Role In Malignant Pleural Mesothelioma Research Articles

PD-L1 expression complements CALGB prognostic scoring system in malignant pleural mesothelioma.

Programmed death ligand-1 (PD-L1) expression is a predictive biomarker in patients with lung cancer, but its role in malignant pleural mesothelioma (MPM) remains unclear. Evidence suggests that higher PD-L1 expression is correlated with worse survival. CALGB is the main scoring system used to predict the benefit of chemotherapy treatment. This study aimed to determine the prognostic value of PD-L1 expression and its addition to CALGB scoring system in patients with MPM. In this retrospective analysis, we evaluated samples with confirmed locally advanced or metastatic MPM. PD-L1 Tumor Proportional Score (TPS) was determined by immunohistochemistry at diagnosis. 73 patients were included in this study. A cutoff value of 15 was set for a high or low PD-L1 TPS. In total, 71.2% (n=52) and 28.8% (n=21) of individuals harbored low or high PD-L1 expression, respectively. PD-L1High was associated with worse median progression-free Survival (mPFS) [4.9 vs. 10.8 months; HR 2.724, 95% CI (1.44-5.14); p = 0.002] and Overall Survival (OS) [6.0 vs. 20.9 months; HR 6.87, 95% CI (3.4-8.7); p<0.001] compared to patients with PD-L1Low. Multivariate analysis confirmed that PD-L1 expression was an independent factor for PFS and OS in patients with MPM and CALGB score of 5-6. PD-L1 addition to CALGB scale improves its prognostic estimation of MPM survival and should be considered in future research.

Epithelioid Mesothelioma Patients with Very Long Survival Display Defects in DNA Repair.

DNA repair has an important role in malignant pleural mesothelioma (MPM) tumorigenesis and progression. Prognostic/predictive biomarkers for better management of MPM patients are needed. In the present manuscript, we analyzed the expression of more than 700 genes in a cohort of MPM patients to possibly find biomarkers correlated with survival. A total of 54 MPM patients, all with epithelioid histology, whose survival follow-up and formalin-fixed paraffin-embedded tumors were available, were included in the study. Gene expression profiles were evaluated using a Nanostring platform analyzing 760 genes involved in different cellular pathways. The percentages of proliferating tumor cells positive for RAD51 and BRCA1 foci were evaluated using an immunofluorescence assay, as a readout of homologous recombination repair status. Patient median survival time was 16.9 months, and based on this value, they were classified as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation in the DNA damage/repair expression score was observed in LS and VLS as compared to SS and VSS. These findings were validated by the lower number of both RAD51 and BRCA1-positive tumor cells in VLS as compared to VSS. The down-regulation of DNA repair signature in VLS was functionally validated by a lower % of RAD51 and BRCA1-positive tumor cells. If these data can be corroborated in a prospective trial, an easy, cost-effective test could be routinely used to better manage treatment in MPM patients.

EP07.01-011 Integrating PD-L1 Expression to the CALGB Prognostic Scoring System in Malignant Pleural Mesothelioma

Programmed death ligand-1 (PD-L1) expression is a predictive biomarker guiding treatment decisions with immunotherapy in lung cancer patients, but its role in malignant pleural mesothelioma (MPM) is unclear. However, evidence suggests that higher PDL-1 expression correlates with worse survival outcomes. The Cancer and Leukemia Group B (CALGB) prognostic scoring system is widely used to define prognosis in MPM. This study aims to assess the prognostic value of PDL-1 expression integrating it into the CALGB scoring system.

NF2 and Canonical Hippo-YAP Pathway Define Distinct Tumor Subsets Characterized by Different Immune Deficiency and Treatment Implications in Human Pleural Mesothelioma.

Simple SummaryIt is a long-held notion that loss-of-function mutations in negative regulators of the Hippo-YAP pathway, such as NF2, LATS1/2, have a similar potential to promote nuclear YAP activity, which is thought to play an essential role in the pathogenesis of MPM. Whether loss-of-function in these individual regulators uniformly affects the Hippo-YAP activity and contributes to a similar disease phenotype has not yet been revealed in MPM. Surprisingly and interestingly, we found in this study that loss-of-function in the upstream regulator NF2 of the Hippo pathway is linked to the aberrant activation of Hippo-YAP-independent signaling. More importantly, our work showed NF2 loss-of-function and dysregulated Hippo-YAP pathway define distinct MPM subsets that differ in molecular features, therapeutic implications, patients’ prognosis, and in particular, infiltrative immune signatures. Our findings in this study may be instrumental for the precise management of immunotherapy and/or targeted therapy for MPM patients.(1) Inactivation of the tumor suppressor NF2 is believed to play a major role in the pathogenesis of malignant pleural mesothelioma (MPM) by deregulating the Hippo-YAP signaling pathway. However, NF2 has functions beyond regulation of the Hippo pathway, raising the possibility that NF2 contributes to MPM via Hippo-independent mechanisms. (2) We performed weighted gene co-expression analysis (WGCNA) in transcriptomic and proteomic datasets obtained from The Cancer Gene Atlas (TCGA) MPM cohort to identify clusters of co-expressed genes highly correlated with NF2 and phospho (p)-YAP protein, surrogate markers of active Hippo signaling and YAP inactivation. The potential targets are experimentally validated using a cell viability assay. (3) MPM tumors with NF2 loss-of-function are not associated with changes in p-YAP level nor YAP/TAZ activity score, but are characterized by a deficient B-cell receptor (BCR) signaling pathway. Conversely, MPM tumors with YAP activation display exhausted CD8 T-cell-mediated immunity together with significantly upregulated PD-L1, which is validated in an independent MPM cohort, suggesting a potential benefit of immune-checkpoint inhibitors (ICI) in this patient subset. In support of this, mutations in core Hippo signaling components including LATS2, but not NF2, are independently associated with better overall survival in response to ICI in patients. Additionally, based on cancer cell line models, we show that MPM cells with a high Hippo-YAP activity are particularly sensitive to inhibitors of BCR-ABL/SRC, stratifying a unique MPM patient subset that may benefit from BCR-ABL/SRC therapies. Furthermore, we observe that NF2 physically interacts with a considerable number of proteins that are not involved in the canonical Hippo-YAP pathway, providing a possible explanation for its Hippo-independent role in MPM. Finally, survival analyses show that YAP/TAZ scores together with p-YAP protein level, but not NF2, predict the prognosis of MPM patients. (4) NF2 loss-of-function and dysregulated Hippo-YAP pathway define distinct MPM subsets that differ in their molecular features and prognosis, which has important clinical implications for precision oncology in MPM patients.

Characterization of soluble PD-L1 in pleural effusions of mesothelioma patients: potential implications in the immune response and prognosis.

Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients' sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients' overall survival (OS). sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model. We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62-1.01, P value = 0.062). Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM.

Extracellular vesicles as biomarkers in malignant pleural mesothelioma: A review.

Extracellular vesicles (EV) are secreted by all cells, including cancer cells, as a mode of intercellular transport and communication. The main types of EV known to date include exosomes, microvesicles and apoptotic bodies, as well as oncosomes and large oncosomes, which are specific to cancer cells. These different EV populations carry specific cargo from one cell to another to stimulate a specific response. They can be found in all body fluids and can be detected in liquid biopsies. EV released from mesothelioma cells can reveal important information about the molecules and signalling pathways involved in the development and progression of the tumour. The presence of tumour-derived EV in circulating body fluids makes them potential novel biomarkers for early diagnosis, prognostication and surveillance of cancer. In this review, we explore the characteristics and functional roles of EV reported in the literature, with a focus on their role in malignant pleural mesothelioma.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of human malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 μg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.

PO-220 RAN, a novel and promising gene for malignant pleural mesothelioma

IntroductionRAN is a member of RAS superfamily of GTPases involved in a varied range of cellular processes. Although it is widely demonstrated RAN is overexpressed in many human tumours having an essential role in malignant cell survival and cancer progression, little is known about its role in Malignant Pleural Mesothelioma (MPM). Previous studies showed the RAN gene is upregulated in mesothelioma tissues and cell lines, so it might be involved in carcinogenesis of MPM. We aimed to explore the functional role of RAN in MPM cell lines and its likely use as co-target in mesothelioma treatment.Material and methodsThe role of RAN in MPM tumorigenesis was investigated through RNA interference, on a panel of one mesothelial cell line (Met-5A) and four MPM cell lines (Mero-14, Mero-25, Istmes-2 and NCI-H28). After monitoring gene knockdown, at both the mRNA and protein levels, a phenotypic study was performed through Caspase-3/7, Sulforhodamine B, Wound-Healing and Colony Formation assays. Flow cytometry was employed to monitor cell cycle. To validate data from siRNA experiments, two different siRNA were independently used to target RAN. The gene was also knocked-out using a lentiviral CRISPR/Cas9 system in Mero-14. Cas9 endonuclease and gRNA were transduced by two different lentiviral transfer vectors.The doxycycline-regulated Cas9 induction was followed by DNA, RNA and proteins extraction to confirm the occurrence of gene disruption. TIDE analysis was carried out to monitor targeted mutations triggered by the genome editing.Results and discussionsThe siRNA-mediated knockdown was confirmed at both the mRNA and protein level in all cell lines. The silencing caused a statistically significant decrease of proliferation rate and clonogenicity in Mero-14, Mero-25 and Istmes-2.The migration ability was affected in Met-5A and Istmes-2. An increase in apoptosis was observed in all cell lines, being statistically significant only in the malignant ones. Flow cytometry analysis showed an increase of cells in G0/G1 phase and a decrease of cells in S phase, being significant in Mero-14 cell line only. RAN knock-out has been confirmed at both the mRNA and protein level, whereas the TIDE analysis is still ongoing.ConclusionThis study showed that MFAP5 is a novel myoepithelial cell marker that appears to be up-regulated in duct epithelium in DCIS and IC-NST during tumourogenesis and that its cytoplasmic expression in invasive tumours seems to have apoor prognostic role manifested by its association with poor prognostic parameters such as high grade, late stage,lymph node invasion and increased MVD.

Malignant pleural mesothelioma immune microenvironment and checkpoint expression: correlation with clinical–pathological features and intratumor heterogeneity over time

BackgroundTumor immune microenvironment (TME) plays a key role in malignant pleural mesothelioma (MPM) pathogenesis and treatment outcome, supporting a role of immune checkpoint inhibitors as anticancer approach. This study retrospectively investigated TME and programmed death ligand 1 (PD-L1) expression in naïve MPM cases and their change under chemotherapy. Patients and methodsDiagnostic biopsies of MPM patients were collected from four Italian and one Slovenian cancer centers. Pathological assessment of necrosis, inflammation, grading, and mitosis was carried out. Ki-67, PD-L1 expression, and tumor infiltrating lymphocytes were detected by immunohistochemistry. When available, the same paired sample after chemotherapy was analyzed. Pathological features and clinical characteristics were correlated to overall survival. ResultsTME and PD-L1 expression were assessed in 93 and 65 chemonaive MPM samples, respectively. Twenty-eight samples have not sufficient tumor tissue for PD-L1 expression. Sarcomatoid/biphasic samples were characterized by higher CD8+ T lymphocytes and PD-L1 expression on tumor cells, while epithelioid showed higher peritumoral CD4+ T and CD20+ B lymphocytes. Higher CD8+ T lymphocytes, CD68+ macrophages, and PD-L1 expression were associated with pathological features of aggressiveness (necrosis, grading, Ki-67). MPM cases characterized by higher CD8+ T-infiltrate showed lower response to chemotherapy and worse survival at univariate analysis. Patients stratification according to a combined score including CD8+ T lymphocytes, necrosis, mitosis, and proliferation index showed median overall survival of 11.3 months compared with 16.4 months in cases with high versus low combined score (P<0.003). Subgroup exploratory analysis of 15 paired samples before and after chemotherapy showed a significant increase in cytotoxic T lymphocytes in MPM samples and PD-L1 expression in immune cells. ConclusionsTME enriched with cytotoxic T lymphocytes is associated with higher levels of macrophages and PD-L1 expression on tumor cells and with aggressive histopathological features, lower response to chemotherapy and shorter survival. The role of chemotherapy as a tumor immunogenicity inducer should be confirmed in a larger validation set.

P3.03-002 Inducible Changes in Cell Morphology and Gene Expression Reflecting the Histological Subtypes of Mesothelioma: Topic: Mesothelioma Transitional

Malignant pleural mesothelioma (MPM) represents an aggressive malignancy with dismal prognosis and limited therapeutic options. MPM occurs in three main histological subtypes: epithelioid, sarcomatoid and biphasic, which are characterized by differences in morphological growth pattern, aggressiveness and patient prognosis. However, the mechanisms and causes responsible for the different cell morphologies are poorly understood. Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression and chemoresistance, but its role in MPM is not well understood. Fibroblast growth factor (FGF) signals promote cell growth, survival and aggressiveness in several tumors including mesothelioma. Aim of this study was to characterize growth factor-induced, EMT-like changes with respect to the MPM histological subtypes. Morphological and behavioral changes of treated cell models were analyzed by morphometry, immunoblotting and functional assays. Alterations in gene or microRNA expression were evaluated via qPCR and array hybridization. Pathway enrichment analysis was based on KEGG. In several cell lines established from biphasic MPM, treatment with FGF2 and EGF induced morphological changes reminiscent of EMT and aggressive behavior such as scattering, increased migration, proliferation and invasiveness. Inhibition of the fibroblast growth factor receptors (FGFR) or the MAPK axis via small-molecule inhibitors could prevent these changes and, in cell lines with sarcomatoid-like shape, reverse scattering and induce a more epithelioid morphology. Comparable results were obtained using an engineered FGFR1 enabling contactless activation via blue light. Analyses of genes and microRNAs regulated by FGF2 or EGF showed an overlap with previously established EMT markers but also identified several novel potential markers such as MMP1, ESM1, ETV4, PDL1, ITGA6 or BDKRB2. Blocking the FGFR or MAPK pathways resulted in the opposite regulation of these genes. Inhibition of MMP1 via siRNAs or pharmacological inhibitors prevented FGF2-induced scattering and invasiveness. In unsupervised clustering, the gene expression profiles of solvent- or cytokine-treated cells were associated with those of epithelioid and sarcomatoid MPM, respectively. Immunohistochemistry showed an association of MMP1 as well as phospho-ERK with the sarcomatoid part of tissue specimens from biphasic tumors. Pathway enrichment analysis of differentially expressed genes as well as the targets of altered microRNAs after FGF2 treatment showed that the regulated genes are assigned to categories important for cell growth and aggressive behavior. Our data characterize FGFR-mediated signals as important players in MPM aggressiveness and the morphological and behavioral plasticity of mesothelioma cells, leading to a better understanding of the link between the MPM histological subtypes and their influence on patient outcome.

A08 - SMO mutations role in malignant pleural mesothelioma: a negative prognostic factor, but possible new target?

A08 - SMO mutations role in malignant pleural mesothelioma: a negative prognostic factor, but possible new target?

Circulating activin A is a novel prognostic biomarker in malignant pleural mesothelioma – A multi-institutional study

IntroductionThe deregulation of activin expression is often observed in various malignancies. Previous studies indicate that activin A plays a protumourigenic role in malignant pleural mesothelioma (MPM). The aim of the study was to evaluate circulating activin A level as a biomarker in MPM. MethodsPlasma samples were collected from 129 MPM patients in four institutions at the time of diagnosis or before surgical resection. Samples from 45 healthy individuals and from 16 patients with non-malignant pleural diseases served as controls. Circulating activin A was measured by enzyme-linked immunosorbent assay and correlated to clinicopathological variables. ResultsPlasma activin A level was significantly elevated in MPM patients (862 ± 83 pg/ml) when compared to healthy controls (391 ± 21 pg/ml; P < 0.0001). Patients with pleuritis or fibrosis only showed a modest increase (versus controls; 625 ± 95 pg/ml; P = 0.0067). Sarcomatoid (n = 10, 1629 ± 202 pg/ml, P = 0.0019) and biphasic (n = 23, 1164 ± 233 pg/ml, P = 0.0188) morphology were associated with high activin A levels when compared to epithelioid histology (n = 94, 712 ± 75 pg/ml). The tumour volume showed a positive correlation with increased circulating activin A levels. MPM patients with below median activin A levels had a significantly longer overall survival when compared to those with high activin A levels (median survival 735 versus 365 d, P < 0.0001). Importantly, circulating activin A levels were exclusively prognostic in epithelioid MPM. ConclusionsOur findings suggest that the measurement of circulating activin A may support the histological classification of MPM and at the same time help to identify epithelioid MPM patients with poor prognosis.

Prognostic value of the lymphocyte-to-monocyte ratio and other inflammatory markers in malignant pleural mesothelioma.

Inflammation plays a role in malignant pleural mesothelioma (MPM) prognosis and symptoms. We investigated the roles of the new and old inflammatory indexes and markers in MPM prognosis. Two hundred and ninety-two MPM patients (167 male and 125 female) were included in this retrospective study. Demographic parameters were collected from the patients' files. Kaplan-Meier curves and multivariate Cox regression analyses were used for the analysis of prognosis. The mean age of the patients was 58.4years. The mean survival time was 14.6±13.0months. Twenty-four potential prognostic factors associated with a poor outcome were calculated in the univariate analysis, and 16 potential prognostic factors were associated with a poor prognosis. These 16 potential prognostic factors were also analyzed in multivariate analysis. Multivariate analysis showed that increased age, stage 3-4 disease, the non-epithelial type, a low Karnofsky performance score, a high white blood cell count, and a low lymphocyte-to-monocyte ratio (LMR) were associated with a poor prognosis. The results of the multivariate analysis showed that a decreased LMR was associated with poor survival. Patients with LMR ≤2.6 had poor survival compared with those with LMR >2.6 (mean 9.6 vs. 17.0months, respectively; p=0.004). LMR is an independent marker of prognosis in patients with MPM and is superior to the other inflammation-based markers. The inexpensive nature and easy reproducibility of the hemogram should encourage the use of the LMR in clinical practice.

F-18 FDG PET-CT Influences Target Delineation When Combined With Standard CT-Based Radiotherapy Planning in the Palliative Setting in Malignant Pleural Mesothelioma; an Exploratory Study

Background: Positron emission tomography computerized tomography (PET-CT) is useful in radiotherapy planning for lung cancer. However, its role in malignant pleural mesothelioma (MPM) is unknown. Objectives: This exploratory study investigated the possible role for PET-CT in radiotherapy planning for MPM. Patients and Methods: Patients receiving radiotherapy for the treatment of pain in MPM, had fluorodeoxyglucose (FDG) PET-CT scanning in addition to their standard CT scan. PET-CT images were then fused with CT planning images, termed Planning-PET-CT. Target volume delineation was undertaken first using CT and subsequently incorporated Planning-PET-CT. Planning treatment volume (PTV), conformity index (CI), mean distance to conformity (MDC), center of gravity distance (CGD) and standard uptake values (SUV) were examined. Results: Sixteen patients were recruited into the study. PET-CT upstaged nine patients. No association between SUV max and either survival or pain response was seen. Volumes contoured using Planning-PET-CT differed markedly from those outlined using CT alone as shown by the following parameters: CI = 0.3 (0.24 - 0.38). MDC = 21.47 (16.73 - 33.70) and CGD = 16.40 (11.80 - 33.87). The median percentage of over contouring was 44.00% (34.33 - 72.50) with 46.67% under contoured (34.00 - 55.00). Conclusions: PET-CT alters the position of the PTV in MPM and upstaged a proportion of patients. Further work to elucidate the role of Planning-PET-CT in target volume definition is justified.

Targeting receptor tyrosine kinases in malignant pleural mesothelioma: Focus on FGF-receptors

Fibroblast growth factor receptors (FGFRs) constitute a subfamily of receptor tyrosine kinases. Four different receptors, FGFR1-4, bind 18 different fibroblast growth factors (FGFs) and signal mainly along the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3 kinase (PI3K) and the phospholipase c gamma (PLCγ) pathway. Physiologically, they are major regulators of embryonic development and metabolism. Deregulation of FGFR signals is increasingly recognized to play important roles in malignant diseases and may constitute a feasible therapeutic target. We recently investigated their role in malignant pleural mesothelioma (MPM), an aggressive malignancy mainly caused by asbestos exposure and with currently limited therapeutic options. We demonstrated high expression of several FGFs/FGFRs, especially FGFR1, FGF2 and FGF18 in cultured tumor cells and tissue specimens and identified FGFR-mediated signals as major driver of MPM cell growth, survival and migration. FGFR blockade by a tyrosine kinase inhibitor or by a dominant-negative receptor construct resulted in reduced MPM growth in vitro and in vivo and, furthermore, enhanced the efficacy of chemo- or radiotherapy. Several other receptor tyrosine kinases, including EGFR, MET and AXL were found to be overexpressed in MPM but translation into clinically successful therapeutic approaches has not yet been achieved. Inhibition of FGF-receptors may have the advantage of targeting both the tumor cells as well as the tumor vasculature and should be further evaluated.

Analysis of expression of programmed cell death 1 ligand 1 (PD-L1) in malignant pleural mesothelioma (MPM).

BackgroundThe increasing incidence and poor outcome associated with MPM requires finding effective treatment for this disease. PD1/PD-L1 pathway plays a central role in tumor immune evasion and appears to be predictive and prognostic marker. PD-L1 is expressed in many different human cancers but its role in MPM has yet to be established. The aim of this study is to evaluate the expression of PD-L1 in MPM.Methods119 MPM patients (p) from two institutions between November 2002 and February 2014 were reviewed. Formalin-fixed, paraffin-embedded tissue was stained with anti-PD-L1 (clone E1L3N). Cases showing more than 1% of tumor cells expression of PD-L1 were considered positive.ResultsPD-L1 was analyzed in 77 p with tumor tissue available and was positive in 20.7% p (14 samples in membrane, 16 in cytoplasm and 4 in immune infiltrate). PD-L1 intensity was weak in 56.2%, moderate in 25% and strong in 18.7% p. There was a significant relationship between PD-L1 expression and histology (PD-L1 expression 37.5% in no-epithelioid tumor and 13.2% in epithelioid; p=0.033). The median survival in p PD-L1 positive was 4.79 vs 16.3 months in p PD-L1 negative (p=0.012).ConclusionsWe have shown PD-L1 is expressed in 20% of patients, associated with no epithelioid histology and poor prognostic in MPM. Our results suggest PD-L1 warrants further exploration in selecting p for immunotherapy.

A novel link between FMR gene and the JNK pathway provides clues to possible role in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive form of thoracic cancer with poor prognosis. While some studies have identified the molecular alterations associated with MPM, little is known about their role in MPM. For example, fragile X mental retardation (FMR) gene is up-regulated in MPM but its role in MPM is unknown. Here, utilizing Drosophila genetics, I investigate the possible role FMR may be playing in MPM. I provide evidence which suggests that FMR may contribute to tumorigenesis by up-regulating a matrix metalloprotease (MMP) and by degrading the basement membrane (BM), both important for tumor metastasis. I also demonstrate a novel link between FMR and the JNK pathway and suggest that the effects of FMR in MPM could in part be mediated by up-regulation of the JNK pathway.

Abstract 4404: CSF-1R signaling drives mesothelioma stemness and chemoresistance

Abstract Clinical management of Malignant Pleural Mesothelioma (MPM) is very challenging because of the uncommon resistance of the tumor to chemotherapy. We started from the observation that 7/7 mesothelioma cell lines tested expressed both the Colony Stimulating Factor Receptor-1 and its -ligands CSF-1 and IL-34, a signaling module mainly characterized in hematopoietic cell subpopulations. Pemetrexed and cisplatin treatment did not affect the number of mesothelioma CSF1R-expressing cells. Additionally, interference with CSF1R expression or function significantly abrogated the resistance of these cells to pemetrexed, suggesting the involvement of CSF-1R signaling in mediating chemoresistance of mesothelioma. This prompted us to isolate and characterize the CSF-1Rpos cells and we found that the purified cells expressed a complex repertoire of pluripotency markers, EMT factors and chemoresistance genes. The functions of the CSF-1R expressing MPM cells relied on the autocrine production of CSF-1 and IL-34, whose downregulation affected the viability of the purified cells. We identified AKT, NFkB and STAT3 activation as distinct features of the CSF-1Rpos cells. We showed that all three pathways contributed to the survival of the CSF1Rpos cells, with AKT exerting the most relevant effects. The forced activation of CSF-1R in untransformed mesothelial cells was sufficient to confer clonogenicity and resistance to pemetrexed, typical feature of transformed mesothelial cells. Furthermore, this induced a gene expression profile very similar to that observed in the FACS sorted CSF-1Rpos cells, suggesting a causal relationship between CSF-1R activation and cell transformation. Active AKT signaling contributed to increased levels of nuclear β-catenin in the CSF-1R expressing cells. Inhibition of AKT reduced the transcriptional activity of β-catenin dependent reporters and sensitized the CSF-1Rpos cells to pemetrexed-induced senescence, thereby reversing the resistance of the CSF1Rpos cells to pemetrexed. This correlated with downregulation of c-MYC, a functional CSF-1R target, and upregulation of p21. Finally, we recapitulated the main findings in primary mesothelioma cultures, which exhibited long-term surviving, chemoresistant CSF-1Rpos cells of mesothelial nature and sensitive to CSF-1R inhibition. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, suggests that CSF-1R signaling may have a critical pathogenic role in malignant pleural mesothelioma and therefore may represent a promising target for therapeutic intervention. Citation Format: Mario Cioce, Claudia Canino, Chandra Goparaju, Haining Yang, Michele Carbone, Harvey I. Pass. CSF-1R signaling drives mesothelioma stemness and chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2014-4404

Abstract 3556: Diagnostic potential of microRNAs (miRs) in malignant pleural mesothelioma (MPM).

Abstract Introduction: MPM is histologically difficult to distinguish from reactive mesothelial proliferations (RMPs), partly because proposed MPM-markers are not specific, reproducible or validated enough (Zimling ZG, Jørgensen A &amp; Santoni-Rugiu E, Histopathology 2012; 60(6B):E96-105). MiRs are small non-coding RNA-strands (∼22 nt) that post-transcriptionally regulate gene-expression, vital cellular processes and oncogenesis. However it is uncertain whether currently published miR-data may provide candidate diagnostic biomarkers for differentiating MPM from RMP. To pursue this goal, we performed a screening of miR-expression in FFPE diagnostic biopsies, surgically resected MPM-specimens and corresponding surrounding non-neoplastic pleura (NP). Materials and Methods: We performed a RT-qPCR-based (Exiqon®) screening of 742 human miRs’ expression in preoperative biopsies, epithelioid MPM-, and NP-specimens from 5 patients treated with extra-pleural pneumonectomy as part of trimodal protocol. The relevant identified differentially expressed miRs were validated on tissue samples from 41 independent MPM-patients by RT-qPCR TaqMan® MicroRNA Assays (Applied Biosystems). Quantification-cycles (Cq) were determined by the related 7500 RT-PCR system software, comparatively analyzing PCR-data with an internal reference-gene (RNU49). Significant (p &amp;lt; 0.05) differences between groups were detected by 1-way ANOVA with Bonferroni post-tests. Validated miR-targets were assessed by immunohistochemistry (IHC). Results: We identified significant difference in expression of 7 cancer-relevant miRs (down-regulation of miR-17-5p, -126, -143, -145 and -652 and up-regulation of miR-221 and -193a-3p) that correctly differentiated MPM from RMPs and was not influenced by chemotherapy (comparable miR-expression-levels in surgical samples and diagnostic biopsies). We furthermore tested if the 7 miRs could fulfill the recommendations of the International Mesothelioma Interest Group (IMIG) for a suitable MPM-marker (sensitivity/specificity &amp;gt; 80%) by generating ROC-curves using the RT-qPCR-data. The IMIG-criteria were not met for miR-17-5p, -221 and -143, while miR-126, -145, -193a-3p and -652 showed high sensitivity and specificity. Preliminary IHC-data of the validated miR-126 target L-type amino acid transporter 1 (LAT1) showed good correlation between miR-126 down-regulation and LAT1 up-regulation in MPM. Conclusion: The cancer-relevant miR-126, -145, -193a-3p and -652 have great potential as biomarkers for distinguishing MPM from RMPs, while miR-17-5p, -143 and -221, previously reported to be specific for MPM's histological subtypes and to play a pathogenic role in MPM, are not entirely suitable for this differential diagnostic purpose. Further studies by in situ-hybridization techniques will additionally corroborate the possible use of these miRs in MPM diagnostics. Citation Format: Morten Andersen, Morten Grauslund, Jesper Ravn, Jens B. Sørensen, Claus B. Andersen, Eric Santoni-Rugiu. Diagnostic potential of microRNAs (miRs) in malignant pleural mesothelioma (MPM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3556. doi:10.1158/1538-7445.AM2013-3556

Gli as a Novel Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.