Biology Of Malignant Pleural Mesothelioma Research Articles

Immunohistochemical expression of cAMP in fluoroedenite‑induced malignant pleural mesothelioma: Preliminary results.

Despite advances in understanding of the biology of malignant pleural mesothelioma (MPM), the prognosis of this malignancy remains poor. Although asbestos still remains the main pathogenic agent of MPM, other asbestos‑like fibers such as fluoro‑edenite (FE) fibers, induce MPM. Notable incidence and mortality rates of MPM have been found in Biancavilla, Italy, where FE fibers have been extracted from building materials for >50years. Cyclic adenosine monophosphate (cAMP) is a secondary messenger that plays a key role in several physiological and pathological mechanisms regulating protein kinase A (PKA) and the CREB pathway. Hyperactivation of the cAMP/PKA/CREB pathway is involved in many neoplastic processes, including tumor cell proliferation, invasion and metastatic spread. The present study investigated immunohistochemical expression of cAMP in patients with FE‑induced MPM, which included six males and four females with an age range of 50‑93years. There was high immunoexpression of cAMP in 5 out of 10 tumors while the remaining 5cases showed low immunoexpression. In addition, there was a correlation between cAMP overexpression and decreased survival times (mean overall survival times, 7.5months in high expression group vs. 18 months in low expression group).

Benefits and Challenges of Inhibiting EZH2 in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer that is mainly associated with prior exposure to asbestos fibers. Despite being a rare cancer, its global rate is increasing and the prognosis remains extremely poor. Over the last two decades, despite the constant research of new therapeutic options, the combination chemotherapy with cisplatin and pemetrexed has remained the only first-line therapy for MPM. The recent approval of immune checkpoint blockade (ICB)-based immunotherapy has opened new promising avenues of research. However, MPM is still a fatal cancer with no effective treatments. Enhancer of zeste homolog 2 (EZH2) is a histone methyl transferase that exerts pro-oncogenic and immunomodulatory activities in a variety of tumors. Accordingly, a growing number of studies indicate that EZH2 is also an oncogenic driver in MPM, but its effects on tumor microenvironments are still largely unexplored. This review describes the state-of-the-art of EZH2 in MPM biology and discusses its potential use both as a diagnostic and therapeutic target. We highlight current gaps of knowledge, the filling of which will likely favor the entry of EZH2 inhibitors within the treatment options for MPM patients.

Isolated BAP1 Genomic Alteration in Malignant Pleural Mesothelioma Predicts Distinct Immunogenicity with Implications for Immunotherapeutic Response.

Malignant pleural mesothelioma (MPM), an aggressive cancer of the mesothelial cells lining the pleural cavity, lacks effective treatments. Multiple somatic mutations and copy number losses in tumor suppressor genes (TSGs) BAP1, CDKN2A/B, and NF2 are frequently associated with MPM. The impact of single versus multiple genomic alterations of TSG on MPM biology, the immune tumor microenvironment, clinical outcomes, and treatment responses are unknown. Tumors with genomic alterations in BAP1 alone were associated with a longer overall patient survival rate compared to tumors with CDKN2A/B and/or NF2 alterations with or without BAP1 and formed a distinct immunogenic subtype with altered transcription factor and pathway activity patterns. CDKN2A/B genomic alterations consistently contributed to an adverse clinical outcome. Since the genomic alterations of only BAP1 was associated with the PD-1 therapy response signature and higher LAG3 and VISTA gene expression, it might be a candidate marker for immune checkpoint blockade therapy. Our results on the impact of TSG genotypes on MPM and the correlations between TSG alterations and molecular pathways provide a foundation for developing individualized MPM therapies.

ER Stress Response and Induction of Apoptosis in Malignant Pleural Mesothelioma: The Achilles Heel Targeted by the Anticancer Ruthenium Drug BOLD-100

Simple SummaryMalignant mesothelioma is a rare cancer arising from the serosal surfaces of the body, mainly from the pleural layer. This cancer, strongly linked to asbestos exposure, shows a very inauspicious prognosis. In fact, there is no efficient therapeutic treatment for malignant pleural mesothelioma (MPM). Thus, there is an urgent need to develop novel therapeutic approaches to treat this form of cancer. Our previous study showed the importance of GRP78 in MPM survival. BOLD-100 is a specific modulator of GRP78 and we have observed that it shows cytotoxicity against MPM cells. In particular, we describe that BOLD-100 increases oxidative stress and deregulates the calcium homeostasis leading to cell stress and, ultimately, to cell death. Our in vitro data strongly suggest that BOLD-100 inhibits the growth of MPM cell lines, proposing the application as a single agent, or in combination with other standard-of-care drugs, to treat MPM.Malignant mesothelioma is a rare cancer arising from the serosal surfaces of the body, mainly from the pleural layer. This cancer is strongly related to asbestos exposure and shows a very inauspicious prognosis, because there are scarce therapeutic options for this rare disease. Thus, there is an urgent need to develop novel therapeutic approaches to treat this form of cancer. To explore the biology of malignant pleural mesothelioma (MPM), we previously observed that MPM cell lines show high expression of the GRP78 protein, which is a chaperone protein and the master regulator of the unfolded protein response (UPR) that resides in the endoplasmic reticulum (ER). Based on our previous studies showing the importance of GRP78 in MPM, we observed that BOLD-100, a specific modulator of GRP78 and the UPR, shows cytotoxicity against MPM cells. Our studies demonstrated that BOLD-100 increases ROS production and Ca2+ release from the ER, leading to ER stress activation and, ultimately, to cell death. Our in vitro data strongly suggest that BOLD-100 inhibits the growth of MPM cell lines, proposing the application as a single agent, or in combination with other standard-of-care drugs, to treat MPM.

KAP1 is a new non-genetic vulnerability of malignant pleural mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is a rare and incurable cancer, which incidence is increasing in many countries. MPM escapes the classical genetic model of cancer evolution, lacking a distinctive genetic fingerprint. Omics profiling revealed extensive heterogeneity failing to identify major vulnerabilities and restraining development of MPM-oriented therapies. Here, we performed a multilayered analysis based on a functional genome-wide CRISPR/Cas9 screening integrated with patients molecular and clinical data, to identify new non-genetic vulnerabilities of MPM. We identified a core of 18 functionally-related genes as essential for MPM cells. The chromatin reader KAP1 emerged as a dependency of MPM. We showed that KAP1 supports cell growth by orchestrating the expression of a G2/M-specific program, ensuring mitosis correct execution. Targeting KAP1 transcriptional function, by using CDK9 inhibitors resulted in a dramatic loss of MPM cells viability and shutdown of the KAP1-mediated program. Validation analysis on two independent MPM-patients sets, including a consecutive, retrospective cohort of 97 MPM, confirmed KAP1 as new non-genetic dependency of MPM and proved the association of its dependent gene program with reduced patients’ survival probability. Overall these data: provided new insights into the biology of MPM delineating KAP1 and its target genes as building blocks of its clinical aggressiveness.

PD-L1, VISTA, and CD47 expression and prognosis impact in malignant pleural mesothelioma.

8562 Background: Malignant Pleural Mesothelioma (MPM) has been characterized by an immune suppressive microenvironment. The immune checkpoint (IC) VISTA is notably expressed in MPM, in contrast to other IC proteins such us PD-L1. Recently, CD47 has been described as a possible diagnostic biomarker for MPM, although its impact in prognosis has not been established yet. Methods: This is a retrospective observational study of immunotherapy naïve MPM patients. Immunochemistry (IHC) assessment of PD-L1, VISTA and CD47 protein expression was performed on tissue microarray of 46 surgical samples. Means were compared using Mann-Whitney U test. Correlation was estimated using Pearson’s coefficients. Overall survival (OS) was assessed using Kaplan–Meier curves and Cox proportional hazard models. A two-sided alpha error of 0.05 was used to assess statistical significance. Statistical analysis was conducted with Stata/SE version 16.1. Results: A total of 46 patients, 71.7% (33/46) male, were included in our study. Among them, 77.8% (35/45) had stage IIIB-IV, 84.8% (39/46) had received systemic therapy and 16.7% (7/42) had undergone radical-intent surgery. Asbestos exposure was confirmed in 65,7% (23/35) patients. Regarding the histological subtype, 71.7% (33/46) were epithelioid (Ep) and 13.0% (6/46) non-epithelioid (NEp), including 5 sarcomatoid and 1 biphasic. In IHC analysis, VISTA and CD47 were expressed in 63.0% (29/46) and 58.7% (27/46), respectively, whereas only 28.3% (13/46) patients had positive PD-L1 expression (≥1%). Median expression of VISTA, CD47 and PD-L1 in tumor samples was 41.8 (95% IC 0.5 - 50.7), 26.3 (95% IC 0 - 45.8) and 0 (95% IC 0-0.5), respectively. VISTA and CD47 expression were significantly higher in the Ep subgroup vs. the NEp subgroup (VISTA 39.4% vs 7.2% p = 0.028; CD47 37.3 vs. 0.8 p = 0.01). Additionally, we found a significant positive correlation between VISTA and CD47 protein expression (Pearson's r = 0.55, p < 0.001), which was consistent with the results we found in an independent MPM patient series from TCGA PanCancer Atlas (N = 87) based on RNA expression (r = 0.46; p < 0.001). Median OS, available in 40/46 cases, was 16.6 months (95% CI 12.03-20.43). On multivariate analysis, CD47 ≥1% expression was significantly associated with longer OS (29.7 vs 10.53 months, HR 0.35, [IC 95% 0.14-0.86]; p = 0.02) after adjusting for histological subtype, PDL1 and VISTA expression. In contrast, PD-L1 ≥1% showed a trend towards worse prognosis (10.3 vs 19.3 months), without reaching statistical significance (HR 2.23 [95% IC 0.95-5.23]; p = 0.065). No OS differences were found regarding VISTA expression. Conclusions: To our knowledge, this is the first study to describe VISTA and CD47 correlation in MPM. Moreover, we demonstrate CD47 expression to be an independent prognostic marker in MPM, suggesting C47 may play a key role in tumor biology of MPM, for which further validation and functional studies are necessary.

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

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

Abstract 1064: Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines

Abstract Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1064.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM)

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM.MethodsKDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways.ResultsLevels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth.ConclusionsThe results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Immunotherapy in malignant pleural mesothelioma: a review of literature data.

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer of the pleural surface, associated with asbestos exposure, whose incidence is still growing in some areas of the world. MPM is still considered a rare and an orphan disease with an unchanged median overall survival (OS) ranging from 8 to 14 months and no treatment advances in the last 15 years both in local and advanced disease. In the recent years, chronic inflammation of the mesothelium together with local tumor suppression plays a major role in the malignant transformation. Also, significant heterogeneity in both tumor and the microenvironment is at the basis of MPM biology. Preclinical data have demonstrated the immunogenicity and the lack of an effective antitumor response by the immune system in MPM thus paving the way to the development of immune therapeutics in this disease. Still there is no clear evidence of any predictive biomarker so that, given the close interaction between the immune infiltrate and mesothelial cells, a number of trials are ongoing to investigate the role and prognostic value of the immune microenvironment. In this review we summarize the rationale for immune therapeutics development in MPM, as well as, the relevant literature and ongoing trials of immune checkpoint inhibitors (ICIs) and vaccines used as both first-line treatment and beyond.

Comprehensive molecular profiling of pleural mesothelioma according to histologic subtype.

8555 Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited therapeutic options. Immunotherapy has emerged as an effective therapeutic strategy, particularly in the non-epithelioid MPM subgroup. An improved understanding of the molecular profile of MPM may inform targeted therapeutic approaches and provide insights into factors underlying differential sensitivity to therapies. Methods: We performed comprehensive genomic profiling of MPMs from 980 patients, with histologic subtyping on a subset (n = 340; n = 235 epithelioid, n = 48 sarcomatoid, n = 57 biphasic). Analyses included quantifying tumor mutational burden (TMB), assessing microsatellite instability (MSI), and evaluating PD-L1 expression (Dako 22C3). Results: Median TMB of MPM was 1.74 (0.87-2.61) mutations per megabase (mut/mb). Median TMB was comparable for non-epithelioid (biphasic, sarcomatoid) vs epithelioid MPM (1.25 mut/mb vs 1.25 mut/mb, p = 0.43). In the overall cohort, inactivating alterations in the following genes were most prevalent: CDKN2A (49%), BAP1 (44%), CDKN2B (42%), MTAP (35%), and NF2 (33%). MTAP loss co-occurred with CDKN2A and CDKN2B loss in > 99% and 94% of cases, respectively. MTAP loss was observed in 28% of epithelioid vs 46% of non-epithelioid MPMs (p = 0.03). BAP1 alterations were detected in 51% of epithelioid vs 36% of non-epithelioid MPMs (p = 0.81). PD-L1 expression was assessed in 308 MPMs, among which 153 (49%) had PD-L1 ≥1%, including 35 (11%) with PD-L1 ≥50%. Among 164 specimens that underwent histologic subtyping and PD-L1 evaluation, PD-L1 was expressed in 46 (71%) non-epithelioid MPMs compared to 49 (51%) epithelioid MPMs (p = 0.03). Given the high prevalence of alterations involving the two genes, we evaluated impact of BAP1 and MTAP status on PD-L1 and TMB. TMB (1.74 mut/mb vs 1.74 mut/mb) and PD-L1 expression (50% vs 54%) were comparable in BAP1-altered vs wildtype specimens. Although TMB was comparable in MTAP-deficient and MTAP-intact tumors (1.25 mut/mb vs 1.74 mut/mb), more tumors with MTAP loss expressed PD-L1 (68% vs 44%, p = 0.0004). Conclusions: Compared to epithelioid MPM, non-epithelioid tumors demonstrate comparable TMB, higher PD-L1 expression, and enrichment for MTAP loss. As MTAP is frequently altered in MPM, further study is indicated to explore the relationship between MTAP status and MPM biology, including sensitivity to therapeutics such as immunotherapy and synthetic lethal approaches.

Large-scale analysis of BAP1 expression reveals novel associations with clinical and molecular features of malignant pleural mesothelioma.

BRCA1‐associated protein‐1 (BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact of BAP1 mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA‐sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT‐qPCR on 483 cases to estimate the relative proportions of epithelial‐like and mesenchymal‐like components in each tumor. Four BAP1 staining patterns were observed: single‐pattern nuclear staining (36%), single‐pattern cytoplasmic staining (25%), single‐pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild‐type BAP1 and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) with BAP1 gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial‐to‐mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra‐tumoral heterogeneity of BAP1 status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non‐epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role of BAP1 in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non‐epithelioid MPM, potentially addressing a critical need in clinical decision‐making in this disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

Metadherin Is a Prognostic Apoptosis Modulator in Mesothelioma Induced via NF-κB-Mediated Signaling

Therapies against malignant pleural mesothelioma (MPM) have yielded disappointing results, in part, because pathologic mechanisms remain obscure. In searching for rational molecular targets, we identified metadherin (MTDH), a multifunctional gene associated with several tumor types but previously unrecognized in MPM. Cox proportional hazards regression analysis delineated associations between higher MTDH expression and lower patient survival from three independent MPM cohorts (n = 349 patients). Through in vitro assays with overexpression and downregulation constructs in MPM cells, we characterized the role of MTDH. We confirmed in vivo the phenotype of altered MTDH expression in a murine xenograft model. Transcriptional regulators of MTDH were identified by chromatin immunoprecipitation. Overexpression of both MTDH mRNA (12-fold increased) and protein levels was observed in tumor tissues. MTDH stable overexpression significantly augmented proliferation, invasiveness, colony formation, chemoresistance, and an antiapoptosis phenotype, while its suppression showed opposite effects in MPM cells. Interestingly, NF-κB and c-Myc (in a feed-forward loop motif) contributed to modulating MTDH expression. Knockdown of MTDH expression profoundly retarded xenograft tumor growth. Thus, our findings support the notion that MTDH integrates upstream signals from certain transcription factors and mediates pathogenic interactions contributing to MPM traits. MTDH represents a new MPM-associated gene that can contribute to insights of MPM biology and, as such, suggest other treatment strategies.

Integrative Molecular Characterization of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of BAP1 alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene VISTA in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options. SIGNIFICANCE: Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene VISTA in epithelioid MPM.See related commentary by Aggarwal and Albelda, p. 1508.This article is highlighted in the In This Issue feature, p. 1494.

P2.06-32 YB-1 - A Key Factor in Mesothelioma Aggressive Growth and Behaviour

Malignant pleural mesothelioma (MPM) is a devastating disease characterized by aggressive growth and local invasion, poor outcome and limited therapeutic options. YB-1 is a multifunctional oncoprotein, which is often up-regulated in cancer and associated with aggressiveness and poor patient outcome. Also, YB-1 is actively secreted by cells upon various stresses. Besides numerous other functions, YB-1 has been described to stimulate cancer cell migration and invasion via regulation of EMT-related factors such as Snail and Twist. Cells were treated with LPS (20 ng/ml) or grown under hypoxic conditions (1% O2, N2 balanced) for 24 hours. YB-1 levels in supernatants were quantified via western blot. Changes in EMT markers were measured by qPCR. Cell migration and cell cycle was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. YB-1 overexpression was achieved by stable transfection with an expression plasmid and confirmed via qPCR and western blot. YB-1 is secreted by MPM cells as well as the immortalised mesothelial cell line Met-5A after exposure to LPS or under hypoxic conditions. When MPM cells as well as primary mesothelial cells derived from pericardial fluid were exposed to soluble YB-1, we observed an upregulation of EMT markers such as SNAIL and TWIST as well as significantly increased migratory capacity. Similar effects were observed in cell lines which overexpress YB-1. YB-1 overexpressing cells migrated at significantly higher speed and covered a larger area. Also, when grown at low density, EMT-like changes in cell morphology as well as scattering was observed. Additionally, while cell divisions occurred at higher frequencies, the duration of the M phase was significantly prolonged. Our data highlight a crucial role of both intracellular and soluble YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, YB-1 also interferes with the cell cycle of MPM cells. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

Abstract B36: Novel miRNAs as tissue-of-origin markers for distinguishing malignant pleural mesothelioma from lung adenocarcinoma

Abstract The outcome of patients with malignant pleural mesothelioma (MPM) is poor, and diagnosis is complicated by a lack of biomarkers capable of distinguishing primary MPM from cancers that have metastasized to the pleura. Clinical diagnosis and tissue of origin is currently assessed through the use of a panel of positive and negative markers; however, there remains a subset of cases that are not identifiable by current clinical biomarkers. Recent studies suggest that the human genome encodes more miRNAs than are currently annotated, and that the novel miRNAs may display enhanced tissue and lineage specificity. We conducted a de novo search for novel miRNAs by applying a prediction algorithm to the small RNA-sequence data in a cohort of MPM tumors (n=87) from The Cancer Genome Atlas (TCGA). This analysis yielded 424 predicted novel miRNA-like sequences, which were subsequently filtered by RNA structure, abundance, and genomic location to identify 154 previously unannotated miRNA sequences. This represents a significant increase to the repertoire of 1,597 annotated miRNAs in MPM. Protein-coding genes predicted to be targeted by these novel miRNAs, using the miRanda algorithm, include genes involved in MPM biology. One of the most highly expressed novel miRNAs identified targets the Ataxia Telangiectasia Mutated (ATM) gene. Another target gene, BRCA1 Associated Protein 1 (BAP1), is also in the DNA damage response pathway. To investigate the ability of these 154 novel miRNAs to distinguish MPM from other thoracic cancers, we assessed their expression in 1,093 lung tumors from four independent cohorts from TCGA and the BC Cancer Agency (BCCA): two adenocarcinoma (LUAD) cohorts (TCGA n=497, BCCA n=94) and two squamous cell carcinoma (LUSC) cohorts (TCGA n=467, BCCA n=35). Principal component analyses revealed that novel miRNA expression was able to unambiguously distinguish MPM from LUAD and LUSC. Furthermore, we developed an miRNA-based classifier model using the weighted voting class prediction method. A 10 novel miRNAs classifier was deduced by comparing MPM and LUAD cases from TCGA and validated by comparing MPM against LUAD cases from the BCCA cohort. Remarkably, this classifier successfully identified 86 out of the 87 MPM cases (98.8%) and 100% of LUAD cases (true positive rate = 98.85%, false positive rate = 1.15%). The strikingly high sensitivity and specificity in distinguishing MPM from LUAD illustrates the potential of using novel miRNAs to supplement current clinical markers to define MPM. Citation Format: Erin A. Marshall, Christine Anderson, Kevin W. Ng, Brenda C. Minatel, Katey S.S. Enfield, Adam P. Sage, Zhaolin Xu, Wan L. Lam, Victor D. Martinez. Novel miRNAs as tissue-of-origin markers for distinguishing malignant pleural mesothelioma from lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B36.

The TCGA malignant pleural mesothelioma (MPM) project: VISTA expression and delineation of a novel clinical-molecular subtype of MPM.

8516Background: To expand the understanding of MPM biology and pave the way for novel therapeutic strategies, The Cancer Genome Atlas (TCGA) Research Network conducted a comprehensive integrated ge...

OA02.05 Expression of miR-223 in Mesothelioma Xenografts Originates from Stromal Cells in the Tumor Microenvironment

Malignant pleural mesothelioma (MPM) is an aggressive cancer caused by asbestos exposure with limited therapeutic options. Dysregulated microRNAs play an important role in MPM biology and candidate microRNAs have been investigated as diagnostic and prognostic biomarkers or as a potential treatment targets. The role of miR-223 has previously been investigated in MPM tumor cells and was shown to act as a tumor suppressor by regulating cell mobility. Previous research indicated miR-223 to be primarily expressed by myeloid progenitor derived cells during differentiation of granulocytes and monocytes. This suggests miR-223 might have a more significant role in the inflammatory response during tumorigenesis. In this study we aimed to investigate the origin of miR-223 using mesothelioma xenograft and syngraft models. Human and mouse mesothelioma cell line-derived xenograft (MSTO-H211 and H226) and syngraft (AB1) models were established. MicroRNA profiles of xenografts were compared against profiles of their corresponding in vitro cultured cells to determine candidates. RT-qPCR using TaqMan MicroRNA assays was used to validate expression levels of miR-143-3p, miR-214-3p and miR-223-3p in tumor xenografts and syngrafts with those in corresponding cell lines in vitro. Species-specific ddPCR analysis was performed on RNA from xenograft tumors to determine the expression of human and mouse pri-miR-223. MicroRNA profiles of xenograft tumors showed significant upregulation (p < 0.05) of miR-143-3p, miR-214-3p and miR-223-3p compared to corresponding in vitro mesothelioma cell lines. Only miR-223 showed significant upregulation in both xenograft and syngraft tumors compared to corresponding in vitro mesothelioma cell lines (>10000-fold increase). Other microRNAs were not significantly different between cell lines and tumors. RNA isolated from xenograft tumors contained significantly more mouse pri-miR-223 than human pri-miR-223 (p < 0.001), with only minimal expression levels of human tumor pri-miR-223 within xenograft tumors. Mature miR-223 is significantly overexpressed in xenograft tumors compared to corresponding in vitro mesothelioma cell lines suggesting stromal contribution. Species-specific pri-miRNA confirmed miR-223 is almost exclusively expressed by the mouse stromal cells in xenograft tumors. Ultimately, localizing the expression of miR-223 to specific cell types (such as myeloid derived cells) through in situ hybridization should help identify a more biologically relevant role for miR-223 in the tumor microenvironment.

Prognostic value of several biomarkers for the patients with malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor of the pleura closely related to asbestos exposure. Rare as it is, the incidence of MPM is predicted to increase mainly as a result of a lengthy latency period from the initial asbestos exposure, making it a public health concern for the next decades. Moreover, the patients with MPM have an extremely poor prognosis due to its high resistance to conventional oncologic treatments and delayed diagnosis. Although the result of current therapeutic modalities based on patient features and clinical stages is very frustrating, great advances have been shown in the knowledge of molecular biology of MPM in recent years. This is accompanied by dozens of putative prognostic biomarkers that are actively involved in tumor biological activities. These prognostic candidates can offer us a new insight into the biological characteristics of MPM, contributing to development of individualized therapeutic strategies directed against oncogenesis and tumor progression. Thus, personalized approaches based on the molecular biology of the patient's tissue or body fluid will potentially improve the present disappointing outcome, bringing new hope for patients with MPM. This article reviews the principal and several novel biomarkers that can have an influence on prognosis, in the hope that they can provide us with a more profound understanding of the biology of this lethal disease.