologic role for and therapeutic significance of miR-215-5p in MPM.

The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma

Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

A combination of MTAP and BAP1 immunohistochemistry is effective for distinguishing sarcomatoid mesothelioma from fibrous pleuritis

ObjectivesHistologic diagnosis of malignant pleural mesothelioma (MPM) is not always straightforward. Loss of BRCA1-associated protein 1 (BAP1) expression as detected by immunohistochemistry (IHC) (BAP1 IHC) and homozygous deletion (HD) of 9p21 as detected by fluorescencein situ hybridization (FISH) (9p21 FISH) are effective for distinguishing malignant mesothelial proliferation from benign proliferation. We have previously reported that immunohistochemical expression of the protein product of the methylthioadenosine phosphorylase (MTAP) gene, which is localized in the 9p21 chromosomal region, is correlated with the deletion status of 9p21 FISH in MPM tissues. In this study, we investigated whether a combination of MTAP and BAP1 IHC could distinguish sarcomatoid MPM from fibrous pleuritis. Materials and MethodsWe examined IHC expressions of MTAP and BAP1 and 9p21 FISH in sarcomatoid/desmoplastic (n = 18) and biphasic MPM (n = 12) and in fibrous pleuritis (n = 17). In biphasic MPM, only sarcomatoid components were evaluated for IHC and FISH. The sensitivity and specificity of each detection assay for discriminating MPM cases from fibrous pleuritis was determined. In addition, we compared the IHC expression of MTAP with the deletion status of 9p21 FISH. ResultsMTAP IHC and BAP1 IHC showed 80% and 36.7% sensitivity, respectively, and both showed 100% specificity in differentiating MPM from fibrous pleuritis. A combination of MTAP and BAP1 IHC yielded greater sensitivity (90%) than that detected for MTAP IHC alone or BAP1 IHC alone. Moreover, a high degree of concordance was observed between the results of MTAP IHC and HD of 9p21 FISH (κ = 0.63). ConclusionsWith an accurate interpretation of results, combined MTAP and BAP1 IHC is a reliable and effective method for distinguishing sarcomatoid MPM from fibrous pleuritis.

Tumor-specific genetic variants can be detected in circulating cell-free DNA of malignant pleural mesothelioma patients

ObjectivesPatients diagnosed with malignant pleural mesothelioma (MPM) face a poor prognosis, with an overall survival plateauing at a median of one year. This can be explained by difficulties in early diagnosis, effective treatment and treatment monitoring. Circulating cell-free tumor DNA (ctDNA) is emerging as an interesting biomarker addressing some of these issues. So far, the development of ctDNA in MPM lags behind that in other tumors. In this study, the possibility of tracing tumor-specific genetic variants, identified in MPM tissue, in circulating DNA of the corresponding patients is investigated. Materials and methodsWhole exome sequencing was performed on paired tumor and germline DNA of ten MPM patients, of which five were treatment naïve. For each patient, a tumor-specific variant was selected and traced in tumor, germline and circulating DNA using droplet digital PCR in two independent runs. ResultsAll but one tumor-specific variants, selected after whole exome sequencing, were validated on primary tumor tissue using droplet digital PCR analysis. Patient-specific, selected variants could be detected in circulating DNA of three MPM patients, either in one or both independent droplet digital PCR runs. Mutated fractions in circulating DNA ranged from 0.28 to 0.9%. Interestingly, all patients whose circulating DNA samples contained tumor-specific variants, were treatment naïve. ConclusionWe demonstrated for the first time the presence of ctDNA within circulating DNA of treatment naïve MPM patients. This finding opens perspectives towards the use of ctDNA as a biomarker for (early and differential) diagnosis, treatment and treatment monitoring of MPM, which all remain challenging.

Abstract 523: miR-206 inhibits pleural mesothelioma by targeting an active KRAS/CDK4/CCND1 pathway

Abstract Introduction: Since there remains no effective, durable chemotherapy against malignant pleural mesothelioma (MPM), we propose to leverage microRNA(miR)-based approaches that recently reached phase I trial testing. We posit there are many miR with therapeutic potential yet to be identified in MPM. Methods/ Results: Our prior miRNA expression profiling results of MPM (GSE40345) were cross-referenced with a novel p-value minimization analytic technique to identify prognostic miRNA. Both MPM cell lines and tissues (tumors and normal pleura) were assessed using quantitative and functional biologic assays. In silico algorithms identified gene targets that were verified by 3'-UTR luciferase assay.In silico analyses showed underexpression of miR-206 by 12.1-fold in MPM tumors compared to normal pleura. Using our p-value minimization technique, we assessed the clinical impact of this underexpressed miR in TCGA data (n=73 MPM) and noted a significant association to worse survival for patients with lower miR-206 expression (p=0.024). We confirmed that miR-206 was significantly underexpressed in tumor tissues by qRT-PCR analysis of a new, randomly selected MPM cohort (n=41) versus normal pleura (n=14). In a set of established MPM cell lines with low endogenous miR-206 level, ectopic re-expression of miR-206 dramatically suppressed cell proliferation, invasiveness, colony foci formation, and growth in soft-agar. Treatment of non-malignant mesothelial cells MeT-5a and LP9 with ectopic miR-206 showed no untoward effects. In silico gene target predictions for miR-206 were summated across 3 databases and screened by prognostic effect based on Kaplan-Meier results from TCGA data (n=87 MPM). Interestingly, we noted several overexpressed MPM-prognostic genes (p&amp;lt;0.05) regulated by miR-206: KRAS, CDK4, and CCND1. qRT-PCR analysis of tissues confirmed elevated transcripts of KRAS, CDK4, and CCND1 in MPM. This signaling axis of KRAS/CDK4/CCND1 is important in MPM as it summates well-known dysregulated tyrosine kinase receptors (EGF, IGF-1, VEGF, MET, etc) that are upstream. The KRAS/CDK4/CCND1 axis is associated with cell cycle progression and survival of cancer cells, but is not an easily druggable target. miR-206 treatment significantly downregulated KRAS, CDK4 and CCND1 genes in MPM cell lines. Conclusion: In vitro, miR-206 exerts tumor suppressive effects in MPM via inhibition of KRAS/CDK4/CCND1 signaling. miR-206 restoration in MPM mimics simultaneous blocking of multiple tyrosine kinases. Loss of miR-206 and concomitant overexpression of KRAS, CDK4 and CCND1 formed a poor prognostic signature of MPM. Our results identify miR-206 as a rational therapeutic agent to studied further in preclinical MPM models. Citation Format: Anand Singh, Roma Pahwa, Li Zhang, Nathanael Pruett, R Taylor Ripley, David S. Schrump, Chuong D. Hoang. miR-206 inhibits pleural mesothelioma by targeting an active KRAS/CDK4/CCND1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 523.

Inhibition of yes-associated protein down-regulates PD-L1 (CD274) expression in human malignant pleural mesothelioma.

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.

Expression analysis of HMGB1 in histological samples of malignant pleural mesothelioma.

High mobility group box 1 (HMGB1) is a chromatin structural protein, expressed ubiquitously in the nuclei of mammalian cells. When transported extracellularly, it acts as a tumour suppressor and oncogenic protein. In malignant pleural mesothelioma (MPM), high serum levels of HMGB1 have been related to a poor prognosis. Conversely, the significance of HMGB1 expression in MPM tissues is still unclear. Biopsy samples from 170 patients with MPM were assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HMGB1 protein and gene expression. The expression level of HMGB1 protein was scored using a semiquantitative system that sums the intensity (0-3) and the percentage (from 0 to 4) of positively stained cells in nuclei, cytoplasm and in both. The final score was considered as high (>3) or low (<3) expression. Gene expression levels were calculated using the ΔΔCt method. High expression levels of HMGB1 as total (P = 0.0011) and cytoplasmic score (P = 0.0462) were related to a worse disease-specific survival (DSS) in the entire cohort and in the clinicopathological subgroups. No significant correlation was found between HMGB1 gene expression and DSS. These findings indicate that HMGB1 may be a useful prognostic biomarker in MPM when detected by immunohistochemistry. Conversely, as it is also expressed in normal and reactive mesothelial cells, HMGB1 cannot be considered a diagnostic biomarker in histological samples of mesothelioma.

Dysregulated Expression of the MicroRNA miR-137 and Its Target YBX1 Contribute to the Invasive Characteristics of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure. On a genomic level, MPM is characterized by frequent chromosomal deletions of tumor suppressors, including microRNAs. MiR-137 plays a tumor suppressor role in other cancers, so the aim of this study was to characterize it and its target Y-box binding protein 1 (YBX1) in MPM. Expression, methylation, and copy number status of miR-137 and its host gene MIR137HG were assessed by polymerase chain reaction. Luciferase reporter assays confirmed a direct interaction between miR-137 and Y-box binding protein 1 gene (YBX1). Cells were transfected with a miR-137 inhibitor, miR-137 mimic, and/or YBX1 small interfering RNA, and growth, colony formation, migration and invasion assays were conducted. MiR-137 expression varied among MPM cell lines and tissue specimens, which was associated with copy number variation and promoter hypermethylation. High miR-137 expression was linked to poor patient survival. The miR-137 inhibitor did not affect target levels or growth, but interestingly, it increased miR-137 levels by means of mimic transfection suppressed growth, migration, and invasion, which was linked to direct YBX1 downregulation. YBX1 was overexpressed in MPM cell lines and inversely correlated with miR-137. RNA interference-mediated YBX1 knockdown significantly reduced cell growth, migration, and invasion. MiR-137 can exhibit a tumor-suppressive function in MPM by targeting YBX1. YBX1 knockdown significantly reduces tumor growth, migration, and invasion of MPM cells. Therefore, YBX1 represents a potential target for novel MPM treatment strategies.

A combination of MTAP and BAP1 immunohistochemistry in pleural effusion cytology for the diagnosis of mesothelioma

Homozygous deletion of 9p21 detected by fluorescence in situ hybridization (FISH) and loss of BRCA1-associated protein 1 (BAP1) expression detected by immunohistochemistry (IHC) are useful for the differentiation between malignant pleural mesothelioma (MPM) and reactive mesothelial hyperplasia. The authors previously described that IHC expression of the protein product of the methylthioadenosine phosphorylase (MTAP) gene, which is localized in the 9p21 chromosomal region, was correlated with the deletion status of 9p21 FISH in MPM tissues. In the current study, the authors investigated whether a combination of MTAP and BAP1 IHC could distinguish MPM from reactive mesothelial cells (RMC) in cell blocks obtained from pleural effusions. The authors examined IHC expression of MTAP and BAP1 in cell blocks obtained from pleural effusions of 45 cases of MPM and 21 cases of reactive mesothelial hyperplasia. Furthermore, IHC expression of MTAP was compared with the deletion status of 9p21 FISH. MTAP and BAP1 IHC differentiated MPM from RMC with 100% specificity for both and sensitivities of 42.2% and 60.0%, respectively. The combination of MTAP and BAP1 IHC yielded a sensitivity of 77.8%, which was higher than that of BAP1 IHC alone or 9p21 FISH alone (62.2%). Moreover, a high degree of concordance was observed between the results of MTAP IHC and 9p21 FISH in cell blocks. A combination of MTAP and BAP1 IHC in cell blocks from pleural effusions appears to be a reliable and useful method for differentiating MPM cells from RMC and can be used in the routine diagnosis of MPM. Cancer Cytopathol 2018;126:54-63. © 2017 American Cancer Society.

Abstract 30: S100A11 is a prominent therapeutic target in malignant pleural mesothelioma

Abstract [Background &amp; Purpose] Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells that show a serious malignancy in thoracic cavity with a median survival time of 9-12 months. The therapeutic approaches in clinical standard are limited to surgery and chemotherapy for early and advanced stages, respectively. The strategies unfortunately provide only palliation, thus further advanced approach is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancer tissues. It has been reported that S100A11 plays an important role in progression of many cancers derived from thyroid, ovarian and lung tissues, but its roles and signature in MPM are poorly understood. In this study, we investigated the impact of S100A11 in MPM cell lines and resected tumors. [Material &amp; Methods] We explored the expression of S100A11 in 7 MPM cell lines (HP-1, H28, MSTO-211H, H2052, H290, H2452, and YUMC44) and 2 normal mesothelial cell lines (Met-5A and LP-9). We analyzed the effect of S100A11 on cell proliferation, colony formation, migration, invasion and downstream signaling. The efficacy of anti-S100A11 antibody on cell proliferation and downstream signaling was also determined. [Results] We found that S100A11 protein was consistently upregulated in 7 MPM cell lines at a significant level in comparison to 2 normal mesothelial cells. The same phenomenon was also confirmed in MPM tissue sections by immunohistochemistry, i.e., S100A11 was strongly stained at MPM cells, but not at surrounding normal lung cells. Specific knockdown of S100A11 by small interfering RNA turned the aggressive cells into the attenuated phenotypes for proliferation, invasion and migration. Those were notably observed in both 2 MPM cell lines (H2052 and H2452). Interestingly, we found that MPM cell lines but not normal cells actively secreted the S100A11 protein. To pursue this, we next tried to inhibit the function of the secreted S100A11. Administration of S100A11 neutralizing antibody significantly inhibited the proliferation of 4 MPM cell lines (H2052, H2452, H28, and H290). The antibody had no effect on the proliferation of only one MPM cell line, MSTO-211H, which showed no secretion of S100A11. Taken together, these results suggest that S100A11 secreted from MPM cells can be a prominent target for effective MPM therapy. [Conclusion] Our results suggest that S100A11 is a possible therapeutic target in MPM. Citation Format: Hiroki Sato, Hiromasa Yamamoto, Kei Namba, Hidejiro Torigoe, Takahiro Yoshioka, Kazuhiko Shien, Junichi Soh, Shinichi Toyooka. S100A11 is a prominent therapeutic target in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2017-30

Integrated analysis of somatic mutations and immune microenvironment in malignant pleural mesothelioma

ABSTRACTTo investigate the link between the genomic landscape of cancer cells and immune microenvironment in tumor tissues, we characterized somatic mutations and tumor-infiltrating lymphocytes (TILs) in malignant pleural mesothelioma (MPM), including mutation/neoantigen load, spatial heterogeneity of somatic mutations of cancer cells and TILs (T-cell receptor β (TCRβ) repertoire), and expression profiles of immune-related genes using specimens of three different tumor sites (anterior, posterior, and diaphragm) obtained from six MPM patients. Integrated analysis identified the distinct patterns of somatic mutations and the immune microenvironment signatures both intratumorally and interindividually. MPM cases showed intratumoral heterogeneity in somatic mutations with unique TCRβ clonotypes of TILs that were restricted to each tumor site, suggesting the presence of a neoantigen-related immune response. Correlation analyses showed that higher neoantigen load was significantly correlated with stronger clonal expansion of TILs (p = 0.048) and a higher expression level of an immune-associated cytolytic factor (PRF1 (p = 0.0041) in tumor tissues), suggesting that high neoantigen loads in tumor cells might promote expansion of functional tumor-specific T cells in the tumor bed. Our results collectively indicate that MPM tumors constitute a diverse heterogeneity in both the genomic landscape and immune microenvironment, and that mutation/neoantigen load may affect the immune microenvironment in MPM tissues.

Inhibition of the platelet-derived growth factor receptor beta (PDGFRB) using gene silencing, crenolanib besylate, or imatinib mesylate hampers the malignant phenotype of mesothelioma cell lines.

Malignant pleural mesothelioma (MPM) is a cancer of the pleural cavity resistant to chemotherapy. The identification of novel therapeutic targets is needed to improve its poor prognosis. Following a review of literature and a screening of specimens we found that platelet-derived growth factor receptor beta (PDGFRB) is over-expressed, but not somatically mutated, in MPM tissues. We aimed to ascertain whether PDGFRB is a MPM-cancer driver gene. The approaches employed included the use of gene silencing and the administration of small molecules, such as crenolanib and imatinib (PDGFR inhibitors) on MPM cell lines (IstMes2, Mero-14, Mero-25). Met5A cells were used as non-malignant mesothelial cell line. PDGFRB-silencing caused a decrease in the proliferation rate, and a reduced colony formation capacity, as well as an increase of the share of cells in sub-G1 and in G2 phase, and increased apoptotic rate of MPM cell lines. Loss of migration ability was also observed. Similar, or even further enhanced, results were obtained with crenolanib. Imatinib showed the least effective activity on the phenotype. In conclusion, our study highlights PDGFRB as target with a clear role in MPM tumorigenesis and provided a rationale to explore further the efficacy of crenolanib in MPM patients, with promising results.

RETRACTED: OA02.03 Circulating Fibroblast Growth Factor 18 is Elevated in Malignant Pleural Mesothelioma Patients - A Multi-Institutional Study

Malignant pleural mesothelioma (MPM) is a rare but devastating malignancy. Despite the search for new promising treatment approaches, the outcome for most MPM patients remains dismal. Therefore, the identification of novel biomarkers is urgently needed in order to identify patients with a better prognosis and to support personalized therapeutic decisions. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens and cell models. The objective of this study was the evaluation of FGF18 as a circulating biomarker in MPM. Plasma was collected from 107 MPM patients at the time of diagnosis or before surgical resection. Samples were included from the Medical University of Vienna, University Hospital Center in Zagreb and from The Concord Repatriation General Hospital and Strathfield Private Hospital in Sydney. Samples from 49 healthy volunteers and from 8 patients with non-malignant pleural diseases served as controls. Circulating FGF18 was measured by enzyme-linked immunosorbent assay and correlated to clinical, pathologic and radiologic parameters. Plasma FGF18 level was significantly elevated in MPM patients vs. healthy controls (P<0.0001). A slight increase of circulating FGF18 level was also detected in patients with pleuritis or fibrosis (vs. control, P=0.0067). Sarcomatoid (n=7) morphology was associated with high FGF18 levels when compared to the epithelioid (n=77) histology (P=0.0064). Importantly, MPM patients presenting with FGF18 levels below the median had a significantly longer overall survival when compared to those with high FGF18 levels (median survival 625 versus 382 d, P=0.0038). Data on multivariate analysis, disease-free survival, correlation with other biomarkers and tumor volume will be presented at the conference. Our findings reveal that FGF18 is a promising blood-derived candidate biomarker in MPM. Furthermore FGF18 may support the histological classification of MPM and the identification of MPM patients with poor prognosis.

P3.03-026 Orotate Phosphoribosyltransferase is Overexpressed in Malignant Pleural Mesothelioma: Dramatically Responds One Case in High OPRT Expression: Topic: Mesothelioma Transitional

Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM. Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases. High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression. The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM.

P3.03-010 Activation of p53 in Malignant Mesothelioma: Topic: Mesothelioma Transitional

Certain microRNA (miRNA)-mRNA interactions are associated with critical biologic processes in malignant pleural mesothelioma (MPM). We wondered how miRNA interact with p53 in MPM since this tumor characteristically retains the wild-type allele which is frequently bypassed by deletion of CDKN2A. This interaction among miRNA and p53 in MPM is poorly understood. Study of this interaction could provide insights on disease mechanisms and/ or novel therapeutic strategies. We retrieved several public miRNA expression MPM data sets to perform a broad survey. We combined two meta-analyses approaches to the normalized data to maximize identifying altered miRNA specific to MPM. Then miRNAs were fit into a network where they inhibited MDM2, releasing inhibition of p53, and in turn were themselves induced by p53 (p53 regulation via a reinforcing loop). Significant miRNA of this screening algorithm were confirmed by qPCR analysis in MPM tissues and cell lines. Specific miRNAs were re-expressed in MPM cells by a lentivirus system or by mimic transfection. p53-luciferase reporter system was used to assess p53 activity. MTS assay assessed cell proliferation. Apoptotic cells were detected by Annexin-V assay. Tumorigenic characteristics of MPM cells were evaluated by clonogenicity, soft agar colony formation and 3D sphere assays. Clinical relevance of these miRNA were assessed in the TCGA MPM cohort (cancergenome.nih.gov). Our meta-analysis, revealed significant changes in several p53-regulated miRNAs. For example, miR-145 expression is repressed by 40% at steady state in MPM specimens (n=38) compared to non-malignant controls (n=21). We directly confirmed in MPM tissues a similar trend of this miRNA, while MDM2 mRNA levels were inversely higher. Next, we assessed the functional role of miR-145 in MPM cell lines with wild-type p53. Using a lentiviral expression system to sustain elevations in miR-145 levels, MDM2 transcript and protein levels were repressed, leading to increases in p53 protein and its transcriptional activity. We observed in miR-145-overexpressed MPM cell lines more apoptosis by Annexin V assay, loss of clonogenicity, growth inhibition, and attenuated tumorigenicity. To confirm that p53 can perpetuate a positive reinforcing loop inducing miR-145, we treated a panel of MPM cells with Nutlin-3a and observed coordinated increases in p53 associated with a rise in miR-145 levels. Interestingly, at least one of these miRNA was prognostic in Kaplan-Meier modeling of overall survival. We have identified candidate miRNAs that, in part, regulate p53 activity in MPM cells. These miRNAs function as tumor suppressors. They are candidates for therapeutic validation.

Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.